JP2005210990A - Method for forming biological scaffold - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for forming a new biological scaffold, meeting requirements of rapid production, energy saving and a low price to overcome the defects of a conventional method. <P>SOLUTION: Different macromolecular materials or different kinds of immobilization agents according to the requirement of application are provided to carry out an immobilization step under a low-temperature condition. The method comprises a dissolving step, a temperature-regulating step, a freezing step and the immobilization step. The immobilization step further includes one of a solid-liquid exchanging step, an acid and alkali-neutralizing step, and a gelatinizing step. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for forming a biological substrate, and more particularly to a method for forming a biological substrate in which a fixing step is performed under low temperature conditions.

The tissue process is one of the areas that have been erected in recent years, and one of the prominent application methods is that a cell is implanted in a bio-degradable material, and the cell grows and functions. The tissue process has already attracted attention as a method for treating defects in organ function or tissue necrosis. The research on organizational processes can be broadly divided into three parts. That is, cells (cells), biocompatible materials (scaffolds), and signals that stimulate the cells to express specific actions (signals). Influence, as well as jointly regulate tissue differentiation and expression. Among these, the role that the biological substrate plays in the tissue process includes the following.
1. Cells or tissue initially cultured are transported to a specific part of the body to prevent the cultured cells or tissue from being destroyed by immune action or other physical actions in the body.
2. Newly created tissues or organs are provided with a solid configuration that meets the requirements, as well as supporting and protecting them.
3. Stimulate cells or tissues to express specific actions. For example, a substance capable of generating a signal is applied to the surface of a biological substrate to achieve a necessary effect.
Biological substrate production methods are generally divided into phase separation methods and non-phase separation methods. The former refers to the state of the polymer solution, and the position of the solution in the thermodynamic phase diagram is modified to enter the phase separation region to achieve the effect of producing pores. Includes thermally induced phase separation, non-solvent incorporation and modification of solution composition. Common non-phase separation process manufacturing methods include fine particle dissolution (addition of pore former), three-dimensional printing (3D printing), high pressure gas or supercritical fluid perforation (gas foaming) and sintering of semi-molten polymer granules. Includes sintering.

In general, the most common method for producing biological substrates by the above-described phase separation method is the freeze drying method, which is divided into two steps. That is, it is freezing and drying. First, the temperature of the blended polymer solution is lowered, and it is made a heterogeneous phase rather than the original homogeneous, and this situation is called phase separation. Is divided into a polymer poor phase and a polymer rich phase, the solvent is removed, pores are formed in the polymer poor phase, and pore walls are formed in the polymer rich phase. That is, when the polymer solution is frozen (phase-separated), a porous structure of the substrate is generated, and therefore the polymer-rich phase form is one of the factors determining the biological substrate structure. As described above, the reason why the freeze-drying method is widely applied to the production of tissue process substrates is that the system is vacuum-dried under low temperature conditions (below the solution melting point) and the solvent is frozen. Then, it is transformed directly into gas and removed. Thus, the original film structure is not affected. However, freeze drying has inherent limitations on conditions, which are as follows.
1. Solvent limitation: It is preferable to use a solvent with high volatility. Otherwise, the vapor pressure of the solvent will be lower than that at room temperature in low temperature conditions, and this will greatly reduce the difficulty of solvent removal. And the problem of solvent residue is likely to occur.
2. Energy resource consumption: The power consumption of refrigeration systems and vacuum pumps is very large, often a bottleneck for large-scale production.
3. Time consumption: Since solvent volatility decreases at low temperatures, the solvent must be removed over a relatively long period of time, and a high boiling solvent system guarantees complete removal of the solvent even in a matter of days Can not.
4). Equipment: Special refrigeration and drying equipment is required and the price is extremely high.

  In view of the above, it can be seen that it is necessary to develop a new biological substrate process to meet the demands for rapid production, energy saving and low cost, lower the production cost and increase the production capacity.

  In order to overcome the disadvantages of traditional methods, the present invention provides a new method of forming a biological substrate that meets the requirements of rapid manufacturing, energy saving and low cost.

  An object of the present invention is to perform a fixing process under low temperature conditions by providing different polymeric materials or different kinds of fixing agents according to the application needs. By removing the polymer poor phase or fixing the polymer rich phase under low temperature conditions, the porous structure of the polymer rich phase is maintained and thereby the biological substrate is formed.

  Another object of the present invention is to increase the type of solvent used so that, for example, a solvent having a high boiling point or a solvent that cannot be processed by a conventional freeze-drying method can be used. By increasing the selection of the solvent to be used, the selection of the polymer material also increases, thereby making it possible to produce biological substrates of different materials.

  Based on the above object, the present invention provides a method for forming a kind of biological substrate. The process steps are simple and do not require the use of special freeze drying equipment. Thus, the present invention is consistent with economic effects and industrial applicability. The above-described method for forming a biological substrate includes the following steps: a dissolution step, a temperature adjustment step, a freezing step, and a fixing step. This fixing step further includes any of the following steps: a solid-liquid exchange step, an acid-alkali neutralization step, and a gelation step.

The invention of claim 1 is a method of forming a biological substrate,
Providing polymer material and solution,
Dissolving the polymer material in the solution to form a solution having a specific viscosity;
A solid state structure including a polymer rich phase substance capable of forming a pore wall portion of a biological substrate by solidifying the solution by performing a freezing process at a specific temperature and a polymer poor phase substance capable of forming a pore of the biological substrate,
Providing a non-solvent and performing a temperature adjustment step, and setting the temperature of the non-solvent to the specific temperature of the freezing step;
A method for forming a biological base material is characterized in that a solid-liquid exchange step is performed, and the biological base material is formed by exchanging and substituting the non-solvent with a polymer poor phase substance.
According to a second aspect of the present invention, in the method for forming a biological substrate according to the first aspect, the freezing step includes a pre-drying step, and the surface of the solid state structure is provided in the pre-drying step at the specific temperature of the freezing step. It is a method for forming a biological substrate, characterized by vaporizing a molecular poor phase substance.
The invention according to claim 3 is the method for forming a biological substrate according to claim 1, wherein the non-solvent can maintain a liquid state at the specific temperature.
According to a fourth aspect of the present invention, in the method for forming a biological substrate according to the first aspect, the non-solvent contains an antifreezing agent, whereby the non-solvent can maintain a liquid state at the specific temperature. The feature is a method for forming a biological substrate.
The invention according to claim 5 is the method for forming a biological substrate according to claim 1, wherein the polymer material is polylactic acid (PLA) and a derivative thereof, the non-solvent is an ethanol aqueous solution, and the ethanol contained in the ethanol aqueous solution The concentration range is 60 wt% to 100 wt%, which is a method for forming a biological substrate.
The invention of claim 6 is the method for forming a biological substrate according to claim 1, wherein the polymer material is a lactic acid glycolic acid polymer (PLGA) and a derivative thereof, and the non-solvent is an ethanol aqueous solution. The ethanol concentration range included is 20 wt% to 50 wt%, which is a method for forming a biological substrate.
The invention of claim 7 is a method of forming a biological substrate,
Providing polymer material and solution,
Dissolving the polymer material in the solution to form a first solution having a specific viscosity;
A solid state structure including a polymer rich phase material capable of forming a pore wall portion of a biological substrate and a polymer poor phase material capable of forming a pore of the biological substrate by solidifying the first solution by performing a freezing process at a specific temperature. Providing a second solution and performing a temperature adjustment step, and setting the temperature of the second solution to the specific temperature in the freezing step,
A method for forming a biological base material is characterized in that an acid-alkali neutralization step is performed with the second solution to solidify a polymer poor phase to form a biological base material.
The invention according to claim 8 is the method for forming a biological substrate according to claim 7, wherein the freezing step includes a pre-drying step, and the surface of the solid state structure is provided at the specific temperature of the freezing step in the pre-drying step. It is a method for forming a biological substrate, characterized by vaporizing a molecular poor phase substance.
The invention according to claim 9 is the method for forming a biological substrate according to claim 7, wherein the second solution can maintain a liquid state at the specific temperature. .
According to a tenth aspect of the present invention, in the method for forming a biological substrate according to the seventh aspect, the second solution contains an antifreezing agent, whereby the second solution can maintain a liquid state at the specific temperature. This is a method for forming a biological substrate.
The invention of claim 11 is the method for forming a biological substrate according to claim 10, wherein the cryoprotectant contains an ethanol component, and the ethanol concentration range is 20 wt% to 70 wt% of the second solution. And a biological substrate forming method.
The invention of claim 12 is the method of forming a biological substrate according to claim 7, wherein the first solution is an acidic solution and the second solution is an alkaline solution. It's a way.
The invention according to claim 13 is the method for forming a biological substrate according to claim 12, wherein the polymer material is chitosan and the concentration range of the alkaline substance contained in the second solution is 0.5N to 3N. A method for forming a biological substrate, characterized in that
The invention according to claim 14 is the method for forming a biological substrate according to claim 7, wherein the first solution is an alkaline solution and the second solution is an acidic solution. It's a way.
The invention of claim 15 is a method of forming a biological substrate,
Providing polymer material and solution,
Dissolving the polymer material in the solution to form a solution having a specific viscosity;
A solid state structure including a polymer rich phase substance capable of forming a pore wall portion of a biological substrate by solidifying the solution by performing a freezing process at a specific temperature and a polymer poor phase substance capable of forming a pore of the biological substrate,
Providing a cross-linking agent and performing a temperature adjustment step to make the temperature of the cross-linking agent the specific temperature of the freezing step;
A method for forming a biological base material is characterized in that a gelling step is performed with the crosslinking agent to form a biological base material by gelling a polymer-rich phase substance.
According to a sixteenth aspect of the present invention, in the method for forming a biological substrate according to the fifteenth aspect, the freezing step includes a pre-drying step, and the surface of the solid state structure is provided in the pre-drying step at the specific temperature of the freezing step. It is a method for forming a biological substrate, characterized by vaporizing a molecular poor phase substance.
The invention according to claim 17 is the method for forming a biological substrate according to claim 15, characterized in that the crosslinking agent can maintain a liquid state at the specific temperature. .
The invention according to claim 18 is the method for forming a biological substrate according to claim 15, wherein the crosslinking agent contains an antifreezing agent, whereby the crosslinking agent can maintain a liquid state at the specific temperature. In this method, the biological substrate is formed.
The invention according to claim 19 is the method for forming a biological substrate according to claim 18, wherein the cryoprotectant contains an ethanol component, and the ethanol concentration range is 20 wt% to 70 wt% of the crosslinking agent. And a biological substrate forming method.
The invention according to claim 20 is the method for forming a biological substrate according to claim 15, wherein the polymer material is alginate and the crosslinking agent is a sodium chloride solution, and the chloride contained in the sodium chloride solution The biological substrate forming method is characterized in that the sodium concentration range is 1 wt% to 20 wt%.

  The present invention provides a method for forming a biological substrate, which provides different polymeric materials or different types of fixatives as needed for the application and performs the fixing step under low temperature conditions. By removing the polymer poor phase or fixing the polymer rich phase under low temperature conditions, the porous structure of the polymer rich phase during freezing is maintained, thereby forming a biological substrate. In the present invention, the type of the solvent to be used is not limited. For example, a solvent having a high boiling point can be used, or a solvent that cannot be treated by a conventional freeze-drying method can be used. By increasing the selection of the solvent to be used, the selection of the polymer material also increases, thereby making it possible to produce biological substrates of different materials.

  In summary, the method for forming a biological substrate of the present invention has a simple manufacturing process and does not require special freeze-drying equipment. Therefore, the present invention is consistent with economic effects and industrial applicability. The above-described method for forming a biological substrate includes the following steps: a dissolution step, a temperature adjustment step, a freezing step, and a fixing step. This fixing step further includes any of the following steps: a solid-liquid exchange step, an acid-alkali neutralization step, and a gelation step.

  The direction that the present invention considers here is a method of forming a biological substrate. In order to fully understand the present invention, a detailed manufacturing process or composition structure is submitted in the following description. However, the practice of the present invention is not limited to special subsections that are familiar to the technician of the biomatrix area. In addition, well-known compositions or manufacturing processes are not described in the subsections, preventing unnecessary limitations from being formed in the present invention. Preferred embodiments of the present invention are described below, but in addition to such detailed descriptions, the present invention can be widely implemented in other embodiments, and the scope of the present invention is not limited thereto. It shall conform to the description of the scope of claims.

  FIG. 1 shows a first embodiment of the present invention. First, a polymer material 110 is provided. The polymer material 110 includes polylactic acid (PLA), lactic acid glycolic acid polymer (Poly (lactide-co-glycolide); PLGA), polyhydroxyalkanoates (PHA), poly- 3-Hydroxybutyric acid (poly (3-hydroxybutyrate); PHB), polycaprolactone (PCL), and derivatives thereof. Polylactic acid (PLA) includes L-type polylactic acid (PLLA), R-type polylactic acid (PDLA) and amorphous polylactic acid (PDLLA), and the commonly used solvents for polylactic acid (PLA) are 1, It is 4-dioxane (1,4-dioxane). Solvents commonly used for lactic acid glycolic acid polymer (PLGA) are dimethyl sulfoxide (DMSO), ethyl acetate, 1,4-dioxane, dimethylformamide (DMF) or N -Methyl-pyrrolidone (NMP). Common solvents for polyhydroxyalkanoates (PHA) are chloroform, acetonitrile, benzene, cyclohexane, 1,4-dioxane, dimethyl sulfoxide (DMSO). ) Commonly used solvents for poly-3-hydroxybutyric acid (PHB) are chloroform, trichloroethylene, 222-trifluoroethanol, 222-trifluoroethanol, dimethylformamide (DMF), ethylacetoacetate and triolein ( triolein). A common solvent for polycaprolactone (PCL) is 1,4-dioxane, chloroform, dimethylformamide (DMF), or dimethyl sulfoxide (DMSO).

  Thereafter, in this embodiment, the polymer material 110 is transported into a mixing apparatus and the dissolution process 135 is performed to dissolve the polymer material 110 in the solution 120 containing the above-mentioned solvent, and the solution 140 having a specific viscosity is prepared. Form. The viscosity of the solution 140 decreases as the addition amount of the polymer material 110 decreases, and increases the pores of the biological substrate to be manufactured. When the polymer material 110 is polylactic acid (PLA) or a derivative thereof, that is, the weight content range thereof is 1 wt% to 15 wt% of the above-described solution 140, and a preferable weight content range is 3 wt% of the above-described solution 140. % To 10 wt%. When the polymer material 110 is a lactic acid glycolic acid polymer (PLGA) or a derivative thereof, the weight content range is 1 wt% to 25 wt% of the above-described solution 140, and the preferred weight content range is the above-described solution 140. 3 wt% to 10 wt%. Subsequently, a freezing step 145 is performed at a specific temperature to solidify the above-described solution 140 to form a solid state structure 150 including a polymer rich phase material and a polymer poor phase material. Among them, the polymer poor phase can form pore portions of the biological substrate, and the polymer poor phase can form pore wall portions of the biological substrate. In addition, the pores of the manufactured biological substrate are reduced by lowering the specific temperature. Next, the non-solvent 130 is provided and the temperature adjustment step 160 is performed to adjust the temperature of the non-solvent 130 to the specific temperature of the freezing step 145. Subsequently, a solid / liquid exchange step 155 is performed, and the above-described non-solvent 130 is used to exchange and replace the polymer poor-phase substance, thereby forming a biological substrate 165.

  In this example, before the freezing step 145, a defoaming step is further performed to remove bubbles contained in the solution 140 described above. In addition, the above-described freezing step 145 further includes a pre-drying step, and this pre-drying step vaporizes the polymer poor-phase substance that the surface of the solid-state structure 150 has at the above-described specific temperature. Further, during the temperature adjustment step 160, the non-solvent 130 described above can maintain a liquid state even at a specific temperature, or the non-solvent 130 further contains an antifreezing agent, and the non-solvent 130 can maintain a liquid state even at a specific temperature. Is done. When the above-described polymer material 110 is polylactic acid (PLA) and its derivatives, the non-solvent 130 is an ethanol aqueous solution, and the ethanol concentration range contained in the ethanol aqueous solution is 60 wt% to 100 wt%, A preferred ethanol concentration range is 75 wt% to 85 wt%. When the polymer material 110 is a lactic acid glycolic acid polymer (PLGA) or a derivative thereof, the non-solvent 130 is an ethanol aqueous solution, and the ethanol concentration range contained in the ethanol aqueous solution is 20 wt% to 50 wt%. The preferred ethanol concentration range is 25 wt% to 35 wt%.

  FIG. 2 shows a second embodiment of the present invention. In this embodiment, first, a polymer material 210 is provided. Among them, the polymer material 210 further includes chitosan and its derivatives. Thereafter, the polymer material 210 is transported into the mixing device and the dissolution process 235 is performed to dissolve the polymer material 210 in the solution 220 containing the above-mentioned solvent, and the first solution 240 having a specific viscosity is formed. The viscosity of the first solution 240 decreases as the addition amount of the polymer material 210 decreases, increasing the pores of the biological substrate to be manufactured. When the polymer material 210 is chitosan or a derivative thereof, that is, the weight content range thereof is 0.5 wt% to 6 wt% of the first solution 240 described above, and a preferable weight content range is the first solution 240 described above. 1 wt% to 3 wt%. Subsequently, a freezing step 245 is performed at a specific temperature to solidify the first solution 240 to form a solid state structure 250 including a polymer rich phase material and a polymer poor phase material. Among them, the polymer poor phase can form pore portions of the biological substrate, and the polymer poor phase can form pore wall portions of the biological substrate. In addition, the pores of the manufactured biological substrate are reduced by lowering the specific temperature. Next, the second solution 230 is provided, and the temperature adjustment process 260 is performed to adjust the temperature of the second solution 230 to the specific temperature of the freezing process 245. Among them, the first solution 240 described above is an acidic solution, and the second solution is an alkaline solution. Alternatively, the first solution 240 described above is an alkaline solution, and the second solution 230 is an acidic solution. When the above-described polymer material 210 is chitosan or a derivative thereof, the first solution 240 is an acidic solution and the second solution 230 is an alkaline solution, and the concentration of the alkaline substance contained in the second solution 230 is included. The range is 0.5N to 3N. Subsequently, an acid-alkali neutralization step 255 is performed to solidify the polymer-rich phase with the above-described second solution 230 and thereby form the biological substrate 265.

  In this embodiment, before the freezing step 245, a defoaming step is further performed to remove bubbles contained in the first solution 240. In addition, the above-described freezing step 245 further includes a pre-drying step, and this pre-drying step vaporizes the polymer poor-phase substance that the surface of the solid-state structure 250 has at the above-described specific temperature. In addition, during the temperature adjustment process 260, the second solution 230 described above can maintain a liquid state even at a specific temperature, or the second solution 230 further contains an antifreezing agent and the second solution 230 maintains a liquid state even at a specific temperature. It is supposed to be possible. The antifreezing agent described above further includes an ethanol component, and the ethanol concentration range is 20 wt% to 70 wt% of the second solution 230.

  FIG. 3 shows a third embodiment of the present invention. In this embodiment, first, a polymer material 310 is provided. Of these, the polymer material 310 is chitosan, alginate, or a derivative thereof. Thereafter, the polymer material 310 is transported into the mixing apparatus and the dissolution process 335 is performed to dissolve the polymer material 310 in the solution 320 containing the above-mentioned solvent, and a solution 340 having a specific viscosity is formed. The viscosity of the solution 340 decreases as the amount of the polymer material 310 added decreases, thereby increasing the pores of the produced biological substrate. When the polymer material 310 is chitosan and its derivatives, that is, its weight content range is 0.5 wt% to 6 wt% of the above-mentioned solution 340, and a preferred weight content range is 1 wt% of the above-mentioned solution 340. 3 wt%. When the polymer material 310 is an alginate and a derivative thereof, that is, its weight content range is 0.5 wt% to 8 wt% of the above-described solution 340, and a preferable weight content range is 1 wt% of the above-mentioned solution 340. 4 wt%. Subsequently, a freezing step 345 is performed at a specific temperature to solidify the above-described solution 340 to form a solid state structure 350 including a polymer rich phase material and a polymer poor phase material. Among them, the polymer poor phase can form pore portions of the biological substrate, and the polymer poor phase can form pore wall portions of the biological substrate. In addition, the pores of the manufactured biological substrate are reduced by lowering the specific temperature. Next, the crosslinking agent 330 is provided and a temperature adjustment step 360 is performed to adjust the temperature of the crosslinking agent 330 to a specific temperature of the refrigeration step 345. When the above-described polymer material 310 is chitosan or a derivative thereof, the crosslinking agent 330 is any one of a sulfuric acid solution, an aldehyde acid solution, a dialdehyde acid solution, and a genipin solution. When the polymer material 310 is an alginate and a derivative thereof, the crosslinking agent 330 described above is a calcium chloride solution, and the concentration range of the calcium chloride contained in the calcium chloride solution is 1 wt% to 20 wt%. The Subsequently, the gelation step 355 is performed to gel the polymer-rich phase substance with the above-described crosslinking agent 330 and to form the biological substrate 365.

  In this embodiment, before the freezing step 345, a defoaming step is further performed to remove bubbles contained in the solution 340. In addition, the above-described freezing step 345 further includes a pre-drying step, and this pre-drying step vaporizes the polymer poor-phase substance that the surface of the solid state structure 350 has at the above-described specific temperature. Further, during the temperature adjustment step 360, the above-mentioned crosslinking agent 330 can maintain a liquid state even at a specific temperature, or further contains an antifreezing agent so that the crosslinking agent 330 can maintain a liquid state even at a specific temperature. The The antifreezing agent described above further includes an ethanol component, and the ethanol concentration range is 20 wt% to 70 wt% of the crosslinking agent 330.

  The embodiments described above do not limit the scope of the present invention, and any modification or alteration in detail that can be made based on these embodiments shall fall within the scope of the claims of the present invention.

It is a flowchart of the formation method of the biological base material provided with the solid-liquid exchange process of 1st Example of this invention. It is a flowchart of the formation method of the biological base material which provided the acid-alkali neutralization process of 2nd Example of this invention. It is a flowchart of the formation method of the biological base material which provided the gelatinization process of 3rd Example of this invention.

Explanation of symbols

110 Polymer material 120 Dissolving solution 130 Non-solvent 135 Dissolving step 140 Solution 145 Freezing step 150 Solid state structure 160 Temperature adjustment step 155 Solid-liquid exchange step 165 Biological substrate 210 Polymer material 220 Dissolving solution 230 Second solution 235 Dissolving step 240 First 1 solution 245 refrigeration process 250 solid state structure 260 temperature adjustment process 255 acid alkali neutralization process 265 biological substrate 310 polymer material 320 solution 330 crosslinking agent 335 dissolution process 340 solution 345 freezing process 350 solid state structure 360 temperature adjustment process 355 gel Conversion process 365 Biological substrate

Claims (20)

In the method for forming a biological substrate,
Providing polymer material and solution,
Dissolving the polymer material in the solution to form a solution having a specific viscosity;
A solid state structure including a polymer rich phase substance capable of forming a pore wall portion of a biological substrate by solidifying the solution by performing a freezing process at a specific temperature and a polymer poor phase substance capable of forming a pore of the biological substrate,
Providing a non-solvent and performing a temperature adjustment step, and setting the temperature of the non-solvent to the specific temperature of the freezing step;
A method for forming a biological substrate, comprising performing a solid-liquid exchange step, and exchanging and substituting with a polymer poor-phase substance with the non-solvent to form a biological substrate.   2. The method for forming a biological substrate according to claim 1, wherein the freezing step comprises a pre-drying step, and the pre-drying step evaporates the polymer poor phase substance which the surface of the solid structure has at the specific temperature of the freezing step. A method for forming a biological substrate, characterized in that   The method for forming a biological substrate according to claim 1, wherein the non-solvent is capable of maintaining a liquid state at the specific temperature.   The method for forming a biological substrate according to claim 1, wherein the non-solvent contains an antifreezing agent, whereby the non-solvent can maintain a liquid state at the specific temperature. Forming method.   The method for forming a biological substrate according to claim 1, wherein the polymer material is polylactic acid (PLA) and a derivative thereof, the non-solvent is an ethanol aqueous solution, and the ethanol concentration range contained in the ethanol aqueous solution is 60 wt% to 100 wt%. %. A method for forming a biological substrate, characterized in that   The method for forming a biological substrate according to claim 1, wherein the polymer material is a lactic acid glycolic acid polymer (PLGA) and a derivative thereof, the non-solvent is an ethanol aqueous solution, and the ethanol concentration range contained in the ethanol aqueous solution is 20 wt. % To 50 wt%, A method for forming a biological substrate. In the method for forming a biological substrate,
Providing polymer material and solution,
Dissolving the polymer material in the solution to form a first solution having a specific viscosity;
A solid state structure including a polymer rich phase material capable of forming a pore wall portion of a biological substrate and a polymer poor phase material capable of forming a pore of the biological substrate by solidifying the first solution by performing a freezing process at a specific temperature. Providing a second solution and performing a temperature adjustment step, and setting the temperature of the second solution to the specific temperature in the freezing step,
A method for forming a biological substrate, comprising performing an acid-alkali neutralization step with the second solution to solidify a polymer poor phase to form a biological substrate.   8. The method for forming a biological substrate according to claim 7, wherein the freezing step includes a pre-drying step, and the pre-drying step evaporates the polymer poor phase substance which the surface of the solid structure has at the specific temperature of the freezing step. A method for forming a biological substrate, characterized in that   8. The method for forming a biological substrate according to claim 7, wherein the second solution can maintain a liquid state at the specific temperature.   8. The method for forming a biological substrate according to claim 7, wherein the second solution contains an antifreezing agent, whereby the second solution can maintain a liquid state at the specific temperature. A method for forming a substrate.   The method for forming a biological substrate according to claim 10, wherein the cryoprotectant contains an ethanol component, and the ethanol concentration range thereof is 20 wt% to 70 wt% of the second solution. Forming method.   8. The method for forming a biological substrate according to claim 7, wherein the first solution is an acidic solution and the second solution is an alkaline solution.   The biological substrate forming method according to claim 12, wherein the polymer material is chitosan and the concentration range of the alkaline substance contained in the second solution is 0.5N to 3N. Material forming method.   8. The method for forming a biological substrate according to claim 7, wherein the first solution is an alkaline solution and the second solution is an acidic solution. In the method for forming a biological substrate,
Providing polymer material and solution,
Dissolving the polymer material in the solution to form a solution having a specific viscosity;
A solid state structure including a polymer rich phase substance capable of forming a pore wall portion of a biological substrate by solidifying the solution by performing a freezing process at a specific temperature and a polymer poor phase substance capable of forming a pore of the biological substrate,
Providing a cross-linking agent and performing a temperature adjustment step to make the temperature of the cross-linking agent the specific temperature of the freezing step;
A method for forming a biological substrate, comprising performing a gelation step with the crosslinking agent to form a biological substrate by gelling a polymer-rich phase substance.   16. The method for forming a biological substrate according to claim 15, wherein the freezing step comprises a pre-drying step, and the pre-drying step vaporizes the polymer poor phase substance that the surface of the solid structure has at the specific temperature of the freezing step. A method for forming a biological substrate, characterized in that   16. The method for forming a biological substrate according to claim 15, wherein the crosslinking agent can maintain a liquid state at the specific temperature.   The method for forming a biological substrate according to claim 15, wherein the crosslinking agent contains an antifreezing agent, whereby the crosslinking agent can maintain a liquid state at the specific temperature. A method for forming a biological substrate.   The method for forming a biological substrate according to claim 18, wherein the cryoprotectant contains an ethanol component, and the ethanol concentration range thereof is 20 wt% to 70 wt% of the crosslinking agent. Forming method. 16. The method for forming a biological substrate according to claim 15, wherein the polymer material is alginate and the crosslinking agent is a sodium chloride solution, and the concentration range of sodium chloride contained in the sodium chloride solution is 1 wt%. A method for forming a biological substrate, characterized in that it is 20 wt%.

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