Direct Human Stem Cell Bioprinting for 3D Expansion Culture and Temporally Controlled Differentiation

Introduction: : The current state-of-the-art of bioprinting involves spatially patterning primary or differentiated cell types within a 3D hydrogel microenvironment. There is a desire for bioprinting to be used to similarly pattern naive stem cells to engineer 3D environments for studying stem cell behavior and expansion in three-dimensional culture as well for studying differentiation and self-organization into precisely designed functional tissues or organoids. We hypothesized that we could develop a hydrogel for encapsulating stem cells that enables the stem cells to proliferate while maintaining their stemness. We have developed a viable method to pattern encapsulated human pluripotent stem cells (hPSCs) within a poly(ethylene glycol)-based hydrogel using digital light projection (DLP)-based bioprinting.

Materials and

Methods: : We optimized a bioink formulation for photoencapsulating hPSCs that can consistently maintain their stemness by developing a hydrogel with a tunable local modulus from 0.5kPa to 5kPa and that has integrin binding sites preferred by stem cells homogenously presented throughout the hydrogel volume.
Fluorescently labeled hydrogels were used for degradation testing. Bioprinted hydrogels were incubated with a 4 mg/mL collagenase solution and imaged every 15 minutes.
For cell-laden hydrogel fabrication, the supernatant from an induced pluripotent stem cell (iPSC) pellet was diluted to a final concentration of 10 – 40 million cells/mL by gently mixing with the PEG prepolymer solution. The mixture was immediately added to the setup to minimize cell exposure to unpolymerized hydrogel precursors before polymerization.
We designed the crosslinking domains to be enzymatically degradable to tune the remodeling rate. We bioprinted the hPSCs to characterize their viability, stemness, proliferation, and distribution over the course of 7 days via live/dead staining, immunofluorescence, confocal imaging, Imaris 3D image analysis, and RNA-Seq.
To evaluate the effect, if any, of the bioprinted 3D culture process on the stem cell chromosomal stability, bioprinted samples were degraded using collagenase to dissociate the cells, and the cells were collected via centrifugation. The dissociated hPSCs were then reprinted; this process was repeated for a total of six (6) “3D” passages. Karyotyping analysis was then performed to compare cells at passage 1 (P1) and passage 6 (P6) under 3D culture conditions. The cells were also passaged into a standard 2D culture.

Results, Conclusions, and Discussions:: Nanoindentation confirmed the average local moduli of the soft and stiff hydrogel constructs to be 0.503 kPa ± 0.208 kPa and 5.56 kPa ± 1.90 kPa. The fluorescently tagged integrin-binding peptide was shown via imaging to be evenly distributed in both soft and stiff conditions. The collagenase enzymatic degradation test demonstrated that both soft and stiff hydrogels fully degrade within 60 minutes. The live/dead staining should that only stem cells on the periphery of the hydrogel construct experienced cell death at 1 DIV; by 7 DIV, the hPSCs proliferated as shown by the live staining with less than ~5% stained for cell death throughout the 2 mm x 2 mm x 250 μm scaffold. On 7 DIV, the stem cell markers NANOG, OCT-4A, and SOX2 were still highly expressed, and the proliferation marker Ki-67 fluorescence signal intensity was persistent. Notably, the inclusion of ROCK inhibitor Y-27632 was not required to achieve these results. RNA-Seq analysis validated that the encapsulated hPSCs have the same gene expression as 2D control for both pluripotent and somatic panels. Both 3D bioprinted cultures with and without initial ROCKi exposure had similar gene expressions for the KEGG Human (2021) “signaling pathways regulating pluripotency of stem cells” at 1 DIV and 4 DIV as the 2D culture control. To further demonstrate the viability and function of the 3D-cultured bioprinted hiPSCs, we passaged the cells by degrading the matrix with collagenase and, after reseeding, observed hiPSC colony formation and proliferation. We repeated this “3D” passaging process 6 times in total, and the karyotyping analysis showed no signs of chromosomal mutations or abnormalities. We also were able to differentiate the 3D encapsulated hPSCs in situ to neuroepithelial cells and cardiomyocytes using commercial kits.
Our results indicate that we have developed a hydrogel formulation compatible with light-based bioprinting to encapsulate hPSCs in a 3D matrix that enables stemness maintenance and proliferation. This work could be translated for bioreactor scale up of stem cell production. Additionally, the ability to 3D pattern stem cells and temporally control differentiation could enable the bottom-up engineering of multicellular tissue constructs.

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