Xenofree Enzymatic Products for the Isolation of Human Adipose-Derived Stromal/Stem Cells

Abstract

Human adipose-derived stromal/stem cells (ASCs) are an abundant, readily available population of adult stem cells that reside in adipose tissue and that have a great potential utility for tissue engineering and regenerative medicine therapeutic applications. Several preclinical studies have shown that ASCs have therapeutic applicability, but a standardized isolation and expansion methodology for clinical cell therapy has yet to be established. ASC are typically isolated and expanded using reagents with xenogenic components and this may pose certain risks and safety issues, such as exposure to infectious agents and immune reactions, creating further obstacles to the translation of ASC-based therapies to clinical scenarios. The objective of this study was to determine the suitability and efficacy of various alternative enzymatic products, CLS1 (Worthington), CLSAFA (Worthington), NB4 (SERVA), and Liberase (Roche), for the digestion of adipose tissue and subsequent isolation of ASCs, assessing cell functionality concerning their proliferation and differentiation ability. Results show that there are no statistically significant differences on yield and proliferation of cells isolated after enzymatic digestion with any of the studied products. The differentiation potential of the cells was not affected, and cell surface marker expression was similar among all products. We concluded that clinical grade products can replace current research-grade products effectively in our cell isolation protocols without any negative effect in the yield or function of human ASCs.

Comparison of intraoperative procedures for isolation of clinical grade stromal vascular fraction for regenerative purposes: a systematic review

“…Fifty‐nine full text studies were assessed on eligibility criteria. Fourteen studies were excluded based on the use of a nonintraoperative protocol for isolation as experimental method (Al Battah et al ., ; Carvalho et al ., ; Chen et al ., ; Condé‐Green and Lamblet ; Doi et al ., ; Escobedo‐Lucea et al ., ; Fink et al ., ; Jiang et al ., ; Okura et al ., ; Pilgaard et al ., ; Seaman et al ., ; Siciliano et al ., ; Vykoukal et al ., ; Yoshimura et al ., ). Seven studies described isolation protocols in general but gave no results (Bernacki et al ., ; Buehrer and Cheatham ; Dubois et al ., ; Hicok and Hedrick, ; Yu et al ., ; Zachar et al ., ; Zhu et al ., ).…”

Production of Human Dental Pulp Cells with a Medicinal Manufacturing Approach

“…For this reason, xeno-free dissociating reagents such as TrypLe or Accutase (Life Technologies, Carlsbad, CA) have been recommended for cell passaging instead of the xenogeneic animal trypsin commonly used (23,24). Likewise, if cell culture medium supplementation with xeno-or allogeneic serum remains permitted, it should be limited to ''cases for which a valid alternative cannot be found'' (European directive 1394/2007).…”

Revisiting the Advances in Isolation, Characterization and Secretome of Adipose-Derived Stromal/Stem Cells

“…Enzymatic activity of collagenase or trypsin is negated by supplementing digested tissue sample with DMEM or α-MEM supplemented with 10% or 20% inactivated fetal bovine serum (FBS) [53,97]. Notwithstanding the enzymatic digestion is a costly method for extraction of ASCs and might affect efficacy and safety [100,101,102]. Therefore, the recent study has explored the economical non-enzymatic method for standardization of ASCs isolation [103].…”