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

Abstract

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.