A cost-effective non-enzymatic isolation method for bovine adipose-derived mesenchymal stem cells: A preliminary study for tropical livestock applications

Abstract

Animal-derived mesenchymal stem cells (MSCs) have potential applications in livestock systems, particularly in regenerative medicine and reproductive biotechnology. In tropical settings, the development of cost-effective and enzyme-free isolation methods is important to support research and applications under limited laboratory resources. This study aimed to preliminarily evaluate bovine adipose-derived mesenchymal stem cells (AD-MSCs) isolated using a non-enzymatic method, focusing on in vitro morphology, marker expression, chromosomal profile, and post-cryopreservation viability. Adipose tissue from Ongole Grade cattle was processed using an explant culture approach. The isolated cells exhibited fibroblast-like morphology, expressed CD44 and CD166, and lacked expression of the hematopoietic marker CD45. Flow cytometry analysis further confirmed CD44 expression at the protein level, with a high proportion of CD44-positive cells. Karyotype analysis showed that most cells retained the normal diploid chromosome number (2n = 60), although minor variations in chromosome morphology were observed. Post-thaw viability remained above 90%, with no observable decline when evaluated using the same cell populations. These findings provide preliminary evidence that non-enzymatic isolation can support the establishment and maintenance of bovine ADMSCs. This approach may serve as a cost-effective and practical alternative for MSC isolation and biobanking in tropical livestock research systems.