E. Brett1, D. Wan1 1Stanford University,Palo Alto, CA, USA
Introduction: The influence of static, 2D culture on cell types has been investigated on cell lines and primary cultures. Long term subcultures of cell lines have shown susceptibility to ‘drift’, both phenotypically and genotypically. CD marker drift is pertinent to primary cell types which require fluorescent assisted cell sorting for isolation. Adipose derived stem cells (ASCs) are an example of such cells, relying on FACS selection of CD45-/CD31-/CD34+ cells from processed lipoaspirate.
Methods: Choosing CD90, CD105, and BMPR1b, we plated FACS-sorted ASCs positive and negative for these markers, and analysed the extent of CD marker drift over 0hr, 12hr, 24hr, 36hr, 48hr, D3, D5, and D7 after standard cell culture. It was consistently observed that ASCs which were sorted for a presence or absence of specific marker at 0 hour have a significantly different CD expression profile after 36 hours of cell culture (*p<0.05). We also analysed the effect of CD positive and negative populations on one another in a co-culture setting. A decrease of CD marker expression was largely observed across the cell populations, caused by originally CD-positive cells drifting towards a negative phenotype. The osteogenic potential of purified positive and negative populations was assessed using a standardized in vitro model of osteogenic differentiation. Significant differences were observed between the osteogenic capacities of fresh ASCs versus ASCs whose CD marker profiles had drifted over 36 hours of culture (*p<0.05). This drift was characterized further using qRT-PCR, which detected concomitant increases in expression of osteogenic and mechanotransductive genes in plated cells.
Results: These observations allude to the volatile nature of CD expression by FACS-sorted ASCs once in culture, and suggest an influence of cell culture on capabilities of ASCs to commit to lineage.
Conclusion: In vitro culture has governance on phenotypic and genotypic profile of ASCs. This has a significant downstream impact on the osteogenic capacity of the cells in vitro; an important consideration for all in vitro assays.
