Breaking down fat: Non-enzymatic “minimal” methods for SVF isolation

by Alexey Bersenev on February 4, 2015 · 1 comment

in adipose

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In the previous post of the series I asked if stromal vascular fraction (SVF) of adipose tissue could be isolated by simple centrifugation? The answer to this question could be a “game changer”, because non-enzymatic methods for SVF isolation is the only possible hypothetical way to avoid FDA regulation as a drug. According to FDA, use of centrifugation fits in “sizing” definition and, therefore, autologous SVF could be exempted as “the same surgical procedure” even if tissue is more than minimally manipulated. In this post, I’ll try to dig into literature to see if non-enzymatic methods can yield “true SVF”.

1. Use of centrifugation supernatant
Gimble’s group reported in 2013 very simple method for isolation SVF by centrifugation of lipoaspirate. Before 3x centrifugation in PBS washing solution, lipoaspirate tissue was “vigorously shaken”. Resulted “wash SVF” was quite different from “digested SVF” phenotypically – lower expression of CD34 (23.7% vs. 81.2%), higher CD45+ (81.7% vs. 27.7%), lower CD90+ (23.2% vs. 80.9%). However, when both types SVF were placed in culture, cell phenotype becomes quite similar in 1-2 weeks (still with big difference in CD34+). The authors conclude, that such “minimal manipulation” could be good alternative to enzymatic method for isolation of adipose stem (stromal) cells (ASC). The drawback of wash supernatant method is very low yield of ASC (almost 20 times less than from enzymatically digested tissue) and longer duration of the culture. Osteo- and adipo- differentiation of ASC, isolated by 2 different methods was quite similar. No CFU assay was performed in the study.

2. Mechanical isolation – shake it!
Edoardo Raposio described a rapid method for SVF isolation by “shaking of lipoaspirate” – use of vibrating shaker at 6000 vibrations/ minute (6 min), then centrifugation at 1600 rpm (6 min). They checked markers and detected “some expression” of CD34 and CD90 on resulted SVF. I’d like to note here that flow plots in the paper are really messy (it would not pass my review) and there is no quantification (%) of expression. This study was recently criticized by 2 other groups – by Aronowitz:

Contrary to the authors’ conclusions, however, their report confirms previous work showing that mechanical methods are not capable of releasing collagen fibers that tightly bind adipose tissue cells together. Only collagenase efficiently allows isolation of a purified population of pluripotential cells from other cell types and lipoaspirate detritus.

and by Bertheuil:

In our opinion, it is necessary to demonstrate that the cells produced by this method meet the in vitro criteria of the mesenchymal stromal cells.
… the authors have limited their inves- tigations to phenotypic characterization without real- izing differentiation in the mesodermal lineage and colony-forming unit-fibroblast experiment.

Another mechanical (automated) method – Fastem/ Corios, was investigated in recent Domenis study. The authors concluded that Fastem process did not enrich significantly clonogenic and multipotent ASC.

Condé-Green described a method, which included “high centrifugation” and vortexing, to isolate SVF. The authors noted low yield, similar viability and difference in cellular composition of SVF, compare to use of enzymatic method:

… the increased hematopoetic and inflammatory cells as well as decreased ADMSCs and endothelial cells in the mechanically isolated cell populations may contribute to reduced herapeutic potential when used in regenerative medicine.

You can also look at Condé-Green method, posted on a blog. I’m not sure how “high centrifugation” (1200g), used by Condé-Green will be compatible with definition of “low centrifugation speed”, cited in recent FDA guidance. I’m also not sure if such processing step as “shaking” (with your hands or on a shaker) and “vibrating” will be classified by FDA as “minimal” and be exempted as “same surgical procedure”.

3. Liposuction aspirate fluid (LAF)
Fluid portion of lipospirate could be used for isolation of SVF. All you need is to let it settle (decantation), collect the fluid separately and centrifuge. At least 2 studies compared fluid portion SVF with fatty portion. Yoshimura reported about compatibale numbers of SVF, isolated from 2 different portions of lipoaspirate. Similarly to Gimble’s study (see above), the authors observed lower CD34+ and CD90+, higher CD45+ on LAF-derived SVF and similarity of markers in culture. Dong studied LAF-derived cells in vitro and in vivo. Cells from both fractions proliferated in culture with similar rate and did not differ in tri-lineage differentiation potential. LAF-derived cells were capable for adipogenic differentiation in vivo. Disadvantage of both studies from clinical stand point is using density gradient centrifugation (ficoll) as a step for isolation of SVF from LAF.

Overall, use of non-enzymatic methods are proposed as “FDA-friendly”, cheap and rapid for SVF isolation from adipose tissue. However, these methods result in lower cell yield, low enrichment for stromal/ progenitor/ vascular cell populations and lower number of stem cells. It comes to the questions: (1) What is the true SVF? and (2) Should we really be care about stem cell content in SVF? Even though, it seem like some non-enzymatic methods for SVF isolation could hypothetically circumvent current FDA requirements and be regulated as medical procedure, one may question a therapeutic potency of such cells compare to well characterized SVF, isolated by enzymatic methods.

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This post is a part of series Breaking Down Fat. In this series we will talk about identification, characterization and clinical processing of potentially therapeutic cell populations from adipose tissue. We started this series in response to the growing trend of wide (mostly uncontrolled) clinical use adipose-derived cells and some controversies/ misconceptions in the field.If you would like to contribute to this series or become a sponsor, please contact us!

{ 1 comment… read it below or add one }

Fas Kuiters February 5, 2015 at 9:56 am

Hi Alexey- thanks for the article, but I do think by virtue of producing SVF one has changed the structural tissue of fat and is a drug or BLA and it does not matter whether it was with enzymatic digestion or without. Or even whether it was by swinging an axe or using a centrifuge.

Cytori came out with an extremely concise analysis of the FDA documents and at least those are the first comments I have seen which seem to make sense. You are invited to review them and comment on them in the light of your preceding articles, which basically do not seem to be correct in its interpretation.

Thanks

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