Introduction: Living tissue is a very complex biological construct made of cells, signaling molecules and multiple extracellular matrix (ECM) components. Each of these elements is equally important to keep the homeostatic balance of tissue and to control the functions of organs. This heterogeneity of living tissues makes their engineering in laboratory conditions challenging 1. Biocompatible hydrogel systems are one of the favored ECM replacement materials, since they provide porous microenvironment with the possibility of nutrient and oxygen exchange. However, it is not possible to employ one hydrogel system for all cell types. Different cell types require specific stiffness, viscoelasticity, or hydrogel chemistry to survive. In this study, we optimize an allyl-modified gelatin (gelAGE) hydrogel to recreate an ideal microenvironment which supports long-term survival and functionality of fibroblast and endothelial cells.
Methodology: GelAGE and multi-arms thiolated polyetylenglycol are photo-crosslinked using thiol-ene click chemistry and characterized using rheology and photo-rheology analysis, as well as swelling and static compression tests. Primary human umbilical vein endothelial cells (ECs) and normal human dermal fibroblasts were casted within the hydrogel precursor solution individually or as co-culture and crosslinking of hydrogels was performed under UVA light. Cell viability and proliferation was determined by live/dead staining and fluorometric measurement of dsDNA content. Cell morphology was visualized with Hoechst and Phalloidin staining. Soluble fibronectin and soluble collagen from all samples were measured by fluorometric methods with respective assay kits. Expression of CD31 and fibronectin were confirmed by immunofluorescent staining. Furthermore, cell spheroids embedded in hydrogel were monitored in real-time using IncuCyte spheroid module, to evaluate the migration of cells out of spheroids through the hydrogel.
Results and conclusion: Two different GelAGE types (G1MM and G2LH), adapted from literature 2, have shown different molecular properties (molecular weight and degree of modification) and physico-chemical properties. G2LH shows longer polymer chains and less substituent moieties. Cell viability and morphology of ECs and fibroblasts were compared between GelAGE1MM and GelAGEG2LH. Both hydrogel types supported cell viability, but cells embedded in GelAGE1MM remained rounded and did not show any morphological alterations until day 28. In contrast, fibroblasts embedded in GelAGEG2LH begun to elongate on the first incubation day, and ECs on day 3. Further biological analyses were therefore performed only with GelAGEG2LH-embedded cells. Biological activity of individual cell types was supported in GelAGEG2LH hydrogels. Time-dependent secretion of soluble collagen and fibronectin was detected in both cell types, but was, expectedly, dramatically higher in fibroblasts than in ECs. Immunocytochemical analysis of co-cultures indicated that endothelial cells expressed CD31 and formed cell-cell contacts. Furthermore, strong fibronectin expression in all cells, as well as vessel-like structural organization at day 14, were observed. Real-time monitoring of cell spheroids embedded in GelAGEG2LH hydrogels showed that cells started to move out of the spheroids into the hydrogel after 5 days of incubation. Taken together, our results demonstrated that GelAGEG2LH hydrogels support cell viability, biological function and motility, and may thus fulfill the demands of different cell types thanks to the respective chemical modifications.