Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(ε-caprolactone) Electrospun Yarns.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(ε-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37 (o)C, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged - the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Cyclic tensile strain upon human mesenchymal stem cells in 2D and 3D culture differentially influences CCNL2, WDR61 and BAHCC1 gene expression levels.

It has been shown that tensile strain can alter cell behaviour. Evidence exists to confirm that human mesenchymal stem cells can be encouraged to differentiate in response to tensile loading forces. We have investigated the short-term effects of cyclic tensile strain (3%, 1 Hz) on gene expression in primary human mesenchymal stem cells in monolayer and whilst encapsulated in a self-assembled peptide hydrogel. The main aims of the project were to gain the following novel information: (1) to determine if the genes CCNL2, WDR61 and BAHCC1 are potentially important mechanosensitive genes in monolayer, (2) to determine if these genes showed the same differential expression in a 3D environment (either tethered to RGD or simply encapsulated within a hydrogel (with RGE motif)) and (3) to determine whether the mesenchymal stem cells would survive within the hydrogels over several days whilst enduring dynamic culture. In the monolayer system, real-time PCR confirmed CCNL2 was significantly downregulated after 1 h strain and 2 h latency (post strain). BAHCC1 was significantly downregulated after 1 h strain (both 2 h and 24 h latency). WDR61 followed the same trend in 2D culture. After 24 h strain and 2 h latency, BAHCC1 was significantly upregulated. We found that both types of peptide hydrogel supported viable mesenchymal stem cells over 48 h. Results of the 3D dynamic culture did not correspond with those of the 2D dynamic culture, where the BAHCC1 gene was not expressed in the 3D experiments. The disparity in the differential gene expression observed between the 2D and 3D culture systems may partly be a result of the different cellular environments in each. It is likely that cells cultured within an intricate 3D architecture respond to mechanical cues in a different and more complex manner than do cells in 2D monolayer, as is illustrated by our gene expression data.