Bioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D

TLDR

The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient‑specific cells, enabling animal‑free drug development and personalized medicine. We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve‑based printing process, and their post‑printing differentiation into hepatocyte‑like cells (HLCs). HLCs derived from hiPSCs and hESCs were bioprinted using a valve‑based process into alginate hydrogel constructs, and their viability, pluripotency, and hepatic marker expression—including albumin secretion—were assessed throughout differentiation. The printed cells expressed hepatic markers such as nuclear factor 4α, secreted albumin, and displayed hepatocyte‑like morphology, with 40‑layer alginate constructs peaking albumin secretion at day 21, and viability and pluripotency were unchanged compared to non‑printed cells, confirming the gentle nature of the valve‑based process.

Abstract

We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs differentiated from both hiPSCs and human embryonic stem cells (hESCs) sources were bioprinted and examined for the presence of hepatic markers to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Examined cells were positive for nuclear factor 4 alpha and were demonstrated to secrete albumin and have morphology that was also found to be similar to that of hepatocytes. Both hESC and hiPSC lines were tested for post-printing viability and pluripotency and were found to have negligible difference in terms of viability and pluripotency between the printed and non-printed cells. hESC-derived HLCs were 3D printed using alginate hydrogel matrix and tested for viability and albumin secretion during the remaining differentiation and were found to be hepatic in nature. 3D printed with 40-layer of HLC-containing alginate structures reached peak albumin secretion at day 21 of the differentiation protocol. This work demonstrates that the valve-based printing process is gentle enough to print human pluripotent stem cells (hPSCs) (both hESCs and hiPSCs) while either maintaining their pluripotency or directing their differentiation into specific lineages. The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine.