This is a great question a wonderful patient asked me recently. The answer is that there are many ways to determine how many cells are in a milliliter of SVF with cell counters, but it is very difficult (outside of precise laboratory studies to my knowledge: see below **) to determine how many viable stem cells are actually present. Technology is almost there where automatic machines can give you such data, but they are still not readily available to doctors and their patients at this time (though I do have 2 calls into companies that are heading there…to see what the starting price tag is for such machines).
**See post https://drcremers.com/2018/02/what-is-best-source-of-stem-cells-bone.html
And search for “Isolation and Preparation of Adipose-derived Stem Cells.”
It is not easy to prove viability of adipose derived stem cells. There are many cell counters, but these count all cells in a sample.
So the question is:
Should patients seek SVF injections or deployments as is currently formulated in its various forms (knowing enzymatic methods have more viable stem cells over manual methods)
Risk further damage of tissue and decline of body function and wait for the technology to get better.
This is a very tough decision.
The technology is almost there, but there are not large scale, prospective randomized, double blinded studies to prove its use in every disease. And it is not cheap.
Why is that? Most feel it is because there are no drug companies involved that could do such a big study. They is no money for them if stem cells work as these cells are coming from inside the patient’s own body.
Many MDs are using the manual method without enzyme use, but this has been proven to yield less viable stem cells. It is easier, though, and cheaper and does work in many studies noted below in certain diseases.
Enzymatic methods of isolating SVF cells from adipose tissue are better and used by the Cell Surgical Network, for instance, but they are more costly and time consuming.
This is a very confusing and frustrating time for patients and surgeons. Doctors want to heal their patients and do no harm. Patients want a guaranteed cure. We are so close, but still not there yet and no guarantee can be given or promises even with the best, purest stem cell isolate.
The first, double blinded study with stem cell injections was done and published by the Mayo Clinic: see here for post.
The below information hopes to educate all patients of where we stand today on Stromal Vascular Fraction which is a more accessible way to inject a patient’s own stem cells into a diseased tissue.
For the dry eye issue, my plan is to use special, sterile filters to isolate out any further blood products and get further purity of the stem cell isolate.
All injection of SVF have risks. The biggest risk is the unknown. The second biggest risk is the risk of no improvement. The risk of infection, tumor growth, cancer appears to be very low if not zero in most situations. Still there is no 100% guarantee it will be a cure for the condition being treated.
Sandra Lora Cremers, MD, FACS
Below are 2 excellent articles about the status of SVF today.
The key points are:
1. The advantage of SVF over ADSCs is believed to be in two fundamental areas. Firstly, although similar in properties such as immunomodulation, anti-inflammatory, angiogenesis, and so forth, the distinctive, heterogeneous cellular composition of SVF may be responsible for the better therapeutic outcome observed in comparative animal studies [9–12].
Digestion of lipoaspirate is achieved by collagenase, and the presence of collagenase in the injectable product does not bode well with regulatory authorities such as the US Food and Drug Administration (FDA) . Consequently, alternative methods are being explored with some encouraging outcomes [22–25].
The surge in clinical applications for ASCs increases the need for clear and reliable information about the efficiency, cost and safety of automated equipment and manual techniques which facilitate separation of the stromal vascular fraction (SVF) from adipose tissue. In clinical practice, adipose-derived stem cells are often not administered as a pure isolate but rather as one constituent of stromal vascular fraction, a heterogeneous mixture of cells resulting from the mechanical or enzymatic processing of aspirated adipose tissue. SVF contains a variety of cells including macrophages, various blood cells, pericytes, fibroblasts, smooth muscle cells, vascular endothelial progenitors and adipose-derived stem cells (Yoshimura et al. 2006; Bourin et al. 2013; Han et al. 2010; McIntosh et al. 2006; Bonab et al. 2006; Yoshimura et al. 2009). Stromal vascular fraction is one component of the heterogeneous mixture of adipose tissue fragments, stromal tissue, blood and tumescent fluid which constitutes lipoaspirate. The ASC content of SVF varies substantially depending on the method employed, with reports from less than 1 % of cells to over 15 % (Table 1). SVF cells can be safely isolated, quantified and characterized at the point of care in approximately 90 min. This is a timeframe which permits isolation and treatment to occur in the same surgical procedure, that is, at the point of care.
Here is what the rep said:
VP, Global Marketing and Business Development
Here is another automatic unit recommended in 2 papers below: waiting to hear from rep on cost.
The main drawback of many of these devices is the cost of operation. The closed, enzymatic systems can be very expensive, with some costing over $50,000 for the system. In addition to purchasing the device, many require single-use disposable kits which can cost hundreds or thousands of dollars for a single disposable kit in some cases. A mechanical system like the StromaCell offers the benefit of a closed sterile system and tends to be more affordable, but does not provide the superior yield afforded by the enzymatic systems such as the Cytori Celution system or the Tissue Genesis Icellator system. All of the systems mentioned here can be operated by a single trained technician at the point of care. The processing times vary between systems, with mechanical systems being in the 15–30 min range and the enzymatic systems ranging from about 60–90 min depending on the amount of tissue processed.
Despite a lack of reported clinical risk, in vitro studies have demonstrated potential oncological risks which clinicians should be cautious of when using SVF based therapies (Bertolini et al. 2012; Bielli et al. 2014). See full reference below.
These studies are also below and the 2014 study concludes:
“Preliminary data describe that SVF/ASCs enrichment did not show increased risk of new cancer or relapse compared with control group.”
Adipose tissue-derived stromal vascular fraction in regenerative medicine: a brief review on biology and translation
Isolation of SVF
Enzymatic isolation of SVF
Non-enzymatic isolation of SVF
Automated devices for point-of-care isolation of SVF
Characterisation of SVF
The curious case of CD34
Current state in the clinic and laboratory
“Fat stem cell” therapies and regulatory scenario
Availability of data and materials
Consent for publication
Ethics approval and consent to participate
|ADSC||Adipose-derived stem/stromal cell|
|BADSC||Brown adipose-derived stem cell|
|BAT||Brown adipose tissue|
|BMMSC||Bone marrow mesenchymal stromal/stem cell|
|CD||Cluster of differentiation|
|EPC||Endothelial precursor cell|
|FDA||Food and Drug Administration|
|HIF1||Hypoxia inducible factor 1|
|HSC||Haematopoietic stem cell|
|IFATS||International Federation for Adipose Therapeutics and Science|
|ISCT||International Society of Cellular Therapy|
|ISSCR||International Society for Stem Cell Research|
|MSC||Mesenchymal stem/stromal cell|
|PDGF||Platelet-derived growth factor|
|REGROW||Reliable and Effective Growth for Regenerative Health Options that Improve Wellness|
|SVF||Stromal vascular fraction|
|VEGF||Vascular endothelial growth factor|
|VLS||Vulvar lichen sclerosus|
|WAT||White adipose tissue|
Mechanical versus enzymatic isolation of stromal vascular fraction cells from adipose tissue
Mechanical isolation methods
Mechanical vs enzymatic methods
Automated/semi-automated devices for SVF isolation
- Aronowitz JA, Ellenhorn JD. Adipose stromal vascular fraction isolation: a head-to-head comparison of four commercial cell separation systems. Plast Reconstr Surg. 2013;132(6):932e–939e. doi: 10.1097/PRS.0b013e3182a80652. [PubMed] [Cross Ref]
- Asatrian G, Pham D, Hardy WR, et al. Stem cell technology for bone regeneration: current status and potential applications. Stem Cells Cloning. 2015;8:39–48. [PMC free article] [PubMed]
- Aust L, Devlin B, Foster SJ, et al. Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy. 2004;6:7–14. doi: 10.1080/14653240310004539. [PubMed] [Cross Ref]
- Baer PC, Geiger H. Adipose-derived mesenchymal stromal/stem cells: tissue localization, characterization, and heterogeneity. Stem Cell Int. 2012;2012:812693. [PMC free article] [PubMed]
- Baptista LS, do Amaral RJ, Carias RB, Aniceto M, Claudio-da-Silva C, Borojevic R. An alternative method for the isolation of mesenchymal stromal cells derived from lipoaspirate samples. Cytotherapy. 2009;11(6):706–715. doi: 10.3109/14653240902981144. [PubMed] [Cross Ref]
- Bertolini F, Lohsiriwat V, Petit JY, et al. Adipose tissue cells, lipotransfer and cancer: a challenge for scientists, oncologists and surgeons. Biochim Biophys Acta. 2012;1862:209–214. [PubMed]
- Bielli A, Scioli MG, Gentile P, et al. Adult adipose-derived stem cells and breast cancer: a controversial relationship. Springerplus. 2014;3:345. doi: 10.1186/2193-1801-3-345. [PMC free article] [PubMed][Cross Ref]
- Biosafe America (2015) Sepax 2. Biosafe Group SA website. Available at: http://www.biosafe.ch/?portfolio=sepax2. Accessed 10 Mar 2015
- Bonab MM, Alimoghaddam K, Talebian F, et al. Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 2006;7:14. doi: 10.1186/1471-2121-7-14. [PMC free article] [PubMed] [Cross Ref]
- Borowski DW, Gill TS, Agarwal AK, et al. Adipose tissue-derived regenerative cell-enhanced lipofilling for treatment of crytpoglandular fistulae-in-ano: the ALFA technique. Surg Innov. 2015;22(6):593–600. doi: 10.1177/1553350615572656. [PubMed] [Cross Ref]
- Bourin P, Bunnell BA, Casteilla L, et al. 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. 2013;15:641–648. doi: 10.1016/j.jcyt.2013.02.006.[PMC free article] [PubMed] [Cross Ref]
- Breite AG, Dwulet FE, McCarthy RC. Tissue dissociation enzyme neutral protease assessment. Transplant Proc. 2010;42:2052–2054. doi: 10.1016/j.transproceed.2010.05.118. [PMC free article] [PubMed][Cross Ref]
- Buschmann J, Gao S, Harter L, et al. Yield and proliferation rate of adipose-derived stromal cells as a function of age, body mass index and harvest site-increasing the yield by use of adherent and supernatant fractions. Cytotherapy. 2013;15(9):1098–1105. doi: 10.1016/j.jcyt.2013.04.009. [PubMed][Cross Ref]
- Conde-Green A, Rodriguez RL, Slezak S, et al. Enzymatic digestion and mechanical processing of aspirated adipose tissue. Plast Recons Surg. 2014;134:54. doi: 10.1097/01.prs.0000455394.06800.62.[Cross Ref]
- Cytori Therapeutics (2015) Clinical trials page. Available at http://www.cytori.com/en/Technology/ClinicalTrials.aspx. Accessed 10 Mar 2015
- Di Rocco G, Gentile A, Antonini A, et al. Enhanced healing of diabetic wounds by topical administration of adipose tissue-derived stromal cells overexpressing stromal-derived factor-1: biodistribution and engraftment analysis of bioluminescent imaging. Stem Cells Int. 2010;2011:1–11. doi: 10.4061/2011/304562. [PMC free article] [PubMed] [Cross Ref]
- Doi K, Tanaka S, Iida H, et al. Stromal vascular fraction isolated from lipo-aspirates using an automated processing system: bench and bed analysis. J Tiss Eng Regen Med. 2013;7:864–870. doi: 10.1002/term.1478. [PubMed] [Cross Ref]
- Domenis R, Lazzaro L, Calabrese S, et al. Adipose tissue derived stem cells: in vitro and in vivo analysis of a standard and three commercially available cell-assisted lipotransfer techniques. Stem Cell Res Ther. 2015;6(1):2. doi: 10.1186/scrt536. [PMC free article] [PubMed] [Cross Ref]
- Eto H, Kato H, Suga H, et al. The fate of adipocytes after nonvascularized fat grafting: evidence of early death and replacement of adipocytes. Plast Reconstr Surg. 2012;129:1081–1092. doi: 10.1097/PRS.0b013e31824a2b19. [PubMed] [Cross Ref]
- FDA (2014a) Medical devices; current good manufacturing practice (CGMP) final rule; quality system regulation. FDA website. Available at http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/PostmarketRequirements/QualitySystemsRegulations/ucm230127.htm. Accessed 10 Mar 2015
- FDA (2014b) Minimal manipulation of human cells, tissues, and cellular and tissue-based products: draft guidance for industry and food and drug administration staff. FDA website http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/ucm427692.htm. Accessed 10 Mar 2015
- FDA (2014c) Human cells, tissues, and cellular and tissue-based products (HCT/Ps) from adipose tissue: regulatory considerations; draft guidance for industry. FDA Website. http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Tissue/ucm427795.htm. Accessed 10 Mar 2015
- Fogarty WM, Griffin PJ. Production and purification of metalloprotease of Baccilus polymyxa. Appl Microbiol. 1973;26(2):185–190. [PMC free article] [PubMed]
- Fraser JK, Hicok KC, Shanahan R, et al. The Celution system: automated processing of adipose-derived regenerative cells in a functionally closed system. Adv Wound Care. 2013;3(1):38–45. doi: 10.1089/wound.2012.0408. [PMC free article] [PubMed] [Cross Ref]
- GID Europe (2015) Complete tissue processing in a single disposable device. Available at. http://www.gideurope.com/gid-svf-1/. Accessed 10 Mar 2015
- Griffin PJ, Fogarty WM. Physiochemical properties of the native, zinc- and manganese-prepared metalloprotease of Bacillus polymyxa. Appl Microbiol. 1973;26(2):191–195. [PMC free article][PubMed]
- Guven S, Karagianni M, Schwalbe M, et al. Validation of an automated procedure to isolate human adipose tissue-derived cells by using the Sepax technology. Tissue Eng Methods. 2012;18(8):575–582. doi: 10.1089/ten.tec.2011.0617. [PMC free article] [PubMed] [Cross Ref]
- Han J, Koh YJ, Moon HR, Ryoo HG, Cho CH, Kim I, et al. Adipose tissue is an extramedullary reservoir for functional hematopoietic stem and progenitor cells. Blood. 2010;115:957–964. doi: 10.1182/blood-2009-05-219923. [PubMed] [Cross Ref]
- Jurgens WJ, Oedayrajsingh-Varma MJ, Helder MN, et al. Effect of tissue-harvest site on yield of stem cells derived from adipose tissue: implications for cell-based therapies. Cell Tissue Res. 2008;332:415–426. doi: 10.1007/s00441-007-0555-7. [PMC free article] [PubMed] [Cross Ref]
- Kakamura T, Ito K. Autologous cell-enriched fat grafting for breast augmentation. Aesthetic Plast Surg. 2011;35(6):1120–1130. [PubMed]
- Kapur SK, Katz AJ. Review of the adipose derived stem cell secretome. Biochimie. 2013;95:2222–2228. doi: 10.1016/j.biochi.2013.06.001. [PubMed] [Cross Ref]
- Kato H, Mineda K, Eto H, et al. Degeneration, regeneration and cicatrization after fat grafting: dynamic total tissue remodeling during the first 3 months. Plast Reconstr Surg. 2014;133:303e–313e. [PubMed]
- Ke Q, Chen A, Minoda M, et al. Safety evaluation of a thermolysin enzyme from Geobacillus stearothermophilus. Food Chem Toxicol. 2013;59:541–548. doi: 10.1016/j.fct.2013.06.046. [PubMed][Cross Ref]
- Lin K, Matsubara Y, Masuda Y, et al. Characterization of adipose tissue-derived cells isolated with the Celution system. Cytotherapy. 2008;10(4):417–426. doi: 10.1080/14653240801982979. [PubMed][Cross Ref]
- LipoKit II infomation (2015) Medi-Kan Int. Website. Available at http://www.medikanint.com/lipokit.html. Accessed 16 Mar 2015
- Marino G, Moraci M, Armenia E, et al. Therapy with autologous adipose-derived regenerative cells for the care of chronic ulcers of lower limbs in patients with peripheral arterial disease. J Surg Res. 2013;185(1):36–44. doi: 10.1016/j.jss.2013.05.024. [PubMed] [Cross Ref]
- Markarian FM, Frey GZ, Silveira MD, et al. Isolation of adipose-derived stem cells: a comparison among different methods. Biotechnol Lett. 2014;36:693–702. doi: 10.1007/s10529-013-1425-x. [PubMed][Cross Ref]
- Matsumoto D, Sato K, Gonda K, et al. Cell-assisted lipotansfer: supportive use of human adipose-derived stem cells for soft tissue augmentation with lipoinjection. Tissue Eng. 2006;12(12):3375–3383. doi: 10.1089/ten.2006.12.3375. [PubMed] [Cross Ref]
- McCarthy RC, Breite AG, Dwulet FE (2010) Biochemical analysis of crude collagenase products used in adipose derived stromal cell isolation procedures and development of a purified tissue dissociation enzyme mixture. Available at. http://www.vitacyte.com/wp-content/uploads/2009/01/ifats-vitacyte.pdf. Accessed 3 Nov 2014
- McCarthy RC, Breite AG, Green ML, Dwulet FE. Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria. Transplantation. 2011;91:137–145. doi: 10.1097/TP.0b013e3181ffff7d. [PMC free article] [PubMed] [Cross Ref]
- McIntosh K, Zvonic S, Garrett S, et al. The immunogenicity of human adipose derived cells: temporal changes in vitro. Stem Cells. 2006;24:1245–1253. doi: 10.1634/stemcells.2005-0235. [PubMed][Cross Ref]
- MicroAire Aesthetics (2013) Stromacell MicroAire Aesthetics Website. http://old.microaire.com/products/microaire-aesthetics/stromacell/. Accessed 10 Mar 2015
- Millan A, Landerholm T, Chapman JR. Comparison between collagenase adipose digestion and Stromacell mechanical dissociation for mesenchymal stem cell separation. McNair Scholars J CSUS. 2014;15:86–101.
- Mitchell JB, McIntosh K, Zvonic S, et al. Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells. 2006;24:376–385. doi: 10.1634/stemcells.2005-0234. [PubMed] [Cross Ref]
- Naderi N, Wilde C, Haque T, et al. Adipogenic differentiation of adipose-derived stem cells in a 3-dimensional spheroid culture (microtissue): implications for the reconstructive surgeon. J Plast Reconstr Aesthet Surg. 2014;67(12):1726–1734. doi: 10.1016/j.bjps.2014.08.013. [PubMed][Cross Ref]
- Nagaishi K, Arimura Y, Fujimiya M. Stem cell therapy for inflammatory bowel disease. J Gastroenterol. 2015;50(3):280–286. doi: 10.1007/s00535-015-1040-9. [PubMed] [Cross Ref]
- Planat-Benard V, Silvestre JS, Cousin B, et al. Plasticity of human adipose lineage cells towards endothelial cells: physiological and therapeutic perspectives. Circulation. 2004;109:656–663. doi: 10.1161/01.CIR.0000114522.38265.61. [PubMed] [Cross Ref]
- Raposio E, Caruana G, Bronomini S, Libondi G. A novel strategy for the isolation of adipose-derived stem cells: minimally manipulated adipose-derived stem cells for more rapid and safe stem cell therapy. Plast Reconstr Surg. 2014;133(6):1406–1409. doi: 10.1097/PRS.0000000000000170. [PubMed] [Cross Ref]
- Rehmam J, Traktuev D, Li J, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation. 2004;109:1292–1298. doi: 10.1161/01.CIR.0000121425.42966.F1.[PubMed] [Cross Ref]
- Savi M, Bocchi L, Fiumana E, et al. Enhanced engraftment and repairing ability of human adipose-derived stem cells, conveyed by pharmacologically active microcarriers continuously releasing HGF and IGF-1, in healing myocardial infarction. J Biomed Mater Res A. 2015 [PubMed]
- Shah FS, Wu X, Dietrich M, Rood J, Gimble J. A non-enzymatic method for isolating human adipose-derived stromal stem cells. Cytotherapy. 2013;15:979–985. doi: 10.1016/j.jcyt.2013.04.001. [PubMed][Cross Ref]
- Suga H, Eto H, Aoi N, et al. Adipose tissue remodeling under ischemia: death of adipocytes and activation of stem/progenitor cells. Plast Reconstr Surg. 2010;126:911–923. doi: 10.1097/PRS.0b013e3181f4468b. [PubMed] [Cross Ref]
- Tissue Genesis (2015) Tissue Genesis Icellator cell isolation system. Tissue Genesis Website. Available at: http://www.tissuegenesis.com/icellator.html. Accessed 10 Mar 2015
- Ude CC, Sulaiman SB, Min-Hwei N, et al. Cartilage regeneration by chondrogenic induced adult stem cells in osteoarthritic sheep model. PLoS One. 2014;9(6):e98770. doi: 10.1371/journal.pone.0098770.[PMC free article] [PubMed] [Cross Ref]
- Vilaboa SD, Navarro-Palou M, Llull R. Age influence on stromal vascular fraction cell yield obtained from human lipoaspirates. Cytotherapy. 2014;12:1092–1097. doi: 10.1016/j.jcyt.2014.02.007. [PubMed][Cross Ref]
- Wang L, Lu Y, Luo X, et al. Cell-assisted lipotransfer for breast augmentation: a report of 18 patients. Zhonghua Zheng Xing Wai Ke Za Zhi. 2012;28(1):1–6. [PubMed]
- Williams SK, Kosnik PE, Kleinert LB, et al. Adipose stromal vascular fraction cells isolated using an automated point of care system improve the patency of expanded polytetrafluoroethylene vascular grafts. Tissue Eng. 2013;19(11, 12):1295–1302. doi: 10.1089/ten.tea.2012.0318. [PubMed] [Cross Ref]
- Yoshimura K, Shiguera T, Matsumoto D, et al. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol. 2006;208:64–76. doi: 10.1002/jcp.20636. [PubMed] [Cross Ref]
- Yoshimura K, Suga H, Eto H, et al. Adipose-derived stem/progenitor cells: roles in adipose tissue remodeling and potential use for soft tissue augmentation. Regen Med. 2009;4(2):265–273. doi: 10.2217/174607126.96.36.1995. [PubMed] [Cross Ref]
- Zimmerlin L, Donnenberg VS, Pfeifer ME, et al. Stromal vascular progenitors in adult human adipose tissue. Cytometry . 2010;77(1):22–30. [PMC free article] [PubMed]
- Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211–229. doi: 10.1089/107632701300062859. [PubMed] [Cross Ref]