A Non-Enzymatic Method to Obtain a Fat Tissue Derivative Highly Enriched in Adipose Stem Cells (ASCs) from Human Lipoaspirates: Preliminary Results

Abstract

Adipose tissue possesses phenotypic gene expression characteristics that are similar to human mesenchymal stem cells (hMSCs). Nevertheless, the multilineage potential may be inhibited, and cells may not expand adequately to satisfy the requirements of Good Manufacturing Practice (cGMP). An autologous hMSC-enriched fat product would fulfil the void from a biomedical and clinical perspective. In this study, we suggest a novel mechanism using a closed system without enzymes, additives or other modifications, which will produce non-expanded, accessible material. This decentralized fat product, unlike unprocessed lipoaspirates, adequately encloses the vascular stroma with adipocytes and stromal stalks along with their vascular channels and lumina. This fat product contained hASCs and fewer hematopoietic elements such as lipoaspirates, which were digested enzymatically according to flow cytometric investigations, and molecular analysis also showed significant hASC uniformity within the cells of the stromal vascular tissue. Moreover, the fat product produced a higher quantity of hASCs similar to hMSCs in isolation with the typical characteristics of an osteogenic, chondrogenic and adipogenic lineage. Interestingly, these properties were evident in the non-enzymatic derived adipose tissue, as opposed to hASCs in isolation from the enzymatically digested lipoaspirates, suggesting that the aforementioned procedure may be an adequate alternative to regenerate and engineer tissue for the treatment of various medical conditions and promote efficient patient recovery.

Keywords: Rigenera protocol; adipose stem cells; adipose tissue; enzymatic digestion; non-enzymatic method.

Figure 1

Panel (A–C) morphological images of Rigenera^® methods at 0 h (A), 72 h (B) and 10 days (C) after 30 s of treatment (scale bar 20 μm). Panel (D–F) morphological images of Rigenera^® methods at 0 h (D), 72 h (E) and 10 days (F) after 45 s of treatment (scale bar 20 μm). Panel G-I morphological images of enzymatic digestion methods at 0 h (G), 72 h (H) and 10 days (I) (scale bar 20 μm).

Figure 2

(A) Cell viability testing by trypan blue shows the average number of cells in the Rigenera^® methods and the enzymatic method at 0 h, 72 h and 10 days. (B) Adipose stem cells (ASCs) obtained from the enzymatic method were able to proliferate at a higher rate with a mean doubling time of 96 h and divided at the same speed as the cells isolated with non-enzymatic method.

Figure 3

Flow cytometry analysis of the non-enzymatic method (A) detected the presence of a cell population that we had previously identified [17,23,32]: including CD34 (35%), CD73 (60%), CD105 (70%) CD90 (70%), CD117 (29%), CD29 (78%), while the cells are negative for CD31, CD45, compared to enzymatic digestion (B).

Figure 4

Gene expression of stemness markers (A,B) and differentiation markers (C) in the two digestion methods. Gene expression profile of mesenchymal stem cell specific markers and differentiation-specific markers of non-enzymatic fat disaggregation are reported as ratios (R) with respect to the mRNA expression of enzymatic fat digestion [13,32].