Macromolecular crowding (MMC) is a biophysical effect that governs biochemical processes inside and outside of cells. As standard cell culture media lacks this effect, the physiological performance of differentiated and progenitor cells, including extracellular matrix 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 up to 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 changed significantly. In addition, we have found that the proliferation rate for bone-marrow derived mesenchymal stem cells 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 allows 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 here a novel promising use in cell culture and tissue engineering.