(770e) Three-Dimensional Tissues Using Human Pluripotent Stem Cell Spheroids As Biofabrication Building Blocks

Qiang Li^a, Haishuang Lin^a, Yuguo Lei

Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, Nebraska, USA

(a): These authors contribute equally

Introduction: A recently emerged approach for tissue engineering is to biofabricate tissues using cellular spheroids as building blocks. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), can be cultured to generate large numbers of cells and presumably be differentiated into all the cell types of human body in vitro, thus are ideal cell source for biofabrication. We previously developed a hydrogel-based cell culture system that can economically produce large numbers of hPSC spheroids. With hPSCs and this culture system, there are two potential methods to biofabricate a desired tissue. In Method 1, hPSC spheroids are first utilized to biofabricate a hPSC tissue that is subsequently differentiated into the desired tissue. In Method 2, hPSC spheroids are first converted into tissue spheroids in the hydrogel-based culture system and the tissue spheroids are then utilized to biofabricate the desired tissue. In this paper, we systematically measured the fusion rates of hPSC spheroids without and with differentiation toward cortical and midbrain dopaminergic neurons and found spheroids’ fusion rates dropped sharply as differentiation progressed.

Materials and Methods: To transfer the culture from 2D to 3D PNIPAAm-PEG hydrogels, hPSCs maintained in Matrigel-coated 6-welll plate were treated with Accutase at 37 °C for 5 minutes and dissociated into single cells. Dissociated cells were mixed with 10% PNIPAAm-PEG solution dissolved in E8 medium on ice and cast on tissue culture plate, then incubated at 37 °C for 10 minutes to form hydrogels before adding warm E8 medium containing 10 μM ROCK inhibitor. Spheroids containing about 5-10 million hPSCs were harvested from the PNIPAAm-PEG hydrogel on day 5, suspended in E8 medium and placed in a 6.5 mm Polyester Membrane Transwell Insert. Cells were then cultured in the cell culture incubator. For some samples, 5 hours later, the medium was removed and a thin layer of ECM (5 μl) was overlaid on top of the fused tissue. Medium was added back to culture the cells. To biofabricate the cortical neural tissue, the fused hPSC tissue was cultured in the cortical neuron induction medium for 11 days and matured in the neural differentiation medium for another 20 days. To biofabricate the ventral midbrain dopaminergic neural tissue, the fused hPSC tissue was cultured in the ventral midbrain neuron induction medium for 11 days and the neural differentiation medium for another 20 days.

Results and Discussion: We biofabricate neural tissues including the cortical neural tissue and midbrain dopaminergic tissue to explore the tissue biofabrication methodology using hPSCs as starting materials in this study. An essential requirement for rapid tissue biofabrication is that spheroids can quickly fuse to form a cohesive tissue. Culturing hPSCs in 3D thermoreversible PNIPAAm-PEG hydrogels allowed the production of spheroids which contained both cells and their native ECM. An important finding of this study is that the fusion rate decreased significantly once hPSC spheroids were differentiated. This is particularly obvious for the cortical neural tissue spheroids. This phenomenon leads us to conclude that a more appropriate method to biofabricate neural tissues is to fuse hPSC spheroids first, followed by differentiating and maturing the hPSC tissue. Importantly, our study also showed that the hPSCs in the hPSC tissue could be differentiated into neurons with protocols similar to these used for differentiating hPSCs into neurons in 2D culture. It should be noted that not all the hPSCs in the biofabricated tissues were differentiated into the desired neurons in this study. These biofabricated tissues are appropriate for modeling diseases and drug discovery. For in vivo application, the differentiation protocols should be optimized to quantitatively convert hPSCs into the desired neurons in the future.

Conclusion: In summary, this study explored some fundamentals and methods for biofabricating tissues using hPSCs as the cell source. We found: (1) the PNIPAAm-PEG hydrogel culture system was excellent for biomanufacturing hPSC spheroids; (2) the spheroid fusion experiment was a quick and efficient method to evaluate the fusion capability of tissue spheroids; (3) the fusion rates of differentiated spheroids were significantly lower than hPSC spheroids; (4) hPSCs could be differentiated into tissue cells in the 3D tissues; and (5) the method by first fusing hPSC spheroids, followed with differentiation and maturation was appropriate for tissue biofabrication.