Study Demonstrates Technique for Freezing Viable Organoid Precursor Cells

Researchers in Ohio report a breakthrough in producing gastrointestinal organoids from thawed cells, overcoming a significant bottleneck that has curtailed the utility of lab-grown human organs for disease and drug development research.

Growing tiny human organs in lab dishes has proven useful as a research tool to test the safety or potency of potential medications or for basic research to delve deeper into the genetic and molecular activities that cause disease.  But in practice, such tissues can be difficult to make. One batch of starting material can generate many tiny spheroids of organ precursor cells, but the next batch may produce few spheroids or none at all. For this reason, labs often experience delays making the organoids they need for pre-clinical experiments.

A new method developed by a team of experts at Cincinnati Children’s Hospital Medical Center and reported in Stem Cell Reports overcomes this bottleneck. Already being used at the hospital’s Center for Stem Cell and Organoid Medicine, the method notably makes it possible to produce high-quality organoids from frozen and thawed material. This makes it possible to ship starter materials to other labs, potentially sparking accelerated use of human gastrointestinal organoids throughout medical research.

More labs will be able to make patient-specific organoids to test drug combinations for precision treatment of complex conditions or rare disease states that require customized care. And scientists doing basic research to learn more about the genetic factors and molecular mechanisms at work in diseases affecting the digestive tract will be able to use organoids by ordering frozen spheroid precursors for their experiments.

“This method can make organoids a more accessible tool,” says first author Amy Pitstick, MS, manager of the Pluripotent Stem Cell Facility at Cincinnati Children’s. “We show that the aggregation approach consistently produces high yields and we have proven that precursor cells can be thawed from cryogenic storage to produce organoids of the small intestine.”

Typically, organoid production requires collecting skin or blood cells that are converted in the lab to make induced pluripotent stem cells (iPSCs). To use these to make intestinal organoids, highly trained lab staff grow a flat layer of organ precursor cells called the mid-hindgut endoderm. Given the right conditions, early-stage organoids called spheroids spontaneously form into 3D balls of cells. These get collected and transferred into a growth medium that provides the necessary signals for the cells to form into the specific cell types of a human organ—in this case the small intestine.

But the number of spheroids generated this way has proven inconsistent. The Cincinnati Children’s team determined it was possible to collect the unused precursor cell layer and use a centrifuge to drive cells into hundreds of tiny wells contained on small plastic plates. This prompts the formation of 3D cell aggregates that can be collected and used for organoid production. Testing detailed in the paper shows that the spheroids made this way have no meaningful differences from those that grew spontaneously.

Next, the team placed samples of the precursor cells into freezers for storage. Once these cells were thawed and aggregated, they also formed viable spheroids.

Chris Mayhew, PhD, director of the Pluripotent Stem Cell Facility, says the approach will make it possible for many research labs to use organoids in their experiments without the time and expense of learning how to grow iPSCs. “The ability to freeze the precursor cells also will allow labs to easily make organoids without having to start each new experiment with complicated and highly variable iPSC differentiation," Mayhew says.

The paper confirms that these spheroids can be reliably grown into more mature organoids that mimic the functions of the small intestine, the large intestine (colon) and the bottom portion of the stomach that connects to the intestines (antrum).

Michael Helmrath, MD, Director of Clinical Translation for the Center for Stem Cell & Organoid Medicine (CuSTOM) at Cincinnati Children’s, has already started using materials made from the new process in his ongoing research to develop transplantable intestinal tissues. “This is a great step forward for the field on many fronts,” he says. “To be able to reduce the complexity of the process and provide higher yields is beneficial to our work. And to be able to translate the methods to other labs will help move regenerative medicine forward.”