Therapeatic evaluation and single cell analysis of adipose stromal vascular fraction isolation from a commercial cell separation system

Abstract

Background In the field of regenerative therapy, the stromal vascular fraction (SVF) extracted from adipose tissue has been widely recognized for its significant benefits. However, the cellular composition and therapeutic effect of SVF products prepared via different methods are unclear. Methods SVF cells were obtained via three approaches: (1) generation of the SVF via mechanical emulsification (M-SVF), (2) generation of the SVF via laboratory enzymatic digestion (L-SVF), and (3) generation of the SVF via commercial cell separation systems (C-SVF). We evaluated their healing effects on mouse wounds. Additionally, we utilized single-nucleus RNA sequencing (snRNA-seq) technology to explore the cellular composition of the C-SVF. Results The cell yield of C-SVF was comparable to that of L-SVF. During in vitro culture, C-SVF exhibited enhanced proliferation and a reduced proportion of apoptotic cells. In a mouse wound model, the application of C-SVF facilitated the closure of mouse wounds and improved collagen remodeling and angiogenesis in the wound area. Additional snRNA-seq analysis revealed that APOE+ adipose-derived stem cells and immune cells, especially M2 anti-inflammatory macrophages, are enriched in C-SVF, which together promote wound repair, and that APOE+ adipose-derived stem cells (ADSCs) and immune cells, especially M2 anti-inflammatory macrophages, are enriched in C-SVF, which jointly regulate and promote wound repair. Conclusion A commercial extraction system is an effective method for isolating viable SVF cells enriched with APOE+ ADSCs and M2 macrophages.

1. Introduction

Stem cell therapy has recently gained widespread acceptance in regenerative medicine, covering a range of treatments for conditions such as stubborn wounds, ischemic conditions, and tissue deficiencies. Adipose-derived stem cells (ADSCs) have increased in prominence as preferred stem cells because of their facile procurement, high storage capacity, and rapid proliferation kinetics [1]. The stromal vascular fraction (SVF), a recently emerged derivative of adipose tissue, has sparked widespread research endeavors. Its abundant bioactive substances, including notably ADSCs and matrix components, have positioned it as a viable solution for treating stubborn wounds [2–4].

At present, there is no broad consensus on the standard separation procedure for SVFs. Typically, separation methods can be classified into two main categories on the basis of whether collagenase is used to assist in the breakdown of the extracellular matrix (ECM) of adipocytes: enzymatic methods and nonenzymatic methods. Nonenzymatic (mechanical) methods rely mainly on physical operations such as emulsification, centrifugation, oscillation, and vortexing to disrupt the ECM and concentrate cellular components [5, 6]. The enzymatic method results in a high yield of SVF cells but is more expensive, has a longer preparation time, and has a greater risk of contamination. In contrast, nonenzymatic methods are less labor intensive, time saving, and easier to use in clinical practice; however, it is still uncertain whether they have regenerative effects comparable to those of enzymatic methods [7].

To obtain SVFs more conveniently and quickly during surgery, a range of semi or fully automatic separation and extraction devices have been introduced to the market [8]. Among these devices, Celution 800 was proven to be more effective with rapid processing time, greater viable cell yield, a lower residual enzyme level and a reduced cost [9]. Celution 800 has been widely used in different indications and has achieved significant results [10–12].