TRANSPLANTATION SURGERY AND RESEARCH

Clinical Safety of Stromal Vascular Fraction Separation

at the Point of Care
Joel A. Aronowitz, MD,*† Ryan A. Lockhart, BS,† Cloe S. Hakakian, BS,† and Kevin C. Hicok, PhD*†
tissue fragment, leads to eventual loss of graft volume.4–6 Mesenchymal
Introduction: Pluripotential cells in adipose tissue may be important in long-
progenitor cells are more likely to survive the physical stress and hyp-
term volume retention and regenerative effects of fat grafting. Unfortunately, graft
oxic insult of transplantation and thereafter play a pivotal role in adipose
harvest with lipoaspiration significantly depletes the population of stromal vascu-
tissue regeneration through adipogenic and vascular differentiation as
lar cells, which includes adipose stem cells. Stromal vascular fraction (SVF) cells
well as the expression of angiogenic, antiapoptotic, and antioxidative
may be isolated from excess lipoaspirate at the point of care and used to replenish
factors.7–13 Lipoaspirate tends to be deficient in these progenitor cells
fat grafts, a technique termed cell-assisted lipotransfer (CAL). Preclinical and
compared to intact adipose tissue because the progenitor cells are
clinical evidence supports the rationale of CAL but clinical adoption of the strat-
concentrated in the perivascular areas of adipose tissue which are not
egy requires evidence of clinical safety. This prospective, level 1 study reports
fully harvested with liposuction. In addition, some progenitor cells
clinical safety of SVF-enhanced fat grafting using a manual, collagenase-based
are released into the fluid portion of the lipoaspirate which is decanted
separation process to isolate autogenous progenitor cells from lipoaspirate at
before grafting.14
the point of care.
These observations are the rationale behind cell-assisted lipo-
Methods: One hundred sixty-four subjects underwent 174 SVF-enhanced autolo-
transfer (CAL),15 a technique in which lipoaspirate fat graft is enriched
gous fat grafting procedures at the university stem cell center between August 2009
with autogenous stromal vascular fraction (SVF). Stromal vascular
and November 2014 for a variety of cosmetic and reconstructive indications.
fraction contains a variety of cells such as pericytes, fibroblasts, and
Results: Cell-assisted lipotransfer was performed for a variety of cosmetic and
macrophages as well as a heterogeneous population of pluripotential
reconstructive indications. The mean time of the SVF isolation process was
adipose stem cells and vascular progenitor cells. Stromal vascular frac-
91 minutes. Because of the frequent concomitant procedures, the average operat-
tion is obtained at the point of care from excess lipoaspirate using a
ing room time increased by only 11 minutes. Mean follow-up was 19.9 months.
collagenase-based process to release the cells from the fibrous stroma.16
There were no major complications and 6 minor complications. No collagenase
A number of studies support the beneficial effects of CAL on
or neutral protease related complications were observed.
long-term volume retention but important parameters, such as the cell
Conclusions: This series of 174 CAL cases demonstrates that SVF cell isolation
dosage versus effect relationship remain to be established. Acceptance
using a standardized, manual, collagenase-based process at the POC is equivalent
in the United States and even clinical research have been hindered by
in safety compared to nonenhanced fat grafting. These results support expanded
issues of cost, regulatory uncertainty, and safety of using a tissue disso-
use of CAL in the clinical research setting.
ciation enzyme mixture,17 that is, collagenase and neutral protease, to
Key Words: stromal vascular fraction, adipose-derived stem cells, clinical safety, isolate SVF from lipoaspirate at the point of care.
SVF isolation, ASC isolation, cell-assisted lipotransfer, breast augmentation, The purpose of this study is to demonstrate the clinical safety of
breast reconstruction CAL in prospective study of 174 consecutive cases of fat grafting using
(Ann Plast Surg 2015;75: 666–671)
progenitor cell enriched lipoaspirate or CAL, using SVF prepared at the
point of care with an enzymatic process.

A utologous fat grafting (AFG) is an increasingly popular technique

in plastic surgery for volume augmentation and improvement of
radiated or damaged tissues. There is a consensus among plastic
METHODS

surgeons that fat grafting is a safe procedure with a low complication
rate and high patient satisfaction useful for a variety of cosmetic and
reconstructive indications.1,2 Fat grafting remains limited however
by unpredictable long-term volume retention, inconsistent results and
insufficient high quality clinical research to help formulate evidence-
based recommendations for fat harvest, graft preparation, and reinjec-
tion best practices.3
One promising avenue for improvement is emerging from a bet-
ter understanding of the microenvironment and cellular dynamics of the
engraftment process. Research suggests that mature adipocytes, due to
their high cytoplasmic oxygen consumption, are particularly suscep-
tible to hypoxia-induced apoptosis after grafting. Apoptosis of mature
adipocytes, especially those located in the center of the lipoaspirate
Received February 18, 2015, and accepted for publication, after revision June 1, 2015.
From the *Cedars-Sinai Medical Center; and †University Stem Cell Center, Los
Angeles, CA.
Conflicts of interest and sources of funding: none declared.
Reprints: Joel A. Aronowitz, MD, Cedars-Sinai Medical Center, Los Angeles, Univer-
sity Stem Cell Center, Los Angeles, 8635 W. Third St. Suite 1090W, Los Angeles,
CA 90048. E-mail: dra@aronowitzmd.com.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
ISSN: 0148-7043/15/7506–0666
DOI: 10.1097/SAP.0000000000000594
Overview
A total of 174 CAL procedures in 164 patients were performed at
the University Stem Cell Center between August 2009 and November
2014. All procedures were conducted under institutional review board
approval, and all patients provided written informed consent. All study
procedures were conducted in accordance with the Declaration of
Helsinki. Lipotransfer procedures were conducted for a variety of re-
constructive and cosmetic indications (Table 1) including breast recon-
struction (Fig. 1A, B), breast and buttock augmentation (Fig. 2A, B),
facial atrophy, correction of lipodystrophy, and contour defects (Fig. 3A,
B), and chronic wound and scar treatment (Fig. 4A, B, C). Detailed data
concerning subject medical history, indications for fat transfer, fat har-
vest, separation technique, process time, and postoperative outcomes
were collected (Table 1). Potential patient safety issues specific
to collagenase and neutral protease include local allergic reaction, in-
fection, evidence of local tissue destruction, and overall complication
rates associated with fat injection were monitored throughout the study.
Inclusion and Follow-Up
Patients with a previous history of malignancy in the last
12 months before treatment were excluded from participation in the
study. Study participants underwent cancer screening by their primary
care physician at regular intervals according to the American Cancer

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Annals of Plastic Surgery • Volume 75, Number 6, December 2015 Clinical Safety of SVF Isolation at POC

included mammogram and ultrasound screenings as well as physical

TABLE 1. Isolation and Demographic Data examinations. Not all patients reported in this study have reached
12 months follow-up. Nonmalignant complications were assessed
Parameter Average (Range) within 3 months after receiving treatment, including complications
Patient age, y 52.2 (19–77) pertaining to fat grafting (ie, oil cyst, necrosis), infection, or residual
BMI, kg/m2 23.4 (14–37) enzyme activity.
Follow-up time, mo 19.9 (3–48)
Volume of lipoaspirate digested, mL 180 (50–800)
No. washes 3 Lipoaspirate Harvest
Processing time, min 91 (45–150)
Final volume of SVF suspension, mL 11.8 (5–25)
Adipose tissue harvest was done under intravenous sedation or
general anesthesia in the operation room. A standard formula tumescent
SVF cells isolated (cells/mL of lipoaspirate) 199,000 (15,000–790,000)
solution with local anesthetic (lidocaine 1% with 1/100,000 epineph-
Implication for CAL No. cases rine and Marcaine 0.5% with 1/200,000 epinephrine) was injected in
Breast augmentation 77 the harvest sites. Adipose tissue for SVF isolation and graft was aspi-
Breast reconstruction 54 rated with 3.0 to 4.0 mm blunt tipped cannula using Wells Johnson
Facial aging 21 Aspirator II apparatus with vacuum lower than 700 mm Hg.11,19
Chronic wounds/scars 8
Buttock enhancement 8
Lipodystrophy/contour defect 6 SVF Isolation
Liposuction Harvest Location No. cases
Abdomen 106 A nonautomated, collagenase-based isolation was performed by
Flanks 55 a technician at the point of care under sterile conditions in a previously
Thighs 37 described19 compact operating room laboratory equipped with a sterile
Complications No. occurrences (%) biohood, centrifuge, and heated shaker. Volume of lipoaspirate digested
Major 0 (0.0%)
ranged from 50 mL to 800 mL (180 mL average). Lipoaspirate was first
washed 3 times using a lactated Ringer solution (130 mmol/L Na+,
Minor 6 (3.7%)
109 mmol/L Cl−, 28 mmol/L lactate, 1.5 mmol/L Ca2+, 4 mmol/L K+)
Oil cyst 3 (1.8%) over a period of 10 to 15 minutes to remove contaminating blood cells
Fat necrosis 0 (0.0%) and tissue debris. Lipoaspirate was then incubated with 35 Wünsch Units
Calcification 0 (0.0%) (U) of collagenase per 50 mL of washed lipoaspirate. Lipoaspirate was
Unrelated 3 (1.8%) incubated with CIzyme AS (Vitacyte, LLC, Indianapolis, IN), a mix-
ture of clostridial collagenase type I and type II (60% type I, 40% type
II) and neutral protease from Bacillus polymyxa resuspended in warm
Society Guidelines for the Early Detection of Cancer. The guidelines (37°C) lactated Ringer solution in a temperature controlled shaker
for early detection of breast cancer include yearly mammograms, clini- at 200 rpm for 20 to 30 minutes at 37°C. The SVF cells were separated
cal breast examinations every 3 years and regular self-examinations.18 from the digested adipose tissue via centrifugation at 2000 rpm
Additional exclusion criteria include pregnancy or suspected preg- for 10 minutes. Isolated SVF cells were then washed a minimum of
nancy, abnormal preoperative breast examination, or known positive 3 cycles using lactated Ringer solution to remove residual enzymes.
BRACA1 or BRCA2 gene mutations. Patients were required to submit The SVF preparation was brought to a final volume of between 5 mL
to preoperative screening for malignancy before participation. and 25 mL (11.8 mL on average) using lactated Ringer solution and
Patients are screened regularly for development of malignancy returned to the surgeon for injection. A small sample of SVF cells
for 12 months after treatment, and any screening thereafter was done (0.1-0.2 mL) was analyzed using a cell counting device (Chemometec
at the discretion of the patient during annual physicals or breast exam- NC 200; Chemometec A/S, Davis, CA) to determine the cell yield
inations. For patients receiving treatment to the breasts, follow-up and viability (Table 1).

FIGURE 1. A, Subject desired breast reconstruction to restore breast symmetry and improve cosmetic appearance of the right breast.
B, 3 months postoperatively: reconstruction was accompanied by autologous SVF-enhanced fat grafting which restored symmetry.

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Aronowitz et al Annals of Plastic Surgery • Volume 75, Number 6, December 2015

FIGURE 2. A, Subject presented with hypoplastic breasts and desired cosmetic augmentation without implants. Subject was injected
with an SVF-enhanced autologous fat graft. B, 6 months after cell-enhanced fat grafting, the breasts were significantly larger while still
presenting with a natural appearance and feeling. C, 13 months after cell-enhanced fat grafting.

Graft Preparation and Injection
Freshly isolated autogenous SVF cells were returned to the sur-
geon in the operating room and recombined with graft material shortly
before injection. The progenitor cell enriched fat graft was injected into
the recipient area using a Cytori Celbrush injector tool (Cytori Therapeu-
tics, inc. San Diego, CA). A layered technique in a grid pattern was
used to ensure even distribution of graft material throughout the recipient
site in a manner identical to non enriched fat grafting procedures.
grafting. The occurrence rates of complications were similar, if not
lower, than those associated with normal AFG.
Of the 164 patients in this study, 54 had a previous history of
breast cancer (32.9%). There were no instances of new malignancy re-
ported at the site of injection in patients followed more than 12 months,
but there was one reported recurrence of breast cancer (1.9%) in a
patient who underwent a unilateral breast reconstruction using a saline
implant accompanied by CAL for contouring.

RESULTS
The average age of the subjects was 52.2 years, with an average
BMI of 23.4 kg/m2. An average of 180 mL of lipoaspirate was used for
isolation of SVF cells. The average number of SVF cells isolated per
mL of lipoaspirate was 1.99  105 cells/mL.
The nonautomated, collagenase-based isolation method took an
average of 91 minutes to complete in the operating room under a
biohood environment. The average graft volume was 223 mL, but
varied from 8 mL to 500 mL depending on the indication and injection
site, with buttock and breast augmentations requiring larger amounts of
fat, whereas facial procedures typically required less than 100 mL. The
average follow-up time was 19.9 months.
Overall, a low complication rate is reported in this study (3.7%).
No reported complications pertain to the use of collagenase or neutral
protease, and no intraoperative complications are reported. In the post-
operative follow-up after fat grafting, there were no major complica-
tions, and 6 minor complications, (3.7%) were observed. Three oil
cysts were observed (1.8%) and 3 other complications were deemed
unrelated to fat grafting (1.8%) (Table 1). The complication spectrum
of SVF-enhanced AFG is the same as that associated with normal fat
DISCUSSION
Experimental as well as clinical studies suggest a positive rela-
tionship between CAL and improved permanent fat graft volume.15,20,21
The potential and theorized negative effects of CAL include local tissue
digestion from residual enzyme activity, possible higher infection and
surgical complication rates and accelerating the growth of an existing
malignancy. The added cost of the procedure and regulatory uncertainty
also hamper adoption of the technique in the United States. Previous
publications have clarified the cost associated with CAL, and residual
enzyme levels are well documented for a variety of different methods
of obtaining SVF in the clinical setting.19
The results of this series suggest that CAL performed routinely
using a manual, collagenase SVF separation method at the point of care
is not associated with an added risk of surgical complications or malig-
nancy. The overall complication rate of 3.7% reported in this series is
comparable to that reported for conventional AFG.22,23 Theorized risks
specific to the SVF separation process, such as allergic reaction to col-
lagenase or neutral protease and tissue necrosis due to residual enzyme,
did not occur in this series. No untoward effects of enzyme activity were
detected. Changes in clinical behavior of adipose cells released from

FIGURE 3. A, Subject had a contour deformity which presented as a concave area on the right buttocks resulting from a complication
from an injection which the subject received as a child. The subject was injected with an autologous SVF-enhanced fat graft to repair
the contour deformity. B, 9 months after cell-enhanced fat grafting. C, 12 months after cell-enhanced fat grafting.

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Annals of Plastic Surgery • Volume 75, Number 6, December 2015
FIGURE 4. A, A 73-year old woman with a chronic wound which presented as non-healing for 22 months. B, The wound was injected
with an autologous SVF cell and fat mixture generated at the point of care. C, The wound was cleared after 9 weeks postoperatively.
their supporting fibrous stroma have been theorized but we detected no
effects attributable to any structural changes produced in adipose tissue
as a result of enzymatic digestion.
Patients were regularly screened for breast cancer with ultra-
sound and mammography for 12 months after the CAL procedure.
Among the 133 patients followed for over a year there were no new neo-
plasms reported, however one patient did have a recurrence of a previ-
ous breast cancer 11 months after receiving CAL. Although none of
the patients followed for less than 12 months (n = 31) have developed
any malignancy, it is too early to make any assumptions based on insuf-
ficient follow up time. Additional studies with long-term follow-up are
needed to rule out the tropic effects of CAL on malignancy.
As mentioned previously, there was 1 recurrence of a previous
triple-negative breast cancer 11 months after treatment. The patient
had undergone chemotherapy and unilateral skin-sparing mastectomy,
but refused radiation therapy. Reconstruction was performed using
350 mL saline implant accompanied by CAL for added contouring.
The recurrence was localized and was excised safely without complica-
tion. No recurrence has been reported in the 2 years since excision of
the recurrent malignancy. It is not certain whether or not pluripotential
cell injection was responsible for this recurrence. The rate of recurrence
in this study (1.9 %) does not suggest an increased risk of recurrence
associated with pluripotential cell injection.24–26
Our data support the notion that CAL does not increase the risk
for development of cancer or accelerate the growth of an existing unde-
tected neoplasm. This study does not nullify the theoretical risks as-
sociated with the pluripotential cell injection theorized from in vitro
studies of adipose-derived stem cell (ASC) trophic factors and their ef-
fects in coculture with breast cancer cells.27,28 Growth factors secreted
by mesenchymal stem cells including vascular endothelial growth factor,
interleukin-6 and platelet-derived growth factor (PDGF) have been
shown to promote growth and metastasis of cancer cells in vitro.29–33
Previous studies suggest that PDGF has been linked to increased risk
of death due to cancer after prolonged exposure, as demonstrated by
the topical recombinant PDGF cream Regranex (becaplermin) which
is used to treat lower extremity diabetic neuropathic ulcers.34 Patients
who used more than 3 tubes of Regranex were linked to a significantly
higher risk or mortality due to cancer.35,36 Patients were informed of
these potential risks before participation.
Another potential risk of CAL is an added rate of infection due
to contamination during the separation process. The manual SVF sepa-
ration technique used in this series was performed under an ultrahigh
filtration biohood to reduce ambient air exposure of the graft material
and SVF cells. There were no cases of graft recipient site infection in
this study.
This report is limited to the issue of safety using manual,
collagenase-based SVF separation at the point of care and does not ad-
dress the potential of CAL compared to non-enhanced fat grafting at
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Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Clinical Safety of SVF Isolation at POC
improving long-term graft volume, predictability or effects on irradiated
or damaged tissues. It also does not provide data regarding other impor-
tant issues, such as the cell dose versus effect relationship and cell sub-
populations within the SVF, that may be more beneficial in adipose
tissue regeneration. The results of this study confirm earlier reports that
a processing time of approximately 90 minutes is necessary to produce
concentrated, autogenous SVF from lipoaspirate at the point of care.
Although a variety of mechanical, non-enzymatic methods have
been proposed for the isolation of ASCs and SVF cells, none pro-
vide the same level of cell recovery achieved using collagenase-based
methods. Mechanical methods have reported cell yields which range
from 6250 to 25,000 ASCs/mL of lipoaspirate processed.37–40 In com-
parison, cell yields of collagenase-based SVF isolation methods have
reported yields of 100,000 to 500,000 viable cells/mL of lipoaspirate
processed compared.15,16,19,37,38 In addition to isolating more viable
cells/mL of lipoaspirate than mechanical methods, enzymatic methods
also differ in composition of the isolated cell population.37 Mechanical
methods have reported a cell composition consisting of only 5%
ASCs,39 whereas enzymatic methods have reported up to 15% ASCs
in the isolated SVF cell population.19 The composition of the cell pop-
ulation acquired mechanically is biased towards hematogenous cells
(CD45+) because there is significantly less tissue disruption to release
stem cells from the perivascular stroma. Mechanical methods offer a
cost-effective way to acquire SVF cells and may be a practical approach
in settings where a large number of ASC cells are not required. How-
ever, without the tissue dissociation afforded by enzymatic digestion
of collagen, all mechanical techniques are limited in the number of vi-
able cells and especially ASCs that can be recovered.
The tissue dissociation enzyme mixture used in this study was
composed of collagenase I and II isoforms derived from Clostridium
histolyticum and neutral protease derived from B. polymyxa, the mixture
of which provides better tissue dissociation than either alone.17,41 The
purity and composition of various tissue dissociation enzyme mixtures
varies from product to product and researchers and clinicians should be
aware of this variability when isolating SVF. Although neutral protease
has been shown to play a critical role in the tissue dissociation pro-
cess,41–44 its exact role is not fully understood. Concerns have been
expressed that unwanted tissue degradation may occur upon reinjection
from miniscule residual neutral protease activity which may be present
in the purified SVF. This study however demonstrated no evidence of
added risk.
Safety of clostridial collagenases has been reviewed, mainly
pertaining to use for treatment of Dupuytren contracture45–47 and
Peyronie disease48,49 in FDA approval process for the drug Xiaflex.50,51
The clinical and non-clinical evidence overwhelmingly supports the
safety of clostridial collagenases. Collagenase was shown to have no
systemic toxicity after local injection and systemic exposure was only
observed if injected into highly vascularized areas.52–55 Collagenase
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Aronowitz et al
was also shown to be removed from the system rapidly, with neither
isoform I nor II being detectable 2 hours after injection. Overall, colla-
genase has a very low level of toxicity.56–58
Safety concerns about residual enzyme activity are negligible at
the levels documented using the manual SVF separation process in this
series. In a previous study conducted by University Stem Cell Center,
we demonstrated that the residual levels of collagenase present using
our manual isolation method is less than 0.01 Wünsch units (U) of
collagenase activity/mL of SVF isolated.19 Even this small enzymatic
activity level drops by a further factor of 10 once the SVF cells are
combined with the lipoaspirate for reinjection. This level of activity is
insignificant when compared for example to 3600 U per dose in
Xiaflex, and 250 U/g in Collagenase Santyl,59,60 another collagenase-
based product topically applied for wound debridement, both of which
have been deemed safe by the FDA. Although limited comparison can
be made between Santyl, Xiaflex and our use of collagenase because
they are administered in different manners, it does offer insight into
the levels of collagenase activity that the FDA has already deemed
safe for human use.
CONCLUSIONS
Enriching fat graft with adipose stem cells contained in SVF of
lipoaspirate, or CAL, is a promising strategy to improve the predictabil-
ity and consistency of fat grafting results. SVF separation techniques
that utilize enzymes are approximately a thousand-fold more effective
in isolating ASCs. Safety concerns related to collagenase use have
inhibited the adoption of CAL as an option in AFG. The results of
this series demonstrate that a nonautomated, collagenase-based SVF
cell isolation process performed at the point of care is equal in safety
to conventional fat grafting. This result supports the expanded use of
this emerging cellular technology in the clinical research setting.
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