Cost-effective strategies for long-term bovine trophectoderm cell culture

Long-term trophectoderm (TE) cell culture provides a powerful model to investigate placenta-specific factors to better understand mechanisms relevant to pregnancy establishment and placental development. However, current TE culture systems rely on costly commercial media and extracellular matrix (ECM) components, which limit their scalability and accessibility. This study evaluated cost-effective alternatives to established conditions by testing modified DMEM/F-12 and a biphasic TE culture system as substitutes for commercial Advanced DMEM/F-12 and for continuous TE culture, and by assessing 0.1% gelatine as an ECM alternative to collagen IV. Trophectoderm outgrowths cultured on collagen IV or gelatine did not differ in attachment timing (P = 0.78) or in expression of placental and differentiation markers PLAC8 (P = 0.78), GATA2 (P = 0.18), and PAG10 (P = 0.39). Similarly, blastocysts cultured in commercial Advanced DMEM/F-12 or base DMEM/F-12 exhibited no differences in attachment day (P = 0.98), TE growth area from days 10-20 (P > 0.05), or expression of PLAC8 (P = 0.35) and PAG10 (P = 0.08), although PAG7 differed between treatments (P < 0.05). Embryos cultured in continuous TE attached later (P = 0.01) than those cultured in TE media, but no differences were observed in TE growth area or expression of PLAC8, PAG7, or PAG10 (P > 0.05). Collectively, these results indicate that affordable media formulations and gelatine-coated cultureware support TE attachment, proliferation, and differentiation. This cost-effective culture framework enables broader application of TE models and supports extended studies of trophoblast function, placental signalling, and early conceptus development.