(B-77) FRESH 3D bioprinting of MIN6 spheroids and their functionality

Introduction:: Type-1 diabetes is an autoimmune disease affecting more than 347 million patients worldwide [1]. In this autoimmune disorder, the body's immune system destroys the insulin-producing beta islet cells in the pancreas. While insulin therapy and islet transplantation are the most common treatments for this condition, donor islet cells do not survive, often due to a lack of revascularization in the patient's body or mechanical stress during the transplantation procedure [2]. Islet transplantation utilizing a tissue engineering-based technique may have practical implications for cell survival and better transplantation methods. However, increasing islet function in 3D constructions remains a significant challenge. 3D Freeform Reversible Embedding Suspended Hydrogel (FRESH) bioprinting technology may offer the innovation for desired structural features such as custom-based construct design, controllable porosity, and cell locations within the tissue-specific microenvironment [3, 4]. In this study, we FRESH printed first-time MIN6 spheroids and collagen to enhance cell survival and functionality.

Materials and Methods:: The MIN6 mouse insulinoma cell line was used in this study to generate spheroids for 3D printing. The spheroids were generated by culturing 400 single cells on a Spherical Plate 5D™ for four days. A custom-made FRESH multimaterial bioprinter was used to 3D print fibrin spheroids bioink and collagen-I. A live/dead assay was performed to assess the cellular viability of the printed constructs for 7 days. Insulin secretion was tested after performing a glucose stimulation insulin secretion assay (GSIS) to test the functionality of the printed constructs at day 7. Compared to static conditions, we cultured printed constructs in custom-designed perfusion bioreactors to establish viability and metabolic activity over time. Peristaltic perfusion was performed at 100 µL/min in a bioreactor.

Results, Conclusions, and Discussions:: We used FRESH bioprinting to create a collagen construct containing MIN6 cell spheroids domain in the middle of the collagen constructs. The spheroid printing was optimized for a spheroid diameter of 104 ± 31 um (Figure 1A), and the spheroid diameter increased to 153 ± 61 um 7 days after printing, demonstrating viability and growth. By utilizing the FRESH bioprinting for the construct, we created the porous scaffold with directly aligned collagen filament and MIN6 spheroids (Figure 1B). Cell viability did not change significantly during the experiment, 83±10% and 86±7% on day 1 and day 7, respectively (Figure 1C). Furthermore, the bioprinted MIN6 spheroids demonstrated a 20-fold higher insulin response to high glucose Krebs solution when compared to low glucose Krebs solution (Figure 1D). We also compared the metabolic activity of the construct in static and perfusion studies and found that the perfusion culture enhanced MIN6 cell activity by 1.25 times more than the static condition (Figure 1E). These results point to the potential of FRESH bioprinting to replicate the natural microenvironment of pancreatic tissues to support function of glucose-response cells and insulin secretion.
In conclusion, we have printed porous 3D collagen constructs with MIN6 spheroids using multimaterial FRESH printing. The MIN6 spheroids retain their shape and functionality during and after printing. The metabolic activity of the spheroids is higher after running perfusion studies for 7 days compared to static controls. The FRESH-printed MIN6 spheroids have the potential for future transplantation techniques. Future studies will focus on the functional analysis of constructs in dynamic conditions and use the islet cells.


Acknowledgements (Optional): : This research was supported by the JDRF.

References (Optional): : (1) J. D. Fernandes, Diabetes Res. Clin. Pract. 117, 48–54 (2016). (2) Stabler, C.L., Nature Reviews Materials, 2019. 4(6): p. 429-450. (3) Hinton, T.J., Science Advances, 2015. 1(9) (4) Lee, A. R. H. A. Science 365.6452 (2019): 482-487.