Perfusion cultures of human embryonic stem cells

Abstract

Human embryonic stem cells (hESC) are self-renewing pluripotent cells capable of differentiating into cells representative of all three embryonic germ layers. Hence, they hold great potential for regenerative medicine. However, significant cell numbers are required to fulfill their potential therapeutic applications. In this study, perfusion with supplemented conditioned media (SCM), produced by mouse embryonic fibroblasts (MEF), was adopted to improve cell densities of hESC cultures. Perfusion enhanced hESC numbers by 70% compared to static conditions, on both organ culture dish (OCD) and petri dish cultures. All cultures maintained healthy expression of the pluripotent marker, Oct-4 transcription factor. In vivo, perfused hESC formed teratomas in severe combined immunodeficiency (SCID) mice models that represent the three embryonic germ layers. When SCM was produced with lower concentrations of MEF, hESC densities and Oct-4 levels were reduced. Hence, perfusion with SCM is a potential feeding method for scale-up production of hESC.

Interwoven Four-Compartment Capillary Membrane Technology for Three-Dimensional Perfusion with Decentralized Mass Exchange to Scale Up Embryonic Stem Cell Culture

“…It has been shown that medium perfusion is beneficial even in 2D cultures [Fong et al, 2005]. Perfusion facilitates continuous nutrient and oxygen supply [Ouyang and Yang, 2008] in larger cell masses.…”

Bioreactor development for stem cell expansion and controlled differentiation

“…For example, Cormier et al attributed the decline in viability of mESC aggregates after 6 days in batch culture to the associated build-up of ammonia (to 2.5 mM) and decrease in pH (from 7.6 to 6.6) [15]. Perfusion, frequent feeding, and/or the use of gas-permeable culture surfaces increased the expansion of MSCs [4,5,6•], ESCs [32,33] and mammary epithelial stem cells [12], while retaining stem cell potential [5,9,32,33]. Under conditions for which oxygen was not limiting, Zhao et al reported greater human MSC expansion at a lower perfusion rate [7].…”

Culture conditions for human embryonic stem cells

“…Understanding the mechanisms of hESC self-renewal and proliferation as non-differentiated cells would greatly facilitate the establishment of defined derivation and culture conditions for hESC (Vallier et al 2005, Wang et al 2005a, Armstrong et al 2006, Rho et al 2006. Recent advantages in the use of biomaterials (Anderson et al 2004) and perfusion culture systems (Fong et al 2005) for the culture of hESC suggest that large-scale culture of hESC in bioreactors under controlled environment using defined culture medium and biomaterial substrates may be feasible in future.…”