4260
Views & Citations3260
Likes & Shares
INTRODUCTION
With a
variety of SVF based therapies beginning to translate into the clinical
setting, it is important for clinicians to be aware of the regulatory
considerations and quality control measures which need to be in place in order
to build reliable data and ensure patient safety. Important factors to consider
are the composition of the SVF output from the isolation method being employed,
the dosing scheme, route of administration appropriate for each specific
treatment, as well as adequate infection control protocols. The methodology
described in this review is only applicable to the autologous use of freshly
isolated SVF cells. Additional quality control and regulation measures are
necessary for other, non point of care uses, such as in a wound care matrix or
expansion of cells in culture.
Brief Regulatory Overview
Current regulation of the clinical use of autologous stromal vascular
fraction cells in the United States remains somewhat convoluted and uncertain.
Use of autogenous cells at the point of care was long considered to fall within
the scope of the practice of medicine and therefore regulated at the state
level. However it is now clear that the field is heading towards an era of
significantly higher regulation at a federal level. The FDA in the United
States issued a series of draft guidance announcements beginning in October
2014 concerning the use of adipose-derived human cellular, tissue, and cellular
and tissue-based products (HCT/Ps) [1-4]. These draft guidance documents are
not binding but are used to express current FDA thinking. A reading of these
draft guidance publications readily reveals the FDA’s position that the
clinical use of autologous SVF cells does not fall under the practice of
medicine or the same surgical procedure exemption. FDA has concluded that the
SVF isolation process, whether enzymatic or purely mechanical, represents more
than minimal manipulation. In this view, the isolation of autologous SVF at the
point of care by whatever means creates a tissue product subject to FDA
oversight. This emerging FDA view means the clinical use of SVF will be
subjected to a rigorous regulatory pathway and enforcement methods unfamiliar
to most clinicians.
A three-tiered system based on level of risk was established by the FDA
to classify the use of HCT/Ps in clinical practice. Table 1 summarizes the FDA
recognized regulatory categories. Previously, it was assumed that the use of
SVF was regulated only under 21 CFR part 1271 and Section 361 of the Public
Health Service Act (PHS), and as such deemed “361 HCT/Ps” [5,6]. When SVF is
considered a 361 HCT/P, it’s use is subject to very little to no FDA regulation
under the assumption that SVF prepared under these criteria qualified for
almost every exemption when used autologously in the same surgical procedure
using surgical practices meeting the local standard of care. The “more than
minimally manipulated” classification of SVF cells subjects them to additional
regulation under Section 351 of the PHS act and/or the Food, Drug and Cosmetics
Act (FD&C), making them “351 HCT/Ps” [6,7]. As a 351 HCT/P, clinical use of
SVF cell-based therapies will be required to be conducted in formal
FDA-approved clinical trials under the regulatory approval of an
Investigational New Drug (IND) application or an Investigational Device
Exemption (IDE) in addition to institutional approval from an Institutional
Review Board (IRB). This is because ultimately these therapies will have to
acquire a Biologics License Application (BLA) before they can reach the market.
FDA-approved clinical trials require extensive amounts of preclinical data and
product characterization as well as a set of quality control measures in place
to ensure patient safety. The cost of the additional analysis as well as the
costs associated with designing, initiating and conducting a clinical trial can
be quite substantial and put an undue financial burden on smaller practitioners
and essentially prevent them from being able to participate in the clinical
research process without government or industry support. The 351 HCT/P
classification of SVF cell based therapies excludes a significant portion of
clinicians from offering these therapies to patients because they lack the
time, money and regulatory knowledge to successfully navigate the complex
regulatory framework involved with establishing a formal clinical trial and
dealing with the FDA.
Infection Control
When it comes to a clinical treatment using SVF cells, infection
control is one of the primary concerns in regards to ensuring patient safety.
There are 2 main tests which need to be run in order to adequately assess the
sterility of the product for human administration. These tests are a STAT gram
stain and aerobic/anaerobic cultures, usually run for 3-5 days [8]. Figure 1 and Table 2 summarizes the suggested quality control assays as well as
the clinical workflow.
The STAT Gram stain and anaerobic cultures will test for the presence
of microbial contaminants. From a practical standpoint, once the final SVF is
isolated, a sample will immediately be sent over to a local pathology lab (or
done in house if proper staffing, facility certifications and equipment is in
order) for gram staining and cultures. The turnaround time for a STAT gram
stain is usually about an hour, but this can differ based on various factors
such as the proximity, workload and staffing available at the pathology lab.
Administration of the therapeutic product cannot proceed until the results of
the gram stain are received and are negative for the presence of bacteria. If a
positive gram stain result is returned from the pathology lab, then the
procedure should not proceed (Figure 2).
The anaerobic and aerobic cultures can be most easily carried out using
a standard blood culture set such as a BD BACTEC aerobic and anaerobic culture
bottle set (Becton, Dickinson and Company, Franklin Lakes, NJ). These can
easily be aseptically inoculated and then sent off to a local pathology lab to
maintain and analyze. As the results from these cultures will not be received
before the treatment occurs, these are not intended to be a clinical deterrent.
Even in the event of a positive culture result, the subject does not
necessarily need to be withdrawn from participation in a clinical trial, but
rather should be monitored closely for potential infection. A positive aerobic
or anaerobic culture does not necessarily mean that the patient is going to
develop an infection. Additionally, pathology or microbiology labs typically
offer microbial identification as well as specificity testing
which can help guide clinicians to the proper measures such as which
antibiotics to use in the event of an infection.
Bacterial Endotoxin Testing
Bacterial endotoxin testing is another important quality control
measure for SVF as well. The US FDA will require this for all SVF and ASC based
therapies [9,10]. Bacterial endotoxins are lipopolysaccharides present in the
cell membrane of gram negative bacteria. Endotoxins are pyrogenic, meaning that
they can potentially cause fever or disease if present at high enough levels in
vivo. While bacterial endotoxins are associated with gram negative
bacteria, the bacteria does not have to be viable in order to exert
pyrogenicity, meaning that even membrane fragments of gram negative bacteria
can cause fever or disease at high enough levels. This is an important parameter
to track in terms of patient safety, especially if the therapeutic product is
to be injected or administered intravenously. SVF can potentially have higher
levels of endotoxin as a result of the tissue dissociation enzyme mixture used
to dissociate the lipoaspirate. The GMP grade tissue dissociation enzyme (TDE)
mixtures tend to contain proteolytic enzymes from bacterial origins. As a
result, there are residual levels of endotoxin present in the final lyophilized
product as a result of the manufacturing process [11]. While there are
acceptable standards for endotoxin levels in the TDE products in order to be
GMP grade, clinicians should be aware of these endotoxin levels which in
combination with other potential endotoxin sources can potentially raise sample
levels above that acceptable threshold for treatment. The certificate of
analysis should provide the endotoxin level present in the TDE product. Ideally,
a STAT endotoxin test sent out to a local pathology laboratory would be the
easiest way to have this conducted because no specialized equipment would need
to be acquired. Unfortunately many microbiology/pathology labs do not offer a
STAT format for this test, but rather the test would have a turnaround time
ranging from a few days to a week. This leaves the alternative of running this
test at the point of care in house. The leading assay used to assess endotoxin
levels is the Limulus Amoebocyte Lysate (LAL) assay. There are systems
available for the point of care endotoxin testing, such as the EndoSafe-PTS100
or EndoSafe NexGenPTS, (Charles River Laboratories International, Inc., San
Diego, CA). These two systems use FDA approved single-use test cartridges to
run point of care endotoxin testing with a 15 minute turnaround time. The
alternative to purchasing the system or sending out the sample to a pathology
lab is to run the LAL assay in house. The necessary reagents may be purchased
and the assay can be run using a microplate reader and minimal other equipment.
There are 3 versions of the LAL assay which can be run: the gel-clot method,
the chromogenic method and the turbidimetric method. All three methods are
approved by the United States Pharmacopeia for use with injectable drugs and
other products [12].
The lot release specifications pertaining to endotoxin are based on the
endotoxin limit. The endotoxin limit is the threshold established for a product
to be safe enough for human use. If the product measures an endotoxin level
above the threshold, clinical treatment should not proceed (Figure 2). The endotoxin limit varies
from patient to patient and is a measurement calculated based on the weight of
the patient, route of administration and the maximum amount of the therapeutic
agent which will be injected. For a detailed explanation of how to calculate
the endotoxin limit, see Chapter 85 of the United States Pharmacopeia [13].
Nucleated Cell Counting
Nucleated cell counting is arguably one of the single most important
aspects of quality control. It will typically be the first checkpoint reached
in terms of lot release specifications for clinical use. From a clinical
workflow perspective, after completing isolation, a sample should be taken and
sent out or set aside for infection control testing and endotoxin testing
followed immediately by cell counting. There will be some downtime before the
results of these tests are received, leaving time to complete cell counting and
other required analysis.
The isolation of SVF cells can be somewhat variable from isolation to
isolation and depends on many factors, some of which cannot be control, mainly
patient to patient variation. Setting lot release specifications is an
essential part of a proper manufacturing process and should not be ignored as
they will be required by the FDA [8] and ensure a minimum level of quality for
cell-based therapies. The SVF cell isolation will need to hit a certain
threshold of viable nucleated cells while also being above the threshold of
acceptable cellular viability (usually ≥70%). If an isolation does not meet the
predetermined lot release criteria, then the clinical treatment should not
proceed.
Nucleated cell counting typically does not require a large sample
volume, typically only a few hundred microliters. Nucleated cell counting can
be conducted in a variety of ways, but it helps to uses those which the FDA has
deemed valid, such as the Chemometec NC-100 or NC-200, which effortlessly can
provide nucleated cell counts and cellular viability in a matter of minutes. A
sample cell count is shown in figure 3. These systems are expensive, so if
funding is not available a cheaper alternative such trypan blue staining and
manual counting using a hemocytometer can be employed. There are a variety of
options available for determining nucleated cell counting and viability, but
typically the ease of use is inversely proportional to the cost to operate.
Dosing Schemes
The FDA will require all clinical trials to contain calculated dosing
schemes [8]. Depending on the indication of use, the dosing pattern will vary.
For example, if using SVF cells for the treatment of a chronic wound or hair
growth, the dose will be calculated as cells per area (ie 500,000 viable nucleated
cells/cm2). Other indications, such as intra-articular injections
for the treatment of osteoarthritis might be established as a set number of
nucleated cells (ie 10 million viable nucleated cells). The difference between
the two being that for the former, the minimum number of cells required to be
obtained from the isolation process can vary based on the size of the treatment
area (a 10cm2 wound will require more cells than a 5cm2
wound), whereas the latter will remain constant. Another aspect which should be
considered is the combination of SVF cells with lipoaspirate tissue for
cell-assisted lipotransfer (CAL) and cell-enhanced fat grafting procedures. For
this, the dose would ideally be calculated as a density of cells per volume of
graft material (ie 10,000 viable nucleated cells per mL of graft material). It
is also important to note that there is very little information currently
available in relation to dose-versus-effect in terms of SVF cell based
therapies. That being so, the FDA will almost certainly require some amount of
dose exploration for all trial, for example a high dose (40 million viable
nucleated cells) and a low dose (20 million viable cells). Controlling the dose
and instituting an aspect of dose exploration is the only way that a valid
dose-vs-effect relationship can be developed. Table 3 summarizes appropriate dosing schemes.
Another
aspect to consider when preparing the dose is that you must take into account
the volume of sample being taken for analysis and not used for treatment.
Typically 1-2 ml of the final output will be used for analysis, including cell
counting, gram staining, 5 day aerobic/anaerobic culture and potentially flow
cytometry and CFU-F assays if being conducted. This will reduce the number of
available viable nucleated cells that clinicians have access to for use in
treatment. There are ways to avoid this which can be built into the isolation
process, such as generating a larger volume of SVF output. It is important to
use a little sample as possible while still meeting the minimum requirements
for each specific test, so as to avoid not being able to meet the dosing
requirement.
Residual Proteolytic Enzymes
If the SVF cell isolation method employs the use of proteolytic
enzymes, there will be a risk of excess levels of residual proteolytic enzymes
in the final isolate, ie collagenase. The use of proteolytic enzymes is an easy
way to drastically increase the yield of nucleated cells recovered during an
isolation process, an aspect which is extremely beneficial if using the isolate
immediately at the point of care [14]. Residual enzymes are a risk because in
theory they can result in allergic reaction or unwanted tissue degradation in
vivo if not adequately removed. The primary enzymes used for isolation of
SVF cells are collagenase and neutral protease, both of bacterial origin.
Collagenase has been fairly well studied in terms of safety for human
use in the two FDA approved iterations of the enzyme, Xiaflex and Collagenase
Santyl. Santyl (250U/g) is a topical wound ointment for use in the nonsurgical
debridement of wounds [15,16]. Xiaflex (3600 U per dose) is a highly
concentrated mixture of type I and type II clostridial collagenases for the
treatment of Dupuytren’s contracture and Peyronie’s disease [17-22].
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 [23-26].
Collagenase was also shown to be removed from the system rapidly, with
neither isoform I nor II being detectable 2 hours after injection. Overall,
collagenase has a very low level of toxicity [27-30]. In a previous study from
our group in which we conducted 174 clinical isolations using
collagenase, we noted no adverse events which could be attributed to excess
residual activity of collagenase or neutral protease. As a note, we conduct 3
washing steps on our SVF isolate [31].
The easiest way to control for excess residual enzymes is to neutralize
all activity of these enzymes. This can be done using autologous serum which
can be isolated from the patient concurrently to the SVF isolation protocol.
Serum has been shown to neutralize the activity of collagenase. This is because
serum contains alpha-2-macroglobulin, which acts as an antiproteinase and
inactivates a variety of proteolytic enzymes, including collagenase [32,33].
Alternatively, a 2013 study by Chang et al. 27 showed that so long as 3 washing
steps are conducted of the resulting SVF isolate, that there is negligible risk
associated with proteolytic enzymes because they are found in such low
concentrations.
This is important because it will be required by the FDA for the
isolation process being used to be evaluated in some way for safety of residual
proteolytic enzymes before an IND/IDE will be granted [8]. This may entail
animal studies or a qualification study measuring the residual enzyme levels in
the final output. Obviously a small qualification study is more favorable than
animal studies for a number of reasons, mainly cost and time. The most common
assays used to assess collagenase activity are the FALGPA assay and the Wunsch
Assay [34,35]. There are a variety of assays which can be used to assess
neutral protease activity [36,37]. The residual enzyme levels in samples can be
easily measured using commercially available kits or manually by purchasing
appropriate reagents. The goal of a qualification study would be to prove that
the levels present in the final output are so low that they are not clinically
significant and do not pose any significant risk to patients.
Flow Cytometry
Product characterization is very important aspect of quality control as
well. Flow cytometry is important for clinical applications because it allows
for identification of the abundance of the various cell types present in the
therapeutic product. For SVF, being that it is a heterogeneous population of
cells, this is important since every SVF isolation is different due to a
variety of factors including differences in isolation techniques, differences
in amount of tissue processed and patient to patient variation. While flow
cytometry is not included in lot release criteria, the cellular composition
will need to be established prior to clinical initiation of a later stage
clinical trial (ie Phase 3) or established as a result of an early stage
clinical trial (ie Phase 1/2) as part of the identity test for the therapeutic
product [38].
There are a number of ways that flow
cytometry can be conducted, but generally the more specific the flow cytometry
analysis is (ie more surface markers), the more information that is ultimately
learned. Ideally, through flow cytometry, the goal would be to identify the
portion of the SVF which is actually composed of adipose-derived stem cells,
which generally is the target therapeutic agent for clinical use. This is a
small fraction of the SVF cell isolate, usually <2%. To do this, a more
targeted flow cytometry protocol is required. Typically additional markers
screened for are CD73, CD90 and CD105 [39-41]. It is crucial to note that the
surface markers for cultured ASCs and uncultured SVF cells differ. Typically,
as SVF cells are cultured, they stop expressing CD34, and therefore express a
different phenotype. Potential clinicians should be cautious not to make this
mistake when reviewing pertinent literature on expected outcomes of cellular
populations and surface markers used to identify them.
Colony Forming Unit-Fibroblast (CFU-F) Assay
The CFU-F assay is a valuable tool and a
definitive method for quantifying the number of adipose-derived stem cells in a
sample of SVF cells. It offers a more specific method for quantification than
using a standard cell counting device, which non-specifically quantifies
nucleated cells. This helps to further characterize the final product. The
CFU-F assay involves culturing a set number of cells (ie 1,000 and/or 2,500
viable nucleated cells) for usually 10-14 days and observing the growth
characteristics. This assay will assess the number of colonies formed, which is
an overall indicator of the frequency of adipose-derived stem cells.
Additionally, this assay determines the population doubling time which assesses
the overall growth characteristics of the adipose stem cells present within the
stromal vascular fraction. Generally, a faster doubling time is preferred
because it means that fewer days can be spent in culture in order to reach confluence.
This saves money, as culture supplies can be expensive, and suggests higher
metabolic activity of the cells. One of the most important considerations when
conducting CFU-F assays is that the culture conditions must be identical from
assay to assay. Results from assays conducted under different culture
conditions are not comparable from an analytical standpoint because cells grow
differently under different growth conditions. Hicok and Hendrick published a
method for conducting a CFU-F assay on SVF cells [42].
This assay is not meant to be a clinical
deterrent, but rather to further develop the identity of the therapeutic
product. As mentioned earlier, the FDA will require the presence of an identity
test in order to proceed with any clinical trial which is phase 3 or later.
Doing a CFU-F in tandem with a 6 marker flow cytometry panel will give an
accurate assessment of the composition of the SVF isolate once the data set is
large enough.
With a large enough data set pertaining to
stem cell content of the SVF, a profile can be generated when comparing a
treatment subject’s CFU-F frequency with the overall success rate of the
procedure. This has been demonstrated with bone marrow aspirates used for
treatment of tibial nonunions. In 2005, Herniguo et al. [43] reported a
relationship between the numbers of CFU-F present in grafted bone marrow
aspirates with the success of the grafting procedure to obtain bone-healing of
tibial nonunions. They observed that more CFU-Fs correlated with increased
volume of mineralized callus formation at 4 months, and low CFU-F counts
correlated with longer healing times. With increasing amounts of data, Herniguo
et al. were able to roughly estimate a relative threshold for success based on
the number of CFU-F present in an aspirate, where a CFU-F level below a certain
number was a strong indicator of slower healing time. The CFU-F assay should be
included in all clinical analysis of SVF in order to build the data in this
manner, not just for the FDA, but for the benefit of the clinician as well.
CONCLUSION
In summary, the regulatory status of SVF based therapies in the United States suggests that an IND or IDE is required in order to proceed clinically. As such, a variety of quality control and analytical measures are required. The goal of these measures is to ensure that clinicians definitively know what they are treating patients with and know that they are not introducing any added risk to the patient in doing so.
1. FDA (2014) Minimal Manipulation of
Human Cells, Tissues, and Cellular and Tissue-Based Products: Draft Guidance
for industry and food and drug administration staff.
2. FDA (2014) Human Cells, Tissues,
and Cellular and Tissue-Based Products (HCT/Ps) from Adipose Tissue: Regulatory
Considerations; Draft Guidance for Industry.
3. FDA (2014) Same surgical procedure
exception under 21 CFR 1271.15(b): questions and answers regarding the scope of
the exception. Draft Guidance for industry.
4. USFDA (2015) Homologous use of
human cells, tissues, and cellular and tissue-based products. Draft Guidance
for Industry and FDA staff..
5. USFDA (2015) 21 CFR part 1271.
6. USFDA (2013) Public Health and
Service Act.
7. USFDA(2015) Food, Drug, and
Cosmetic Act. U.S. Food and Drug Administration.
8. USFDA (2008) Guidance for FDA
Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and
Control (CMC) Information for Human Somatic Cell Therapy Investigational New
Drug applications (INDs).
9. USFDA (2015) Bacterial
Endotoxins/Pyrogens. Inspection Technical Guides. U.S. Food and Drug
Administration.
10. USFDA (2012) Guidance for
Industry: Pyrogen and Endotoxins Testing: Questions and Answers. U.S. Food and
Drug Administration.
11. Williams KL (2007) Pyrogens, LAL
Testing and Depyrogenation. (3rd edn), Informa Healthcare.
12. Joiner TJ, Kraus PF, Kupiec TC
(2002) Comparison of endotoxin testing methods for pharmaceutical products. Int J Pharm Comp 6: 408-409.
13. United States Pharmacopeia (2012)
Bacterial Endotoxins Test. Chapter 85, United States Pharmacopeia.
14. Aronowitz JA, Lockhart RA,
Hakakian CS (2015) Mechanical versus enzymatic isolation of stromal vascular
fraction cells from adipose tissue. Springer
Plus 4: 713.
15. Shi L, Carson D (2009) Collagenase
Santyl ointment: A selective agent for wound debridement. J Wound Ostomy Continence Nurs 36:
512-516.
16. Smith, Nephew (2014) Exploring
Clinical Data for Collagenase Santyl Ointment.
17. Nunn AC, Schreuder FB (2014)
Dupuytren’s contracture: emerging insight into a viking’s disease. Hand Surg 19: 481-490.
18. Gilpin D, Coleman S, Hall S,
Houston A, Karrasch J, Jones N (2010) Injectable collagenase clostridium
histolyticum: a new nonsurgical treatment for Dupuytren’s disease. J Hand Surg Am 35: 2027-2038.
19. Sood A, Therattil PJ, Paik AM,
Simpson MF, Lee ES (2014) Treatment of Dupuytren’s contracture with injectable
collagenase in a veteran population: A case series at the department of
veterans affairs new jersey health care system. Eplasy 14: e13.
20. Egui Rojo MA, Moncada Iribarren I,
Carballido Rodriguez J, Martinez-Salamanca JI (2014) Experience in the use of
collagenase histolyticum in the management of peyronie’s disease: Current data
and future prospects. Ther Adv Urol
6: 192-197.
21. Gelbard M, Goldstein I, Hellstrom
WJ, et al. (2013) Clinical efficacy, safety and tolerability of collagenase
clostridium histolyticum for the treatment of peyronie disease in 2 large
double-blind, randomized, placebo controlled phase 3 studies. J Urol 190: 199-207.
22. AAC (2009) Briefing Document for
Collagenase Clostridium Histolyticum (AA4500).
23. Miyabayashi T, Lord PF, Dubielzig
RR, Biller DS, Manley PA (1992) Chemonucleolysis with collagenase. A
radiographic and pathologic study in dogs. Vet Surg 21: 189-194.
24. Friedman K, Pollack SV, Manning T,
Pinnell SR (1986) Degradation of porcine dermal connective tissue by
collagenase and by hyaluronidase. Br J
Dermatol 115: 403-408
25. Bromley JW, Hirst JW, Osman M,
Steinlauf P, Gennace RE, et al. (1980) Collagenase: an experimental study of
intervertebral disc dissolution. Spine
5: 126-132.
26.
Bromley
JW (1982) Intervertebral discolysis with collagenase. Arzneimittelforschung 32: 1405-1408.
27. Chang H, Do BR, Che JH, et al.
(2013) Safety of adipose-derived stem cells and collagenase in fat tissue. Aesthetic Plast Surg 37: 802-808.
28. Zook BC, Kobrine AI (1986) Effects
of collagenase and chymopapain on spinal nerves and intervertebral discs of
Cynomolgus monkeys. J Neurosurg 64:
474-483.
29. Sussman BJ, Bromley JW, Gomez JC
(1981) Injection of collagenase in the treatment of herniated lumbar disk:
Initial clinical report. JAMA 254:
730-732.
30. Garvin PJ (1974) Toxicity of
collagenase: the relation to enzyme therapy of disk herniation. Clin. Orthop Relat Res 101: 286-291.
31. Aronowitz JA, Lockhart RA,
Hakakian CS, et al. (2015) Clinical Safety of Stromal Vascular Fraction
Separation at the Point of Care. Ann
Plast Surg 75: 666-671.
32. Curry TE Jr, Mann JS, Estes RS, et
al. (1990) Alpha 2-macroglobulin and tissue inhibitor of metalloproteinases:
collagenase inhibitors in human preovulatory ovaries. Endocrinol 127: 63-68.
33. Pedersen JZ, Franck C (1986)
Increased serum levels of alpha-2-macroglobulin in severe chronic airflow
obstruction. Eur J Respir Dis 68:
195-199.
34. Wünsch E, Heidrich HG (1963) On
the Qualitative Determination of Collagenase, Z. Physiol. Chem
333: 149.
35. Van Wart HE, Steinbrink DR (1981)
A continuous spectrophotometric assay for Clostridium histolyticum
collagenase. Analytical Biochem
133: 356-365.
36. Breite AG, Dwulet FE, McCarthy RC
(2010) Tissue dissociation enzyme neutral protease assessment. Transplantation Proceedings 42:
2052-2054.
37. Lockhart RA, Hakakian CS,
Aronowitz JA (2015) Tissue dissociation enzymes for adipose stromal vascular
fraction cell isolation: A review. J
Stem Cell Res Ther 5: 12
38. USFDA (2013) Guidance for
industry: Preclinical assessment of investigational cellular and gene therapy
products.
39. Mitchell JB, McIntosh K, Sanjin Z,
et al. (2006) Immunophenotype of human adipose-derived cells: temporal changes
in stromal-associated and stem cell-associated markers. Stem Cells 24: 376-385.
40. Bourin P, Bunnell BA, Casteilla L,
et al. (2013) Stromal cells from the adipose tissue-derived stromal vascular
fraction and culture expanded adipose tissue-derived stromal/stem cells: a
joint statement of the International Federation for Adipose Therapeutics and
Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy 15: 641-648.
41. Lin G, Garcia M, Ning H, et al.
(2008) Defining stem and progenitor cells within adipose tissue. Stem Cell Dev 17: 1053-1064.
42.
Hicok
KC, Hendrick MH (2001) Automated isolation and processing of adipose-derived
stem and regenerative cells. Methods
Mol Biol 702: 87-105.
43.
Herniguo
P, Poignard A, Beaujean F, et al. (2005) Percutaneous autologous bone-marrow
grafting for nonunions. Influence of the number and concentration of progenitor
cells. J Bone Joint Surg Am 87: 1430-1437.
44. Bipartisan Policy Center (2015) Advancing
regenerative cellular therapy: Medical innovation for healthier americans.
QUICK LINKS
- SUBMIT MANUSCRIPT
- RECOMMEND THE JOURNAL
-
SUBSCRIBE FOR ALERTS
RELATED JOURNALS
- Dermatology Clinics and Research (ISSN:2380-5609)
- Journal of Clinical Trials and Research (ISSN:2637-7373)
- International Journal of Surgery and Invasive Procedures (ISSN:2640-0820)
- Journal of Spine Diseases
- Ophthalmology Clinics and Research (ISSN:2638-115X)
- Journal of Cell Signaling & Damage-Associated Molecular Patterns
- Journal of Immunology Research and Therapy (ISSN:2472-727X)