As a graduate student at the Institute of Molecular Biotechnology (IMBA) nearly a decade ago, Pablo Hofbauer studied cardiac development and generated self-organizing cardiac organoids.1 He immediately grasped the implications of this system—which offered an in vitro human heart chamber model—beyond studying cardiac development.
Several drugs cause functional impairment in the heart, but studying this remains challenging because cell culture and animal models do not capture the complex human heart physiology. Research into drug-induced cardiotoxicity is important, because “it's a classical drug discovery problem [that causes] so many potential drugs fail in the early stages,” Hofbauer explained.
To explore the utility of their cardiac chamber-like organoids to study cardiotoxicity, Hofbauer and his advisor at IMBA, Sasha Mendjan, cofounded HeartBeat.bio about five years ago and teamed up with scientists at Molecular Devices, LLC. to scale the platform. Now, the two companies have developed a kit that can help eliminate variability in generating beating cardiac organoids, a challenge that had impeded widespread adoption of the model. This offers an accessible, reproducible, and functional in vitro platform to study cardiovascular disease and screen drugs as well as test toxicity.
Building Functional Cardiac Organoids from Stem Cells
When Hofbauer, Mendjan, and their team started developing cardiac organoids in 2016, not much was known in the field. “The only thing we knew, more or less, were the signals that are relevant for heart development,” said Hofbauer. They tweaked these chemical signals to activate or inhibit certain developmental pathways, yielding a method to coax human pluripotent stem cells—including embryonic and induced pluripotent stem cells (iPSCs)—into cardiac chamber-like structures.
3D reconstruction of a cardiac organoid reveals internal lumen-like cavities. Nuclei are stained in blue. Qualitative analysis and 3D reconstructions suggest the presence of an internal hollow chamber that is consistently observed across multiple reconstructed volumes.
Axxam S.p.A.
Next, the researchers tackled a challenge that could hinder the organoids’ broader adoption. “If you think about the process [of] taking iPSCs and differentiating them…there [are] lots and lots of media components, lots and lots of growth factors that drive that differentiation process,” said Nikki Carter, Director of Scientific Applications at Molecular Devices, LLC.
Sourcing these individually from different manufacturers would introduce experimental variability, so the researchers eliminated this process. “We productized that to make an easy-to-use kit that the customer can buy off the shelf and it's got all the components that they need to do the differentiation process,” explained Carter. “The one big aspect that the kit tries to solve is the variability that is usually inherent to organoids,” agreed Hofbauer.
Putting Cardiac Organoids to the Test
The journey to making this product was a long and winding one. Carter and her team first tested the reproducibility of Hofbauer and Mendjan’s protocol, followed by exploring ways to scale the pipeline. Throughout the process, they had to ensure consistency.
To this end, the researchers relied on assays like testing calcium flux—which drives contraction and relaxation—to characterize cardiac cell functioning and validating cells’ identity with PCR. They also tested the consistency of their approach to yield similar results irrespective of the iPSC lines used.
The resulting standardized approach, which guided the development of the ready-to-use kit, helped the research team generate functional, beating cardiac organoids from human iPSCs using both manual and automated workflows.
The kit offers two options: Users can either create single-lineage cardiomyocyte organoids to study muscle cells in the heart or multi-lineage organoids containing cardiomyocytes and supporting cells. These can help researchers study fibrosis and chronic toxicity. Screening drugs using both these models could offer a clearer picture of whether the drug affects only cardiomyocyte function or if it also influences other cells in the heart, said Carter.
According to Hofbauer, the kit offers an alternative to animal models for cardiac testing. While animal models cannot be replaced in biomedical research yet, Hofbauer said, “Our product…is one of those building blocks that [will] slowly help phase out the need for animal models altogether.”
- Hofbauer P, et al. Cardioids reveal self-organizing principles of human cardiogenesis. Cell. 2021;184(12):3299-3317.e22.
