Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: the noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces.

A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive culture dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted dishes merely by reducing the temperature. However, during the early stage of culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other culture dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.

[1]  N. Papadopoulos,et al.  An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry. , 1994, Journal of immunological methods.

[2]  K. Henle,et al.  Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures. , 1990, Cryobiology.

[3]  Teruo Okano,et al.  Temperature dependence of swelling of crosslinked poly(N,N′-alkyl substituted acrylamides) in water , 1990 .

[4]  J. Leighton,et al.  A cell line derived from normal dog kidney (MDCK) exhibiting qualities of papillary adenocarcinoma and of renal tubular epithelium , 1970, Cancer.

[5]  J. Gallardo,et al.  The making of a tight junction , 1993, Journal of Cell Science.

[6]  M. Heskins,et al.  Solution Properties of Poly(N-isopropylacrylamide) , 1968 .

[7]  T. Okano,et al.  Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface. , 2000, Biomacromolecules.

[8]  J. T. Crawford,et al.  Apoptosis induced by cold shock in vitro is dependent on cell growth phase. , 1987, Biochemical and biophysical research communications.

[9]  M. Takeichi,et al.  Cadherin cell adhesion receptors as a morphogenetic regulator. , 1991, Science.

[10]  T. Okano,et al.  Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces. , 1995, Biomaterials.

[11]  T. Okano,et al.  Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets. , 2000, Journal of biomedical materials research.

[12]  A. Harris,et al.  Silicone rubber substrata: a new wrinkle in the study of cell locomotion. , 1980, Science.

[13]  H. Aberle,et al.  Cadherin‐catenin complex: Protein interactions and their implications for cadherin function , 1996, Journal of cellular biochemistry.

[14]  T. Okano,et al.  Two-dimensional manipulation of confluently cultured vascular endothelial cells using temperature-responsive poly(N-isopropylacrylamide)-grafted surfaces. , 1998, Journal of biomaterials science. Polymer edition.

[15]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[16]  H. Mannherz,et al.  Cold‐induced apoptosis in cultured hepatocytes and liver endothelial cells: mediation by reactive oxygen species , 1999, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  T. Okano,et al.  Signal transduction and cytoskeletal reorganization are required for cell detachment from cell culture surfaces grafted with a temperature-responsive polymer. , 1999, Journal of biomedical materials research.

[18]  T. Okano,et al.  Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces. , 1999, Journal of biomedical materials research.

[19]  T. Okano,et al.  A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). , 1993, Journal of biomedical materials research.

[20]  D E Ingber,et al.  Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis. , 1994, International review of cytology.

[21]  M. Yamato,et al.  Condensation of collagen fibrils to the direct vicinity of fibroblasts as a cause of gel contraction. , 1995, Journal of biochemistry.