Isolation, Characterization, and Cryopreservation of Human Adipose Tissue-Derived Mesenchymal Stem Cells Using the Enzymatic Method

Raziyeh Taghizadeh  Ghavamabadi; Hassan  Morovvati; Zahra  Taghipour

doi:10.18502/ssu.v34i3.21723

Raziyeh Taghizadeh Ghavamabadi Department of Basic Science, Faculty of Veterinary Medicine, University of Tehran, Iran.
Hassan Morovvati Department of Basic Science, Faculty of Veterinary Medicine, University of Tehran, Iran.
Zahra Taghipour Department of Anatomical Sciences, Faculty of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.

DOI: https://doi.org/10.18502/ssu.v34i3.21723

Keywords: Stem cell, Cell isolation, Collagenase enzyme, Cell differentiation.

Abstract

Introduction: Mesenchymal stem cells (MSCs) play a significant role in regenerative research due to their strong self-renewal capacity, their ability to differentiate into various cell lineages, and their secretion of trophic and regenerative factors. Among different cell sources, adipose tissue is considered particularly valuable because of its easy accessibility and high yield. The aim of this study was to isolate adipose-derived mesenchymal stem cell using collagenase type I and to characterize the isolated cells for establishing a cell bank at the Cell Culture Center of the Department of Anatomy

Methods: In this study, adipose tissue samples obtained through liposuction were enzymatically digested using collagenase type I to isolate the cells. The isolated cells were cultured in an appropriate culture medium, and the expression of mesenchymal surface markers was then analyzed by flow cytometry. In addition, the differentiation potential of the cultured cells toward adipogenic and osteogenic lineages was also evaluated through lineage-specific induction followed by appropriate staining procedures. Finally, the cultured cells were cryopreserved and stored in a liquid nitrogen for long-term preservation.

Results: The mesenchymal phenotype of the isolated cells was confirmed by the high expression of CD90 and CD73 and the low expression of hematopoietic markers CD34 and CD45. Oil Red O and Alizarin Red staining further verified the adipogenic and osteogenic differentiation of the cells, respectively.

Conclusion: The results of this study demonstrated that the cells isolated using the collagenase enzymatic method exhibited morphological, immunophenotypic, and functional characteristics consistent with the standard criteria for mesenchymal stem cells, indicating their strong potential for applications in tissue engineering and regenerative medicine.