(471c) 3D Bioprinting of iPSC Derived Islet Organoids in Hydrogel Constructs

One of the largest medical problems in the world is the lack of organs available for transplantation. In the US alone, as of February 2021, over 107,000 people were waiting for a transplant, and the organ need increases while the supply remains stagnant(1). 3D bioprinting is designed to fight this issue by printing biomaterial layers embedded with cells that can work to recreate a tissue construct. Primarily bioprinting has focused on constructing hard tissues, with less work going towards supplementing a soft tissue environment(2). Soft tissue requires bioinks that can act as support structures, while maintaining the correct biophysical properties for human cells. Another technology used to address organ shortages is induced pluripotent stem cells (iPSCs), as they can be differentiated to almost any tissue and tailored to patients. The current aim of this project is combining the advantages of bioprinting and iPSC organoids to form a complex soft tissue environment. In this study, iPSC islets were bioprinted with the goal of engineering endocrine pancreas tissue that can remain viable and retain functionality.

The ideal bioink was determined by optimizing the ink composition to produce a structure that provided the desired stability and compatibility with iPSC culture. A combination of 3% w/v alginate, and 6% w/v methylcellulose crosslinked with 50 mM CaCl2 was able to achieve these attributes, which was first confirmed with primary human islets. Cadaveric human islets (Prodo lab) were bioprinted in the ink and maintained in culture for over 3 days. The islets remained viable within the strand and exhibited the appropriate islet markers, c-peptide and glucagon as confirmed by immunofluorescent imaging. Islet function was confirmed by Glucose Sensitive Insulin Secretion (GSIS) assay, where the printed islet response was found comparable to control islets, with a stimulation index (Insulin at high glucose/ Insulin at low glucose) of 20.

Having confirmed retention of primary islet function in the bioink and printing configuration, we next adapted the procedure for iPSCs and iPSC-derived islet organoids. As a first step, we printed undifferentiated iPSCs which were suspended in the bioink as single cell (SC). The SC iPSCs self-assembled to form small aggregates during post-print culture within the print strand. The aggregates retained high viability over a 9-day culture, however, the process of self-aggregation resulted in cell debris within the strands, likely from the cells which failed to aggregate. As an alternative, we pre-aggregated the undifferentiated iPSCs in agarose microwells, and printed at an average size of 350 μm and cultured for 3 days. LiveDead imaging showed the aggregates maintained viability, while fluorescent staining showed the presence of Oct4 and Nanog, confirming pluripotency. Importantly printing of preformed aggregates significantly reduced the observed debris while printing single cells.

We next advanced with printing iPSC derived islet organoids. These organoids were derived using an organoid culture method established previously, where the islets exhibit pancreatic and islet markers, c-peptide and glucagon(3). iPSC islet organoids thus derived were suspended in the bioink and printed using the same procedure and maintained in culture for 7 days. LiveDead imaging showed the iPSC islets sustaining viability, while fluorescent staining indicated that they also retained expression of the pancreatic markers, NKX6.1 and PDX1, and islet markers c-peptide and glucagon. Work is currently ongoing to see if iPSC aggregates can undergo differentiation post-printing, and if after printing, iPSC islets can demonstrate proper islet function when observed under GSIS testing conditions. This project acts as proof of concept for bioprinting a functional pancreatic environment using iPSC islets, with future work focused on organ transplantation.

(1)“Organ Donation Statistics.” OrganDonor.gov, Health Resources and Services Administration, 25 Feb. 2021, www.organdonor.gov/statistics-stories/statistics.html.

(2)Mandrycky, Christian, et al. "3D bioprinting for engineering complex tissues." Biotechnology advances 34.4 (2016): 422-434.

(3)Richardson, Thomas et al. “Alginate encapsulation of human embryonic stem cells to enhance directed differentiation to pancreatic islet-like cells.” Tissue engineering. Part A vol. 20,23-24 (2014): 3198-211.