Web Date: November 20, 2015
Temperature-Sensitive Gel Grows Cells In 3-D
Biologists have a new alternative to fickle protein gels for culturing cells in three dimensions: a polymer gel that not only supports cell growth but handily transforms from gel to liquid when the temperature drops for easy cell harvesting (Biomacromolecules 2015, DOI: 10.1021/acs.biomac.5b01266).
In a 2012 lecture in Coventry, England, George M. Whitesides of Harvard University challenged polymer chemists to come up with a synthetic replacement for animal-protein-based hydrogels such as Matrigel for growing cells in 3-D. Cells grown in a 3-D matrix better represent body tissues than flat cell cultures. Protein-based gels have poor batch-to-batch reproducibility, Whitesides said, and cell biologists need hydrogels that will produce the same cell-growing environment every time.
Steven P. Armes of the University of Sheffield was in the audience. His group had just made synthetic temperature-responsive gels, which he realized might be a ready-made replacement for Matrigel.
After the lecture, Armes caught up with Whitesides, and they began a collaboration to explore if the new gels would support 3-D cell culture.
Armes’s gel is constructed from a diblock copolymer, a covalently linked chain of two separate polymers: poly(glycerol monomethacrylate) (PGMA) and poly(2-hydroxypropyl methacrylate) (PHPMA). PGMA is water soluble, and PHPMA is hydrophobic. In water, the hydrophobic sides of the chains cluster together to form shapes that differ depending on the temperature. At refrigerator temperature (4 °C), the copolymer forms spherical nanoparticles that stay suspended in the water. At and above room temperature (25 °C), the spheres fuse to form very long wormlike structures, which entangle to form a soft hydrogel. The change from warm gel to cool liquid is fully reversible.
The researchers quickly saw that this low-cost polymer might make a uniform gel free of animal proteins, a necessity for some applications, such as human therapeutics. In addition, researchers could recover the cells from the scaffold simply by lowering the temperature, which would avoid the use of potentially damaging enzymes.
To test the “worm gel,” Whitesides’s group embedded it, along with mouse lung cancer cells, in the spaces of hole-punched sheets of thick polyvinyl chloride sandwiched by polyester mesh. Cells have access to air and nutrients through the breathable mesh while they grow inside the 3-D gel.
After initial tests, the team needed a stronger gel. So the polymer chemists added disulfide groups to the water-soluble chains, which eventually lead to bonding between neighboring worms and stiffen the gel.
In the disulfide-reinforced gel, the cells grew for 12 days, even without any of the growth factors and adhesion proteins within Matrigel. After 12 days, the researchers recovered 91% of the cells by cooling the worm gel in refrigerated buffer for an hour. Cells grown in Matrigel required a bath in degradation enzymes to recover all of the cells.
Buddy D. Ratner of the University of Washington doesn’t expect this gel to replace Matrigel just yet but says its simple thermoreversibility and predictable structure offer distinct advantages potentially helpful in tissue engineering and cancer research.
Armes and his group are now designing new generations of worm gel not just for cell culture but for other biological needs. “We just need more cell biologists to tell us about their problems so that we can try and address them,” he says.
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