Background: This study explores the potential of discarded male layer embryos as a sustainable and non-GMO cell source for cultivated chicken meat production. The research aims to identify efficient methods for isolating muscle progenitor cells (MPCs) with high proliferative potential by conducting transcriptome analysis on thigh muscle tissues from both male and female chick embryos. Methods: Transcriptome analysis was performed on the thigh muscle tissues of male and female chick embryos, aged 12-13 days, (n = 4 each), to investigate the gene expression profiles and identify strategies for efficiently isolating MPCs. This approach aims to pinpoint techniques that would allow for the selection of MPCs with optimal growth and proliferation capabilities. Results: Using heatmap, hierarchical clustering, and multidimensional scaling (MDS), we found no significant sex-based differences in gene expression, except for the overexpression of the female-specific gene LIPBLL. The expression of muscle stem cell factors, including PAX3, PAX7, and other myogenic regulatory genes, showed no significant variation. However, to recover MPC-rich cells isolated from male thigh muscle, we found that by the pre-plating 7 stage, myogenesis-related genes, MYHs and MUSTN1 were minimally expressed, while the cell cycle arrest gene CDKN1A sharply increased. Conclusions: Our findings suggest that simple cell isolation directly from tissue is a more scalable and efficient approach for cultivated meat production, compared to labor-intensive pre-plating methods, making it a viable solution for sustainable research and resource recycling.

Tissue engineering (TE) has been developed to create functional organs and tissue by combining 3D matrix and cells in vitro. Vascularization and angiogenesis are utmost important for supply of nutrients and oxygen in tissue engineered organs. The present study was performed to isolate and characterize primary endothelial cells (EC) from aorta of alpha 1, 3-enzyme galactosyltransferase knock out (GalT KO) pig, to minimize immune rejection and analyze body immune system for future xenotransplantation studies. Isolation of primary EC from aorta were performed by incubation with dispase for 8-10 min at 37°C. Primary EC were cultured in EC growth medium on different extra cellular matrix (ECM), either collagen or gelation. Primary EC exhibits morphological characteristics and showed positive expressions of EC specific marker proteins i.e. PECAM1, KDR and VWF despite of their ECM surface; however, on collagen based surface they showed increase in mRNA level analyzed by qPCR. Primary EC cultured on collagen were sorted by flow cytometer using KDR marker and cultured as KDR positive cells and KDR negative cells, respectively. KDR positive cells showed dramatically increased in PECAM1 and VWF level as compared to KDR negative cells. Based on the above results, primary EC derived from GalT KO are successfully isolated and survived continuously in culture without becoming overgrown by fibroblast. Therefore, they can be utilize for xeno organ transfer, tissue engineering, and immune rejection study in future.

There is a growing interest in the application of primary hepatocytes for treatment of liver diseases in humans and for drug development. Several studies have focused on long-term survival and di-differentiation blocking of primary hepatocytes in an in vitro culture system. Therefore, the present study also aimed to optimize an in vitro culture system using primary rat hepatocytes. Primary rat hepatocytes from 6-week-old male Crl:CD rats were isolated using a modified two-step collagenase perfusion. Healthy 3.5 × 106 primary rat hepatocytes were seeded into a 2 dimensional (2D) culture in a 25T culture flask coated with collagen type I or into a 3D culture in a 125-ml spinner flask for 7 days. Production of plasma protein (ALB and TF), apoptosis (BAX and BCL2), and CYP (CYP3A1) related genes were compared between the 2D and 3D culture systems. The 3D culture system had an advantage over the 2D system because of the relatively high expression of ALB and low expression of BAX in the 3D system. However, the level of CYP3A1 did not improve in the 3D culture with and without the presence of a dexamethasone inducer. Therefore, 3D culture has an advantage for albumin production and primary rat hepatocyte survivability, but a low expression of CYP3A1 indicated that primary rat hepatocytes require a high–density culture for stress reduction by continuous flow.