Cell sheets

Yang J, Yamato M, Nishida K, Ohki T, Kanzaki M, Sekine H, Shimizu T, Okano T (2006) Cell delivery in regenerative medicine the cell sheet engineering approach. J Control Release 116 193-203. 

Epithelial cells are cells covering the body surface and bounding cavities, e.g. the gut or kidney tubules. They are bound together laterally by tight junctions to form sheets of cells and their apical and basolateral surfaces differ in composition and are kept apart by the tight junctions. They have the ability to transport solutes across the cell sheet from the apical surface. Of course, in culture a single epithelial cell cannot exhibit these properties but as the cells divide they form stable clusters of tightly associated cells and monolayers of such cells do show polarity. This is most easily achieved in serum-free, hormonally defined medium ( 5.8) when differentiated... 

Remove the medium and wash the cell sheet twice with 4 ml of acetate buffer (0.1 M NaCl 0.05 M NaAc pH5.8). 

After 15 min at 37 °C remove the lysolecithin and wash the cell sheet twice with complete medium and finally return to the incubator with 4 ml complete medium (Croce et al., 1971 Keay et al., 1972). 

Harvest by shaking to dislodge the cell sheet into the medium. (If the cells do not come off the glass some versene may be added but NOT trypsin.)... 

The method depends on infecting a small number of cells in a confluent monolayer. The virus produced in the infected cells will move laterally to infect adjacent cells and various techniques are used to prevent further spreading. The degenerative effect on the cells spreads until a visible area of dead cells (a plaque) is apparent. Staining of the cell sheet makes the colourless plaque more easy to see. 

Figure 3 Photograph of the Cellmate robot built by The Technology Partnership, Cambridge, UK. This robot performs cell seeding in either large Tflasks or roller bottles, exchanges media, rinses cell sheets, purges gases, harvests cells by trypsinization or scraping, and supports transfections...

SaladikDT, Soler AP, Lewis SA, and Mullin JM.Cell Division Does Not Increase Transepithelial Permeability of LLC-PKl Cell Sheets. Experimental Cell Research 220 446-455,1995. 

Pour off spent medium into a discard bottle, then wash cell sheet with 20 ml of PBS and discard immediately. 

Add 10 ml of warm trypsin solution using a 10-ml plastic pipette and pipetter. Gently roll the solution over the cell sheet and pour off the trypsin into the discard bottle. 

Fig. 1. Plaque reduction assay of varicella-zoster virus. (A) Varicella-zoster virus plaques in Mewo cell sheet. The upper row has no drug present. Note toxicity at 100 xM for all compounds tested. (B) Efficacy of compounds A, B, and C. All have IC50 values of <1 pM.

Where a stromal surface meets the epithelium, the fibroblasts interact with epithelial cells to form a basal lamina. This thin, flexible layer of specialized extracellular matrix (40 to 120 nm thick) underlies all epithelial cell sheets or tubes and is detected by... 

Functional biomaterial surfaces have been created to change between being hydrophobic and hydrophilic, in response to external signals, such as differences in temperature, solvent environment, light, or electrical current. Temperature-sensitive poly(A/-isopropylacrylamide) (PNIPAm)-coated substrate is hydrophobic at a cell culture temperature of 37°C that favors cell adhesion and becomes hydrophilic at a lower temperature of 20°C, causing the detachment of cell sheets. These reversible responsive surfaces have been used to culture and harvest layers of endothelial, epithelial, lung, liver, cardiac, and kidney cells, and could eventually enable assembly of complex tissues and organs. 

Shimizu, T. Yamato, M. Kikuchi, A. Okano, T. Cell sheet engineering for myocardial tissue reconstruction. Biomaterials 2003, 24 (13), 2309-2316. 

Dang JM, Leong KW (2007) Myogenic induction of aligned mesenchymal stem cell sheets by culture on thermally responsive electrospun nanofibers. Adv Mater Deerfield 19( 19) 2775-2779... 

Titration ef viruses. HSV-1 was assayed by using monolayer cultures of BSC-1 cells grown in 6-well multidishes. The cells were plated 3x10 cells/well in MEM(E) with 10% FBS and 1.1 g/1 sodium bicarbonate. After 24 hours, the cell sheet was about 75% confluent and was inoculated with 0.2 ml of the virus suspension to be assayed and incubated for 1 hour to permit viral adsorption. The cell sheets were then overlaid with 3 ml medium containing 0.5% methocel and incubated for another two days. 

Harvesting the hRPE membrane from donated eyes of premature newborns (gestational age approximately 20 weeks or more) is followed by dissociation and growth in culture until confluence of a monolayer cell sheet is reached in the culture flask. 

Ide T, Nishida K, Yamato M et al (2006) Structural characterization of bioengineered human comeal endothelial cell sheets fabricated on temperature-responsive culture dishes. Biomaterials 27 607-614... 

Kanzaki M, Yamato M, Hatakeyama H et al (2006) Tissue engineered epithelial cell sheets for the creation of a bioartificial trachea. Tissue Eng 12 1275-1283 Kiick KL (2008) Peptide- and protein-mediated assembly of heparinized hydrogels. Soft Matter 4 29-37... 

Kraehenbuehl TP, Zammaretti P, Van der Vlies AJ et al (2008) Three-dimensional extracellular matrix-directed cardioprogenitor differentiation systematic modulation of a synthetic cell-responsive PEG-hydrogel. Biomaterials 29 2757-2766 Kwon OH, Kikuchi A, Yamato M et al (2000) Rapid cell sheet detachment from poly (N-isopropylacrylamide)-grafted porous cell culture membranes. J Biomed Mater Res 50 82-89... 

Kwon OH, Kikuchi A, Yamato M et al (2003) Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes. Biomaterials 24 1223-1232 Lai JY, Chen KH, Hsu WM et al (2006) Bioengineered human comeal endothelium for transplantation. Arch Ophthalmol 124 1441-1448... 

Nitschke M, Zschoche S, Baier A et al (2004) Low pressure plasma immobilization of thin hydrogel films on polymer surfaces. Surf Coat Technol 185 120-125 Nitschke M, Gramm S, Gotze T et al (2007a) Thermo-responsive poly(NiPAAm-co-DEGMA) substrates for gentle harvest of human comeal endothelial cell sheets. J Biomed Mater Res A 80 1003-1010... 

Nitschke M, Gotze T, Gramm S et al (2007b) Detachment of human endothelial cell sheets Irom thermo-responsive poly(NiPAAm-co-DEGMA) carriers. Express Polym Lett 1 660-666 Pan YV, Wesley RA, Luginbuhl R et al (2001) Plasma polymerized N-isopropylacrylamide synthesis and characterization of a smart thermally responsive coating. Biomacromolecules 2 32-36... 

Shimizu T, Yamato M, Isoi Y et al (2002) Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ Res 90 E40-E48... 

Tsuda Y, Kikuchi A, Yamato M et al (2005) The use of patterned dual thermoresponslve surfaces for the collective recovery as co-cultured cell sheets. Biomaterials 26 1885-1893 Tsuda Y, Shimizu T, Yamato M et al (2007) Cellular control of tissue architectures using a three-dimensional tissue fabrication technique. Biomaterials 28 4939 946 Van Tomme SR, Hennink WE (2007) Biodegradable dextran hydrogels for protein delivery applications. Expert Rev Med Devic 4 147-164... 

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