Maximizing pediatric stem cell harvest: A novel two-step digestion approach for adipose tissue

Abstract

BACKGROUND

Stromal vascular fraction (SVF) derived from adipose tissue (AT) is a heterogeneous cell mixture. SVF is commonly prepared via enzymatic digestion of AT. Typically, only the initial SVF (SVF1) is harvested, and the remaining AT is discarded. However, residual AT may contain viable regenerative cells.

AIM

To explore a novel approach involving a second enzymatic digestion to maximize stem cell yield, particularly relevant for pediatric patients or those with limited adipose reserves.

METHODS

AT samples obtained via liposuction were digested twice using collagenase type IV to yield SVF1 and SVF2 (after 1^st and 2^nd digestions, respectively). Both fractions were assessed for cell viability, mononuclear cell count, plating efficiency, and the expression of surface markers via flow cytometry. Gene expression analysis of vascular endothelial growth factor, stromal cell-derived factor, nitric oxide synthase-3, CD14, matrix metalloproteinase 3, interleukin-4 receptor, P16, and P53 was performed using quantitative reverse transcription polymerase chain reaction. Histology was performed on AT samples to assess structural integrity and cellular composition across different digestion stages.

RESULTS

Results demonstrated that SVF2 contained a significant number of mononuclear cells and exhibited a high plating efficiency. Flow cytometry and gene expression analyses further confirmed that SVF2 retained regenerative cells. Furthermore, histological evaluation using hematoxylin and eosin staining revealed the presence of intact cells within the residual tissue post-first digestion.

CONCLUSION

In conclusion, secondary digestion of AT (typically discarded after 1^st digestion), can yield additional regenerative cells. This method enhances stem cell number for stem cell-based therapies and is particularly valuable for pediatric patients and those with low-AT reserves.

Keywords: Stromal vascular fraction; Adipose tissue derived stem cells; Adipose tissue; Regenerative medicine; Two-step digestion approach

Core Tip: This study presents a novel dual-digestion method to isolate stem cells from small adipose tissue samples, optimizing cell yield for regenerative medicine applications. Traditionally, only the first stromal vascular fraction is retained for research or therapeutic purpose, while remaining undigested tissue is discarded. However, a second enzymatic digestion (yielding stromal vascular fraction 2) revealed a significant number of viable regenerative cells. They presence of viable regenerative cells was confirmed by plating efficiency, flow cytometry, gene expression analysis and histological studies. This approach is especially beneficial for pediatric patients or individuals with limited adipose reserves, enhancing the number of autologous stem cell by maximizing the utility of small-volume adipose tissue samples.