Motohiro Hirose

Novel patterned cell coculture utilizing thermally responsive grafted polymer surfaces

Here we demonstrate a novel cell coculture method without any apparent limitation in cell-type combinations that exploits thermally responsive polymer-grafted patterns to alter cell-cell and cell-surface interactions. Thermally responsive acrylamide polymer is first covalently patterned onto culture surfaces by masked electron beam irradiation. One cell type is then cultured to confluency at 37 degrees C. Reducing cell culture temperature below 32 degrees C selectively swells temperature sensitive polymer-grafted domains, detaching adherent cells only from these grafted patterns. Another cell type is then seeded over the same surface at 37 degrees C. These subsequently seeded cells adhere only to the now-exposed polymer-grafted domains. Initially seeded cells remaining adherent on nonpatterned surfaces and cells added in the second seeding are then cocultured at 37 degrees C in well-ordered patterns.

Two-dimensional cell sheet manipulation of heterotypically co-cultured lung cells utilizing temperature-responsive culture dishes results in long-term maintenance of differentiated epithelial cell functions

Here we report two-dimensional cell sheet manipulation (2D CSM) of heterotypically co-cultured lung cell sheets and the maintenance of differentiated phenotypes of lung epithelial cells over prolonged periods of up to 70 days. This was facilitated by poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture dishes. PIPAAm-grafted dishes are responsive to temperature changes and offer a unique surface on which cells adhere and multiply like on ordinary tissue culture dishes under the permissive temperature of 37 degrees C, but on lowering of temperature resulting in changes in hydration of the polymer the cells spontaneously detach from the surface without use of enzymes like trypsin which is the common procedure. It has been well documented that type II pneumocytes of the lung lose many of their special features rapidly in culture. The culture system detailed here comprises random co-culture of epithelial and mesenchymal cells of lung. The heterotypic cell culture system promotes cell-cell interactions maintaining a harmonized physiology. When this heterotypic monolayer on PIPAAm-grafted dishes was subjected to lower temperature of 20 degrees C and 2D CSM we were able to transfer the monolayer as a single contiguous sheet with cell-cell connections intact to other surfaces. This non-invasive transfer of cell sheet resulted in shrinkage of the monolayer, enabling the type II cells to regain their cuboidal morphology and specialized characters like Maclura pomifera lectin binding and surfactant protein A (SP-A) expression. The active dome formation also observed subsequent to transfer reaffirms the uniqueness of the culture conditions and 2D CSM in future for developing tissue like architecture in vitro.

Cell type specific recognition of the reconstituted aggregates from isolated type I collagen, type IV collagen, and type V collagen

The ultrastructure of animal tissues has a graded distribution that is reminiscent of the reconstituted aggregate structures from isolated type I collagen, type V collagen, and type IV collagen aligned in a gradient manner. We can assume that mesenchymal cells with different functions are also located in a graded way in accord with the graded distribution of collagen supramolecular aggregates. It was already known that proliferation, shape, migration, gene expression, and responses to growth factors of the cultured fibroblasts were greatly affected by the type I collagen fibrils. Other differentiated cells such as endothelial cells and smooth muscle cells are surrounded by type IV collagen and/or type V collagen more closely. The reconstituted type IV collagen aggregates in a gel form were tested as culture substrates for these differentiated cells. Of particular interest was the examination of myofibroblasts that are thought responsible for the pathogenesis of fibrotic or cirrhotic tissues. It was found that the type IV collagen gel appeared to restore the behaviors of smooth muscle-related cells including the myofibroblasts into the contractile stage. The whole cells cultured on the type IV collagen gel were totally quiescent and formed a multicellular network. The findings suggest that interrelationships in a graded way between differentiated cells and the specific collagen aggregates might be involved in the homeostasis of tissue structure and function.

Regulation of Phenotypes of Human Aorta Endothelial Cells and Smooth Muscle Cells in Culture by Type IV Collagen Aggregates

Publisher Summary This chapter discusses the regulation of phenotypes of human aorta endothelial cells and smooth muscle cells in culture by type IV collagen aggregates. Type IV collagen comprises the skeletal structure of basal lamina, which functions as a physical barrier for biological macromolecules and cells. Basal lamina affects the most fundamental cell functions, including cell proliferation and differentiation. Vascular endothelial cells and smooth muscle cells produce and deposit basal lamina components in extracellular surroundings. Reciprocal interactions of the cells with the basal lamina may result in a vicious or favorable spiral. The favorable spiral is a dynamic steady state of tissue structure and function. Incubation of the type IV collagen solution in neutral pH and physiological ionic strength causes two macroscopically different forms of type IV collagen aggregates to form, depending on the temperature of incubation. Proliferation and differentiation of human aorta endothelial cells and smooth muscle cells in culture are dependent on the aggregate forms or non gel and gel form as well as collagen types. Extracellular environments are one of the most fundamental elements for the maintenance or the restoration of human aorta endothelial tissues and smooth muscle tissues.