Cryopreservation and validation of differentiated PDGFRα-positive cells for long term usage in experimentation

Abstract

Abstract Objective Differentiation of immortalized Mesenchymal Stromal Cells (iMSCs) into PDGFRα-positive cells under controlled growth conditions has several vital implications in functional studies concerned with the pathogenesis of Diabetic Gastroparesis (DGP). A study published previously by our research group demonstrated the importance of these cells as a novel, in-vitro model for investigating the functional role of neuronal nitric oxide synthase. The currently available methods require fresh differentiation of PDGFRα-positive cells for each round of experimentation. This leads to longer delays, higher usage of reagents, and inconsistency in reproducibility of experiments frequently. We thus aimed to establish through validation that cryopreserving and maintaining the iMSC-derived PDGFRα-positive cells for functional investigations help us to overcome these challenges. Results We demonstrated for the first time that the differentiated PDGFRα-positive cells from iMSCs can be cryopreserved and thawed to be used as per the experimental requirements with prolonged preservation of their characteristics. We assessed the viability of differentiated PDGFRα-positive cells pre- and post-freezing with the subsequent validation of their functional features using flow cytometry, qRT-PCR, and western blotting. We have been successful in demonstrating for the first time that the cryopreservation of previously differentiated PDGFRα-positive cells can be used as a feasible and cost-effective model for experimental reproducibility in functional studies of Diabetes Gastroparesis. Graphical Abstract

Keywords

• iMSCs
• Stem cell differentiation
• PDGFRα-positive cells
• qRT-PCR
• Flow cytometry
• Western blotting