Fat Injection From Filling to Regeneration

100
Part I • Principles and Basic Concepts
Fig. 6-1
T is scanning electromicroscope image o adipose tissue shows how large-sized spherical adipocytes are
packed in the adipose tissue. T ere is signif cant vasculature and a capillary branch network running
between adipocytes. (Scale bar = 4 µm.)
Fig. 6-2
T is image shows whole-mount staining o adipose tissue. Harvested, unf xed adipose tissue was stained
with BODIPY (green, adipocytes), lectin (red, endothelial cells), and Hoechst 33342 (blue, nuclei).
Whole-mount histologic evaluation o the living adipose tissue showed adipocytes and the capillary
network. Note that every adipocyte directly attaches to a capillary (scale bar = 3 µm).

101
Chapter 6 • Adipose issue and the Stromal Vascular Fraction
C el
u a r C om p onent s o f A d i p ose T i ssue
Adipose tissue has many cells other than adipocytes, although adipocytes constitute more than 9 %
o adipose tissue volume. Our estimate o cellular components through ow cytometry and two- and
three-dimensional histologic evaluation is as ollows: cm3 o adipose tissue contains 5 to million cells,
including million adipocytes, million ADSCs, million VECs, and to million other cells (such as
adipose tissue–resident macrophages and lymphocytes, pericytes, and f broblasts).2,4,6
Red blood cells
White blood cells
Fresh stromal
vascular fraction
Endothelial
cells
Adipose-derived
stem/stromal cells
Others
Flow
cytometry
Cell processing center (CPC)
Liposuction
Digestion with collagenase
Stromal vascular fraction and
adipose-derived stem/stromal cells
Endothelial
cells
ADSCs
Others
Fig. 6-3
T is series o images demonstrates the isolation method and cellular composition o the stromal vascu-
lar raction (SVF) o harvested at. SVF is isolated rom lipoaspirates through collagenase digestion. SVF
contains erythrocytes, CD 5+ blood cells (leukocytes), CD + /CD − adipose-derived stem/stromal
cells (ADSCs), CD + /CD + endothelial cells, and CD 5−/CD −/CD − other cells.
ADSCs are f broblast-like cells that localize between adipocytes (co-localize with capillaries), in the ves-
sel wall and in the connective tissue; most o them show perivascular localization.7 ADSCs are consid-
ered to be progenitors o both adipocytes and vascular endothelial cells and are thus responsible or
adipose tissue turnover. ADSCs are heterogeneous and are composed o monopotent progenitor cells
and multipotent stem cells.8

102
Part I • Principles and Basic Concepts
Fig. 6-4
Localization o adipose-derived stem/stromal cells is shown. Whole-mount staining CD34 (green, both
ADSCs and endothelial cells), lectin (red, endothelial cells), and Hoechst 3334 (blue, nuclei) reveals that
ADSCs are perivascularly localized like pericytes in the capillary network, running between adipocytes.
Very recently, the existence o another type o multipotent or pluripotent stem cells (multilineage-
dif erentiating stress-enduring [Muse] cells) in human adipose tissue, labeled by SSEA-3, was suggested9;
Muse cells are not localized in the connective tissue near the vessels, or are between adipocytes very
sparsely. T ese f ndings suggest that there may be two types o adipose stem/progenitor cells: multipo-
tent “stem” cells (Muse cells) located near but outside o larger vessels, and adipose “progenitor” cells
(corresponding to ADSCs) located around capillaries.
P h ysi o o i c a n d B i o o g i c F uncti on s
o f A d i p ose- D eri ved S tem/ S troma C el
s
ADSCs are generally regarded as monopotent progenitors o adipocytes, or bipotent progenitors o ad-
ipocytes and valvular endothelial cells (VECs).8 ADSCs are also main players in any kind o adipose
tissue remodeling, such as developmental growth, obesity-related hyperplasia, and tissue repair a er
accidental damage or injury.1,2,10 Adipocyte and VEC regeneration a er at gra ing and tissue expan-
sion a er application o internal/external mechanical orce can result rom the contribution o activated
ADSCs. In experimental in vitro conditions, ADSCs can di erentiate into multiple lineages such as adi-
pocytes, chondrocytes, osteoblasts, muscle cells, and neural-like cells (Fig. 6-5). T us ADSCs are consid-
ered to be an essential element o the remodeling and repairing capacity o adipose tissue and a potential
tool or a wide spectrum o regenerative medicine.1

103
Chapter 6 • Adipose issue and the Stromal Vascular Fraction
Adipogenic

Endothelial

Osteogenic

Chondrogenic
ADSC
Fig. 6-5
ADSCs are multipotent; they can be expanded by adherent culture. Experimentally, ADSCs can in vitro
di erentiate into angiogenetic, adipogenic, osteogenic, and chondrogenic lineages by culturing in each
di erentiating medium.
S troma l V a scul a r F ra cti on F rom A d i p ose T i ssue
T e SVF is a heterogeneous cell mixture and contains all cell populations in the adipose tissue other than
adipocytes. T e SVF can be cryopreserved, although only ADSCs can generally be recovered a er thaw-
ing. I we seed SVF on the culture dish, only ADSCs will grow in regular culture media such as DMEM
and F . With such adherent culture methods, ADSCs can be expanded almost without limit and be
cryopreserved i needed.
SVF contains not only adipose tissue-resident cells such as ADSCs, VECs, pericytes, adipose-resident
macrophages and lymphocytes, as well as hemorrhage-derived blood cells.11 T ere ore the cell com-
position o SVF largely depends on the extent o hemorrhage contamination, because only a part o
adipose-derived cells are collected in SVF, but all contaminated peripheral blood cells are collected. Most
o the contaminated RBCs can be removed by a hemolysis process (using hypotonic water or a bu er), but
un ortunately all WBCs are included in the f nal product o SVF. Using ow cytometry, SVF cells are clas-
sif ed into our major populations by phenotyping (sur ace marker expression). CD45+ cells are leuko-
cytes, which contain both adipose-resident and hemorrhage-derived ones. CD45−/CD + /CD45+ cells
are VECs, while CD45−/CD −/CD 4+ cells are ADSCs (see Fig. - ). CD45−/CD −/CD 4− cells
are other cells, such as pericytes and f broblasts.6,11 T e phenotype o ADSCs will change a er culture ex-
pansion, and CD 4 expression ades away during the f rst weeks when ADSCs are cultured in DMEM.12

104
Part I • Principles and Basic Concepts
I sol a ti on M
eth od s o f th e S troma l V a scul a r F ra cti on
Collagenase (pure or crude products) is the most commonly used enzyme or SVF isolation rom li-
poaspirates. Enzymatic SVF isolation can be done within 2 hours, but the e ciency o isolation is gen-
erally less than 5 % to date. Although g o adipose tissue or lipoaspirates originally contains several
million SVF cells, the number o isolated SVF cells is less than 2 million (usually .3 to .8 million).
Liposuction product
Stromal vascular fraction
Filtration plus hemolysis

Counting of nucleate cells
Centrifugation plus
washing three times
90 minutes in total
Collagenase digestion
30 minutes
Fig. 6-6
T e standard method o SVF isolation rom lipoaspirates by collagenase digestion is shown. T e ex-
tracted cell pellet is collected, washed, and rinsed af er 3 minutes o digestion. T e SVF cells obtained
are counted with a cell counter or hemocytometer.
Some nonenzymatic methods have been reported in the literature or obtaining SVF or ADSCs. Actu-
ally, SVF can be obtained even rom the uid portion o lipoaspirate, although most o the SVF cells are
blood cells, and the number o ADSCs is very limited.11
Mechanical crushing through such methods as regular or powered liposuction can release some stromal
cells (such as ADSCs and VECs) into the aspirated uid. Similarly, shaking, mincing, or cutting the in-
tact or aspirated at can release stromal cells, although the most e cient method or device or this has
not been established. Ultrasonic cavitation has also been used or this purpose, but easible technology
or this is yet to be developed. T us collagenase digestion has been the benchmark method or extract-
ing SVF rom adipose tissue in either manual or semiautomated isolation. A number o automated or
semiautomated isolation systems are commercially available (see Chapter 7). T e number o nucleated
cells in the SVF obtained should be counted either manually or with an automated hemocytometer or

105
Chapter 6 • Adipose issue and the Stromal Vascular Fraction
nucleocell counter. T e viability o SVF cells is usually higher than 8 %. ADSCs easily grow a er plating
the SVF. A colony- orming assay can be used or quality control o the cultured ADSCs; colony- orming
cells are regularly contained at % to % in cultured ADSCs.12
C i ni ca A d mi ni stra ti on o f S troma V a scu a r F ra cti on
a n d A d i p ose- D eri ved S tem/ S troma C el
s
In cell-assisted (cell-enhanced) lipotrans er, reshly isolated SVF was originally combined with regular
at gra ing,13 and a recent study used purif ed ADSCs expanded by adherent culture.14 T e combination
o SVF or ADSCs with at gra ing is an example o homologous use. T e ADSCs are expected to di er-
entiate into adipocytes or VECs and/or release trophic actors to promote tissue repair or regeneration
a er gra ing.
In some clinical applications, SVF or ADSCs are injected into the skin or subcutaneous/muscular layer
to promote vascularity, immune-modulation or suppression, or rejuvenation and revitalization. I SVF
cells or an ADSC suspension is injected locally, the cells easily di use and migrate into the surrounding
tissue or enter the lymphatic circulatory system.15 Such un avorable migration as well as di erentiation
into unexpected lineages has to be avoided; one solution is to prepare cell aggregation/spheroids or cell
sheets be ore injection or transplantation.16 Intravenous in usion o SVF or ADSCs is also per ormed in
some clinical trials, such as or neurogenic disorders and systemic autoimmune diseases, but the asso-
ciated sa ety concerns, including embolization, are being discussed. SVF or ADSC cells have also been
injected into the joint cavities as a treatment or osteoarthritis to reduce chronic in ammation.
C onc usi on
Fundamental knowledge o the anatomy and physiology o adipose tissue is essential or plastic surgeons
per orming at gra ing and related regenerative surgery. SVF and ADSCs appear to have the potential to
promote wound healing and vascularity o ischemic tissue, revitalize stem cell–depleted f brotic tissue,
and treat the symptoms o chronic in ammatory diseases.16 Plastic surgeons need to collaborate with
pro essionals in cell biology and related f elds to establish clinical evidence o the e cacy o these treat-
ments, observing appropriate ethical care under the regulations o each country.
REFERENCES
. Yoshimura K, Suga H, Eto H. Adipose-derived stem/progenitor cells: roles in adipose tissue remodeling
and potential use or so tissue augmentation. Regen Med : , 9.
. Mashiko , Yoshimura K. How does at survive and remodel a er gra ing? Clin Plast Surg : 8 , .
. Spalding KL, Arner E, Westermark PO, et al. Dynamics o at cell turnover in humans. Nature :78 ,
8.
. Eto H, Suga H, Matsumoto D, Inoue K, Aoi N, Kato H, Araki J, Yoshimura K. Characterization o adipose
tissue structure and cellular components: di erences between aspirated adipose tissue and excised adi-
pose tissue. Plast Reconstr Surg : 87, 9.
. Pasarica M, Sereda OR, Redman LM, et al. Reduced adipose tissue oxygenation in human obesity: evi-
dence or rare action, macrophage chemotaxis, and in ammation without an angiogenic response. Dia-
betes 8:7 8, 9.

106
Part I • Principles and Basic Concepts
. Suga H, Matsumoto D, Inoue K, Shigeura , Eto H, Aoi N, Kato H, Abe H, Yoshimura K. Numerical mea-
surement o viable and nonviable adipocytes and other cellular components in aspirated at tissue. Plast
Reconstr Surg 22: 3, 2 8.
7. raktuev DO, Mer eld-Clauss S, Li J, et al. A population o multipotent CD34-positive adipose stromal
cells share pericyte and mesenchymal sur ace markers, reside in a periendothelial location, and stabilize
endothelial networks. Circ Res 2:77, 2 8.
8. Planat-Benard V, Silvestre JS, Cousin B, et al. Plasticity o human adipose lineage cells toward endothelial
cells: physiological and therapeutic perspectives. Circulation 9: 5 , 2 4.
9. Kuroda Y, Kitada M, Wakao S, et al. Unique multipotent cells in adult human mesenchymal cell popula-
tions. Proc Natl Acad Sci U S A 7:8 39, 2 .
. Yoshimura K, Eto H, Kato H, et al. In vivo manipulation o stem cells or adipose tissue repair/reconstruc-
tion. Regen Med ( Suppl):33, 2 .
. Yoshimura K, Shigeura , Matsumoto D, et al. Characterization o reshly isolated and cultured cells de-
rived rom the atty and uid portions o liposuction aspirates. J Cell Physiol 2 8: 4, 2 .
2. Bourin P, Bunnell BA, Casteilla L, Domonici M, Katz AJ, March KL, Redl H, Rubin JP, Yoshimura K,
Gimble JM. Stromal cells rom the adipose tissue-derived stromal vascular raction (SVF) and culture
expanded adipose tissue-derived stromal/stem cells: a joint statement o the International Federation or
Adipose T erapeutics and Science (IFA S) and the International Society or Cellular T erapy (ISC ).
Cytotherapy 5: 4 , 2 3.
3. Yoshimura K, Sato K, Aoi N, et al. Cell-assisted lipotrans er (CAL) or cosmetic breast augmentation:
supportive use o adipose-derived stem/stromal cells. Aesthet Plast Surg 32:48, 2 8.
4. Kølle SF, Fischer-Nielsen A, Mathiasen AB, et al. Enrichment o autologous at gra s with ex-vivo ex-
panded adipose tissue-derived stem cells or gra survival: a randomised placebo-controlled trial. Lancet
382(9898): 3, 2 3.
5. Yoshimura K, Sato K, Aoi N, et al. Ectopic f brogenesis induced by transplantation o adipose-derived
progenitor cell suspension immediately a er lipoinjection. ransplantation 85: 8 8, 2 8.
. Kuno S, Yoshimura K. Condensation o tissue and stem cells or at gra ing. Clin Plast Surg 42: 9 , 2 5.

107
C h a p t e r 7
T v (SVF) p w z -
, w p p . T stromal vascular
fraction w M R 1 9 , p - p
w p w . A p -
f , SVF p p ( p , ,
f ) v ( p ), w
w . I p
v .
I p p p w SVF p p
p x v p v , v w -
v .2,3 O , w p - v
(ADSC ). T p -
(MSC ), f , , v v
k xp -
p f .4 I p , q w k w SVF xp
ADSC w q p
MSC w.5,6
F w k, v v p p SVF
v p .7-10 A , -
SVF, p - -p v ,
Aut omat ed Syst ems or
Pr ocessing t he St r omal
Vascul ar Fr act ion and
Cal cul at ing t he Number
o St em Cel l s
John K. Fraser, Zeni Alfonso

108
Part I • Principles and Basic Concepts
harvest techniques and tissue processing methods make accurate characterization o SVF exceptionally
challenging. T e ability to conf dently characterize SVF is a key to interpretation o clinical results, par-
ticularly when di erent tissue collection and processing approaches are applied.
In this chapter we will describe the automation o adipose tissue processing and easily adoptable and
validated characterization strategies or SVF cells that we have used in f lings with regulatory authorities
in multiple jurisdictions.
A utoma ti on a nd S ta n d a rd iza ti on o f th e S troma l
V a scul a r F ra cti on
Clinical application o SVF cells requires that they be prepared using a clinical-grade processing system
and reagents within a standardized methodology. Ideally, the system, reagents, and methods used will
generate a consistent, reproducible cell SVF cell product with a reliable sa ety and e cacy prof le. We
re er to cells processed in such a manner as adipose-derived regenerative cells (ADRCs) as a means o
distinguishing them rom the cell population obtained using non-clinical-grade enzyme reagents, open
non-automated system processes, or where consistent cell product composition has not been validated.
Fig. 7-1
Celution System 800 Device
A
Celution 805 Consumable Set
B
Celase Reagent
C
Our approach led to the development o the Celution System, an automated system designed to digest,
extract, wash, and concentrate SVF cells rom adipose tissue. T e Celution System is a CE marked med-
ical device in the European Union and an investigational device in several FDA-approved clinical trials
in the United States. T is system was designed to provide high-quality, reproducible, standardized, val-
idated cell processing so that the end product could be relied on to meet a clearly def ned composition
and thus a more predictable sa ety and e cacy prof le. T e Celution System consists o a stand-alone
reusable hardware unit: the Celution Device, the Celution Consumable Set, and Celase Reagent. T e
Celase Reagent is a proprietary enzymatic reagent that digests the extracellular matrix to release the

109
Chapter 7 • Automated Systems or Processing Stromal Vascular Fraction and Calculating Stem Cells
entrapped SVF cells. In accordance with the need to apply only medical-grade reagents, Celase is manu-
actured as a sterile product under pharmaceutical standards using a mammalian product- ree system to
eliminate concern regarding transmissible spongi orm encephalopathic agents. T is ensures sa ety and
provides consistent, reproducible tissue digestion activity. Adipose tissue is introduced into the tissue
collection chamber o the Celution Device, where it is washed to remove red blood cells and debris, and
then digested with Celase. T e device subsequently moves cells and uids through a closed system o
tubing and reservoirs, concentrates cells through the use o centri ugation technology, and subsequently
removes the waste through the use o the same pumps and tubing used to deliver the uids and cells.
Since the release o the Celution System, many other automated, semiautomated, and manual adipose
tissue processing systems have been developed, such as the Cha-Station (CHA Biotech, Kangnamgu,
Republic o Korea) and Icellator ( issue Genesis, Honolulu, HI). T e Sepax system (Biosa e, Eysins,
Switzerland), a product originally designed to process umbilical cord blood, has also been used to con-
centrate adipose tissue cells a er digestion with collagenase.11 A comparison o these our systems is pro-
vided in able - . It is important to note that although each o these systems processes adipose tissue,
intrinsic di erences in their design and reagents mean that the cell product prepared by one system is
o en very distinct rom any other in terms o yield, composition o cell types, and in residual enzyme
level.12 T ese reasons make it extremely di cult to extrapolate sa ety and e cacy data obtained with one
system to that o another and emphasizes the importance o accurate characterization o the product.
Table 7-1 Summary of Characteristics of Four Automated Processing Systems
Celution 800/CRS Icellator

Sepax

Cha-Station
Process

Enzymatic
centri ugation
Enzymatic
centri ugation
Centri ugation
ollowing
manual digestion
Enzymatic
centri ugation
Enzyme

GMP, sterile

GMP

Not provided

Not provided
issue input
- 6 cc

5-6 cc

5 -5 cc
5 ml × 4 = cc
Process time -8 min

65-85 min

min

-4 min
NO E: T e systems cited here are subject to ongoing development and the processing characteristics o some may change over time as
design enhancements are introduced.
GMP, Good manu acturing processes.
Although many systems are now available, very ew studies have evaluated di erent devices in a con-
trolled, head-to-head comparison. Furthermore, di erences in the techniques used to characterize the
cell population produced by these approaches requently con ound comparison between published stud-
ies. T e most comprehensive head-to-head comparison so ar was published by Aronowitz and Ellen-
horn in .12 In their study, the authors compared our di erent systems: Multi Station, Cha-Station,
Celution System, and Lipokit with MaxStem (Medi-Khan, West Hollywood, CA). T e results showed
considerable di erences in cell yield (viable nucleated cells per gram o input adipose tissue), residual
enzyme levels, and what is very important, in the composition o the cell product. For example, CD 4+
stromal cells (def ned as cells expressing CD 4 but not the endothelial marker CD or the hemato-
poietic marker CD45) composed, on average, approximately 4 % o the cell product produced by the
Celution System, but only approximately % to 8% o the cells with the other systems. Similarly, the
requency o CD 4+ /CD + vascular endothelial cells in the cell product prepared with the Celution
System was, on average, our old higher than that obtained with the other systems. Stem cell requency,
as assessed by the CFU-F assay, was also our old higher. T e most important conclusion o this paper
was that when using the same starting adipose tissue and the same cell characterization assays, di erent
processing systems yield very di erent products. T is clearly demonstrates the importance o standard-
ized characterization approaches. T is need has also been recognized by the pro essional societies in-
volved in bringing this orm o cell therapy to the clinic.13

110
Part I • Principles and Basic Concepts
D etermi na ti on o f C el
Yi e d a n d V i a bi i ty
Unlike the relatively homogeneous nature o in vitro–expanded cell therapies, primary isolates rom
adipose tissue are complex mixtures o multiple cell types ranging in diameter rom 5 to 3 µm. T e
presence o red blood cells (RBCs) at concentrations o to 2 logs greater than nucleated cells and o
lipid droplets ormed during cell processing adds a urther layer o complexity to determining what is
and what is not a viable cell. Furthermore, incomplete proteolytic cleavage o extracellular matrix and
DNA released rom damaged adipocytes can yield cell clusters. As a result, wildly disparate claims re-
garding cell yield have been made at scientif c meetings, in the literature, and in marketing materials.
Consequently, the ability to conf dently compare and interpret preclinical and clinical results between
research groups is critically diminished.
In our experience, conventional hemocytometer-based cell counting methodology using simple dye ex-
clusion or determining the cell viability proved unreliable because o the requent di culty in distin-
guishing between nucleated SVF cells, RBCs, cell-sized lipid droplets, and other actors. reatment with
commercially available ammonium chloride solutions to lyse red blood cells can help but can also lead
to arti acts, since at least some SVF cells in the immediate postdigestion period appear to be amenable
to lysis as well.
For similar reasons, particle-counting systems using image analysis–based object counts or electrical
current disruption tend to overestimate the number o nucleated cells, because the counting algorithms
used to identi y a cell are not sensitive enough to distinguish between the nucleated cells and other sim-
ilarly sized or charged acellular particles within the solution. Additionally, technical issues can arise as a
result o the larger cell clusters or ragments o extracellular matrix clogging the uid pathways o these
instruments.
Fig. 7-2
We have ound that the most reliable approach to count nucleated cells is to apply methods that stain
and identi y the nucleus. In this respect we have validated both a manual method using a uorescent
microscope and a commercially available automated system. T e manual “live/dead” method incorpo-
rates acridine orange dye that penetrates and stains nuclei o both intact viable cells and o dead cells
and ethidium bromide which does not penetrate living cells, and hence stains only dead or dying cells.
Cells are loaded onto a hemocytometer and observed by uorescent microscopy using a dual excitation/
emission f lter to simultaneously observe green and orange wavelength light. T e example above is a u-
orescent image o ethidium bromide (red, dead) and acridine orange (green, live) stained ADRCs using

111
Chapter 7 • Automated Systems or Processing Stromal Vascular Fraction and Calculating Stem Cells
the manual “live/dead” assay at an objective power o 0× . Although it is time consuming, consistency
and accuracy o the results using this technique were high. However, the reproducibility o this manual
method is dependent on the experience and technical skill level o the technician, so or more wide-
spread utility, such as in a clinical laboratory setting, a aster, less subjective, semiautomated approach is
pre erred.
Semiautomated nuclei-based optical counting methods use either a one- or two-step process. Cytori has
validated the two-step nuclei counting process using Chemometec’s NucleoCounter device. T e f rst step
measures the number o dead nucleated cells by aspirating the sample into a micro uidic cassette con-
taining uorescent propidium iodide DNA–binding dye, which can only penetrate dead cells. In the sec-
ond step, the total number o cells is determined by lysing the cell membranes to allow the dye to stain
all nuclei by adding equal volumes o a cell lysis bu er and a postlysis stabilization bu er be ore sample
acquisition into the micro uidic cassette. Digital images o uorescent nuclei are taken in an observation
window within the microcassette, and the total number o viable cells is determined by subtracting the
number o dead cells rom the total cell number. We have previously reported validation o this approach
or SVF cells prepared using the Celution System.14
In summary, the complexity o the cell product obtained rom adipose tissue, whether by enzymatic
techniques or by other means, demands the implementation o counting methods validated to ensure
accurate and reproducible results. In our experience, nuclei-based methods reliably meet this challenge,
whereas other approaches can generate inaccurate data, potentially con ounding the conclusions drawn.
D etermi na ti on o f S tem C el
F re q uency i n th e S troma
V a scu a r F ra cti on
As noted previously, SVF contains a substantial number o adipose-derived stem cells. T e requency o
these cells is estimated by applying a standard clonogenic assay known as colony orming units–f broblast
(CFU-F). In this assay, SVF cells are cultured at relatively low density (usually about 00 cells/cm2) un-
der conditions that allow the adherence and proli eration o individual cells into identif able colonies.
T e medium is replaced approximately days a er initial plating and every 3 to days a er that until
the end o the assay, usually at day a er plating. At that time the cultures are f xed, stained, and
enumerated. Because o the low plating density, the colonies grow in an isolated ashion whereby each
colony represents the clonal expansion o a single stem cell (CFU-F) in the original cell suspension. In
our laboratory, it is standard to per orm the CFU-F assay with two cell concentrations in sextuplicates
and average the our middle values to minimize assay variation. For reproducible results, it is essential
that cell count and viability be ore plating is accurately measured using DNA-based methods, as detailed
earlier.
M
u ti p a ra metri c F ow C ytometry
T e use o ow cytometry (FCM) to identi y cell sur ace proteins expressed on both mature and stem/
progenitor cells in uids such as peripheral blood, bone marrow, or umbilical cord blood has long been
considered the gold standard. T is approach has also proved use ul or characterization o the sur ace
protein phenotype o culture-expanded ADSCs.4,15 However, ew publications have rigorously evaluated
the composition o reshly isolated SVF cells. Unlike blood and marrow, SVF cells are not natively a sin-
gle cell suspension. Furthermore, the intrinsic auto uorescent properties o cells reshly isolated rom
adipose tissue are distinct rom those obtained rom cell culture or rom blood and marrow. For these
reasons, the use o a meticulously def ned and comprehensive antibody panel is essential to accurately
def ne the multiple cell subpopulations ound in SVF.

112
Part I • Principles and Basic Concepts
Fig. 7-3
Unlike whole blood, bone marrow, or cultured cells, SVF cells do not have an easily recognizable light
scatter pattern. T e population can include a signif cant amount o red blood cells, cellular debris, and
aggregates as well as a wide range o cell types o various sizes, shapes, and granularity. All o this con-
tributes to a sample in which components will generally scatter more light than samples derived rom
blood-related tissues. One o the f rst challenges in ow cytometry o SVF cells is to correctly capture
their light scatter prof le. Leukocytes (cells expressing CD45) are the most easily recognizable nucleated
cell type within dot plot prof les; there ore we generally use these cells as a re erence point to set the ini-
tial voltages or light scattering by back-gating CD45+ cells onto FSC vs SSC plots. T e example above
shows a typical ADRC dot plot demonstrating distribution o CD45+ cells (blue).
o ensure that all cell types are included in the scatter gate, the voltages are adjusted to place the lym-
phocytes in the lower le corner o the scatter plot, and then gate both smaller events as well bigger
and more re ractive (excluding the o -scale aggregates). In addition, a viability dye should be included,
particularly in preparations o SVF where the requency o dead cells is signif cant, such as greater than
0% to 0%.13
Stringent nonspecif c negative controls are needed to account or the auto uorescence o SVF. Addi-
tionally, single compensation controls are needed to correct or spillover o uorescent emissions when
multiple probes are used in a single assay tube. An accurate way to identi y positive events is the use
o a uorescence minus one (FMO) control. However, in our experience, nonidiotypic interactions are
common in some SVF preparations. In this case, an isotype control can be added to the FMO control to
veri y nonspecif c staining in any given cell population within the sample.
T e inclusion or exclusion o the “dim” population when reporting the requency o some events can also
be credited or the large variability noted in the literature regarding the percent expression reported or
CD and CD 4 markers. One should be aware that CD , or example, is also expressed by monocyte/
macrophage cells, and there ore it should not be used as the only parameter used to phenotype endo-
thelial cells.16
In conclusion, or ow cytometric characterization o SVF cells, one must take into consideration the
sensitivity o the reagents used, the combination o antibodies/ uorochromes, sample preparation, very
stringent controls, and correct compensation.17 With an appropriate selection o well-def ned antibodies,
implementation o an optimal gating strategy as described herein, and the use o appropriate controls,
identif cation o subpopulations within the SVF populations can be clearly and reproducibly achieved.

113
Chapter 7 • Automated Systems or Processing Stromal Vascular Fraction and Calculating Stem Cells
Table 7-2 Major Cell Type/Phenotype Pairs in Adipose Tissue
Cell Surface Phenotype

Probable Cell Type

Mean Frequency (%)
CD45–/CD 4bright/CD bright/CD 46+
Endothelial cells–related


5- 5
CD45–/CD 4–/CD –/CD 46+
Smooth muscle/pericytes


5-
CD45–/CD 4bright/CD –/CD 46–
Fraction containing ADSCs
5-5
CD45+ /CD 46–/CD 4–/CD –
White blood cells


5-55
Average requencies o major cell populations ound in ADRCs isolated rom adipose tissue collected rom 12 di erent consented patients
rom various local aesthetic clinics.
aking this into consideration, and having tested more than antibodies, Cytori has established a
methodology that uses our uorescent parameters; the method provides su cient capacity to subdi-
vide the SVF cells into at least our major cell subpopulations: leukocytic cells, endothelial cells, smooth
muscle–related cells, and the CD 4bright stromal raction that includes, among several other cell types,
the stem cells.
Fig. 7-4
Typical ADRC dot plot showing staining
with CD146 and CD34
T ese our major subpopulations can be resolved by the combination o our robust CD markers: CD45,
CD , CD 46, and CD 4. T is panel is consistent with the recommendations o pro essional societies
in this f eld.13,18 T e major cell types/phenotype pairs ound in adipose tissue are summarized in the
table above. T e ranges shown represent inherent donor-to-donor di erences present even when using
a standardized processing approach. Some o this variability may also be attributed to actors such as
the products and technique used or tissue harvesting, the wetting or tumescent solution composition,
and potentially the anatomic site rom which at is aspirated. Levels o epinephrine in the tumescent
uid, or instance, impact the amount o peripheral blood within the tissue and can lead to changes in
the requency o CD45+ cells. It is important to note that while CD45+ cells represent as many as ap-
proximately hal o the SVF cells, many o these cells are tissue-derived macrophages rather than blood
cells present rom incomplete washing o the tissue prior to digestion or released rom vessels within the
tissue. T at is in agreement with data reported in the literature that in healthy mammals, macrophages
can account or approximately % o adipose tissue cellularity,19 which makes them the largest group o
immune cells within adipose tissue.20

114
Part I • Principles and Basic Concepts
C oncl u si on
Current automated plat orms or processing o adipose tissue have improved and will continue to im-
prove the procedures to obtain SVF cells, as well as signif cantly reducing procedure time and the vari-
ability introduced by manual methodologies. T e goal o the eatures described is to bring standardiza-
tion to the isolation and characterization o SVF cells.
With all o this in mind, it is easy to overlook the act that there is more to SVF than just the cell com-
ponents and that actors such as the presence o bacterial endotoxin ( rom crude enzyme preparations)
and the residual level o the processing reagent also have an impact on sa ety and e cacy potential.12
Standardization o all o these actors will greatly enhance preclinical and clinical research and help to
accelerate the f eld o translation medicine using SVF cells.
REFERENCES
. Rodbell M. Metabolism o isolated at cells. I. E ects o hormones on glucose metabolism and lipolysis. J
Biol Chem 39:375, 96 .
. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilin-
eage cells rom human adipose tissue: implications or cell-based therapies. issue Eng 7: , 00 .
3. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Al onso ZC, Fraser JK, Benhaim P, Hed-
rick MH. Human adipose tissue is a source o multipotent stem cells. Mol Biol Cell 3: 79, 00 .
. De Ugarte DA, Al onso Z, Zuk PA, Elbarbary A, Zhu M, Ashjian P, Benhaim P, Hedrick MH, Fraser JK.
Di erential expression o stem cell mobilization-associated molecules on multi-lineage cells rom adipose
tissue and bone marrow. Immunol Lett 89: 67, 003.
5. Fraser JK, Wulur I, Al onso Z, Hedrick M. Fat issue: an underappreciated source o stem cells or bio-
technology. rends Biotechnol : 50, 006.
6. Caplan AI. Why are MSCs therapeutic? New data: new insight. J Pathol 7:3 8, 009.
7. Feng Z, ing J, Al onso Z, Strem BM, Fraser JK, Rutenberg J, Kuo HC, Pinkernell K. Fresh and cryopre-
served, uncultured adipose tissue-derived stem and regenerative cells ameliorate ischemia-reper usion
induced acute kidney injury. Nephrol Dial ransplant 5:387 , 0 0.
8. Houtgraa JH, den Dekker WK, van Dalen BM, Springeling , de Jong R, van Geuns RJ, Geleijnse
ML, Fernandez-Aviles F, Zijlsta F, Serruys PW, Duckers HJ. First experience in humans using adipose
tissue-derived regenerative cells in the treatment o patients with S -segment elevation myocardial in-
arction. J Am Coll Cardiol 59:539, 0 .
9. Gotoh M, Yamamoto , Kato M, et al. Regenerative treatment o male stress urinary incontinence by peri-
urethral injection o autologous adipose-derived regenerative cells: -year outcomes in patients. Int J
Urol : 9 , 0 .
0. Marino G, Moraci M, Armenia E, et al. T erapy with autologous adipose-derived regenerative cells or the
care o chronic ulcer o lower limbs in patients with peripheral arterial disease. J Surg Res 85:36, 0 3.
. Güven S, Karagianni M, Schwalbe M, et al. Validation o an automated procedure to isolate human adi-
pose tissue-derived cells by using the Sepax technology. issue Eng Part C Methods 8:575, 0 .
. Aronowitz JA, Ellenhorn JD. Adipose stromal vascular raction isolation: a head-to-head comparison o
our commercial cell separation systems. Plast Reconstr Surg 3 :93 e, 0 3.

115
Chapter 7 • Automated Systems or Processing Stromal Vascular Fraction and Calculating Stem Cells
3. Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, Redl H, Rubin JP, Yoshimura K,
Gimble JM. Stromal cells rom the adipose tissue-derived stromal vascular raction and culture expanded
adipose tissue-derived stromal/stem cells: a joint statement o the International Federation or Adipose
T erapeutics and Science (IFA S) and the International Society or Cellular T erapy (ISC ). Cytotherapy
:64 , 20 3.
4. Hicok KC, Hedrick MH. Automated isolation and processing o adipose-derived stem and regenerative
cells. In Gimble JM, Bunnell BA, eds. Adipose-Derived Stem Cells: Methods and Protocols, Methods in
Molecular Biology, vol 02. New York: Springer Science+ Business Media, 20 .
. Mitchell JB, McIntosh K, Zvonic S, et al. Immunophenotype o human adipose-derived cells: temporal
changes in stromal-associated and stem cell-associated markers. Stem Cells 24:3 6, 2006.
6. Bourlier V, Zakaro -Girard A, Miranville A, et al. Remodeling phenotype o human subcutaneous adi-
pose tissue macrophages. Circulation :806, 2008.
. Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay. Clin Lab Med 2 :469,
2003.
8. Corselli M, Crisan M, Murray IR, et al. Identif cation o perivascular mesenchymal stromal/stem cells by
ow cytometry. Cytometry A 83: 4, 20 3.
9. Weisberg SP, Hunter D, Huber R, et al. CCR2 modulates in ammatory and metabolic e ects o high- at
eeding. J Clin Invest 6: , 2006.
20. Schipper HS, Prakken B, Kalkhoven E, et al. Adipose tissue-resident immune cells: key players in immu-
nometabolism. rends Endocrinol Metab 23:40 , 20 2.

(Trang không có chữ)

117
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Det er minat ion o Fat Viabil it y
Lee L.Q. Pu, Kotaro Yoshimura
117

118
Part I • Principles and Basic Concepts
L a bora tory T ests
Laboratory tests that have been used to determine the viability o at gra s include viable adipocyte
counts, colorimetric assays, adipocyte-specif c enzyme assays, routine histologic examination, and im-
munohistochemical staining. Each test must be processed in vitro and may be time consuming; each has
been selected by investigators as a single test or in combination to determine the viability o at gra s
experimentally.
Table 8-1 Commonly Used Laboratory Tests to Determine Fat Viability
Name of the Test

What to Measure?
Viable adipocyte counts

o quantitatively detect viable adipocyte within at; enzyme digestion is
required
Colorimetric assay

A cell proli eration assay to indirectly determine viability o any living cells
in a tissue
Glycerol-3-phosphate
dehydrogenase (G3PDH) assay
An adipose-specif c test to assess the cellular unction o at using a
spectrophotometric assay
Routine histologic examination o evaluate the structural integrity o at but cannot be used to assess the
actual viability o at
Whole-mount staining

o visualize viable adipocytes and capillary network a er staining the living
adipose tissue
Immunohistochemical staining
or perilipin
An immunohistochemical method to specif cally evaluate viability o
adipocytes; it is the most reliable way to histologically visualize viable
adipocytes
VIABLE ADIPOCYTE COUNTS
Viable adipocyte counts through tryptan blue staining is a well-established test or detecting viable cells
within a specif c tissue. T e test requires enzyme digestion to release adipocytes rom a parcel o gra ed
at. However, enzyme digestion can damage many adipocytes, which may lead to the underestimation
o the viable adipocyte number. Each viable adipocyte with an intact membrane should be stained blue
under microscopy. All blue-stained cells are counted as viable adipocytes. Clearly, more blue-stained
adipocytes in a given volume should represent better viability o at gra s.
In general, g o a at gra specimen is collected. Each specimen is washed with phosphate-bu ered
solution (PBS) three times. It is then mixed with mg/cc o type I collagenase (Sigma, St. Louis, MO)
in PBS containing 5% bovine serum albumin (Sigma) or digestion and is incubated at 3 ° C in a CO2
incubator. A er -hour incubation, the digestion is terminated with 0% (v/v) etal cal serum (Sigma),
and any remaining tissue ragments are removed by straining the digested atty tissue through a piece o
large-weave gauze. T e digested at gra s are ractionated into mature adipocytes (top layer) and stromal
pellet (bottom) a er centri ugation at 200 g or 0 minutes. T e viable adipocytes are determined a er
0.4% trypan blue vital stain (Sigma) rom a 00-microliter sample with : dilution with trypan blue.
T e number o viable adipocytes is then counted with a hemocytometer under a microscope with 400×
magnif cation.7,8

119
Chapter 8 • Determination o Fat Viability
Fig. 8-1
T e normal appearance o intact viable adipocytes is shown. T is cell counting technique can be di cult
in adipocytes because o the scant cytoplasm in mature adipocytes.9 In addition, since a cell isolation
procedure is used in the test, it may not accurately re ect the viability o at gra s in the intact tissue,
because isolation o adipocytes eliminates the stromal component surrounding the adipocytes.10 Never-
theless, this test can provide meaning ul in ormation about the viability o at gra s and can serve as a
baseline test in determining the viability o at gra s.
COLORIMETRIC ASSAY
A colorimetric assay uses tetrazolium salt, 2,3-bis(2-methoxy- -nitro-5-sul ophenyl)-5-[(phenyl-amino-
carbonyl]-2H-tetrazolium hydroxide (X ), which can be reduced by active mitochondrial dehydroge-
nase in viable cells, yielding a highly colored ormazan product. Even newly dead cells will not cleave
X , so the assay is sensitive to the presence o living cells. T ere ore a quantitative in vitro colorimetric
assay o cell proli eration has been used to determine the viability o at gra s.4,6,9
Rohrich et al4 described the colorimetric assay as ollows:
T e colorimetric assay using X [2,3-bis(2-methoxy- -nitro-5-sul ophenyl)-5-[(phenyl-amino)-
carbonyl]-2H-tetrazolium hydroxide; Roche Molecular Biochemicals, Branchburg, NJ] is designed
or spectrophotometric quantif cation o cell growth and viability. T e tetrazolium salt X is me-
tabolized into a water-soluble ormalin dye through mitochondrial dehydrogenase activity and is
measured at 5 to 5 nm. T e colorimetric assay system using X (Roche) is designed or spec-
trophotometric quantif cation o cell growth and viability. T e Cell Proli eration Kit II X (Roche
Diagnostics No. 5 5) can be used to assay the viability o adipocytes through the ollowing pro-
tocol. First, the X labeling reagent and the electron-coupling reagent ( rom the kit) are prepared
in a 37° C waterbath. Culture medium (Dulbecco’s modif ed eagle medium, pH 7. ) is trans erred
( . ml) into each well (2 -well plate). One well is designated as control (media + X + no cells).
Duplicates o each sample are per ormed. A heat-treated at sample ( ° C, minutes) serves as a
negative control. Specimen aliquots ( .2 ml) are placed into each well, except or control wells. X
labeling reagent (5. ml) and electron-coupling reagent ( . ml) are mixed, and .5 ml o the mixed
X reagents is added to each well. Absorbance readings are per ormed at , , 2, and 3 hours at
2 nm and nm. At each time point, aliquots ( .75 ml) are trans erred rom each well into a
-well plate or readings (Spectramax Plus 3 ; Molecular Devices, Sunnyvale, CA). Between read-
ings, the 2 -well plate is incubated at 37° C.
T e X assay provides a good correlation between the number o viable cells within at gra s and the
resulting ormazan absorbance values.4,6,9,11 Because the assay does not rely on a cell isolation procedure,
it may accurately re ect viability o at gra s with the intact stromal component surrounding the at
cells. However, the assay is not specif c or adipocytes. Any viable cells within at gra s—blood-derived
cells, endothelial cells, adipose-derived stem cells, and f broblasts—can in uence the total absorbance
values.9

120
Part I • Principles and Basic Concepts
GLYCEROL-3-PHOSPHATE DEHYDROGENASE ASSAY
T e glycerol-3-phosphate dehydrogenase (G3PDH) assay is a relatively simple, adipose-specif c test that
can be used to assess the cellular unction o adipose tissue. T e enzyme G3PDH is normally present in
the cytoplasm o mature adipocytes, and it is released to the extracellular compartment when adipocytes
are damaged. T ere ore, the level o G3PDH can be measured either extracellularly or within the cyto-
plasm o adipocytes.12
Although the level o extracellular G3PDH rom damaged adipocytes can be measured through the use
o a washing-out technique and spectrophotometric assay,12 measurement o the enzyme level in the
cytoplasm o adipocytes has been proposed by many investigators, and a specif c G3PDH assay kit has
been developed by the manu acturer (Kamiya Biomedical, Seattle, WA) to measure the level o G3PDH
in the cytoplasm o adipocytes. T e higher level o the enzyme should represent a better cellular unction
o adipose tissue.8,9 According to the manu acturer’s instructions, G3PDH activity within at gra s is
evaluated using a spectrophotometric assay. Brie y, g o a at gra specimen is mixed with 4 ml o . 5
M cane sugar solution and homogenized. T e mixture is then spun at 7 g at 4° C or minutes, and
the supernatant is taken to the special centri uge tube, which is again spun, but at 54, g or 6 min-
utes. T e supernatant obtained a er the second centri ugation is diluted approximately to times
with an enzyme-extracting reagent.
Fig. 8-2
Final procedures o the assay are as ollows: the substrate reagent (4 microliters) is dispensed into an
assay well and heated to 5° C; the diluted supernatant is also heated to 5° C, and microliters o it is
added to the well and mixed with the substrate reagent. T e optical absorption at 34 nm is measured or
3 to minutes and plotted on a graph; the change in optical density (Δ OD) per minute rom the linear
position o the curve is obtained; and G3PDH activity is calculated based on this ormula:
G3PDH activity (U/ml) = Δ OD @ 34 nm/min × .4
with the value expressed as U/ml.

121
Chapter 8 • Determination o Fat Viability
A recent study demonstrates that although both the X and G PDH assays provide good correlations
between the number o viable adipocytes and resulting values, only the G PDH assay is strictly specif c
or adipocytes.9 Because the ratio o adipose stromal cells to adipocytes has been ound to be much
larger than previously described, a G PDH assay should be a pre erred laboratory test when determin-
ing the viability o at gra s. However, the assay can be per ormed only a er enzyme digestion and cen-
tri ugation, which may partly destroy adipocytes, and the direct assessment o the original adipocytes
within at gra s without tissue dissociation remains di cult.9 Any assays without a requirement or
tissue dissociation, such as the glucose transport test, should be urther investigated or a potential role
in assessing the viability o at gra s.13
ROUTINE HISTOLOGY
Histology has been used primarily in evaluating the structural integrity o at gra s. It cannot be used to
assess the actual viability o at gra s, because adipocytes can maintain their histologic structure while
having less than optimal cellular unction.8,14-16 It should be used as a supplement test and can conf rm
the architecture o the gra ed at.
Fig. 8-3
Each at gra specimen (about g) is f xed immediately in 0% bu ered ormalin, concentrated by
gravity f ltration through a porous paper, processed through graded alcohols and xylene, embedded in
para n, sectioned at 5 microns in thickness, and stained with hematoxylin and eosin (H&E) staining.
All histologic slides should be examined by an experienced pathologist or architectural disruption, ad-
ipocyte degeneration or necrosis.8 A normal histologic specimen o “excised” at gra s with H&E stain-
ing is shown above. Other types o tissue staining, such as oil red staining, can also be used or the same
purpose but rozen sections are required or such kind o histology.
WHOLE-MOUNT STAINING
Whole-mount staining o the living adipose tissue (within a ew hours rom sampling) has some advan-
tages. T ree-dimensional cellular structure can be visualized including adipocytes and capillary net-
work, which cannot be obtained by any other methods. Adipocytes are visualized by Bodipy staining,
but any other lipid droplets including dead adipocytes can be positively stained by this method and we
need to be care ul in the evaluation o viability o adipocytes.17

122
Part I • Principles and Basic Concepts
Fig. 8-4
Lectin (Capillary)
Bodipy (Adipocyte)
Hoechst (Nuclei)
A
ASC
Lectin (Capillary)
CD34 (ASC)
Hoechst (Nuclei)
B
Whole-mount staining o adipose tissue is shown. T e three-dimensional structure is clearly shown (A)
with staining or adipocytes (green with Bodipy), capillary network (red with isolectin), and nuclei (blue
with Hoechst). A three-dimensional image o adipose tissue (B) shows that every adipocyte is attached
to a capillary network, which occasionally has CD34-positive perivascular adipose-derived stem/stromal
cells (ADSCs).
Fig. 8-5
Whole-mount staining o adipose tissue. Living intact adipose tissue was visualized with whole-mount
staining or adipocytes (yellow with Bodipy), capillary network (red with isolectin), and nuclei (blue with
Hoechst). Large vasculature and the capillary network running between adipocytes were clearly visualized.
T e original staining and visualization method was reported by Eto et al.17 T e adipose tissue was cut
into 3 mm pieces within hours af er sampling and incubated with the ollowing reagents or 30 min-
utes: Bodipy 558/568 or Bodipy-FL (both rom Molecular Probes, Eugene, OR) to stain adipocytes,
Alexa Fluor 488-conjugated isolectin GS-IB4 (Molecular Probes) to stain vascular endothelial cells, and
Hoechst 3334 (Dojindo, Kumamoto, Japan) to stain all nuclei, or propidium iodide (PI; Sigma) to stain
nuclei o necrotic cells. T e sample was then washed and directly observed with a con ocal microscope
system (Leica CS SP , Leica Microsystems, Wetzlar, Germany). en images acquired with every 3 µm

123
Chapter 8 • Determination o Fat Viability
interval were used or reconstructing a 0 µm-thick three-dimensional image. Immunostaining such
as CD 4 ( or ADSCs), MAC- ( or macrophages) or Lyve- ( or lymphatic cells) can be combined, de-
pending on the purpose o the assay.
IMMUNOHISTOCHEMICAL STAINING FOR PERILIPIN
Perilipin is a protein coating lipid droplets in the cytoplasm o adipocytes. Immunostaining or perilipin
is very specif c to adipocytes and will be gradually lost within several days a er they die. T us this is cur-
rently the most reliable way to histologically visualize viable adipocytes. T is staining is very use ul or
evaluating the tissue viability o gra ed at, which usually includes many dead adipocytes (lipid drops).
o evaluate at gra retention, the gra ed at should be evaluated by both weight/size and viability, and
perilipin staining is required to see the latter.18
Fig. 8-6
H&E staining

Immunostaining for perilipin
Serial histologic sections o adipose tissue were stained with H&E staining and immunostaining or
perilipin. T ere are many round lipid droplets in the H&E image, but the perilipin staining revealed that
only some o these are viable adipocytes. T us viable and dead adipocytes are very clearly di erentiated
in the perilipin staining.
Fig. 8-7
Gra ed at was immunostained or perilipin (green, viable adipocytes), MAC- (red, macrophages) and
Hoechst (blue, nuclei) weeks a er gra ing. Many new adipocytes/preadipocytes emerged between
dead adipocytes (note the unstained round area) surrounded by inf ltrated macrophages or phagocyto-
sis o lipid droplets.

124
Part I • Principles and Basic Concepts
Harvested adipose tissue is f xed in zinc f xative (BD Biosciences, San Jose, CA), embedded in para n,
and sectioned at µm or immunohistochemistry. Guinea pig antiperilipin (Cedarlane Laboratories,
Burlington, Ontario, Canada) is used as the primary antibody. Isotypic antibody was used as a negative
control or each staining. For visualization, Alexa Fluor - or Alexa Fluor -conjugated secondary
antibodies (Molecular Probes) are used. Nuclei are stained with Hoechst . In addition, another pri-
mary antibody can be combined, such as CD (ADSCs), MAC- (macrophages), and von Willebrand
actor (vascular endothelial cells), depending on the staining purpose.
P re f erred P rotocol
Over the years, a pre erred protocol or determining the viability o at gra s has been developed in
each author’s laboratory. We have used a combination o viable adipocyte count, G PDH assay, and
regular histologic examination to determine the viability o at gra s and ound such a combination
is clearly better than any o the tests used alone. Our pre erred protocol was used to evaluate the via-
bility o at gra s obtained by direct excision, conventional liposuction, a at gra harvest and trans er
device, and the Coleman technique.8,14,15 In addition, this protocol was used to evaluate the viability o
adipose tissues be ore and a er cryopreservation.16 Recently, the whole-mount staining method enabled
three-dimensional visualization o adipose tissue and close relationship between adipocytes and cap-
illary network was clearly visualized.17,18 Perilipin staining, as an immunohistochemical method, can
clearly di erentiate viable adipocytes rom dead ones and can contribute to elucidating adipocyte ate
a er at gra ing; this is the most reliable method to evaluate adipocyte viability among all in vitro tests.19
However, all tests used in our protocol are in vitro–based studies; it would be ideal i a laboratory test
were developed that can be per ormed in vivo as well as in a clinical setting and that will allow analysis
without tissue dissociation. Such a test may be used in the uture to predict the clinical outcome o struc-
tural at gra ing in cosmetic or reconstructive surgery.
C oncl u si on
It is essential that at gra s harvested and processed by whatever technique maintain their viability be-
ore they are transplanted in patients. Our recent study avors the Coleman technique as a pre erred
method or at gra harvesting and processing, because this technique yields a greater number o viable
adipocytes and sustains a more optimal level o cellular unction within harvested and processed at
gra s.15 Several methods have been described or potentially optimizing at gra s be ore they are trans-
planted in vivo; or example, pretreatment o at gra s with a culture medium, insulin, or polymer.20-22
In addition, cell-assisted lipotrans er has been introduced and conf rmed clinically, with promising pre-
liminary results.23,24

125
Chapter 8 • Determination o Fat Viability
T ech ni ca l P ea rl s
• Long-term survival o transplanted atty tissue depends on the harvesting and processing technique so
that adipocytes should remain viable be ore implantation.
• Laboratory tests that have been used to determine the viability o at gra s include viable adipocyte
counts, colorimetric assays, adipocyte-specif c enzyme assays, and routine histologic studies, whole-
mount staining, and immunohistochemical staining or perilipin.
• A combination o viable adipocyte count, G3PDH assay, and histologic examination should be per-
ormed to determine the viability o at gra s; such a combination is clearly better than any o the tests
used alone.
• An advanced immunohistochemical technique such as perilipin staining has a clear advantage or
evaluating the viability o at gra s histologically, because this test can truly di erentiate live and dead
adipocytes within adipose tissue.
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Part I • Principles and Basic Concepts
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mice. Plast Reconstr Surg 0 : 940, 998.
. Yuksel E, Wein eld AB, Cleek R, et al. Increased ree at-gra survival with the long-term, local delivery o
insulin, insulin-like growth actor-I, and basic f broblast growth actor by PLGA/PEG microspheres. Plast
Reconstr Surg 05: 7 , 000.
. Medina MA, Nguyen JT, Kirkham JC, et al. Polymer therapy: a novel treatment to improve at gra viabil-
ity. Plast Reconstr Surg 7: 70, 0 .
3. Yoshimura K, Sato K, Aoi N, et al. Cell-assisted lipotrans er or cosmetic breast augmentation: supportive
use o adipose-derived stem/stromal cells. Aesthetic Plast Surg 3 :48, 008.
4. Kolle SF, Fischer-Nielsen A, Mathiasen AB, et al. Enrichment o autologous at gra s with ex-vivo ex-
panded adipose tissue-derived stem cells or gra survival: a randomised placebo-controlled trial. Lancet
38 (9898): 3, 0 3.

Gr o t h Fact or s
in t he Lipoaspir at e
Norbert Pallua, Benedikt Schäfer, Bong-Sung Kim
F g g u d m y d d u d b m l d g
l l u g y b u l b l y d y. H w v , m y l g- m ud
v w l v lum l d u lly ly g g -
g. A umb v b d u d d ud d l u l .
L l , w f l d d w ll by m y
l u g , v b d d l v d m d g v b l y. A um-
b ubl v u d f u . Al ug L v ğlu l1 d S
l2 d d b v g v l d d k u v v l
g , K k l3 d d d -d v d m ll (ADSC) v b l y. Gug ll
l4 d G d l5 u b v d d m l l d ADSC .4,5 k -
g , b ul d u u l l x x
g , lly d g ll .
A m mm ly d x l d m d g v b l y u xyg
u ly y u . T u ly xyg by d u l m d w m l-
l m , w u w y v bly b m g y x . A qu l g d
y x , l d ll u d g d , ul g l v lum d
z . I x , K l6 d b d -z m d l u f -
l u v v g z , g g z , d l z g z . T u u v -
ul z d bl l u v v l d bl ul . T m bl d
v l m x g v ul u , l ll d angiogenesis, g ly m l x
lly d d my d . A g g l d ly d d
g w .
T w ll v d g u k wl dg b u g w du g g -
g, umm z l k , d v d u l k u u d v l m f ld.
127
C h a p t e r 9

128
Part I • Principles and Basic Concepts
G row th F a ctors
Growth actors are naturally occurring proteins or steroid hormones that act by autocrine or paracrine
signaling. T ey bind to specif c receptors o target cells and stimulate, among other processes, cellular
growth, proli eration, di erentiation, and, what is essential, angiogenesis.
One o the most prominent growth actors involved in angiogenesis is the vascular endothelial growth
actor (VEGF). VEGF knockout in mice is lethal, underscoring the overall importance o this protein.
During angiogenesis, VEGF initiates the proli eration, migration, and di erentiation o endothelial cells
and mediates tubulogenesis by regulating vascular permeability. An important stimulus or an increased
VEGF production is, among other actors, tissue hypoxia. It has also been ound that VEGF secretion
positively correlates with the rate o adipocyte di erentiation.7
Another crucial actor or angiogenesis and adipose tissue expansion is the basic f broblast growth actor
(bFGF), which is known to stimulate proli eration and migration o endothelial cells. Moreover, bFGF
enhances the proli eration o ADSCs and the mitosis o adipocytes.
Insulin-like growth actor (IGF- ) contributes to angiogenesis by stabilizing new blood vessels. In
addition, it possesses a positive e ect on the survival o adipocytes and thereby enhances the viability o
at gra s a er transplantation.
Platelet-derived growth actor BB (PDGF-BB) is involved in angiogenesis through recruitment o peri-
cytes, which contribute to stabilization and maturation o newly ormed blood vessels. Moreover, it has
an antiapoptotic e ect on existing cells in the stromal vascular raction (SVF), stimulates the proli era-
tion o ADSCs, and acts as a survival actor.
Other important growth actors in adipose tissue are stromal cell–derived actor- -alpha (SDF- -alpha)
and the epidermal growth actor (EGF). SDF- -alpha plays an important role in angiogenesis, while EGF
is an important actor or the induction o mitosis.
aken together, growth actors such as VEGF, bFGF, IGF- , PDGF-BB, SDF- -alpha, and EGF acilitate
angiogenesis through cell recruitment and urther maturation as well as stabilization o neovessels. T ey
represent important survival actors or transplanted tissue and promote adipose tissue expansion.

129
Chapter 9 • Growth Factors in the Lipoaspirate
G row th F a ctors a n d L i p oa sp i ra tes
Adipose tissue is known to be a rich reservoir or growth actors. T ere ore at gra ing encompasses the
trans er not only o tissue mass with a volumetric e ect, but also growth actors and growth actor–pro-
ducing cells. However, during at harvesting, processing, storage, and injection, adipose tissue undergoes
signif cant manipulation, which may impose unknown e ects on the growth actors o the lipoaspirate.
We previously published a report demonstrating that lipoaspirates contain signif cant amounts o growth
actors.8 In our study, lipoaspirates were harvested and processed according to the Coleman protocol.
As a result o centri ugation, the lipoaspirate can be divided into our ractions: an upper oily raction,
a layer o the purif ed at, a watery raction, and a cellular pellet.9 T e cellular pellet consists o various
cells that some plastic surgeons use as the "red pellet" to enrich the at gra , although other surgeons do
not use it. T e literature also speaks o the cellular pellet as a distinct raction/layer. T e highest concen-
trations o growth actors were detected in the purif ed adipose tissue raction a er centri ugation or in
native adipose tissue, which was not urther centri uged a er harvest.
[
p
g
/
m
g
]
250
200
150
100
50
0
Native
Purified fat
Pellet
Watery

BFGF
IGF
VEGF
PDGF
* indicates p ≤0.05.
Fig. 9-1
Considerable quantities o VEGF, bFGF, IGF- , and PDGF-BB were ound in native adipose tissue and
purif ed at samples in di erent concentrations. T e highest growth actor content was measured in
purif ed at, whereas the pellet as well as the watery raction contained signif cantly lower amounts. T e
level o IGF- was highest, ollowed by bFGF, whereas VEGF and PDGF-BB were present in signif cantly
lower concentrations.

130
Part I • Principles and Basic Concepts
Furthermore, growth actors were analyzed rom native adipose tissue or purif ed adipose tissue a er
incubation in growth medium or and 5 days, respectively. Importantly, purif ed and native at secreted
signif cant amounts o growth actors even a er and 5 days o culture.8
40
Proliferation 3
Proliferation 6
Differentiation 3
Differentiation 6
Leptin
30
10
20
0
p
g
/
m
g
200
Proliferation 3
Proliferation 6
Differentiation 3
Differentiation 6
MMP9
150
50
100
0
p
g
/
m
g
10
Proliferation 3
Proliferation 6
Differentiation 3
Differentiation 6
PDGF
6
8
2
4
0
p
g
/
m
g
300
400
Differentiation 3
Differentiation 6
FGF
200
100
0
p
g
/
m
g
500
Proliferation 3
Proliferation 6
Differentiation 3
Differentiation 6
VEGF
300
400
100
200
0
300
Proliferation 3
Proliferation 6
Differentiation 3
Differentiation 6
IGF
200
100
0
Fig. 9-2
In a recent study we ocused on the content o growth actors in the SVF.10 T e SVF was incubated
in either di erentiation or proli eration media, and levels o VEGF, IGF, PDGF, leptin, and matrix
metallopeptidase-9 (MMP-9) were measured by ELISA a er and 6 days. We ound that the type o
culture medium had a signif cant in uence on growth actor secretion. While the concentration o
VEGF increased rom day to 6 in di erentiation and proli eration media, leptin levels decreased
over the same period in both media types. Levels o IGF, PDGF, and MMP-9 increased only when
cultured in di erentiation medium. We concluded that cultivation o the SVF with di erentiation
medium results in elevated growth actor concentrations and thus promises a more stable outcome.
T is aspect is o importance or in vitro SVF expansion in tissue engineering approaches and stem cell
therapy.
Graded densities is a new concept introduced by Allen et al.11 T e authors hypothesized that centri uga-
tion leads to a separation o the purif ed at raction into phases o graded densities. Highest and lowest
density atty tissue was injected into mice and harvested a er 2 weeks or short-term ollow-up and a er
weeks or long-term ollow-up, respectively. Survival o highest density at was higher than lowest
density at a er both 2 and weeks. Subsequent analysis revealed that the count o ADSCs as well as
the concentrations o the VEGF, SDF- -alpha, PDGF, and adiponectin were signif cantly higher in the
highest density at compared with the lowest density at raction. T e authors concluded that the highest
density phase o lipoaspirate resembles the most avorable raction because o its high growth actor and
progenitor cell content and yields longer-lasting results.

131
Chapter 9 • Growth Factors in the Lipoaspirate
T e technique used or harvesting and processing lipoaspirate urther alters the content o growth ac-
tors in the lipoaspirate. oday a great variety o cannulas are available so that plastic surgeons can adapt
to the specif c needs o each procedure. T e micro at gra ing procedure consists o tissue harvest with
a multiper orated cannula, leading to a more liquid lipoaspirate that allows at gra ing into superf cial
dermal layers.
Notwithstanding the unique benef ts o this technique, Alharbi et al12 reported that lipoaspirates har-
vested and processed by the conventional Coleman protocol contained signif cant higher amounts o
VEGF and IGF- compared with lipoaspirates harvested by the micro at harvesting technique. No di -
erence was seen in the concentrations o bFGF or PDGF.
T ese issues can be addressed by using di erent cannulas or distinct purposes. Large cannulas or har-
vest and injection according to the Coleman protocol ensure a high level o angiogenic actors, which
has been ound to lead to a higher gra take. T is technique is avored when maximal gra take is the
primary goal, as in breast augmentation. On the other hand, Magalon’s microgra ing technique13 can be
used to thicken and repair the dermis with increased numbers o ADSCs.
R esul ts
In a clinical retrospective study including patients, acial scars a er at gra ing were examined by a
patient and observer scar assessment scale (POSAS), photo documentation, and laser Doppler spectros-
copy (O C).14 Scar pain, color and pigmentation, sti ness and pliability, and irregularity were improved
a er at gra ing, leading to an overall high satis action rate among patients. Moreover, O C measure-
ments showed an increased hemoglobin level in the early stages a er gra ing.
Fig. 9-3

T is young woman sustained a dog bite injury. It was subsequently treated with at gra ing, which led to
a signif cant improvement o her scar.15
Lipof lling also e ectively addressed age-related lipodystrophy o the ace in patients undergoing a lipo-
aceli .16 Fat and stem cell based procedures consist o a high superf cial musculoaponeurotic system
(SMAS) ace-li and an ancillary lipof lling. Very satis ying results in long-term ollow-ups a er these
kinds o aceli procedures could be reached. Follow-up included comparison o pre- and postoperative

132
Part I • Principles and Basic Concepts
photographs as well as O C analysis. Besides angiogenic growth actors, the di erentiation o ADSCs
into collagen producing f broblasts represents a easible explanation or the marked improvement in the
overall quality o the skin. An example or the rejuvenating e ect o autologous adipose tissue in age-
related lipodystrophy is illustrated in Fig. 9-4.
Fig. 9-4
Female patient be ore (lef ) and months a er (right) treatment o age-related lipodystrophy by autol-
ogous at gra ing.16
P otenti a l C om p l i ca ti on s
Fat gra ing is generally considered to be a sa e procedure and in most cases entails manageable compli-
cations i the per orming specialist respects basic standards. But with regard to growth actors, there are
specif c risks that need to be considered.
One o the most avorable aspects o growth actors is their ability to stimulate the growth o blood
vessels. T is being said, angiogenesis also plays a pivotal role in tumorigenesis since rapidly expanding
cancer masses are strictly dependent on potent vasculature. T is relationship between tumor growth
and angiogenesis was f rst described by Folkman et al.17 Further studies have demonstrated that certain
growth actors directly stimulate the proli eration o neoplastic cells and promote their survival. T is
inevitably leads to the question o whether growth actors within the lipoaspirate also can cause and uel
existing cancer or increase cancer recurrence.
So ar, a great number o in vitro studies as well as animal experiments have indicated increased growth
o tumor cells that were stimulated by the introduction o adipose tissue or ADSCs. By contrast, clinical
observations have not conf rmed these experimental results. In multiple clinical studies including a high
number o breast cancer patients, no adverse e ect o at gra ing had been documented. T e sole excep-
tion o clinical studies that actually conf rms a carcinogenic potential was ound in cases o ductal and
lobular intraepithelial neoplasia. Petit et al18 observed that the requency o these pathological entities
signif cantly increased a er at trans er. One possible explanation is that these neoplastic cells are less
dedi erentiated and thus more responsive to paracrine signals such as growth actors.
Based on the current literature no def nite guideline or this delicate issue can be provided.
T e loss o volume remains an important disadvantage, because the take rate o the transplanted tissue is
unpredictable and can lead to additional gra ing procedures and decreased patient satis action.

133
Chapter 9 • Growth Factors in the Lipoaspirate
D i scussi on
Lipoaspirates contain important growth actors like IGF- , VEGF, bFGF and PDGF-BB and a er cen-
tri ugation the content o growth actors is higher in the purif ed transplant. Consequently, during the
at gra ing procedure growth actors also are trans erred to the recipient site.
T e secretion o growth actors separates lipoaspirates rom synthetic f llers. In contrast to synthetic
f llers, lipoaspirates are regenerative and dynamic f llers that can actively secrete growth actors and re-
spond to external stimuli such as tissue hypoxia or cytokines. Another important aspect is that lipoaspi-
rates deliver a physiological mix o growth actors. Studies ocusing on the distribution o single growth
actors so ar show rather moderate results. While each growth actor surely has its distinct unction it
is also known that they work in a costimulatory manner in uencing and ampli ying their distinctive
e ects.19
Proangiogenic growth actors guarantee a su cient vascularization o the at gra and thereby optimize
the f nal outcome a er transplantation. A su cient capillary network, which o ers adequate oxygen
supply, alleviates the degree o necrosis and apoptosis o the transplanted cells. In other words, at gra s
prevent volume loss as a result o tissue hypoxia.
Di erent studies have shown the angiogenic potential o at gra s. T e pivotal role o VEGF was shown
in a study using positron emission tomography-computed tomography (PE -C ).20 T e authors har-
vested adipose tissue rom the epididymal at pad o mice, treated it by transiently trans erred VEGF-A,
and f nally transplanted the gra into the orehead area. PE -C allowed an in vivo monitoring o
transplant volume and metabolic activity. Four and twelve weeks a er transplantation, no statistical
signif cant di erence in uorodeoxyglucose uptake as an indicator or metabolism was seen between the
VEGF-A and control group. However, histological analysis o the at pads a er twelve weeks revealed a
signif cantly increased capillary density in the VEGF-treated transplants. Furthermore, at weight and
volume were elevated in the VEGF-A group.
Besides VEGF, EGF was identif ed as a potent stimulator o neovascularization in a recent study.21 Fat
was harvested rom the inguinal region o rabbits, injected with EGF in the experimental group and
saline in the control group, and eventually gra ed into the ear. A er three months the harvested trans-
plants showed a signif cantly higher mass in the EGF- experimental group. In addition, ears o rabbits o
the control group experienced a higher degree o adhesion and f brosis. T e number o blood vessels was
markedly higher in the EGF-treated group. Finally, transplanted adipocytes o the EGF-treated animals
showed less de ormation or necrosis with higher density, all indicating the avorable e ect o EGF on
transplant vascularization and survival.
T e a orementioned in vivo studies20,21 clearly underscore the benef cial e ect o growth actors rom the
at gra on the vascularization and survival o at gra s.
Besides the benef ts o growth actors on the survival o the gra s itsel , the regenerative e ect o growth
actors derived rom the gra ed tissue on the recipient site is a pivotal point. Furthermore, the e ects
o at gra ing in the treatment o critical wounds have been under intense scrutiny. Zogra ou et al22
reported that skin gra s showed signif cantly ameliorated survival, angiogenesis, and epithelialization
in diabetic rats when they were simultaneously treated with cultured ADSCs. Expanded expression o
VEGF as well as increased capillary density and collagen intensity were detected.
Sultan et al23 investigated the e ect o at gra s in a murine burn model. A ull-thickness burn injury
was in icted on the dorsum o mice. A er weeks, human at (experimental) or saline solution (con-
trol) was transplanted under the burned area ollowed by analysis o the scars a er our and eight weeks.
Burn scars o at-gra ed animals showed a signif cantly higher ux in Doppler analysis a er 4 weeks.

134
Part I • Principles and Basic Concepts
T e proangiogenic markers VEGF and EGF were upregulated in at-gra ed animals, whereas the f brotic
markers MMP-9 and tumor growth actor-beta ( GF-beta) were downregulated. Moreover, histologic
staining revealed improved vascularity and an ameliorated scar index.
A promising approach to enhance at gra viability is enrichment o the lipoaspirate with growth actors;
intense research over the past decade has solidif ed this concept. Many di erent concepts have been
developed to allow a continuous and reliable delivery o growth actors. A simple coinjection o growth
actors is not a reasonable option because o the early degradation processes ( or instance, only 0 min-
utes or VEGF), which quickly nulli y their e ects.
Studies show that VEGF-trans ected ADSCs improve the survival o at gra s. Some investigators have
researched the use o SVF cells or lipoaspirates with VEGF encapsulated in nanospheres, which pro-
tects VEGF rom early denaturation. Another approach is genetic modif cation o ADSCs to improve
at viability. For example, VEGF was success ully transduced in ADSCs and injected in mice, leading to
a higher density o capillaries. Besides simultaneous injection o nanospheres/microspheres or growth
actor trans ected cells, the preconditioning o the recipient site with VEGF is an approach with prom-
ising outcomes.24
T e importance o FGF- and erythropoietin was studied in similar experimental settings and promoted
gra vascularization and survival. It was urther reported that erythropoietin increased expression o
proangiogenic actors, leading to less apoptosis and to alleviation o in ammation.25
However, an excess o growth actors can potentially cause deleterious e ects, as reported by Hiraoka
et al.26 Adipose tissue regeneration was monitored in collagen matrices containing bFGF-loaded mi-
crospheres. Histologic analyses were per ormed and revealed that f brosis and tissue in ammation in-
creased with rising growth actor concentrations, whereas low concentrations had no e ect.
It seems to be essential to determine the appropriate transplant-to-growth actor ratio or bioengineer-
ing purposes through uture studies to f nd a balance between benef cial and detrimental e ects.
C oncl usi on
Lipoaspirates contain signif cant amounts o growth actors and growth actor producing cells. T e con-
tent o growth actors in the lipoaspirate critically depends on harvesting and processing techniques.
T ere is a wide body o literature demonstrating that growth actors have a benef cial e ect on gra re-
tention. One major mechanism is the enhanced survival o the adipose tissue gra by promoting angio-
genesis and thereby securing the oxygen supply. Furthermore, growth actors secreted rom the gra ed
tissue improve the recipient site by increasing the ormation o blood vessels and decreasing cell death
as well as cicatrization. New tissue engineering approaches promise even higher gra viability through
growth actor supplementation o at gra s. Still, caution is recommended, especially in the treatment
o cancer patients, where at gra s may promote tumorigenesis through their angiogenic properties.
Further studies are required to produce a clearer understanding o the physiology o growth actors to
increase the sa ety o at gra ing.
REFERENCES
. Livaoğlu, M, Buruk CK, Uragloğlu M, et al. E ects o lidocaine plus epinephrine and prilocaine on autol-
ogous at gra survival. J Cranio ac Surg : 0 , 0 .
. Shoshani O, Berger J, Fodor L, et al. T e e ect o lidocaine and adrenaline on the viability o injected
adipose tissue—an experimental study in nude mice. J Drugs Dermatol : , 00 .

135
Chapter 9 • Growth Factors in the Lipoaspirate
. Keck M, Janke J, Ueberreiter K. [T e in uence o di erent local anaesthetics on the viability o preadipo-
cytes] Handchir Mikrochir Plast Chir :2 , 2007.
4. Gugerell A, Kober J, Schmid M, et al. Botulinum toxin A and lidocaine have an impact on adipose-de-
rived stem cells, f broblasts, and mature adipocytes in vitro. J Plast Reconstr Aesthet Surg 67: 276, 20 4.
. Girard AC, Atlan M, Benchari K, et al. New insights into lidocaine and adrenaline e ects on human ad-
ipose stem cells. Aesthetic Plast Surg 7: 44, 20 .
6. Kato H, Mineda K, Eto H, et al. Degeneration, regeneration, and cicatrization a er at gra ing: dynamic
total tissue remodeling during the f rst months. Plast Reconstr Surg : 0 e, 20 4.
7. Berendsen AD, Olsen BR. How vascular endothelial growth actor-A (VEGF) regulates di erentiation o
mesenchymal stem cells. J Histochem Cytochem 62: 0 , 20 4.
8. Pallua N, Puls ort AK, Suschek C, et al. Content o the growth actors bFGF, IGF- , VEGF, and PDGF-BB
in reshly harvested lipoaspirate a er centri ugation and incubation. Plast Reconstr Surg 2 :826, 200 .
. Condé-Green A, Baptista LS, de Amorin NF, et al. E ects o centri ugation on cell composition and via-
bility o aspirated adipose tissue processed or transplantation. Aesthet Surg J 0:24 , 20 0.
0. Pallua N, Serin M, Wolter P. Characterisation o angiogenetic growth actor production in adipose tis-
sue-derived mesenchymal cells. J Plast Surg Hand Surg 48:4 2, 20 4.
. Allen RJ Jr, Canizares O Jr, Schar C, et al. Grading lipoaspirate: is there an optimal density or at gra -
ing? Plast Reconstr Surg : 8, 20 .
2. Alharbi Z, Opländer C, Almakadi S, et al. Conventional vs. micro- at harvesting: how at harvesting tech-
nique a ects tissue-engineering approaches using adipose tissue-derived stem/stromal cells. J Plast Re-
constr Aesthet Surg 66: 27 , 20 .
. Mojallal A, Lequeux C, Shipkov C, et al. Improvement o skin quality a er at gra ing: clinical observa-
tion and an animal study. Plast Reconstr Surg 24:76 ; discussion 77 , 200 .
4. Pallua N, Baroncini A, Alharbi Z, et al. Improvement o acial scar appearance and microcirculation by
autologous lipof lling. J Plast Reconstr Aesthet Surg 67: 0 , 20 4.
. Pallua N, Kim BS. Fat gra ing and adipose tissue engineering. Plastic Surgery Hyperguide Online uto-
rial. Copyright by Vindico Medical Education.
6. Pallua N, Wolter . T e lipo- aceli : merging the ace-li and liposculpture: eight years experience and a
preliminary observational study. Aesthetic Plast Surg 7: 07, 20 .
7. Folkman J, Merler E, Abernathy C, et al. Isolation o a tumor actor responsible or angiogenesis. J Exp
Med :27 , 7 .
8. Petit JY, Rietjens M, Botteri E, et al. Evaluation o at gra ing sa ety in patients with intraepithelial neopla-
sia: a matched-cohort study. Ann Oncol 24: 47 , 20 .
. Sundaram H, Mehta RC, Norine JA, et al. opically applied physiologically balanced growth actors: a
new paradigm o skin rejuvenation. J Drugs Dermatol 8( Suppl Skin Rejuvenation):4, 200 .
20. ervala V, Grönroos J, Hartiala P, et al. Analysis o at gra metabolic adaptation and vascularization
using positron emission tomography-computed tomographic imaging. Plast Reconstr Surg :2 , 20 4.
2 . Park B, Kong JS, Kang S, et al. T e e ect o epidermal growth actor on autogenous at gra . Aesthetic
Plast Surg :7 8, 20 .
22. Zogra ou A, Papadoupoulos O, sigris C, et al. Autologous transplantation o adipose-derived stem cells
enhances skin gra survival and wound healing in diabetic rats. Ann Plast Surg 7 :22 , 20 .
2 . Sultan SM, Barr JS, Butala P, et al. Fat gra ing accelerates revascularisation and decreases f brosis ollow-
ing thermal injury. J Plast Reconstr Aesthet Surg 6 :2 , 20 2.
24. opcu A, Aydin OE, Ünlü M, et al. Increasing the viability o at gra s by vascular endothelial growth
actor. Arch Facial Plast Surg 4:270, 20 2.
2 . Hamed S, Egozi D, Kruchevsky D, et al. Erythropoietin improves the survival o at tissue a er its trans-
plantation in nude mice. PLoS One :e 86, 20 0.
26. Hiraoka Y, Yamashiro H, Yasuda K, et al. In situ regeneration o adipose tissue in rat at pad by combin-
ing a collagen sca old with gelatin microspheres containing basic f broblast growth actor. issue Eng
2: 47 , 2006.

(Trang không có chữ)

137
C h a p t e r 1 0
I l b z d d l f ll m b dy d w
m y. Ad l d b d ly,
p dly, d ly.1 M y d d f m
b d l p d m l w l y d
l - m by m m d p y b l y, p p p ,
m j , d mp ll y p d .
I p w w ll p d w l
b d m f d by m y . T b mm z
p d , b b b ly l f d l
mp : ( ) l ; ( ) p d l p p ; ( ) p p -
p ; d (4) j p f d m ll p l p f w y. W w ll
l dd mb mp d . A mp m
d l p b p bl d d z d f lly d d
pp d by m w ll- d d d .
DIFFERENT TECHNIQUES BASED ON THE
VOLUME REQUIRED
F b d d d b d l m -
: ( ) m ll- l m (l 00 ) p m d p m ly l
j p p ; ( ) l - l m ( 00 00 ) p -
m d p m ly b d b dy; d m l m (m
00 ) p m d p m ly b k m d b m -
.2 H w , p p w p , w w ll dd
p m ly m ll - l - l m .
An Over vie o Fat Gr a t ing
Techniques
Lee L.Q. Pu, Jeng-Yee Lin
137

138
Part I • Principles and Basic Concepts
B a si c F a t G ra f ti ng T ech ni q ues
Basic at gra ing techniques include at harvesting, processing, preparation o the recipient site, and
placement o at gra s. However, at gra ing technique can also be divided into smaller and larger at
gra ing based on the volume needed. Each category may have its own respective technique that is spe-
cif cally developed or the procedure.3
HARVESTING
Small-Volume Graf Harvesting Technique: Atraumatic Low Negative Pressure Method
Syringe aspiration rather than the standard liposuction approach is supported by more recent studies as
a relatively less traumatic method or harvest o at or autologous gra ing and should be considered as a
standardized technique o choice, at least or small quantities o lipoaspirate.4,5 However, this technique
can be time consuming even or experienced surgeons, and a large quantity o at gra s may not easily
be obtained with this technique. T e appropriate size o cannula and syringe used or aspiration to har-
vest at gra s remains controversial. A comprehensive study has concluded that the viability o at gra s
is signif cantly better when at is harvested with a 2 mm diameter cannula with a blunt tip and several
side-holes connected to a 0 cc syringe. T e use o a smaller syringe ( 0 cc) is advisable to maintain a
minimal negative pressure during harvesting.4
T e syringe aspiration method, better known as the Coleman technique, is especially use ul and pre-
dictable or small-volume at gra ing and has been widely used by many surgeons.3,5,6 Placement o 2
to mm incisions or harvesting at can be done with a No. blade in locations in which the scars can
easily be concealed. Fat gra s can be harvested through the same incision made or inf ltration o the
anesthetic solution. A small clamp is used to dilate the underlying subcutaneous tissue through the inci-
sion to allow insertion o the harvesting cannula with ease. T e anesthetic solution is then inf ltrated to
the donor site 0 to minutes be ore at extraction, which makes harvesting o at gra easier and less
traumatic. T e ratio o aspirated at to tumescent solution should be about : so that each pass o at ex-
traction can be more e cient. Pretunneling can be per ormed a er inf ltration o an anesthetic solution
to make at harvesting easier and less time consuming.

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Chapter 10 • An Overview o Fat Gra ting Techniques
Fig. 10-1
Di erent sizes and shapes o cannulas are used as needed or at harvest (small volume) and injection;
the orked-tip cannula can be used to release f brotic tissue, scar, or adhesion.
Fig. 10-2
A cc Luer-Lok syringe is connected to a harvesting cannula. We pre er a cc syringe to a larger one,
because the cc syringe is less cumbersome in the hand. We use a harvesting cannula that is 5 cm
long with a blunt tip and has dual openings like the shape o a bucket handle. Gently pulling back on

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Part I • Principles and Basic Concepts
the plunger creates a cc space vacuum negative pressure in the syringe. With a gentle back-and- orth
movement o the syringe, the at is gradually collected inside the syringe; this method causes minimal
trauma to the lipoaspirate. I little at with too much uid is drawn up in the syringe, the uid can be eas-
ily pushed out rom the bottom o the syringe in a vertical position, and at extraction can continue. A -
ter harvest, all incision sites are closed with interrupted sutures once excess tumescent uid or blood has
been milked out. In general, at gra s o less than cc can be harvested e ectively with this method.
Large or Megavolume Graf Harvesting: Low Suction Pressure Liposuction Technique
For harvest o a large quantity o atty tissue, one study showed that at harvested with low-pressure aspi-
ration (lower than 5 mm Hg) yields a higher viable adipocyte count than lipoaspirate harvested with a
high-pressure method (greater than 6 mm Hg).7 Several manu acturers have attempted to develop an
“ideal” device that combines at harvest, processing, and trans er.8 Un ortunately, only a ew such devices
have been studied comprehensively or their reliability and superiority, and the f ndings o these studies
are still less than convincing.9,10
One example is the LipiVage device or at harvest, washing, and trans er (Genesis Biosystem, Inc., Lew-
isville, X).8 It may have certain benef ts or less-experienced surgeons or harvesting and processing
at gra s so that their results can be more consistent in terms o adipocyte viability and intact struc-
ture. Several other closed devices have recently been developed and used clinically by surgeons. T ey
are the PureGra system (a closed system with washing and f ltration rom Cytori T erapeutics, San
Diego, CA),9 the Revolve system (a closed at-processing system rom Li eCell, Branchburg, NJ),10 and
the LipoKit (a closed and manual system with centri ugation rom Medi-Khan, West Hollywood, CA).11
Each o these devices, which combine at harvesting with processing, may have the potential or use in
large-volume at gra ing in the uture. However, the use ulness o these devices has not been well ac-
cepted by surgeons because o their cost and unconf rmed e ectiveness.
Khouri et al12 advocated low-pressure (3 mm Hg) vacuum liposuction with a specially designed device
(the K-VAC syringe rom Lipocosm, Key Biscayne, FL) to harvest at with constant low pressure and a
low g orce ( 5 g or to 3 minutes) using hand-cranked centri ugation or megavolume at harvesting
and processing.
Fig. 10-3
A newly designed harvesting cannula by Khouri et al may have many advantages because o its e ciency
and less trauma to adipocytes. Such a cannula can be incorporated into a large-volume at gra ing in-
strument set and can be used in conjunction with lower suction pressure or large-volume at gra har-
vest.

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Chapter 10 • An Overview o Fat Gra ting echniques
PROCESSING
Several methods or processing harvested at have been proposed, including decanting, cotton gauze
rolling, centri ugation, and washing and f ltration, to e ectively remove the inf ltrated anesthetic solu-
tion and cell debris within the lipoaspirate and to obtain more concentrated at or gra ing.13 T e best
method or per orming this stage is the most controversial topic in at gra ing. Most debate concerns
whether centri ugation o the lipoaspirate is necessary or at gra processing.
Processing Harvested Fat With Centrifugation
Centri ugation, as part o the Coleman technique, is our pre erred method or processing lipoaspirate,
since most studies support its clinical application.5,6 T ere are several advantages to centri ugation. More
viable adipocytes are ound at the bottom o the middle layer a er centri ugation even with a orce o
g or minutes based on viable cell counts, and this makes manipulation o the at gra easier and re-
sults in measurably more reliable viability o the gra ed tissue.14,15 Recent studies have shown that proper
centri ugation can concentrate not only adipocytes and ADSCs but also several angiogenic growth ac-
tors within the processed lipoaspirate.11,16 Because a higher content o stem cells or angiogenic growth
actors positively correlates with at gra survival both in experimental and clinical studies,17 centri uga-
tion at rpm (about g) or minutes appears to o er more benef ts, because it is e ectively con-
centrating adipocytes, ADSCs/progenitor cells, and angiogenic growth actors. Allen et al,18 investigating
optimal density in an animal study, reported that higher-density at gra s have been ound to have better
survival than lower-density gra s. T us higher or lower density o at gra s can be selected accordingly
or di erent but more predictable outcomes a er at gra ing. Appropriate centri ugation should be a
valid method o choice or processing at gra s, especially or small-volume at gra ing.2-6
Fig. 10-4
T e Luer-Lok aperture o the cc syringe is locked with a plug at completion o harvest, and the li-
poaspirate is then ready or centri ugation. Each syringe is covered to prevent prolonged exposure to
air and possible contamination. When the needed volume o at has been harvested, the plunger is care-
ully removed, and all lipoaspirate-f lled cc syringes are placed into a centri uge and centri uged at
rpm ( g) or minutes. Greater g orce or longer duration o centri ugation may be harm ul
to the adipocytes and is not recommended.19

142
Part I • Principles and Basic Concepts
Fig. 10-5
Af er being centri uged, lipoaspirate within the syringe will have separated into three layers: the oil con-
tent in the upper layer, atty tissue in the middle layer, and the uid portion at the bottom. T e oil can
be decanted rom the Luer-Lok syringe. T e residual oil is wicked with a cotton strip or swab. T e uid
at the bottom can be easily drained once the plug at the Luer-Lok aperture is removed. T rough centri -
ugation, three distinct ractions are ormed: oil, at, and a liquid layer that includes tumescent uid and
debris.

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Chapter 10 • An Overview o Fat Gra ting echniques
Fig. 10-6
T e concentrated at in the syringe can then be trans erred to a cc syringe (our pre erred size or at
injection) with an adapter. T e cc acrylic syringe is pre erred or small-volume at gra ing; it creates
little riction or resistance during injection, and thus the surgeon can easily control the injected volume.
T e air bubble inside the syringe should be removed so that quantif cation o the volume injected can be
recorded precisely. We have also used a cc syringe or large-volume at gra ing.
Processing Fat With a Device, Filtration, or Gravity Sedimentation
T e manu acturers o several at processing systems (such as the Revolve System, Li eCell, Branchburg,
NJ; PureGra , Cytori T erapeutics, San Diego, CA; or LipoKit, Medi-Khan, West Hollywood, CA)9-11
have claimed that their devices produced equivalent or higher concentrations o purif ed at with reduced
red blood cell debris and ree oil compared with alternative methods such as centri ugation. However,
these studies in act might have problems in their experiment designs and were not tested independently.
In addition, they ailed to prove their superiority in at gra processing in terms o the concentration o
viable adipocytes and in vivo at gra retention rates.

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Part I • Principles and Basic Concepts
Some surgeons advocate the use o a f ltration technique or even just gravity sedimentation to process
large or megavolume harvested at gra s. However, without proper centri ugation, at gra s processed
with f ltration only or sedimentation only may not be pure enough or trans er; they might contain po-
tentially in ammatory materials such as ree atty acids and ragmented RBCs. T ere ore proper centri -
ugation, even or large or megavolume at gra ing, may be considered an advisable technique.
One technique or large or megavolume at gra processing has been described by Khouri et al.12 Li-
poaspirate is harvested with low (3 mm Hg) vacuum pressure using a -gauge cannula with side-
holes. T e lipoaspirate is then collected into IV bags, and each bag is centri uged at 5 g or 3 minutes,
using a centri ugation machine or a hand-cranked centri uge. Once the in ranatant uid is drained,
the supernatant at is concentrated in the same collection container, which becomes the gra delivery
container. Compared with the pastelike, compacted gra obtained rom g centri ugation, a dilute
slurry o gra material is acquired rom low g manual bag centri ugation. It is a pre erred method by its
advocates or large or megavolume at gra ing with the specif cally designed device because o its speed
and e ectiveness.
Fig. 10-7
Alternatively, standard centri ugation can still be per ormed to process lipoaspirate harvested with
low-pressure suction and a multiple side-hole cannula.2 T is is the method used in the senior author’s
practice (L.L.Q.P.) or large-volume at harvest and processing. In this system, a cannula with multiple
side-holes (see Fig. -3) is used to harvest at under low-suction pressure. Lipoaspirate is f rst collected
into the canister and is then trans erred into multiple cc syringes or centri ugation.
PREPARATION OF RECIPIENT SITES WITH EXTERNAL
VOLUME EXPANSION
Preparation o the recipient site to create an optimal environment or survival o at gra s is an essential
actor or increasing gra retention. One innovative way o achieving this, f rst proposed by Khouri
et al,20 is preexpansion o the recipient site using an external device that applies a continuous negative
pressure on the recipient site skin, similar to the negative pressure wound (Wound VAC) therapy used to
promote wound healing. Since high gra retention is critical and di cult to achieve in megavolume at

145
Chapter 10 • An Overview o Fat Gra ting echniques
gra ing in cases o breast augmentation or reconstruction, a specially designed external preexpansion
device or the breast was f rst used in at gra ing to the breast. T is preexpansion device, or external vol-
ume expansion (EVE), or use on breast tissue has proved success ul in increasing long-term at reten-
tion in the breasts, as conf rmed in scientif c studies. Khouri et al hypothesized that EVE may potentially
increase the thickness o tissue and improve angiogenesis in the recipient site or the gra ed at. It was
also reported by other investigators that EVE induces adipose tissue enlargement by activating resident
progenitor cells, combined with controlled release o basic f broblast growth actor, as well as vascular
remodeling.21,22 Another study ound that external volume expansion induces mechanical stimulation,
edema, ischemia, and in ammation in the recipient site, thus maintaining an environment conducive
to cell proli eration and angiogenesis over distinct time periods. Disadvantages o the use o external
volume expansion include skin rashes, dermatitis, hyperpigmentation, and the necessity o patient com-
mitment and compliance to its continued and uninterrupted use.23
Preexpansion o breast tissue with an external breast tissue expander (BRAVA) be ore an augmentation
or reconstruction procedure has been used success ully by a number o surgeons.20,24 T e device consists
o a rigid plastic dome with silicone gel cushioning at the base and tubing connected to a negative pres-
sure pump. During the expansion period the vascularity o the gra site is increased. T e patient is asked
to wear the BRAVA device or hours per day or or more weeks. For the last to 8 hours be ore
surgery, the patient is asked to maintain uninterrupted expansion and to come to the operating room
still wearing the expander, which is inducing an immediate temporary three-dimensional enhanced en-
largement o the subcutaneous tissue matrix. However, the expansion program must be individualized
or each patient, and the duration o expansion can be extended to achieve proper preexpansion. Some
concerns about the sa ety o BRAVA’s clinical use have been raised.
PLACEMENT OF FAT GRAFTS
Fig. 10-8
One o the most important techniques in at gra ing is how to place at gra s in the recipient site or
sites. T e key to a success ul at gra injection is to achieve an even distribution o at gra s. By doing
so, the injected at will have maximal contact with the tissue in the recipient site or better gra survival
through plasmatic imbibition and neovascularization.6

146
Part I • Principles and Basic Concepts
≤ 2 mm
Evenly disperse microribbons
Avoid > 2 mm collections
Avoid cavities
Fig. 10-9
Gra ing with a small volume in each pass will not only produce a better surgical outcome but also can
help to avoid such complications as f brosis, oil cyst ormation, calcif cation, and even in ection that have
been associated with large-bolus gra ing. o achieve this goal, a small volume (no more than 0. cc) o
at gra s should be injected in each pass. However, the equivalent amount o at can be injected in each
pass or large-volume at gra ing.12 Slow injection o 0. to cc per second should be injected during the
withdrawal phase in each pass to minimize trauma to the at gra .25 T ese small aliquots o at should be
placed in multiple passes within multiple tissue planes and tunnels, in multiple directions.1,2,6,12,26
Injection should be as gentle as possible to avoid possible injury to vessels or nerves. Injection with re-
sistance would compromise the result and increase the chance o associated complications. T e injecting
cannulas usually range rom 0 gauge to gauge in diameter; the tip o the injection cannula is usually
blunt and has one opening on the side. T ey vary in length and shape according to the volume and area
to be gra ed. T e most commonly used cannulas are to 9 cm long or a acial procedure and 9 to cm
or a body procedure. In general, a smaller cannula should be used or at gra ing to such areas as the
periorbital region, where only a small volume o at is injected in each pass. Small cannulas also allow
the surgeon to have more precise control over the volume when an extremely tiny amount o at gra s is
injected. Cannulas may be straight or curved with a blunt or orked tip to meet di erent needs. T e can-
nula with a orked tip can cut through tissues and can be used to release f brotic tissue or scar, adhesion,
or ligamentous attachments.
≤2 mm

147
Chapter 10 • An Overview o Fat Gra ting echniques
Recently an injection device has been proposed by some surgeons to acilitate at gra s placement and
to ensure accurate delivery o small at parcels each time. Each delivered at volume can be set at a cer-
tain amount, and multiple injections can be per ormed in a more evenly distributed ashion.27,28 Such a
device has primarily been used in the ace because o its accuracy and sa ety and may also be used or
at gra ing to other areas o the body such as in the breast or buttock to ensure even distribution and to
avoid a bolus injection o at gra s. It can be used by less experienced surgeons to produce a better out-
come and greater sa ety or their patients. However, the use ulness o such a device, as well as its added
cost, is still a source o debate among experienced surgeons.
Preoperative photographs with detailed planning marks on the patient are use ul or intraoperative com-
parison, because the changes that need to be made with at gra ing in the operating room are usually
quite subtle. T e surgeon must be certain o where the cannula tip is during the entire injection process.
I there is any doubt about the tip location, the cannula tip should be tented toward the skin; blanch-
ing o the skin overlying the advanced cannula will reveal its exact location. I at gra s are placed in a
correct location, the “augmented” e ect in the gra ed area can easily be assessed. I the volume is not
increased even though gra ing has been done in the correct site, other actors may be restricting volume
enlargement, such as a f brotic adhesion or a tight skin envelope. Fibrosis or adhesions can be dissected
with an -gauge needle or a orked-tip cannula. A tight skin envelope may require preexpansion be ore
at gra ing.
For large or megavolume at gra ing, the principle or the placement o at gra s should be the same
as small-volume at gra ing, except that more volume will be required, since the recipient site is much
larger12; a cc aliquot o at can be injected in each pass. Attention should be made to avoid a “bolus”
injection. Again, the basic principles or placement o at gra s should be ollowed to ensure a better
outcome and avoid at necrosis.
O th er I m p orta nt C on si d era ti on s
DONOR SITE SELECTION
Fig. 10-10
A variety o body areas have abundant or excess at and are suitable as donor sites or the harvest o at
gra s; these include the abdomen, anks, buttocks, medial and lateral thighs, and medial knees. As a
general rule, donor sites are selected or which the body contour will be enhanced by liposuction. Sites

148
Part I • Principles and Basic Concepts
chosen should be easily accessible with the patient in the supine position, which is the position that is
used or almost all acial and body augmentation procedures. Although there is no evidence o a single
optimal donor site or harvest, because the viability o adipocytes within the at gra s rom di erent
donor sites may be considered equal, one study ound higher concentrations o ADSCs in the lower
abdomen and inner thigh in patients under 5 years o age.29 With what we know about the potential
role o ADSCs in at gra ing,30 the lower abdomen and inner thighs should there ore be chosen as the
“better” donor sites or at transplantation.31 T ese donor sites are not only easily accessible by the sur-
geon with the patient in the supine position, but also are scientif cally sound as long as the patient has
an adequate amount o adipose tissue in those areas. I the patient is placed in the prone position, the
posterior medial thigh, lateral thigh, and ank can be accessed as the primary donor sites or harvest
o at gra s. Palm and pinch tests should be per ormed in the donor tissue to determine whether there
is adequate at reserve and to quickly estimate the amount o at that can be harvested.12 A palm size is
roughly measured as cm2; the pinch test predicts the layer thickness o the at gra to be harvested.
For example, even in a thin patient, a . 5 cm layer thickness o at harvested over the sur ace area o a
palm will yield 5 cc o at ( cm2 × . 5 cm = 5 cc). T ere ore 5 cc o at can be harvested solely
rom the anterior sur ace o a woman’s thigh that has f ve palm measures.
ANESTHESIA
Harvest o lipoaspirate or at gra ing can be per ormed with the use o a general, epidural, or local an-
esthetic, with or without sedation. Intravenous sedation is routinely used in conjunction with regional
or local anesthesia i the patient requests it. T e tumescent solution used or donor site analgesia and
hemostasis should contain the lowest concentration o lidocaine possible, because a high concentration
could have a detrimental e ect on adipocyte unction and viability.32 In general, we o en use . % o
lidocaine in Ringer’s lactate solution i the at gra ing procedure is to be per ormed under general anes-
thesia and . 5% i local anesthesia will be used or the procedure, with or without sedation. T e tumes-
cent solution also contains epinephrine with a concentration o : , . Epinephrine can precipitate
vasoconstriction in the donor sites as well as the recipient sites, which may decrease blood loss, bruising,
and hematoma, thus reducing the possibility o intraarterial injection o the transplanted at especially
when injecting around the periorbital areas or elsewhere in the ace.
OVERCORRECTION
Whether overcorrection is necessary or at gra ing remains uncertain. Because viable at gra s are
only observed in the peripheral zone approximately .5 mm rom the edge o the gra area, and the
percentage o gra viability depends on its thickness and geometric shape,33 overcorrection or better
gra survival in the recipient site appears to lack scientif c support. In addition, signif cant overcorrec-
tion may increase the incidence o at necrosis and subsequent calcif cation or even severe in ection.34
T ere ore signif cant overcorrection should be avoided until its necessity and sa ety can be conf rmed
by uture studies. Interstitial uid pressure in the tissue can be monitored to prevent overgra ing during
the procedure.12