title: 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.
abstract: 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.
authors.affiliation: Institute of Biomedical Engineering, Tokyo Women's Medical University, Japan.
authors.forename: Maya A
authors.initials: MA
authors.lastname: Nandkumar
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamato
authors.forename: Ai
authors.initials: A
authors.lastname: Kushida
authors.forename: Chie
authors.initials: C
authors.lastname: Konno
authors.forename: Motohiro
authors.initials: M
authors.lastname: Hirose
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Teruo
authors.initials: T
authors.lastname: Okano
mesh_terms: D000180:Acrylic Resins; D000199:Actins / Q000378:metabolism; D000818:Animals; D001672:Biocompatible Materials; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D007150:Immunohistochemistry; D007633:Keratins / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D008168:Lung / Q000166:cytology* / Q000378:metabolism; D008422:Materials Testing; D010641:Phenotype; D037121:Plant Lectins; D011510:Proteolipids / Q000378:metabolism; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000378:metabolism; D051381:Rats; D013696:Temperature; D014746:Vimentin / Q000378:metabolism
chemical_list: D000180:Acrylic Resins; D000199:Actins; D001672:Biocompatible Materials; D037102:Lectins; D037121:Plant Lectins; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D014746:Vimentin; C052970:poly-N-isopropylacrylamide; C034551:maclurin; D007633:Keratins