A comprehensive study on optimization of proliferation and differentiation potency of bone marrow derived mesenchymal stem cells under prolonged culture condition

Abstract

Bone marrow derived stem cells (BMSC) have paved way to clinical approaches for its utilization in a variety of diseases due to its ease of isolation combined with its multilineage differentiation capacity. However, the applicability of BMSC is not successful due to the lesser number of nucleated cells obtained from large samples. Hence, culture expansion of BMSC is a prerequisite, as high numbers of stem cells are needed to meet the standards of clinical advancement. There are attempts on optimizing culture condition for large scale production of BMSC. It was believed that, prolonged culture of BMSC is difficult since they tend to lose their characteristics and differentiation potential. Hence, our study aims to determine whether BMSCs could retain its proliferative and differentiation capacity in prolonged in vitro culture by a comparative study on extensive culturing of BMSC with the following four media, DMEM LG (DMEM-Low Glucose), DMEM KO (DMEM-Knock Out), Alpha MEM (Alpha Minimal Essential Medium), DMEM F 12. We found that two samples among the three cultured tend to lose their property in long term culturing. Besides, we also found that DMEM LG and Alpha MEM were the optimal media for in vitro culturing of BMSC. Overall, it was concluded that BMSC can be cultured until passage 15 without losing its characteristics. However, its potency beyond passage 15 has to be further elucidated for utilization of the ex vivo expanded BMSC for subsequent cellular therapies.

Polycaprolactone scaffold engineered for sustained release of resveratrol: therapeutic enhancement in bone tissue engineering

“…20 The mononuclear cell layer was carefully aspirated and transferred to a tube containing 0.7% ammonium chloride solution and incubated at room temperature for 5 minutes to lyse any residual red blood cells. The cells were centrifuged at 450 rpm and 20°C for 10 minutes, the pellet was suspended in phosphate-buffered saline, and viability of the cells was determined using trypan blue exclusion staining.…”

“…Further, analyses were carried out using a FACSAria™ flow cytometer (BD Biosciences) and data acquisition was done using FACSDiva™ software (BD Biosciences). 20 …”

“…20 The subconfluent cells were treated with osteogenic medium (DMEM-LG containing 10% fetal bovine serum, 1% antibiotic, 0.1 µM dexamethasone, Sigma-Aldrich, India), 10 mM glycerophosphate (Sigma-Aldrich, India), and 2 mM ascorbic acid (Sigma-Aldrich, India) and cultured for 15 days. The culture was washed with phosphate-buffered saline (pH 7.4) and fixed with formalin (10%).…”

“…20 The adipogenic medium contained DMEM-LG, 10% fetal bovine serum, 1% antibiotic-antimycotic solution, 0.1 µM dexamethasone (Sigma-Aldrich), 0.5 mM isobutyl methylxanthine (SigmaAldrich), 1 µg insulin (Sigma-Aldrich), and 200 mM indomethacin (Sigma-Aldrich). Cells were cultured in adipogenic medium for 14 days and fixed with 10% formalin in saline.…”

Lineage depletion of stromal vascular fractions isolated from human adipose tissue: a novel approach towards cell enrichment technology

“…The SVF was isolated from subcutaneous fat according to our previously published protocol (Dhanasekaran et al 2012a). The isolated SVF was analysed for a cell viability testing and lineage depleted using human lineage cell depletion kit (Cat No.…”