Isolation of equine multipotent mesenchymal stromal cells by enzymatic tissue digestion or explant technique: comparison of cellular properties

Abstract

BackgroundThe treatment of tendon lesions with multipotent mesenchymal stromal cells (MSCs) is widely used in equine medicine. Cell sources of MSCs include bone marrow, as well as solid tissues such as adipose tissue. MSCs can be isolated from these solid tissues either by enzymatic digestion or by explant technique. However, the different preparation techniques may potentially influence the properties of the isolated MSCs. Therefore, the aim of this study was to investigate and compare the effects of these two different methods used to isolate MSCs from solid tissues.Equine adipose tissue, tendon and umbilical cord matrix served as solid tissue sources of MSCs with different stiffness and density. Subsequent to tissue harvest, MSCs were isolated either by enzymatic digestion with collagenase or by explant technique. Cell yield, growth, differentiation potential and tendon marker expression were analysed.ResultsAt first passage, the MSC yield was significantly higher in enzymatically digested tissue samples than in explanted tissue samples, despite a shorter period of time in primary culture. Further analysis of cell proliferation, migration and differentiation revealed no significant differences between MSCs isolated by enzymatic digestion and MSCs isolated by explant technique. Interestingly, analysis of gene expression of tendon markers revealed a significantly higher expression level of scleraxis in MSCs isolated by enzymatic digestion.ConclusionsBoth isolation techniques are feasible methods for successful isolation of MSCs from solid tissues, with no major effects on cellular proliferation, migration or differentiation characteristics. However, higher MSC yields were achieved in a shorter period of time by collagenase digestion, which is advantageous for the therapeutic use of MSCs. Moreover, based on the higher level of expression of scleraxis in MSCs isolated by enzymatic digestion, these cells might be a better choice when attempting tendon regeneration.

State of the art: Stem cells in equine regenerative medicine

“…Investigative results confirm undifferentiated cell migration in vitro and in vivo . Cell migration from spheroids generated with passage 3 MSCs from adipose tissue, superficial digital flexor tendon and umbilical cord matrix was measured after 24 h of spheroid culture in standard culture medium on adherent cultureware .…”

“…Explant isolation, the process of MSC migration from minced tissue in ideal culture conditions, was proposed to reduce the potential effects of tissue digestion on the cells. Collagenase digestion of equine adipose tissue, tendon and umbilical cord matrix resulted in higher MSC yield from all tissues than from explant isolation, and there were no differences in cell proliferation or differentiation characteristics between the techniques . Future work to optimise cell isolation that includes determination of cell phenotype will be important to elucidate the effects of gradients, digestion and associated protocols on cells.…”

“…In another study using the same cell passage and similar techniques, MSCs from adipose tissue and tendon migrated faster than those from umbilical cord tissue and bone marrow [43]. The authors suggested that MSC migration may be fundamental to systemic application [43] and graft integration into recipient tissue [43,75].…”

GMP-Compliant Human Fetal Skin Fibroblasts for Wound Healing

“…Enzymatic digestion with consequent mechanical damage during centrifugation times seems as a laborious procedure that may lead to reduced cell viability (31). It has been indicated that the characteristics of isolated cells may be altered by the use of enzymatic digestion method as a repercussion of different environment of the cells (32). In explant culture method, the processing time for the tissue is faster than conventional enzymatic method.…”

Calcium-Sensing Receptor-Mediated Osteogenic and Early-Stage Neurogenic Differentiation in Umbilical Cord Matrix Mesenchymal Stem Cells from a Large Animal Model

“…Several groups reported success in isolating and establishing MSCs cultures from UCM in human [2]–[8] as well as in large animal models, such as horses [9]–[16], pigs [17], [18], and dogs [19]–[22].…”