3D Culture of rat embryonic stem cells and rat induced pluripotent stem cells in a novel PGmatrix hydrogel (LB20)

Abstract

Expanding embryonic stem cells (ESCs) in a synthetic 3D microenvironment in vitro while maintaining the undifferentiated state has been challenging. It would be advantageous to identify a substrate which could be modified such as by changing ligands that interact with cell surface receptors or by changing the substrate’s stiffness. We developed a synthetic PGmatrix hydrogel to include these design specifications and determined whether it could support the expansion of undifferentiated ESCs in a 3D microenvironment. Rat ESCs (rESCs) and rat induced pluripotent stem cells (riPSCs) were used for evaluation. The cells were grown both in 2D and 3D in PGmatrix hydrogel containing 2 inhibitors. Dynamic rheological tests of PGmatrix hydrogel in 2i medium showed a rapid increase of storage moduli (G’ = 600 Pa for 3mM in 1 hr) indicating gelation after mixing. The mechanical strength of hydrogel was full recovered after shear‐thinning into liquid phase.Also, the hydrogel reached a self‐supporting strength within 1000sec which is a reasonable rate to ensure cells were suspended in the hydrogel matrix before they reached the bottom of the plate. Both cell lines were cultured for more than 30 passages using regular trypsinization method to produce single cells at passage prior to plating. We harvested cells from the hydrogel for passaging by shearing the gel to liberate the cells. Phase contrast images of cells grown in 3D showed similar morphology which had rounded phase‐bright borders compared to cells grown on murine embryonic fibroblast (MEF) feeder layer. They retained pluripotent stem cell properties and showed positive staining for alkaline phosphatase, Oct‐4, Nanog, Sox‐2 and SSEA‐1. Karyotypes of pluripotent stem cells cultured in 3D were normal after 20 passages. Embryoid bodies (EBs) could be formed by hanging drop after the cells were trypsinized from 3D cultures. We plan to compare the gene expression profiles of 2D and 3D cells and determine if the 3D cultured cells can undergo differentiation into all the 3 germ layers and form teratomas. Growing ESCs in a fully synthetic 3D culture can significant advantages for directing differentiation and examining the interactions with surface receptors. Such knowledge will enable 3D culture for their use in drug development or for encapsulation of cells for clinical use.