One of the most challenging tasks in biomedical sciences is finding the experimental systems that are most informative about in vivo biological processes. In most instances, the road is long and arduous, and the implementation of better methodologies often takes time, even after their superiority is demonstrated and validated. Cell culture provides a fascinating example in this respect.
For decades, cells, often of a single type, have been cultured as monolayers growing in two dimensions in Petri dishes. While it is convenient and reproducible as a technique, in live organisms cells do not grow in two dimensions. Cells in live organisms are instead embedded, together with other cell types, in the extracellular matrix (ECM) to form 3-D structures. Two-dimensional cell culture appears, therefore, to be less than suitable as an in vitro tool seeking to accurately characterize the biology of tissues and organs.
“Three-dimensional cell culture is telling us much more about the physiology of normal organs and tumors than was available before,” said Mina J. Bissell, Ph.D., distinguished scientist at the Lawrence Berkeley National Laboratory.
Approximately three decades ago, Dr. Bissell was one of the first investigators to develop the concept of 3-D cell culture. In 1982, Dr. Bissell and collaborators introduced the concept of dynamic reciprocity, which proposed that the ECM, instead of just providing a structural anchor to the cells, signals through cell-surface receptors and the cytokeleton to the nucleus and chromatin, affecting gene expression. At the same time the nucleus signals back, establishing a highly dynamic and reciprocal interaction. This model was validated repeatedly.
Using 3-D cell cultures, investigators in the Bissell lab were able to dissect interactions between the microenvironment that surrounds mammary cells and cell behavior, showing that cells signal very differently in 3-D and 2-D.
One of the challenges of 3-D culture systems revolves not only around growing cancer cells but around how to grow non-neoplastic cells. Each type of cell possesses its own challenges. Cancer cells often produce their own ECM and the compounds they require for survival; they also form 3-D tumors when grown on agar. Non-neoplastic cells need the right substratum to form 3-D structures.
“During work with non-neoplastic cells, one needs to provide what is necessary for them to form structures that will look and behave like a normal structure,” explained Sophie A. Lelièvre, Ph.D., associate professor at Purdue University.
The majority of cancers have epithelial origins, and to experimentally reproduce the phenotype of non-neoplastic epithelial cells, it is imperative to check for several things.
“One of them is polarity,” noted Dr. Lelièvre. All epithelial cells are polarized, with a basal side facing the ECM and an apical side facing the outside, which can be the blood vessel lumen, the lung alveoli, or the breast ductal system.
Dr. Lelièvre and collaborators have shown that maintaining apical polarity in epithelial cells is more sensitive to culture conditions than maintaining basal polarity. The full polarity of non-neoplastic cells is one of the most difficult aspects to preserve in vitro but represents one of the most important requirements to faithfully reproduce tissues phenotypically in vitro.
If cultures are grown with cells that are only partially differentiated, which means that they have basal but not apical polarity, those cells are already in a different state, they are already primed into the cell cycle, and the experimental conditions are not exploring a fully differentiated system.
“That is an important concept when studying cancer, because non-neoplastic tissue is fully polarized,” said Dr. Lelièvre. “In order to understand cancer, it is essential to first understand how a fully polarized cell becomes partially polarized. Otherwise, by starting with cells that are not fully polarized, one could miss an important step,” she added.
Dr. Lelièvre and collaborators have developed what they say is the first high-throughput culture system for non-neoplastic cells. This valuable tool will make it possible to study that impact of carcinogenic factors, such as environmental exposures, an important facet of preventive research.