Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier

Abstract

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries.

Recent Advancements in Hydrogel Biomedical Research in Italy

“…The PF gels created a suitable environment for cell growth and protein production, resulting in significantly higher purified Cripto-1 yields compared to traditional two-dimensional (2D) culture systems. The Cripto-1 produced by cells grown on the 3D microcarriers had similar bioactivity to commercially available Cripto-1, demonstrating a promising potential for hydrogel-based approaches in improving biomanufacturing processes for therapeutic proteins [67].…”

Harnessing three-dimensional porous chitosan microsphere embedded with adipose-derived stem cells to promote nerve regeneration

“…In this way, cells and microcarriers can combine to form an excellent 3D microenvironment and improve the efficiency of cell expansion. Microcarrierbased cell transplantation strategies have been reported to play a beneficial role in bone and cartilage tissue defects [23], muscle repair [24], and skin wound healing [25]. However, there are few studies on using microcarrier complex cells in tissue engineering nerve conduits to treat peripheral nerve defects.…”