# Trillion cell culture to fuel organ biofabrication

> **NIH NIH DP2** · STANFORD UNIVERSITY · 2022 · $1,416,600

## Abstract

PROJECT SUMMARY
The convergence of human induced pluripotent stem cells (hiPSCs), organoids, synthetic biology and 3D
bioprinting promises a future of patient-specific lab-grown organs for patients suffering from organ failure.
However, to realize this organ engineering vision, biofabrication researchers sorely need thousand-liter-scale
cultures of hiPSCs to generate enough material to begin high-throughput experimentation. Solving the myriad
challenges in organ construction, vascularization, maintenance, maturation, and characterization will require
decades of painstaking research. Yet, deriving patient-specific cells at this scale remains two orders of
magnitude too expensive for academic laboratories due, in large part, to the expensive growth factors required
for hiPSC maintenance and differentiation. Furthermore, existing protocols to generate organoids from stem cells
are cumbersome, slow, and inefficient, limiting the number of organoids that can be derived for 3D bioprinting
applications. In these proposed studies, we detail novel methods to dramatically reduce the cost of stem cell
maintenance and increase the scale of organoid production. To reduce the costs of large-scale hiPSC growth
by two orders of magnitude, we propose to engineer growth factor-free hiPSCs by programming them to express
constitutively-active growth factor receptors which can be excised prior to differentiation. To enhance the scale
and throughput in generating multicellular cardiac organoids, we propose engineering hiPSCs to undergo
simultaneous multicellular differentiation without requiring growth factors. To achieve this, we propose a novel
stochastic Cre-lox recombination system to upregulate one-of-three transcription factors, EOMES, Nkx3.1, or
ETV2, to generate tri-cellular synthetic cardiac organoids containing cardiomyocytes, fibroblasts, and endothelial
cells, respectively. By culturing millions of these synthetic cardiac organoids in suspension culture, we will derive
therapeutically-relevant quantities of densely cellular myocardial bioink for 3D bioprinting. We will next use
synthetic cardiac organoid bioink to derive a human-scale, thick-walled, and vascularized ventricle model. These
bioprinted ventricles will be housed in a custom perfusion bioreactor for studying how mechanical and electrical
stimulation can maintain vascular perfusion, enhance cardiomyocyte maturation and alignment, and affect organ-
scale contractility and ejection fraction. The highly scalable stem cell and organoid culture methods presented
here are applicable across many organ systems, and could revolutionize the scale and pace of organ
biofabrication research.

## Key facts

- **NIH application ID:** 10473259
- **Project number:** 1DP2HL168563-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Mark A. Skylar-Scott
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $1,416,600
- **Award type:** 1
- **Project period:** 2022-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10473259

## Citation

> US National Institutes of Health, RePORTER application 10473259, Trillion cell culture to fuel organ biofabrication (1DP2HL168563-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10473259. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*