JPWO2020027575A5 - - Google Patents

Description

One embodiment of the present invention comprises: (a) a first cell on a first substrate that has a melting point or is converted from hydrophobic to hydrophilic at a temperature anywhere from 0°C to 30°C; (b) forming a second cell layer on a second substrate that is enzymatically degraded; (c) separating said first cell layer and said second cell layer; (d) providing a temperature below the melting point of the first substrate or a temperature at which the first substrate is converted to hydrophilic to selectively remove the first substrate; and (e) contacting the second substrate with a solution containing an enzyme to selectively remove the second substrate.

One embodiment of the present invention comprises: (a) a first cell on a first substrate that has a melting point or is converted from hydrophobic to hydrophilic at a temperature anywhere from 0°C to 30°C; forming a layer;

(d) selectively removing the first substrate by providing a temperature below the melting point of the first substrate or at which the first substrate is converted to hydrophilic;

The method for producing a multilayered cell sheet according to the present invention has the advantage of being able to produce a cell sheet in which multiple cell layers are laminated by a simple method. The method for producing a multilayer cell sheet includes a first substrate that has a melting point at a temperature between 0° C. and 30° C. or that is converted from hydrophobic to hydrophilic, and a first substrate that is decomposed by an enzyme. After stacking cell layers using two substrates, either substrate can be selectively removed. This can be used to overlay additional cell layers on top of the cell layers exposed by the selectively removed substrate. In addition, since the cell layer is laminated on the non-removed base material, it can be easily transferred, and after the transfer, the remaining base material can be selectively removed to allow multi-layered cell layer transplantation. be.

According to one embodiment of the present invention, steps (d) and (e) are performed sequentially, and between steps (d) and (e), (d′) the exposed first cell layer and an additional first cell layer provided on an additional first substrate at a temperature below the melting point of the first substrate or the first substrate converts to hydrophilic and selectively removing said additional first substrate. Specifically, the laminated structure of "first base material/first cell layer/second cell layer/second base material" formed by the step (c) includes steps (d) and ( The step d′) may form a laminated structure of “additional first cell layer/first cell layer/second cell layer/second substrate”.

According to one embodiment of the present invention, steps (a) and (c) are carried out in an atmosphere of 35°C to 40°C, specifically 36°C to 38°C, more specifically 36°C. It may be performed in an atmosphere of ~37°C. This is because the first substrate has a melting point at a temperature of any point from 0° C. to 30° C. or has a property of being converted from hydrophobic to hydrophilic, so the temperature range (that is, This is because the first base material maintains a solid phase form or hydrophobicity in an atmosphere of 35° C. to 40° C., and the cells are actively activated to promote the formation of a cell layer. Similarly, since the additional first substrate in step (d') also has the same properties as the first substrate, the process of forming an additional first cell layer and stacking the additional first cell layer may be performed in an atmosphere of 35°C to 40°C. Furthermore, in step (b), an atmosphere of 35° C. to 40° C., specifically an atmosphere of 36° C. to 38° C., more specifically an atmosphere of 36° C. to 37° C. is used to promote the formation of the cell layer. may be performed in

According to one embodiment of the present invention, the polyphosphazene-based hydrogel may comprise a temperature-sensitive polyphosphazene-based compound and have a melting point adjusted to between about 4°C and about 10°C. For example, temperature-sensitive and crosslinkable phosphazene-based hydrogels disclosed in Korean Patent Publication No. 10-2017-0061530, ionic groups capable of controlling the decomposition rate disclosed in Korean Patent Publication No. 10-2014-0016521 may be included in the polyphosphazene-based hydrogel of the present invention, such as the biodegradable temperature-sensitive polyphosphazene-based hydrogel disclosed in Korean Patent Publication No. 10-2007-0076386.

According to one embodiment of the present invention, the Pluronic-based hydrogel may have a melting point temperature adjusted to about 0° C. to 30° C. using a Pluronic polymer. Any polymer having a polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO) structure (PEO-PPO-PEO) may be used as the pluronic polymer. For example, F38, F68, F77, F98, F108, F127 derivatives starting with F, L31, L42, L43, L44, L62, L72, L101 derivatives starting with L, and P75, P103, P104 derivatives starting with P etc. (all product names) may be included. More specifically, F68 with a molecular weight of about 8,700 daltons and F127 with a molecular weight of about 12,600 daltons, which are approved by the US Food and Drug Administration (FDA), are used among the pluronic polymers. good too.

However, the first base material is not limited to polyphosphazene-based hydrogels and pluronic-based hydrogels, and any hydrogel having a melting point at a temperature of 30° C. or lower can be used as the first base material. and/or may be applied as an additional first substrate.

According to one embodiment of the present invention, step (d) includes adding the first or lowering the ambient temperature to a temperature below the melting point of said first substrate or a temperature at which said first substrate is converted to hydrophilic. Specifically, the process of selectively removing the first base material in step (d) includes the "first base material/first cell layer/second cell layer" formed by step (c). The laminated structure of "/second substrate" is immersed in a solution having a temperature below the melting point of the first substrate or a temperature at which the first substrate is converted to hydrophilic, and the first substrate It may be one that selectively removes the substrate. In addition, the process of selectively removing the first base material in step (d) is performed by removing the "first base material/first cell layer/second cell layer/second cell layer" formed by step (c). The ambient temperature (ambient temperature) of the laminated structure of the "substrate" is lowered to a temperature below the melting point of the first substrate or to a temperature at which the first substrate is converted to hydrophilic, and the first substrate may be selectively removed. At this time, the solution having a temperature below the melting point of the first base material may be physiological saline having a temperature of about 4°C to about 30°C. Also, the ambient temperature (ambient temperature) may be from about 4°C to about 30°C. Similarly, the step of selectively removing the additional first substrate in step (d') comprises immersing the laminated structure in a saline solution having a temperature of about 4°C to about 30°C to selectively Removing the additional first substrate or lowering the ambient temperature (ambient temperature) to a temperature below the melting point of the first substrate or at which said first substrate is converted to hydrophilic. may If the solution has a temperature below the melting point of the first substrate or additional first substrate, or if the ambient temperature is less than 20° C. and the cell layer of the laminated structure may be damaged, poly(N -isopropylacrylamide) substrates or substrates surface treated with poly(N-isopropylacrylamide) can be used to selectively remove said first substrate at 20°C to 30°C.

Claims (10)

(a) forming a first cell layer on a first substrate that has a melting point or is converted from hydrophobic to hydrophilic at a temperature anywhere from 0° C. to 30° C.;
(b) forming a second cell layer on a second substrate that is enzymatically degraded;
(c) contacting the first cell layer and the second cell layer;
(d) selectively removing the first substrate by providing a temperature below the melting point of the first substrate or at which the first substrate is converted to hydrophilic;
(d′) after contacting the exposed first cell layer with an additional first cell layer provided on an additional first substrate, below the melting point of the additional first substrate or a temperature at which the additional first substrate is converted to hydrophilic and selectively removing the additional first substrate;
(e) selectively removing the second substrate by contacting the second substrate with a solution containing an enzyme ;
(d) step, (d') step and (e) step are performed sequentially,
(d') The method for producing a multilayered cell sheet, wherein step (d') is repeated twice or more to form a plurality of additional first cell layers on the first cell layer . The step (d) includes contacting the first substrate with a solution having a temperature below the melting point of the first substrate or a temperature at which the first substrate is converted to hydrophilicity, or 2. The method for producing a multilayer cell sheet according to claim 1, wherein the temperature is lowered to a temperature below the melting point of the first substrate or a temperature at which the first substrate is converted to hydrophilicity. The multilayer according to claim 1, wherein the first cell layer and the second cell layer are formed by culturing on the first substrate and the second substrate, respectively. A method for producing a cell sheet. The method for producing a multilayer cell sheet according to claim 1, wherein steps (a) and (c) are performed in an atmosphere of 35°C to 40°C. The first base material comprises at least one of polyphosphazene-based hydrogel, pluronic-based hydrogel and poly(N-isopropylacrylamide) ,
The pluronic hydrogel according to claim 1, which is a material that utilizes a polymer having a polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO) structure (PEO-PPO-PEO) . A method for producing a multilayer cell sheet. 2. The method for producing a multilayer cell sheet according to claim 1, wherein the first base material is surface-treated with poly(N-isopropylacrylamide). 2. The method for producing a multilayer cell sheet according to claim 1, wherein the second base material is a hydrogel base material containing carboxymethylcellulose or alginate. [8] The method of claim 7 , wherein the carboxymethylcellulose or alginate is conjugated with tyramine. 2. The method for producing a multilayer cell sheet according to claim 1, wherein the enzyme is carboxymethylcellulose-degrading enzyme or alginate-degrading enzyme. The first cell layer and the second cell layer contain mesenchymal stem cells (MSCs), myocyte precursor cells, myocytes, and fibroblasts, respectively. , chondrocytes, endothelial cells, epithelial cells, embryonic stem cells (ESCs), hematopoietic stem cells, anchorage-dependent cell precursors 2. The multilayered cell sheet according to claim 1, comprising cells selected from the group consisting of cell precursors, induced pluripotent stem cells (iPSCs), and cardiomyocytes. manufacturing method.