One of the biggest advancements in stem cell research in recent years has been the development of organoids—three-dimensional cell cultures that recapitulate some key features of actual organs. These mini-organs could especially be a boon to regenerative medicine. However, they are currently grown using Matrigel, an extracellular matrix that is derived from a mouse tumor cell line, and are not suitable for clinical use. 

As a Matrigel substitute, researchers have now engineered a synthetic hydrogel whose properties can be easily fine-tuned to match specific applications. As demonstrated recently in Nature Cell Biology, the synthetic hydrogel was used to culture human intestinal organoids (HIOs). These HIOs were transplanted into the guts of mice to improve wound healing, suggesting that the hydrogel may one day help provide a treatment for intestinal conditions like inflammatory bowel disease. 

“We were able to engineer a fully synthetic matrix that serves as an alternative to Matrigel,” says study lead author Andrés J. García, a bioengineer at the Georgia Institute of Technology. “And because we can control its polymerization, we can use that matrix as a delivery vehicle for organoids, which are able to repair intestinal tissue in mice.”

Researchers have previously used Matrigel to create numerous types of organoids, such as cerebral, renal (kidney), and intestinal organoids. These structures—derived from tissue cells, embryonic stem cells, or induced pluripotent stem cells (a type of cell that can be coaxed to develop into different types of tissue)—have been used for a variety of research, including for disease modeling, basic physiological studies of cells and tissues, and drug testing. If placed in the body, the organoids can mature further and integrate into host tissues, helping to repair tissue damage. 

But the reliance on Matrigel for organoid development is problematic, García says. Matrigel is a complex mixture derived from murine malignant (sarcoma) cells, which are cultured in vitro and then removed to leave behind a gelatinous protein-rich extracellular matrix. The potency and efficacy of the matrix varies from batch to batch and researchers do not know what is exactly in the mixture when they receive it. Furthermore, Matrigel’s use of animal tumor cells poses a potential risk of disease transmission if the organoids are used in humans for regenerative medicine. 

García and his colleagues sought to develop a completely synthetic matrix system that resolves Matrigel’s key issues, in particular its lot-to-lot variability and use of mouse tumor material. They selected a hydrogel platform based on a four-arm poly(ethylene glycol) macromer with maleimide groups at each terminus (PEG-4MAL). This platform is biocompatible, exhibits low toxicity and inflammatory responses in vivo, and has a well-defined structure, tunable reaction timescales, and can incorporate bio-functional groups. The researchers functionalized the PEG-4MAL macromers with various adhesive peptides and added in crosslinking peptides to create PEG-4MAL hydrogels. They grew HIOs from human pluripotent stem cells in the hydrogels, just like scientists have previously done with Matrigel. 

The mechanical properties and stiffness of the hydrogel are easily tunable by varying the macromer concentration. The researchers found that HIO viability in the hydrogels decreased with increasing hydrogel stiffness, with optimal stiffness being one similar to Matrigel (at 4% PEG-4MAL and 96% water). The adhesive peptides also had a large impact on the growth and viability of organoids—in particular, a peptide containing RGD (Arg-Gly-Asp), a cell attachment sequence also found in the cell adhesion molecule fibronectin, was an important component. The proliferation, morphology, and organization of the structural proteins within HIOs grown in Matrigel were the same as those grown in the optimized PEG-4MAL matrix containing the RGD peptide. 

The researchers also found that the PEG-4MAL hydrogel was the perfect vehicle to deliver organoids into the injured colons of immunocompromised mice. To deliver the organoids, they used a colonoscope to separately inject HIOs in functionalized PEG-4MAL and a protease-degradable crosslinking peptide, which polymerized inside of the mouse intestines. The HIOs developed and integrated into the murine colon such that the cells could only be identified by looking for human-specific biomarkers, and the synthetic hydrogel was replaced by the extracellular matrix produced by the cells. The HIOs significantly increased wound repair by day 5, García says, adding that the wound healed completely by 28 days. 

“This is a timely and interesting paper,” Fredrik Holmberg and Ole Haagen Nielsen of the University of Copenhagen’s Center for Inflammatory Bowel Disease wrote in an email. Aside from the lack of malignant cells, the scalability, relatively low manufacturing costs, batch-to-batch consistency, and malleability of the hydrogel are key advantages, they say. However, they note, induced pluripotent stem cells might carry a risk of malignancy (if reprogramming genes fail to turn off or cancer-causing genes are incidentally switched on).

“I think for the future of regenerative medicine, [the synthetic hydrogel] is a big step forward,” adds Mark Donowitz, LeBuff Professor of Medicine and Physiology at Johns Hopkins School of Medicine who was not involved in the study. “It’s a real breakthrough, but it is the first of more to come,” he says, stressing that other groups are also working on Matrigel alternatives.  

The research is a proof-of-concept, García says, and the next steps are to use larger animal models and test the hydrogel in the context of diseases, such as inflammatory bowel disease (Crohn’s disease and ulcerative colitis). Importantly, García and his colleagues also showed that the hydrogel formulation that worked for HIOs can be used to develop human lung organoids, and other types of organoids likely only require the formulation to be tweaked. In particular, the team is interested in developing organoids for pancreatic islets (insulin-releasing cells), which could help people with diabetes. García says: “We think this is really a platform technology that can be used for many applications.”

Read the article in Nature Cell Biology.