3D in vitro approaches, such as organoids grown from tumor cells derived from mice and patients, are essential for studying therapeutic approaches to cancer control due to their ability to mimic the pathological properties of an environment real tumor in the laboratory.
The organoids are grown in a tissue-derived gel substrate placed in a laboratory dish. Traditional gels – comprising a complex mixture of proteins, proteoglycans, and growth factors derived from actual murine tumors – can be finicky in use, with the quality and purity of the gel varying from batch to batch. There is no guarantee that a specific type of cell will grow in a particular batch of gel.
“The issue of reproducibility is a major one,” said co-author Linda Griffith, PhD, professor of biological and mechanical engineering at the Massachusetts Institute of Technology (MIT), in a statement. “The research community has been looking for ways to make more methodical cultures of these types of organoids, and in particular to control the microenvironment.”
Organoid models for pancreatic cancer are particularly difficult to develop. First, traditional gels do not easily support the growth of cancer cells and their environment. Second, pancreatic cancer cells lose properties, such as their distinct stiffness, once they are removed from the body.
Griffith’s lab at MIT has been working for 10 years on designing a synthetic gel that could be used to grow epithelial cells with more desirable and predictable properties.
Griffith and his colleagues formulated a gel based on polyethylene glycol (PEG), a polymer often used for medical applications because it does not interact with living cells. By studying the biochemical and biophysical properties of epithelial and stromal cells and their interactions with the extracellular matrix (which surrounds the body’s organs), the researchers identified characteristics that they could incorporate into the PEG gel to facilitate coherent cell growth.
The key biophysical property required for the growth of pancreatic cancer cells has been shown to be a strong adhesion between the gel and the organoid cells. The researchers found that incorporating small synthetic peptides derived from fibronectin and collagen into the gel allowed them to achieve the necessary “sticking” required by integrins, cell surface proteins that are involved in cell adhesion. The strong adhesion with integrins allowed cells to firmly adhere to the gel and form organoids.
Another key property of the gel formulation was its adjustable “stiffness”, which helps recreate the stiffness characteristic of pancreatic tumors. By adjusting the properties of the gel, Griffith and his colleagues reliably generated gel scaffolds that mimicked the stiffness profiles of normal and tumor pancreatic tissue.
After developing the PEG gel, Griffith collaborated with Claus Jorgensen, PhD, who leads a team of researchers studying pancreatic cancer at the Cancer Research UK Manchester Institute, to test the gel and use it to grow pancreatic organoids derived from mice. .
Jorgensen and his students were able to produce the gel and use it to grow healthy and cancerous pancreatic cells.
“We got the protocol from Linda and we got the reagents and then it worked,” Jorgensen said. “I think that says a lot about the robustness of the system and its ease of implementation in the lab.”
When Jorgensen and his colleagues compared pancreatic organoids grown in gel to tissues they had studied in living mice, they found that tumor organoids expressed many of the same integrins seen in pancreatic tumors. In addition, many supporting cells that normally surround pancreatic tumors were also present.
After successfully growing mouse cells, the scientists verified that the PEG gel would also promote the in vitro growth of pancreatic cells of human origin. They seeded the gel scaffold with established human pancreatic ductal organoids and monitored their growth. Immunofluorescence analysis indicated that the organoids grew continuously over several days, eventually developing into fully mature spheres.
Researchers believe the gel, which can be easily made in the lab, will prove useful in studying lung, colorectal and other cancers.
Griffith plans to use the gel to grow and study tissue from patients with endometriosis, a painful condition in which uterine tissue grows outside the uterus. It has filed a patent on the gel technology and is in the process of clearing it for commercial production.
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