Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells

Abstract

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a non enzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44 + cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

Dancing with the Cells: Acoustic Microflows Generated by Oscillating Cells

“…We examine this hypothesis by replacing the culture medium with 0.25% trypsin‐EDTA and monitoring the streaming flow for 4 min. We limit the experiments to 4 min because longer exposure times can cause detachment of the cells from the substrate, and also permanently damage the cells surface proteins, which can eventually cause cell death …”

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

“…This can be achieved by a combination of mechanical and enzymatic methods, but should be carefully performed, as excessive exposure to enzymatic digestion may result in destruction of antibody-binding epitopes, thus decreasing the effectiveness of downstream immunostaining for cytometric analysis (6)(7)(8). For immuno-oncology research, sample sources primarily consist of blood, tumor tissue (liquid or solid), or bone marrow.…”

“…For immuno-oncology research, sample sources primarily consist of blood, tumor tissue (liquid or solid), or bone marrow. It is important to consider, however, that cryopreservation may induce functional and phenotypic changes and may significantly affect the relative frequencies of viable peripheral blood mononuclear cell (PBMC) subpopulations (7)(8)(9)(10)(11)(12)(13). Bone marrow and peripheral blood samples often undergo standardized Ficoll gradient separation through which mononuclear cells are harvested.…”

“…The first hurdle is tissue disaggregation, which is highly dependent on successful disruption of extracellular matrix, a mixture of cohesive factors and structural proteins. This can be achieved by a combination of mechanical and enzymatic methods, but should be carefully performed, as excessive exposure to enzymatic digestion may result in destruction of antibody-binding epitopes, thus decreasing the effectiveness of downstream immunostaining for cytometric analysis (6)(7)(8). Furthermore, unlike cell isolation from blood samples, tissue dissociation is a multi-step process that involves serial washes for removal of reagents, clumps, and debris in order to produce a single-cell suspension that can subsequently either be prepared for analysis or frozen for later use.…”

“…Cryopreservation is often the method of choice, and typically consists of re-suspension of cell pellet in 10% Dimethyl sulfoxide (DMSO) either in total fetal bovine serum (FBS) or in a medium formulation comprising high FBS concentration. DMSO serves as a cryoprotectant during the freezing process, and upon thawing and removing DMSO, viable cells can be recovered for analysis or subsequent culture (6,7). It is important to consider, however, that cryopreservation may induce functional and phenotypic changes and may significantly affect the relative frequencies of viable peripheral blood mononuclear cell (PBMC) subpopulations (7)(8)(9)(10)(11)(12)(13).…”