Development of a Chlorella-based serum-free culture formulation for HaCaT cells with metabolic and attachment support

Abstract Background Fetal bovine serum (FBS) provides diverse soluble and extracellular functions that are difficult to reproduce in serum-free culture, particularly for anchorage-dependent cells. This study aimed to develop a microalgae-based serum-free formulation for HaCaT keratinocytes by evaluating microalgae-derived soluble components, selected culture supplements, and an attachment-supporting component. Results Among low-molecular-weight fractions (LFs) prepared from green Chlorella vulgaris and white and yellow Auxenochlorella protothecoides , green C. vulgaris LF produced the highest MTT-derived metabolic activity. Supplementation with insulin-transferrin-selenium, albumin, and ascorbic acid, designated as the supplement mixture (SM), further increased the MTT response, but the resulting LF + SM formulation did not maintain observable cell attachment after passage. Addition of fish gelatin to SM generated SMG, and the resulting LF + SMG formulation improved post-passage cell retention. Under standardized reseeding conditions, direct viable-cell counting showed that LF + SMG supported cell expansion through four consecutive passages, reaching 7.84 cumulative population doublings by P4. Hoechst-normalized F-actin-associated fluorescence was also maintained during later passages under LF + SMG conditions. Conclusions The final LF + SMG formulation supported post-passage cell retention and HaCaT cell expansion through four consecutive passages by integrating microalgae-derived soluble components, selected culture supplements, and attachment support. These findings demonstrate the feasibility of a Chlorella -based serum-free formulation that provides metabolic and attachment support for serial HaCaT cell culture.