Differential Viability in Alpha-MEM Culturing Media May Predict Alternative Media Responsiveness in Dental Pulp Stem Cell (DPSC)

Objective: Dental pulp stem cells (DPSC) are the subjects of new and emerging fields of clinically applied biotechnology. However, much remains unknown regarding the most effective and appropriate methods for isolation, expansion and culture techniques for DPSC. To address these deficiencies, the primary objective of this study was to evaluate any effects of the major, commercially available cell culture media on DPSC phenotypes, such as growth, viability and biomarker expression.

Methods: This Institutional Review Board (IRB) approved study involved previously collected and cryopreserved DPSC isolates that were identified, thawed and cultured for this study (n=16). Each DPSC isolate was plated into 96-well assays under each of the experimental conditions (DMEM, DMEM:F12, RPMI, alpha-MEM) to determine any effects on cellular growth and viability. RNA was extracted from all DPSC isolates under the optimal growth conditions for screening using qPCR primers specific for DPSC biomarkers, such as Sox-2, Oct-4 and NANOG.

Results: Comparison of the standard DPSC cell culture media alpha-MEM to DMEM revealed differential results. Comparison of alpha-MEM to DMEM:F12 revealed no change among some DPSCs (n=3), decreased viability (n=8) or increased viability (n=5) - similar to the comparisons with RMPI demonstrating no change (n=5), decreased viability (n=6) or increased viability (n=5). Further analysis revealed that DPSC with low viability (<50%) in alpha-MEM responded positively to one or more of the culture media alternatives, while virtually none of DPSC with high viability (>50%) responded to any of the other experimental conditions. Screening of mRNA using qPCR revealed most DPSC isolates continued to express one or more of the pluripotent stem cell biomarkers (Oct4, Sox2, Nestin, NANOG), but no clear pattern of growth with the optimal media type correlated with viability.

Conclusions: These results demonstrated that many DPSC isolates responded positively to one or more of these media, including DMEM, DMEM:F12, RPMI when viability was <50% using the standard DPSC culture media alpha-MEM, but not when viability was >50%. These findings may be broadly applicable and add significantly to the evidence regarding the potential culturing methods that may be employed in various ex vivo and in vitro DPSC studies.