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Materials

Stem Cells Look Good On Paper

Biomaterials: Engineered porous cellulose films encourage stem cells to develop into heart tissue

by Katherine Bourzac
October 2, 2015

PAPER THIN
Schematic of coculturing stem cells and mature cells with a cellulose membrane
Credit: ACS Nano
Engineered cellulose films with newly differentiated stem cells growing on their surface can be peeled off an underlying sheet of mature heart cells (left). Placing the paper cell-side-down in a new culture dish, lowering the temperature, and lifting the paper off leaves a sheet of new heart cells behind (right).

Specially designed sheets of paper can help scientists grow stem cells into differentiated tissues. The paper membranes allow stem cells to grow in close contact with mature cells beneath them, encouraging differentiation and providing an easy way to separate the new tissue once it’s grown (ACS Nano 2015, DOI: 10.1021/acsnano.5b03823). The researchers demonstrated the approach by growing heart tissue from human stem cells.

Scientists use various methods to coax stem cells to differentiate into a particular kind of tissue in the lab. One method involves growing stem cells in a dish with the desired cell type. For example, to get cardiac tissue, scientists can coculture the stem cells with cardiomyocytes from an established cell line. But to use heart tissue derived from a patient’s own stem cells for transplantation, the patient’s newly differentiated cells would need to be separated from the mature, foreign cells they were cultured with. This typically requires dissolving the supportive protein network, called the extracellular matrix, that the cells have grown around themselves and is necessary to form tissue.

Kookheon Char and Byung-Soo Kim of Seoul National University, in South Korea, and colleagues wanted to try a new approach for making differentiated cells by using porous cellulose films that Char had developed in previous work for culturing cells. First, Char made films of varying thicknesses and porosities, both in the hundreds-of-nanometers range, by spin-coating cellulose fibers onto a substrate under controlled conditions. He then coated these films with a thermally responsive polymer that swells when the temperature is lowered and releases the sheets of cells, without the need to chemically dissolve the extracellular matrix.

Next Kim’s group seeded human bone marrow stem cells, which can differentiate into many cell types, on one side of the cellulose sheets, and placed them on culture dishes seeded with rat cardiac muscle cells. They watched how the two cell types interacted with each other through the film. They measured how closely the two cell types came to one another, what protein signals they exchanged, and whether they formed an intimate physical contact between cells called a gap junction. On all measures, the coculture films seemed to facilitate better cell connections than a commercial coculture system, which uses a porous, 10-μm-thick sheet of polycarbonate to separate the two types of cells.

After one week, they assessed the degree of stem cell differentiation by measuring the cardiac gene expression and protein levels in the stem cell layer. For example, expression of all cardiac cell genetic markers was five to eight times higher in cells grown in the cellulose system, compared with stem cells grown on their own. In the commercial system, only two of five markers showed an increase in gene expression relative to controls, and these increases were only two- and fourfold. Finally, they showed that new heart cells could be separated from the mature cells without a chemical step. The researchers peeled off the film carrying the differentiated stem cell sheet, placed it cell-side-down in a new culture dish at 37 °C for four hours, and then lowered the temperature to 20 °C for 30 minutes. Removing the cellulose paper left the new sheet of differentiated cardiac cells behind.

No commercial coculture system allows this intimacy of connection between two cell types, says James L. McGrath, a biomedical engineer at the University of Rochester, in New York, who is working on nanoporous silicon membranes for cell culture. The cellulose system allows cells to behave more naturally, sitting very close to and exchanging chemical signals with their neighbors on the other side of the sheet of paper. The high levels of stem-cell differentiation demonstrate the advantage of this, says McGrath.

A CLEAN LIFT
Fluorescence images of a coculture of human stem cells and rat cardiac cells.
Credit: ACS Nano
Fluorescence images show newly differentiated human heart cells (green) growing in a coculture with rat cardiac muscle cells (red) separated by a cellulose film. After the film is peeled away, only the differentiated human cells remain on the sheet.

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