Optimisation of Isolation of Amniotic Membrane-derived Mesenchymal Stem Cells and Studying their Immunosuppressive Potency

Aparajeet, Amar (2025) Optimisation of Isolation of Amniotic Membrane-derived Mesenchymal Stem Cells and Studying their Immunosuppressive Potency. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Amar Aparajeet (20MS143)) 20MS143_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (5MB)

Abstract

Mesenchymal stem cells (MSCs) are a popular tool in regenerative and immune therapy. Their ability to differentiate into multiple cell types to replace cells and modulate the functioning of immune cells has been studied for a long time. Big strides have been made, and clinical trials might soon be possible. However, a big pool of MSCs is required to perform either in vitro or animal model studies. So, isolation and expansion of these cells is a vital task. Different sources have been used to isolate MSCs, but most of them are based on the enzymatic digestion method. The amniotic membrane (AM), is a popular source for isolating MSCs. It is birth-associated tissue, which is discarded as medical waste, and hence, its collection comes without the ethical concerns associated with other sources. In this project, we tried to standardise a protocol to isolate MSCs from AM using the explant culture method. We found that 14% fetal bovine serum (FBS) and normoxic conditions were ideal for the isolation and expansion of amniotic membrane-derived mesenchymal stem cells (AM-MSCs). Furthermore, from the isolated MSCs, we also standardised a protocol to quantify the immunosuppressive potency of the conditioned media generated by culturing these AM-MSCS using an in vitro assay. We found that when these cells were plated at a density of 1×104 cells/cm2 and cultured in FBSsupplemented media for 48 hours, followed by a 48-hour culture in serum-free media, they were able to suppress the activation of peripheral blood mononuclear cells (PBMCs) to their maximum extent.

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