Controlled 3D Stem Cell Culture Using Noise-free and Specific Signaling Culture Systems

Introduction:: Human pluripotent stem cells (hPSCs) hold broad capabilities for clinical applications as they can undergo multi-lineage differentiation into various cell and organoid types for disease modeling, patient-specific drug screening, and transplantation [1]. The most widely used biomaterial for expanding and differentiating iPSCs in 2D is Matrigel, a reconstituted basement-membrane matrix harvested from mouse tumors that suffers from batch-to-batch variability, xenogenic contaminants, and nonspecific protein interactions [2]. 2D coatings also do not allow for physiological conditions, increased cell-cell interactions, and the high cell yields required for clinical translation (i.e. ~10 billion iPSC-cardiomyocytes required for one cardiac implantation) [3]. This has motivated the development of 3D synthetic alternatives which are chemically-defined, xenogenic-free microenvironments optimized to fit a specific cell type and yield reproducible results. The most common synthetic alternative is poly(ethylene glycol) (PEG)-based scaffolds [4], however, hPSCs can spontaneously differentiate and suffer from reduced long-term proliferative capacity [2]. We propose the use of zwitterionic materials, specifically poly(carboxybetaine) (PCB), a unique super-hydrophilic synthetic polymer that achieves extremely low protein adsorption [5] for controlled 3D hPSC culture. PCB is an ideal material for creating a “noise-free”, clean background in which to engineer specific signaling cues such as cell adhesive ligands for hPSC study. While other synthetic materials like PEG can also be used to control 3D material properties, they are not capable of providing a truly “clean” background as they are amphiphilic (i.e., partially hydrophilic and hydrophobic) and are not as nonfouling as PCB.


Materials and Methods:: Human iPSC line, iPSCORE_5_1, obtained from WiCell Research Institute, was cultured on Matrigel in mTeSR Plus medium supplemented with 50µg/mL Normocin. Cells were passaged with Accutase for 7 min at 37ºC. Medium was changed daily. To encapsulate cells in hydrogels, iPSCs on Matrigel were dissociated, centrifuged, and the pellet was resuspended in the polymer and MMP-degradable crosslinker. This solution was plated as 20µL droplets and gelled in 10 minutes at 37°C. Assays were conducted after cell release from hydrogel. The viability of encapsulated iPSCs was monitored with Invitrogen’s live/dead cell imaging kit and imaged using a BioTek Cytation 7. The fold expansion and total cell yield was measured using a Nexcelom cell counter after dissociating to single cells using Accutase. Karyotype analysis was performed by KaryoLogic Inc. (Durham, NC). For immunofluorescence staining, iPSCs from PCB hydrogels were fixed with 4% PFA, permeabilized with 0.5% Triton-X, and blocked with 10% goat serum. Spheroids were incubated with primary antibodies overnight and a 3hr incubation of secondary antibodies. Spheroids were imaged using a Zeiss LSM880 Confocal Microscope at 63x magnification. For alkaline phosphatase staining, cells were stained for 15 min following kit instructions (Sigma, SCR004) and imaged using a BioTek Cytation 7. RT-qPCR measured gene expression levels for NANOG and OCT4 using GAPDH as the reference gene, ΔΔC_t analysis, and cells cultured on Matrigel as the control. Future characterization will include flow cytometry of pluripotency markers, embryoid body differentiation, and teratoma assay.


Results, Conclusions, and Discussions:: The biocompatibility and pluripotency maintenance of the PCB hydrogel with hPSCs is fundamental to the use of PCB hydrogels to support the culture of hPSCs. Preliminary results indicate that after only 5 days of culture, iPSCs cultured in PCB hydrogels generate uniformly sized spheroids of diameter ~100um with excellent cell viability (Fig. 1a). Moreover, when PCB hydrogels are compared to PEG hydrogels with the same modulus, same cell adhesive ligand (RGD), and same crosslinker, iPSCs in PCB hydrogels exhibit a fold expansion of ~15 after 7 days which is significantly greater than PEG which achieves ~7 fold expansion (Fig. 1b). PCB hydrogels also result in a final cell count of nearly 50 million cells per mL hydrogel (Fig. 1c). Additionally, karyotype analysis shows that iPSCs cultured in PCB hydrogels maintain a normal karyotype (Fig. 1d). Moreover, the evaluation of four hPSC pluripotency markers, OCT4, TRA-1-60, SSEA-3, and SSEA-4, by immunocytochemistry revealed high expression of all markers in the iPSCs cultured in PCB suggesting that pluripotency is not lost with high cell yields (Fig. 1e). This is further confirmed by alkaline phosphatase staining in nearly all spheroids (Fig. 1g) and RT-qPCR results showing increased expression of NANOG and OCT4 in iPSCs cultured in the PCB hydrogel (Fig. 1f). Future work involves integrating other cell adhesive ligands into the PCB hydrogel to create an optimized 3D microenvironment for hPSC expansion and pluripotency maintenance that leverages PCB’s noise-free properties, characterizing the long-term culture of 4 hPSC cell lines over 25 passages in the PCB hydrogel, and investigating how the PCB hydrogel affects hPSCs mechanistically. This cell culture system has several key advantages including (a) noise-free background, (b) ability to incorporate specific cell-adhesive ligands to recapitulate in vivo niche conditions, (c) cell-mediated hydrogel degradation, (d) easy and biocompatible release of encapsulated cells from hydrogel when desired by the user, (e) ability to passage cells repeatedly for long-term culture, (f) ability to mimic 3D physiological conditions, and (g) flexibility to adjust polymer and peptide formulations as needed.


Acknowledgements (Optional): : We acknowledge support from Cornell University, including Robert Langer Professorship, Cornell NEXT Nano Initiative, and Seed Grant for Intercampus Collaborative Projects.


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