The effect of macromolecular crowders as a supplement to serum free media on human corneal stromal cells proliferation and marker expression.

Native human corneal stromal keratocytes (HCSKs) deposit extracellular matrix (ECM) to maintain corneal homeostasis, repair, and regeneration. Adding macromolecular crowders (MMCs) to culture media encourages HCSKs to produce ECM in vitro. However, MMCs may also be used as serum or growth factor replacement for the in vitro culture of cells. This could control phenotype and reduce the cost of future industrial-scale production of cells such as HCSKs for predicted therapeutic applications in corneal repair. To identify the effects of adding (MMCs) to a serum-free medium and the possible role of MMCs in supporting the growth of HCSKs in an undifferentiated phenotype. Primary HCSKs isolated from human corneas were cultured in serum-free media supplemented with different concentrations of MMCs additive (0%, 4%, and 8%). The influence of the MMCs on proliferation and protein expression was investigated using quantitative colorimetric analysis. The addition of MMCs increased cell proliferation and enhanced expression of quiescent keratocyte markers such as ALDH3A1 and Collagen V. It also downregulated stromal fibroblasts’ specific marker, metalloprotease 2 (MMP2), and α smooth muscle actin. These results indicate that adding MMCs to serum-free media significantly supported cell behaviour. In addition, its presence suggested the retention of native quiescent markers when expanded ex vivo. Moreover, the addition of MMCs to serum-free media enhances human corneal keratocyte proliferation and ECM content, but its effects vary based on media glucose concentration. In standard glucose media, MMCs boost cell proliferation and increase collagen V expression as an indicator for robust ECM; however, there is a higher risk of fibroblastic transformation. In serum-free media, MMCs support a quiescent keratocyte phenotype, reduce metabolic stress, and better mimic the native corneal environment, making it more suitable for regenerative applications. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-39340-7.