RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four β-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution—pronase—was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed

Enzymatic catalysed synthesis and gelation of ionic peptides for 3D cell culture applications

The enzymatic catalyzed synthesis and gelation of an ionic peptide and its use to create hydrogels for 3D cell culture is discussed. Time resolved small angle scattering in conjunction with imaging technique allowed the structural changes occurring through this enzymatic reaction to be assessed. In turn, the structural information about the fibrillar network and its local density proved key in facilitating the understanding of the relationships between self-assembly behavior, local nanostructure and final physical properties of the materials. The understanding of the gelation process of these materials allowed the design of a simple and efficient methodology to prepare gels for cell culture. Tetrapeptide/enzyme solution containing cells could be injected into cell culture plate with subsequent gelation of the materials leading to encapsulation of the cells into a 3D network. This system was evaluated for the 3D cell culture of human dermal fibroblasts (HDF). Microscopy showed that cells were uniformly distributed within the gel matrix. Cell counting and live/dead staining showed proliferation of HDF with limited cell death over 10 days