Abstract
Macromolecular crowding (MMC) is a biophysical effect that governs biochemical processes inside and outside of cells. Since standard cell culture media lack this effect, the physiological performance of differentiated and progenitor cells, including extracellular matrix (ECM) deposition, is impaired in vitro. To bring back physiological crowdedness to in vitro systems, we have previously introduced carbohydrate-based macromolecules to culture media and have achieved marked improvements with mixed MMC in terms of ECM deposition and differentiation of mesenchymal stem cells (MSCs). We show here that although this system is successful, it is limited, due to viscosity, to only 33% of the fractional volume occupancy (FVO) of full serum, which we calculated to have an FVO of approximately 54% v/v. We show here that full-serum FVO can be achieved using polyvinylpyrrolidone (PVP) 360 kDa. Under these conditions, ECM deposition in human fibroblasts and MSCs is on par, if not stronger than, with original MMC protocols using carbohydrates, but with a viscosity that is not significantly changed. In addition, we have found that the proliferation rate for bone marrow-derived MSCs and fibroblasts increases slightly in the presence of PVP360, similar to that observed with carbohydrate-based crowders. A palette of MMC compounds is now emerging that enables us to tune the crowdedness of culture media seamlessly from interstitial fluid (9% FVO), in which the majority of tissue cells might be based, to serum environments mimicking intravascular conditions. Despite identical FVO's, individual crowder size effects play a role and different cell types appear to have preferences in terms of FVO and the crowder that this is achieved with. However, in the quest of crowders that we have predicted to have a smoother regulatory approval path, PVP is a highly interesting compound, as it has been widely used in the medical and food industries and shows a novel promising use in cell culture and tissue engineering.