Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures.
It is well-known that 3D in vitro cell cultures provide a much better model than 2D cell cultures for understanding the in vivo microenvironment of cells. However, significant technical challenges in handling and analyzing 3D cell cultures remain, which currently limits their widespread application. Herein, we demonstrate the application of wholly synthetic thermoresponsive block copolymer worms in sheet-based 3D cell culture. These worms form a soft, free-standing gel reversibly at 20-37 °C, which can be rapidly converted into a free-flowing dispersion of spheres on cooling to 5 °C. Functionalization of the worms with disulfide groups was found to be essential for ensuring sufficient mechanical stability of these hydrogels to enable long-term cell culture. These disulfide groups are conveniently introduced via statistical copolymerization of a disulfide-based dimethacrylate under conditions that favor intramolecular cyclization and subsequent thiol/disulfide exchange leads to the formation of reversible covalent bonds between adjacent worms within the gel. This new approach enables cells to be embedded within micrometer-thick slabs of gel with good viability, permits cell culture for at least 12 days, and facilitates recovery of viable cells from the gel simply by incubating the culture in buffer at 4 °C (thus, avoiding the enzymatic degradation required for cell harvesting when using commercial protein-based gels, such as Matrigel).
Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
Karen A
KA
Simon
Department of Chemistry, University of Sheffield , Dainton Building, Brookhill Sheffield S37H, United Kingdom.
Nicholas J
NJ
Warren
Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.|Wyss Institute for Biologically Inspired Engineering, Harvard University , 60 Oxford Street, Cambridge, Massachusetts 02138, United States.
Bobak
B
Mosadegh
Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.|Wyss Institute for Biologically Inspired Engineering, Harvard University , 60 Oxford Street, Cambridge, Massachusetts 02138, United States.
Marym R
MR
Mohammady
Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.|Wyss Institute for Biologically Inspired Engineering, Harvard University , 60 Oxford Street, Cambridge, Massachusetts 02138, United States.
George M
GM
Whitesides
Department of Chemistry, University of Sheffield , Dainton Building, Brookhill Sheffield S37H, United Kingdom.
Steven P
SP
Armes
D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D045744:Cell Line, Tumor; D002470:Cell Survival; D003094:Collagen / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004338:Drug Combinations; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020100:Hydrogels / Q000737:chemistry* / Q000494:pharmacology; D007797:Laminin / Q000737:chemistry; D044367:Phase Transition; D011109:Polymethacrylic Acids / Q000737:chemistry* / Q000494:pharmacology; D011509:Proteoglycans / Q000737:chemistry; D013696:Temperature
D004220:Disulfides; D004338:Drug Combinations; D020100:Hydrogels; D007797:Laminin; D011109:Polymethacrylic Acids; D011509:Proteoglycans; C059485:matrigel; D049452:Green Fluorescent Proteins; D003094:Collagen