Culture Encapsulations with Alginate Affect Cytodifferentiation and 3D Craniofacial Development

Our lab has successfully cultured alginate encapsulated E11.5 mouse heads in a rotating bioreactor and obtained development of cartilage, vibrissae (whiskers), and possibly tooth buds, and maintained development of eyes. However, the internal tissues of the heads appeared necrotic, which we attributed to a lack of oxygenation and nutrient exchange. This hindered exchange only allowed for nourishment to external cellular structures such as vibrissae and eye. Internally, cartilage thrived since cartilage development does not require as much oxygenation as other structures. This lack of nutrient exchange could possibly be due to the thickness of the alginate encapsulation and/or the concentration of the alginate mixture. Objective: to determine the concentration of alginate that will prevent collapse of internal cavities and allow for consistent growth and differentiation of craniofacial structures, including teeth, when used to encapsulate embryonic heads for culture in the bioreactor. Method: Embryos from E12.5 ICR timed pregnant mice were dissected above the forelimbs, and the heads were divided into four equal groups. Groups were embedded in varying alginate concentrations (0.4%, 0.8%, or 1.2%) or were not embedded to serve as controls. Heads were cultured in the rotating bioreactor for a total of 4 days, then fixed and processed for microscopic evaluation and histology. Results: Internal cavities were maintained more effectively with the 1.2% alginate concentration than with the lower alginate concentrations, but specimen quality increased with lower alginate concentrations. Cartilage, tooth, eye, ear, or vibrissae were found in most samples, however the 0.4% alginate concentration had more types of structures. Conclusion: A lower concentration of alginate for tissue encapsulation allows for more cytodifferentiation and corresponding craniofacial development of internal structure compared to a higher alginate concentration. (Supported in part by AADR 2003 Fellowship to V. Leonhart)