Designing a hepatocellular microenvironment with protein microarraying and poly(ethylene glycol) photolithography.
In this study, robotic protein printing was employed as a method for designing a cellular microenvironment. Protein printing proved to be an effective strategy for creating micropatterned co-cultures of primary rat hepatocytes and 3T3 fibroblasts. Collagen spots (ca. 170 microm in diameter) were printed onto amino-silane- and glutaraldehyde-modified glass slides. Groups of 15-20 hepatocytes attached to collagen regions in a highly selective manner forming cell clusters corresponding in size to the printed collagen domains. Fibroblasts, seeded onto the same surface, adhered and spread around arrays of hepatocyte islands creating a heterotypic environment. The co-cultured hepatocytes produced and maintained high levels of liver-specific biomarkers, albumin and urea, over the course of 2 weeks. In addition, protein printing was combined with poly(ethylene glycol) photolithography to define intercellular contacts within the clusters of hepatocytes residing on individual collagen islands. Glass slides, treated with 3-acryloxypropyl trichlorosilane and imprinted with 170 m diameter collagen spots, were micropatterned with a high-density array of 30 microm x 30 microm poly(ethylene glycol) (PEG) wells. As a result, discrete groups of ca. 9 PEG microwells became functionalized with the cell-adhesive ligand. When exposed to micropatterned surfaces, hepatocytes interacted exclusively with collagen-modified regions, attaching and becoming confined at a single-cell level within the hydrogel wells. Micropatterning strategies proposed here will lead to greater insights into hepatocellular behavior and will benefit the fields of hepatic tissue engineering and liver biology.
Revzin
Alexander
A
Rajagopalan
Padmavathy
P
Tilles
Arno W
AW
Berthiaume
François
F
Yarmush
Martin L
ML
Toner
Mehmet
M
D016475:3T3 Cells; D000818:Animals; D018920:Coculture Techniques; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D011092:Polyethylene Glycols; D040081:Protein Array Analysis / Q000379:methods*; D012371:Robotics / Q000379:methods
D011092:Polyethylene Glycols