Optimization of a protocol to isolate bovine adipose tissue-derived mesenchymal stromal cells

Subtitle:Development of an optimized protocol to isolate bovine adipose tissue-derived mesenchymal stromal cells

A wide diversity of isolation procedures for mesenchymal stromal cells (MSCs) from adipose tissue (AT) has been described in different species, including cattle. Although a high number of MSCs is often required for various tissue engineering approaches, isolation efficiency is rarely evaluated. In the present study, diverse enzymatic digestion methods and one culture explant method were compared using subcutaneous AT samples of 5 adult cows. Within the enzymatic digestion methods, different enzymes (collagenase I, collagenase I + trypsin, liberase, collagenase IV and MMP-12 catalytic domain), enzymatic concentrations (0.25%, 0.1%, 0.04% and 400 ng/mL) and incubation times (3h, 6h, overnight and 24h) were evaluated. Plastic adherent cells were observed using most isolation methods except when cells were isolated with: (i) 0.1% collagenase I for 24h, (ii) 0.1% collagenase I + 0.25% trypsin overnight, (iii) 0.1% collagenase I + 0.25% trypsin for 24h, (iv) 0.04% collagenase IV for 3h and (v) 0.04% collagenase IV overnight. Cells were collected in at least 4 out of 5 isolation repeats for the following methods: (i) 3u coll I 0.1%, (ii) 3u lib 0.1%, (iii) 3u lib 0.04%, (iv) 6u lib 0.1% and (v) 6u lib 0.04%. Enzymatic treatment with 0.1% liberase for 6h resulted in the highest average cell concentration (3.1 x 106 cells/mL) and cell viability (98.84%) when compared to the other methods. These cells were subsequently immunophenotyped and differentiated towards the osteogenic, chondrogenic and adipogenic lineage to confirm their MSC identity. In conclusion, an optimized protocol to ensure a maximum cell yield of bovine MSCs from subcutaneous AT has been developed.

Publication year:2021