A novel method to prepare size‐regulated spheroids composed of human dermal fibroblasts

Abstract

A simple method to prepare size-regulated spheroids has been successfully developed by combining a temperature responsive polymer, poly-N-isopropyl-acrylamide (PNIPAAm), conjugated with collagen and ultraviolet (UV) irradiation with photomasks. The coating layer composed of PNIPAAm conjugated with collagen functions as a cell substratum at 37 degrees C, then when lowering the temperature of culture medium the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature LCST; about 30 degrees C, but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. On the other hand, UV effectively immobilized collagen in the coating layer because UV generates crosslinkages in collagen molecules. Crosslinkages were quantitatively introduced by controlling the energy of UV-irradiation thus the ability of human dermal fibroblasts to attach to and detach from the surface was tightly controlled. When the collagen content in the coating layer was 9 microg/cm(2) (collagen ratio, 4.5%), UV-irradiation energy of 2000 J/m(2) was suitable to obtain 100% of the attachability and detachability. However, the cells did not attach to the nonirradiated surface at this collagen content because insufficient collagen was immobilized. Using photomakes to apply UV-irradiation, it was possible to obtain cell-adhesive areas(irradiated areas) and nonadhesive areas (nonirradiated areas) on the same surface. Consequently, spheroids of any size and in any number from one dish were prepared. The viability of cells in spheroids 350 microm in diameter was maintained at a high level for 28 days; however, viability of spheroids 800 microm in diameter rapidly decreased for 2 days. The size was very important to maintain the viability. This novel method is useful to develop size-regulated spheroids for different applications; for example, in toxicology tests.