Application of a cell sheet–polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability

Abstract

We have succeeded in fabricating a cell sheet-polymer film complex involving a temperature-sensitive polymer that has enough mechanical strength that can be manipulated even by forceps. The polymer film can be removed by lowering the temperature after transplantation, demonstrating its potential use in regenerative medicine. Recently, tissue engineering involving cell sheets was developed, tissues being fabricated by layering of these cell sheets. This technique promises high density cell packing, which is important for native cell functions, and successful heart therapy using cardiac cell sheets has been reported. On the other hand, the fabrication of a large tissue using cell sheets is difficult because of fragility of the cell sheets. Here, we have developed a novel method in which cells are attached to a temperature-sensitive poly-N-isopropylacrylamide film mixed with laminin and collagen IV, and report that the cell sheet-polymer film complex can be manipulated with forceps. A cell sheet can be removed from the polymer film by lowering the temperature after the manipulation. We have utilized this technique for the primary myocardium and fabricated a physiologically active multi-layered cardiac cell sheet. By applying a micropattern to this polymer film, we have succeeded in making a skeletal muscle cell sheet in which myotubes are oriented in the desired direction. Overall, we showed that this method is useful for cell sheet manipulation, morphogenesis, and transplantation.

Characterization of Partially Lifted Cell Sheets

“…Color images available online at www.liebertpub.com/tea monolayer. 2,10,45,46 As far as we know, the partial-lift method represents the first technique for achieving this goal, as current work is primarily limited to simple, short-term manipulations. 21 We additionally demonstrate that since the lifted cells exhibit vastly different physiologies, in terms of total cellular content of actin and plakoglobin (and likely other proteins), prelifting the cells may offer an opportunity for the cells establish a new, different distribution of junctional proteins, before use.…”

“…For example, modified thermo-responsive polymeric surfaces [2][3][4] are expensive to purchase (or labor intensive to prepare) and can have a long harvesting time ( > 1 h). Another approach uses magnetic beads 49 ; however, this technique requires a peptide-conjugated magnetite cationic liposome coating, a magnetic field, and potential dose-dependent detrimental effects on cell proliferation and metabolism.…”

“…[2][3][4][5] In addition, cell sheet tissue engineering bears a striking resemblance to the embryonic cell sheet building machinery. In early development, embryonic morphogenesis results largely from deformation of analogs of cell sheets, via internally generated forces.…”

Industrial bioprocesses: Beyond routine applications of established methodologies

“…To begin the journal's sixth decade of publication, a novel ''solely industrial'' article was published in early 2009, entitled ''Application of a cell sheet-polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability'' (Fujita et al, 2009). This article described the development of a physiologically active, multi-layered, cardiac cell sheet for regenerative medicine applications.…”

Efeito da Laserfototerapia associada ou não à Vitamina C na indução de membranas celulares (cell sheets) de células-tronco da polpa dentária humana

“…Visando transpor a necessidade de utilização desse aparato, Teruno Okano e colaboradores (1993) desenvolveram uma placa de cultivo especial (UpCell dish) (CellSeed Inc., Shinjukuku, Tóquio, Japão), que possibilita a formação de uma membrana celular organizada capaz de soltar-se da placa praticamente intacta (Cell Sheet), sendo possível o transporte e implantação dessas células sem a necessidade de um scaffold mediador (Okano et al, 1993;1995;Yang et al,2005;Wang et al, 2014;Long et al, 2014 A engenharia tecidual mediada por MCs já tem sido aplicada em diversos tecidos / órgãos como córnea, miocárdio, fígado e tecidos periodontais (Nishida et al, 2004;Shimizu et al, 2006;Ohashi et al, 2007;Ding et al, 2010;Zhao et al, 2013).Também já foram desenvolvidas culturas em 3D, onde a sobreposição das monocamadas de MCs, do mesmo tipo celular ou não, possibilitou a formação de estruturas funcionais complexas, mimetizando, neste caso, o tecido cardíaco (Haraguchi et al, 2012). Dentro da Odontologia regenerativa, a Periodontia tem se destacado na aplicação das MCs, principalmente na tentativa de repor o tecido periodontal perdido ao redor de raízes dentárias (Fujita et al, 2009;Kelm;Fussenegge, 2010;Wei et al, 2012).…”