Title : Optimized protocol for generating clinical grade umbilical cord tissue mesenchymal stem cell for cell therapy in various disease
Abstract:
Mesenchymal stem cell is being used for therapeutic purpose because of their self-renewal, Immunomodulatory capability and mitochondrial transfer signalling. Mesenchymal stem cells can be isolated from various sources eg, Umbilical Cord, Bone Marrow, Adipose Tissue, Dental Pulp and amniotic fluid. Most frequently used is umbilical cord tissue mesenchymal stem cell due to easily available. In this study three different methods and media are evaluated for isolation and expansion and to check regenerative properties in animal model of acute liver injury. Healthy umbilical cord tissue was collected in PBS containing 10%FCS with 2% Antibiotics. Umbilical cord tissue washed and cleaned in Povidone. MSCs are isolated by three different methods, UC Dissociation Kit (Millitenyi), Explant Culture and Collagenase from 1 gram of cord tissue. These isolated MSC were grown in three different medias GMP Basal Brew Media, Stempeutics (Stem cells Technology) and alpha MEM with 10% FCS (Gibco Life Technology). All cells were characterised for morphology, surface marker expression, population doubling time, CFU-F, Bioenergetics. In vivo therapeutic potential of Umbilical Cord MSC compared with adult Bone Marrow MSC s in drug induced liver injury. Dissociation Kit method gave more yield of MSCs (3×10^5) in shortest time as compare to Collagenase (2.3×10^5) and Explant (1.5×10^5). There is no significant difference are observed in Surface Markers present on MSC Surface. MSCs isolated from all three different methods showed similar characteristic surface marker expression of MSCs. There is significant difference were found in Population Doubling Time and CFU as per shown in Graph. MSCs isolated from Dissociation kit have lowest population doubling dime and have increased CFU ability then others Methods. From cultured MSC in three different medias, BREW media provide best culture condition for expansion of MSCs in vitro wrt clear spindle shape morphology and have more OCR (Oxygen Consumption Rate) by Bioenergetics assay. Mice treated with UC-MSCs showed better therapeutic potential then BM-MSCs in terms of survival and reduction of liver injury. From this study concluded Umbilical Cord Dissociation Kit and Basal Brew media was best among for isolation and expanding clinical grade MSC. MSC isolated from all three methods showed similar characteristic surface marker expression of MSC. MSC from Umbilical Cord Dissociation kit have lowest population doubling time and have increases CFU ability then Mice treated with Umbilical cord tissue MSC shows better therapeutic than Bone Marrow MSC in terms of survival and reduction of liver injury.
