Method for manufacturing porous structure

Abstract

To stably produce a porous structure having a large number of pores with uniform pore diameters.SOLUTION: A method for manufacturing a porous structure is used, comprising the following steps (a) to (e): (a) forming a hydrogel; (b) freezing the hydrogel; (c) drying a freeze-drying apparatus; (d) placing the frozen hydrogel after step (b) in the freeze-drying apparatus previously dried in step (c); and (e) freeze-drying the hydrogel after step (d).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a porous structure. [Background technology]

[0002] In the fields of regenerative medicine and drug discovery screening, interest in tissue engineering is growing, and this is leading to an increased need for three-dimensional cell culture. Typically, three-dimensional cell cultures have the problem that oxygen and nutrients do not reach the center, making the cells in the center prone to death. One method known to solve this problem is to use a porous structure as a cell culture carrier. Non-Patent Document 1 reports a method for producing a porous structure by crosslinking gelatin using transglutaminase to form a gel, followed by freeze-drying. Patent Document 1 discloses a method for producing a porous structure by crosslinking gelatin chemically with carbodiimide or physically with UV curing to form a gel, followed by freeze-drying. Summary of the Invention [Problem to be solved by the invention]

[0003] Porous carriers for cell culture have been produced as described in Patent Document 1 and Non-Patent Document 1. However, there was a problem in that even if the techniques in Patent Document 1 and Non-Patent Document 1 were applied as they were, it was not always possible to stably obtain a porous structure with a uniform pore size and a large number of pores.

[0004] An object of the present invention is to provide a method for producing a porous structure that can stably produce a porous structure having a large number of pores with uniform pore diameters. [Means for solving the problem]

[0005] The present invention provides the following: <Method of manufacturing porous structure> A method for producing a porous structure, comprising the following steps (a) to (e): (a) forming a hydrogel; (b) freezing the hydrogel; (c) drying the freeze-drying device; (d) placing the frozen hydrogel after step (b) in the freeze-drying apparatus previously dried in step (c); and (e) freeze-drying the hydrogel after step (d). <Porous structure> A porous structure manufactured by a method for manufacturing a porous structure, comprising the following steps (a) to (e): (a) forming a hydrogel; (b) freezing the hydrogel; (c) drying the freeze-drying device; (d) placing the frozen hydrogel after step (b) in the freeze-drying apparatus previously dried in step (c); and (e) freeze-drying the hydrogel after step (d). [Effects of the Invention]

[0006] According to the present invention, it is possible to provide a method for producing a porous structure that can stably obtain a porous structure having a large number of pores with uniform pore diameters. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a flow diagram of an example of a method for producing a porous structure of the present invention. [Figure 2] 1 shows images of the porous structure of Example 1 observed by a scanning electron microscope (SEM). A is a photograph of the top surface of the entire gel, and B is a 5x magnified photograph of A. [Figure 3] 1 shows SEM images of the porous structure of Example 2. A is a photograph of the top surface of the entire gel, and B is a 5x magnified photograph of A. [Figure 4] 1 shows SEM images of the porous structure of Comparative Example 1. A is a photograph of the top surface of the entire gel, and B is a 5x enlarged photograph of A. In the figure, the circled areas are pores. [Figure 5] 1 shows SEM images of the porous structure of Comparative Example 2. A is a photograph of the top surface of the entire gel, and B is a photograph of A enlarged five times. DETAILED DESCRIPTION OF THE INVENTION

[0008] 1. Structure and Definitions In this specification, the expression "A to B" (A and B are both numerical values) means "A or more and B or less" unless otherwise specified.

[0009] As used herein, the term "porous structure" refers to a three-dimensional structure having one or more pores. Furthermore, as used herein, the term "porous structure" refers to a three-dimensional structure having a porous structure. The use of the porous structure of this embodiment is not particularly limited, but it is typically used as a cell culture carrier.

[0010] As used herein, the term "gel" refers to a polymer having a three-dimensional network structure or a swollen body thereof, typically a hydrogel. As used herein, the term "hydrogel" refers to a gel that retains a large amount of water within the spaces of its network structure, also known as a "water-containing gel."

[0011] In this specification, "room temperature air" refers to air that is distinct from the air inside a sealed container under a certain environment, such as a freeze-drying apparatus, refrigerator, or freezer, and refers to the air inside a room, such as a laboratory or workroom, which may contain outside air, i.e., air from outside, and is at room temperature. In this specification, "room temperature" refers to 15°C to 35°C.

[0012] As used herein, unless otherwise specified, "refrigeration" refers to lowering the temperature of an object to a refrigeration temperature. Here, "refrigeration temperature" refers to a temperature above 0°C and below 10°C. As used herein, "freezing" refers to lowering the temperature of a liquid or gel-like object to a freezing temperature to solidify it, unless otherwise specified. Here, "freezing temperature" refers to a temperature below -15°C. As used herein, "cooling" refers to lowering the temperature of an object relatively, regardless of the target temperature.

[0013] In this specification, unless otherwise specified, "humidity" refers to relative humidity (RH%), i.e., water vapor amount / saturated water vapor amount x 100 (%). Unless otherwise specified, humidity refers to humidity measured with a digital hygrometer (e.g., CUSTOM CTH-202).

[0014] 2. Method for manufacturing porous structure A first embodiment of the present invention is a method for producing a porous structure. The method of this embodiment is characterized by including the following steps (a) to (e): (a) forming a hydrogel; (b) freezing the hydrogel; (c) drying the freeze-drying device; (d) placing the frozen hydrogel after step (b) in the freeze-drying apparatus previously dried in step (c); and (e) freeze-drying the hydrogel after step (d).

[0015] The method of this embodiment includes at least step (c), and therefore, compared to conventional manufacturing methods, it is possible to stably manufacture a porous structure having a large number of pores with uniform pore diameters.

[0016] The present inventors have discovered that, when manufacturing a porous structure, the uniformity of pore size and the number of pores in the resulting porous structure vary depending on the manufacturing date. The inventors speculated that this is due to differences in indoor air conditions such as temperature and humidity, and more specifically, differences in conditions inside the freeze-drying chamber (hereinafter simply referred to as "chamber") immediately after the start of freeze-drying. They then discovered that the quality (uniformity of pore size and number of pores) of the resulting porous structure can be maintained at a certain level or higher by thoroughly drying the chamber until just before placing the hydrogel before the start of freeze-drying.

[0017] During the production of porous structures, it was hypothesized that residual moisture remaining in the freeze-drying chamber after operation until the chamber is sufficiently dried comes into contact with the hydrogel placed inside, melting the frozen hydrogel surface, leading to nonuniform pore size and a reduction in the number of pores in the porous structure. Porous structures are obtained by freezing the water contained in the hydrogel to form fine ice particles within the hydrogel, which are then sublimated under reduced pressure to form numerous pores. Therefore, if the frozen surface of the hydrogel melts due to moisture, the ice particles are lost, preventing the formation of pores of sufficient quality and number. By thoroughly drying the chamber from the initial stage of placing the hydrogel in the freeze-drying chamber, porous structures can be produced under consistent conditions, with little influence from the humidity and temperature of the air outside the chamber.

[0018] 1 is a flow diagram of each step in an example of the method of this embodiment. In the figure, steps enclosed in a solid-lined square are essential steps, and steps enclosed in a dashed-lined square are optional steps. Below, the method of this embodiment will be described step by step.

[0019] 2-1 Step (a) Hydrogel formation step The method of this embodiment includes a step of forming a hydrogel. The material of the hydrogel is not particularly limited and can be appropriately selected depending on the purpose. When used for cell culture, a biodegradable material and a material to which cells can adhere are preferable. For example, protein gels such as cross-linked polyethylene glycol, Matrigel™ basement membrane matrix, gelatin, collagen, and fibrin gel, polysaccharide gels such as hyaluronic acid and agar, and polymer gels such as acrylic acid polymers can be used. The method of forming the hydrogel can be appropriately selected from known methods depending on the material and intended use. In many cases, hydrogel formation involves preparing a sol-like material solution, subjecting it to enzyme treatment (e.g., transglutaminase), chemical cross-linking, UV treatment, etc., and then cooling it to refrigeration temperatures.

[0020] In the method of this embodiment, the hydrogel formed in this step may be immediately subjected to the subsequent hydrogel freezing step (b), but it is preferable to carry out the following steps (a-1) and (a-2) before the freezing step.

[0021] 2-2 Step (a-1) Hydrogel Cooling Step The method of this embodiment may include a step of cooling the formed hydrogel to refrigeration temperature. This cooling step may be performed in conjunction with the formation of the hydrogel in step (a) when the formation is performed at refrigeration temperature. The means for cooling to refrigeration temperature is not particularly limited, and any known cooling means such as a refrigerator, water cooling, ice cooling, or a Peltier element may be used. The cooling time is not particularly limited as long as the temperature of the entire hydrogel is reduced to the refrigeration temperature, but can be, for example, 5 minutes to 4 hours, 30 minutes to 3 hours, or 1 hour to 2 hours.

[0022] 2-3 Step (a-2) Hydrogel Exposure Step The method of this embodiment may include a step of exposing the hydrogel cooled to a refrigeration temperature to room temperature air. When this exposing step is performed, the hydrogel must be cooled to the refrigeration temperature. There are no particular limitations on the method for exposing the hydrogel, as long as it can be brought into contact with room temperature air after being cooled to the refrigeration temperature.

[0023] The present inventors have found that when a hydrogel at refrigerated temperature is brought into contact with air having a temperature and humidity normally present in a laboratory, workroom, etc., fine condensation occurs on the surface of the hydrogel, and this condensed moisture can form a large number of pores with relatively large diameters on the surface of the porous structure in the subsequent freeze-drying step. This is thought to be because the water droplets formed by condensation on the hydrogel surface turn into ice particles in the subsequent freezing step, and these ice particles further sublimate in the subsequent freeze-drying step.

[0024] The temperature of the room temperature air is room temperature, i.e., 15°C to 35°C, preferably 18°C ​​to 32°C, and more preferably 20°C to 30°C. The humidity of the room temperature air is not particularly limited as long as it is within the range of normal indoor humidity, and can be appropriately adjusted by air conditioning, etc. The humidity of the room temperature air can be, for example, 25% to 85%, 30% to 80%, 40% to 75%, or 50% to 70%.

[0025] The exposure time of the hydrogel in room temperature air can be 15 seconds to 5 minutes or 20 seconds to 1 minute. Typically, it is about 30 seconds. The "exposure time" here refers to the time it takes for a hydrogel to be taken out of a refrigerator, for example, into room temperature air, and then cooled again. If the exposure time is less than 15 seconds, sufficient condensation may not occur. Furthermore, if the exposure time is longer than 5 minutes, the condensed water droplets may aggregate to form larger droplets, resulting in a decrease in their number. Setting the exposure time within the above range allows for the desired number and size of water droplets to form on the hydrogel surface.

[0026] 2-4 Step (a-3) Hydrogel re-cooling step After the exposure time, the hydrogel that has formed water droplets may be immediately subjected to the subsequent freezing step, or may be cooled again to refrigeration temperatures before the freezing step. Cooling again to refrigeration temperatures has the advantage that the water droplets that condense on the hydrogel surface grow using the moisture in the vicinity, forming larger ice particles in the subsequent freezing step. If the hydrogel is immediately subjected to the subsequent freezing step without being cooled again to refrigeration temperatures, the cooling rate is too fast, resulting in little growth of the ice particles and the formation of pores with relatively small diameters. When cooling again to refrigeration temperatures, the cooling time is not particularly limited, but can be, for example, 5 minutes to 1 hour or 15 minutes to 45 minutes.

[0027] 2-5 Step (b) Hydrogel freezing step The method of this embodiment includes a step of freezing the hydrogel. Specifically, it includes a step of cooling the hydrogel to a freezing temperature to solidify it. As described above, the freezing temperature is not particularly limited as long as it is −15° C. or lower, and can be, for example, −80° C. to −15° C. Typically, it can be about −20° C.

[0028] The freezing time is not particularly limited as long as it is a time that allows the entire hydrogel to be frozen, but it can be, for example, 5 minutes to 4 hours, 30 minutes to 3 hours, or 1 hour to 2 hours. The "freezing time" here refers to the time from when the hydrogel is placed under freezing temperature conditions, for example, in a freezer, until it is removed.

[0029] 2-6 Step (c) Pre-drying step of freeze-drying device The method of this embodiment includes a step of drying the interior of the freeze-drying apparatus prior to the subsequent hydrogel placement step. This step may be performed in parallel with step (b) above. Any known freeze-drying apparatus may be used. For example, a freeze-drying apparatus may be used in combination with a freeze-drying machine (e.g., FDS-1000 (Tokyo Rikakikai Co., Ltd.)) as the main body and a dry chamber (e.g., DRS-1 (Tokyo Rikakikai Co., Ltd.)) as the freeze-drying chamber. These may also be used in combination with an existing vacuum pump (e.g., GLS-051XF (Tokyo Rikakikai Co., Ltd.)).

[0030] The pre-drying of the freeze-drying apparatus is performed by operating the freeze-drying apparatus in an empty state before placing the hydrogel inside the chamber. Specifically, this is performed by reducing the air pressure inside the chamber. The air pressure inside the chamber is not particularly limited as long as it allows drying inside the chamber, but can be, for example, 100 Pa or less, 50 Pa or less, or 10 Pa or less.

[0031] If the freeze dryer is equipped with a cold trap, the cold trap may be operated simultaneously. Here, the cold trap refers to a mechanism that cools, freezes, and traps the moisture (water vapor) discharged from the chamber. The temperature of the cold trap is not particularly limited as long as it can rapidly cool and freeze the moisture, but can be, for example, −20°C or below, −50°C or below, −70°C or below, or −80°C or below.

[0032] The pre-drying time is not particularly limited and can be changed appropriately depending on the type of freeze-dryer, the size of the freeze-drying chamber, and the internal air pressure and temperature. For example, in the case of the freeze-drying machine and freeze-drying chamber described above, the pre-drying time can be 30 minutes to 2 hours or 45 minutes to 90 minutes. The pre-drying time here refers to the time from the start of operation, including the time required for depressurization and cooling from normal pressure and temperature to the target air pressure and temperature, until the depressurization is stopped.

[0033] After drying, the pressure inside the freeze-drying device must be returned to normal (atmospheric) pressure in order to install the hydrogel in the subsequent stage. The pressure inside the device can be returned to normal by stopping the decompression of the freeze-drying device and purging the gas into the device.

[0034] 2-7 Step (d) Hydrogel installation in freeze-drying device The method of this embodiment includes a step of placing the hydrogel frozen in step (b) in a freeze-drying apparatus that has been pre-dried in step (c). Although the interior of the freeze-drying apparatus after pre-drying is sufficiently dry, the structure of a typical freeze-drying apparatus requires the freeze-drying chamber to be opened to place the hydrogel, making it difficult to completely prevent the intrusion of outside air. Therefore, when placing the hydrogel, it is preferable to minimize the open area of ​​the freeze-drying chamber and shorten the open time. For example, since the above-mentioned dry chamber has an open area at the bottom, after removing the dry chamber from the freeze-drying machine, it can be immediately placed on a workbench so that the bottom is closed, and then the bottom can be partially opened, allowing the hydrogel to be placed on the internal shelf. The method of placing the hydrogel in the freeze-drying apparatus can be modified as appropriate depending on the type and shape of the freeze-drying apparatus used. However, regardless of the type, the open time is preferably within 3 minutes, 2 minutes, 1 minute, or 30 seconds.

[0035] Immediately after the hydrogel is placed, i.e., immediately before the subsequent freeze-drying step, the relative humidity in the freeze-drying apparatus, particularly the freeze-drying chamber, is preferably 50% or less, particularly 30% or less, 20% or less, or 10% or less. The temperature in the freeze-drying chamber is preferably 15°C to 35°C, particularly 20°C to 30°C.

[0036] 2-8 Process (e) Freeze-drying process The method of this embodiment includes a step (d) of freeze-drying the hydrogel placed in the freeze-drying apparatus. The freeze-drying conditions are not particularly limited and can be any known freeze-drying conditions for hydrogels. The air pressure inside the chamber is not particularly limited as long as the chamber can be dried, but can be, for example, 100 Pa or less, 50 Pa or less, or 10 Pa or less. If the freeze-drying apparatus has a cold trap, the cold trap may be operated simultaneously. In this case, the temperature of the cold trap can be, for example, -20°C or less, -50°C or less, -70°C or less, or -80°C or less. The freeze-drying time is not particularly limited as long as it allows the hydrogel to be sufficiently dried, and can be varied appropriately depending on the type of hydrogel, etc., but can be, for example, 1 to 120 hours, and may be, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 15 hours or more, 18 hours or more, 21 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, 48 hours or more, 60 hours or more, or 72 hours or more, or, for example, 120 hours or less, 108 hours or less, 96 hours or less, 84 hours or less, 72 hours or less, 66 hours or less, 60 hours or less, 54 hours or less, 48 ​​hours or less, 42 hours or less, 36 hours or less, 30 hours or less, 24 hours or less, 18 hours or less, 12 hours or less, 9 hours or less, 6 hours or less, or 3 hours or less. The freeze-drying time here refers to the time until the pressure reduction is stopped, including the time for reducing the pressure and cooling from normal pressure and temperature at the start of operation to the target pressure and temperature.

[0037] In the porous structure obtained through the freeze-drying step, the average pore size is preferably 5 μm to 1000 μm, particularly 5 μm to 100 μm, 5 μm to 30 μm, or 10 μm to 20 μm. The coefficient of variation of the pore size (standard deviation / average value×100(%)) is preferably 150% or less, 100% or less, 80% or less, or 70% or less. Furthermore, although it depends on the average pore size, the number of pores is preferably 10 / mm 2 ~100 pieces / mm 2 , especially 20 pieces / mm 2 ~50 pieces / mm2 It is preferable that:

[0038] 3 Porous structure A second embodiment of the present invention is a porous structure. The porous structure of this embodiment is characterized by being manufactured by any of the methods described in Section "2. Method for manufacturing a porous structure." Unless otherwise specified or unless there is any particular contradiction, the materials (raw materials), configuration, etc. of the porous structure of this embodiment are the same as those described in Section "2. Method for manufacturing a porous structure."

[0039] The porous structure of this embodiment is not particularly limited, but is preferably one that is suitable as a carrier for cell culture, and is particularly preferably a porous structure having a large number of pores with uniform pore diameters.

[0040] In the porous structure of this embodiment, the average pore size is preferably 5 μm to 1000 μm, particularly 5 μm to 100 μm, 5 μm to 30 μm, or 10 μm to 20 μm. The coefficient of variation of the pore size (standard deviation / average value×100(%)) is preferably 150% or less, 100% or less, 80% or less, or 70% or less. Furthermore, although it depends on the average pore size, the number of pores is preferably 10 / mm 2 ~100 pieces / mm 2 , especially 20 pieces / mm 2 ~50 pieces / mm 2 It is preferable that: [Example]

[0041] The present invention will be specifically described below by showing examples, but the following description is not intended to limit the scope of the present invention to the scope of the examples.

[0042] [Example 1] (1) Preparation of hydrogel Gelatin (Gelatin LET-NP250, Nitta Gelatin Co., Ltd.) was dissolved in PBS(-) (Dulbecco's Phosphate Buffered Saline: DPBS(1x), Gibco) to prepare a 1.0 (w / v)% gelatin solution. 1 μL of 0.5 mg / mL laminin solution (iMatrix-511, Nippi Co., Ltd.) was added to 100 μL of the gelatin solution and mixed using a vortex mixer (Corning LSE). 1 μL of transglutaminase solution (Bacterial transglutaminase, Zedira Co., Ltd.) adjusted to 25 U / mL in PBS(-) was then added and mixed using a vortex mixer. The solutions were added and mixed in a 1.5 mL microtube in an aluminum block incubator (Petit Cool MiniT-C, Wakembee Tech Co., Ltd.) set at 37°C.

[0043] A 1 mm thick silicone rubber sheet (Dow, SYLGARD 184) with a 6 mm diameter hole was attached to a 35 mm dish, and 30 μL of a gelatin solution containing laminin and transglutaminase was dropped into the hole. The dish containing the gelatin solution was then left to stand in a refrigerator (Panasonic, MPR-312DCN) at 4°C for 2 hours to obtain a hydrogel.

[0044] (2) Exposing the hydrogel to room temperature air and freezing it The dish containing the gelatin gel was removed from the refrigerator and placed in a room-temperature cooling device (TAISEI Peltier Temperature Controller Model TA-PB-1). The time from the refrigerator to the cooling device was approximately 30 seconds. The cooling device was then turned on, and the dish was cooled from room temperature to 4°C, after which it was held for 30 minutes. It was then cooled to -20°C and held for 2 hours until frozen. The actual room temperature had a minimum of 20.5°C and a maximum of 27.5°C. The relative humidity of the air at the room temperature was 31.3% minimum and 81.1% maximum.

[0045] (3) Pre-drying of the freeze-drying device A freeze dryer (FDS-1000, manufactured by Tokyo Rikakikai Co., Ltd.) equipped with a dry chamber (DRS-1, manufactured by Tokyo Rikakikai Co., Ltd.) was operated to reduce the pressure to 100 Pa or less, and simultaneously, the cold trap inside the freeze dryer body was cooled to -80°C, freezing the vapor and drying the inside of the dry chamber. The drying time, including the time from starting the device to reducing the pressure to 100 Pa or less and cooling the cold trap to -80°C, was approximately 1 hour. With the cold trap inside the freeze dryer still cooled, the pressure reduction was stopped, and the pressure inside the dry chamber and freeze dryer was returned from a vacuum state to normal pressure (atmospheric pressure).

[0046] (4) Freeze drying The dish containing the frozen hydrogel was removed from the cooling device and placed in the dry chamber of the freeze-dryer that had been pre-dried. The dish was placed as follows: The dry chamber was removed and placed on a workbench with the open side of the dry chamber facing downwards. The dish containing the hydrogel was placed in the dry chamber, taking care to prevent outside air from entering the dry chamber as much as possible, and the dry chamber was then returned to the freeze-dryer. This operation took about 1 minute. After the dry chamber was returned, the freeze-dryer was depressurized again, and freeze-drying was carried out for 4 hours at a reduced pressure of 10 Pa or less, yielding a porous structure.

[0047] (5) Observation of the porous structure surface using a scanning electron microscope (SEM) The surface of the obtained porous structure was observed using an SEM (Keyence Corporation, Model No. VHX-D510). Figure 2 shows the SEM observation images. A is a photograph of the top surface of the entire hydrogel, and B is a 5x enlarged photograph of A. The SEM observation images were analyzed, and the number of pores on the 6 mm diameter surface and the average pore diameter were counted and measured.

[0048] [Example 2] (1) Preparation of hydrogel Gelatin (Gelatin LET-NP250, Nitta Gelatin Co., Ltd.) was dissolved in PBS(-) (Dulbecco's Phosphate Buffered Saline: DPBS(1x), Gibco) to prepare a 1.0 (w / v)% gelatin solution. 1 μL of 0.5 mg / mL laminin solution (iMatrix-511, Nippi Co., Ltd.) was added to 100 μL of the gelatin solution and mixed using a vortex mixer (Corning LSE). 1 μL of transglutaminase solution (Bacterial transglutaminase, Zedira Co., Ltd.) adjusted to 25 U / mL in PBS(-) was then added and mixed using a vortex mixer. The solutions were added and mixed in a 1.5 mL microtube in an aluminum block incubator (Petit Cool MiniT-C, Wakembee Tech Co., Ltd.) set at 37°C.

[0049] A 1 mm thick silicone rubber sheet (Dow, SYLGARD 184) with a 6 mm diameter hole was attached to a 35 mm dish, and 30 μL of a gelatin solution containing laminin and transglutaminase was dropped into the hole. The dish containing the gelatin solution was then left to stand in a refrigerator (Panasonic, MPR-312DCN) at 4°C for 2 hours to obtain a hydrogel.

[0050] (2) Freezing of the hydrogel The dish containing the gelatin gel was removed from the refrigerator and immediately transferred to a freezer at -20°C, where it was left to freeze for 2 hours.

[0051] (3) Pre-drying of the freeze-drying device A freeze dryer (FDS-1000, manufactured by Tokyo Rikakikai Co., Ltd.) equipped with a dry chamber (DRS-1, manufactured by Tokyo Rikakikai Co., Ltd.) was operated to reduce the pressure to 100 Pa or less, and simultaneously, the cold trap inside the freeze dryer body was cooled to -80°C, freezing the vapor and drying the inside of the dry chamber. The drying time, including the time from starting the device to reducing the pressure to 100 Pa or less and cooling the cold trap to -80°C, was approximately 1 hour. With the cold trap inside the freeze dryer still cooled, the pressure reduction was stopped, and the pressure inside the dry chamber and freeze dryer was returned from a vacuum state to normal pressure (atmospheric pressure).

[0052] (4) Freeze drying The dish containing the frozen hydrogel was removed from the cooling device and placed in the dry chamber of the freeze-dryer that had been pre-dried. The dish was placed as follows: The dry chamber was removed and placed on a workbench with the open side of the dry chamber facing downwards. The dish containing the hydrogel was placed in the dry chamber, taking care to prevent outside air from entering the dry chamber as much as possible, and the dry chamber was then returned to the freeze-dryer. This operation took about 1 minute. After the dry chamber was returned, the freeze-dryer was depressurized again, and freeze-drying was carried out for 4 hours at a reduced pressure of 10 Pa or less, yielding a porous structure.

[0053] (5) Observation of the porous structure surface using SEM The surface of the obtained porous structure was observed using an SEM (Keyence Corporation, Model No. VHX-D510). Figure 3 shows the SEM observation images. A is a photograph of the top surface of the entire hydrogel, and B is a 5x enlarged photograph of A. The SEM observation images were analyzed, and the number of pores on the 6 mm diameter surface and the average pore diameter were counted and measured.

[0054] [Comparative Example 1] (1) Preparation of hydrogel Gelatin (Gelatin LET-NP250, Nitta Gelatin Co., Ltd.) was dissolved in PBS(-) (Dulbecco's Phosphate Buffered Saline: DPBS(1x), Gibco) to prepare a 1.0 (w / v)% gelatin solution. 1 μL of 0.5 mg / mL laminin solution (iMatrix-511, Nippi Co., Ltd.) was added to 100 μL of the gelatin solution and mixed using a vortex mixer (Corning LSE). 1 μL of transglutaminase solution (Bacterial transglutaminase, Zedira Co., Ltd.) adjusted to 25 U / mL in PBS(-) was then added and mixed using a vortex mixer. The solutions were added and mixed in a 1.5 mL microtube in an aluminum block incubator (Petit Cool MiniT-C, Wakembee Tech Co., Ltd.) set at 37°C.

[0055] A 1 mm thick silicone rubber sheet (Dow, SYLGARD 184) with a 6 mm diameter hole was attached to a 35 mm dish, and 30 μL of a gelatin solution containing laminin and transglutaminase was dropped into the hole. The dish containing the gelatin solution was then left to stand in a refrigerator (Panasonic, MPR-312DCN) at 4°C for 2 hours to obtain a hydrogel.

[0056] (2) Freezing of the hydrogel The dish containing the gelatin gel was removed from the refrigerator and immediately transferred to a freezer at -20°C, where it was left to freeze for 2 hours.

[0057] (3) Freeze drying The dish containing the frozen hydrogel was removed from the refrigerator and placed in the dry chamber of a freeze-drying apparatus (Tokyo Rikakikai Co., Ltd., FDS-1000) equipped with a dry chamber (Tokyo Rikakikai Co., Ltd., DRS-1) without prior drying. After the dry chamber was returned, the freeze-drying machine was operated and freeze-drying was carried out under conditions of -80°C and 10 Pa or less for 4 hours to obtain a porous structure.

[0058] (4) Observation of the porous structure surface using SEM The surface of the obtained porous structure was observed using an SEM (Keyence Corporation, model number VHX-D510). Figure 4 shows the SEM observation images. A is a photograph of the top surface of the entire hydrogel, and B is a photograph of A enlarged 5 times. In the image, the circled areas are pores. Almost no pores were observed within one field of view. The SEM observation image was analyzed, and the number of pores on the 6 mm diameter surface and the average pore diameter were counted and measured.

[0059] Comparative Example 2 (1) Preparation of hydrogel Gelatin (Gelatin LET-NP250, Nitta Gelatin Co., Ltd.) was dissolved in PBS(-) (Dulbecco's Phosphate Buffered Saline: DPBS(1x), Gibco) to prepare a 1.0 (w / v)% gelatin solution. 1 μL of 0.5 mg / mL laminin solution (iMatrix-511, Nippi Co., Ltd.) was added to 100 μL of the gelatin solution and mixed using a vortex mixer (Corning LSE). 1 μL of transglutaminase solution (Bacterial transglutaminase, Zedira Co., Ltd.) adjusted to 25 U / mL in PBS(-) was then added and mixed using a vortex mixer. The solutions were added and mixed in a 1.5 mL microtube in an aluminum block incubator (Petit Cool MiniT-C, Wakembee Tech Co., Ltd.) set at 37°C.

[0060] A 1 mm thick silicone rubber sheet (Dow, SYLGARD 184) with a 6 mm diameter hole was attached to a 35 mm dish, and 30 μL of a gelatin solution containing laminin and transglutaminase was dropped into the hole. The dish containing the gelatin solution was then left to stand in a refrigerator (Panasonic, MPR-312DCN) at 4°C for 2 hours to obtain a hydrogel.

[0061] (2) Exposing the hydrogel to room temperature air and freezing it The dish containing the gelatin gel was removed from the refrigerator and placed in a room-temperature cooling device (TAISEI Peltier Temperature Controller Model TA-PB-1). The time from the refrigerator to the cooling device was approximately 30 seconds. The cooling device was then turned on, and the dish was cooled from room temperature to 4°C, after which it was held for 30 minutes. It was then cooled to -20°C and held for 2 hours until frozen. The actual room temperature had a minimum of 20.5°C and a maximum of 27.5°C. The relative humidity of the air at the room temperature was 31.3% minimum and 81.1% maximum.

[0062] (3) Freeze drying The dish containing the frozen hydrogel was removed from the refrigerator and placed in the dry chamber of a freeze-drying apparatus (Tokyo Rikakikai Co., Ltd., FDS-1000) equipped with a dry chamber (Tokyo Rikakikai Co., Ltd., DRS-1) without prior drying. After the dry chamber was returned, the freeze-drying machine was operated and freeze-drying was carried out under conditions of -80°C and 10 Pa or less for 4 hours to obtain a porous structure.

[0063] (4) Observation of the porous structure surface using SEM The surface of the obtained porous structure was observed using an SEM (Keyence Corporation, Model No. VHX-D510). Figure 5 shows the SEM observation images. A is a photograph of the top surface of the entire hydrogel, and B is a photograph of A enlarged 5 times. Almost no pores were observed within the field of view. The SEM observation images were analyzed, and the number of pores on the 6 mm diameter surface and the average pore diameter were counted and measured.

[0064] [result] Table 1 shows the appearance evaluation, number of pores, and average pore diameter of each of the porous structures of Examples 1 and 2 and Comparative Examples 1 and 2. The criteria for the appearance evaluation of the SEM observation images are as follows. ◎: Many pores of uniform diameter are observed (number of pores: more than 1000); ○: Many pores are observed (number of pores: more than 500 and less than 1000); △: Multiple pores with clear diameters are observed (number of pores: over 100 and up to 500); ×: Almost no pores with a clear pore size are observed.

[0065] [Table 1]

[0066] The results in Table 1 confirmed that pre-drying the gel in the freeze-drying apparatus before freeze-drying resulted in the formation of numerous pores in the resulting porous structure. In contrast, few pores were observed in the porous structure produced without pre-drying. This is thought to be because the lack of pre-drying of the gel before freeze-drying caused a portion of the frozen surface of the gel to melt due to the influence of moisture remaining in the chamber immediately after starting operation. Meanwhile, the porous structure (Example 1) exposed to room temperature air before freezing the gel showed a greater number of uniform pores than the porous structure (Example 2) that was not exposed to room temperature air. This is thought to be because numerous tiny droplets formed on the gel surface during exposure to room temperature air, which then froze to form tiny ice particles that remained on the surface and evaporated, forming pores in those areas.

[0067] As described above, when freeze-drying a gel to produce a porous structure, it was demonstrated that pre-drying in a freeze-dryer can stably produce a porous structure with a large number of pores with uniform pore sizes. Furthermore, it was demonstrated that cooling the gel to refrigeration temperature and exposing it to room temperature air before freezing can produce a porous structure with a larger number of pores with more uniform pore sizes.

[0068] The present invention includes the following embodiments. [1] A method for producing a porous structure, comprising the following steps (a) to (e): (a) forming a hydrogel; (b) freezing the hydrogel; (c) drying the freeze-drying device; (d) placing the frozen hydrogel after step (b) in the freeze-drying apparatus previously dried in step (c); and (e) freeze-drying the hydrogel after step (d). [2] The method according to [1], further comprising the following steps (a-1) and (a-2) after step (a) and before step (b): (a-1) cooling the hydrogel to a refrigeration temperature; and (a-2) A step of exposing the hydrogel cooled in step (a-1) to room temperature air. [3] The method according to [1] or [2], wherein the step (c) is a step of drying under a reduced pressure of 100 Pa or less in a freeze-drying apparatus. [4] The method according to any one of [1] to [3], wherein the relative humidity inside the freeze-drying apparatus immediately after installation in step (d) is 50% or less. [5] The method according to any one of [1] to [4], wherein the average pore size of the porous structure is 5 μm to 1000 μm. [6] A porous structure manufactured by the method described in any one of [1] to [5]. [Prior art documents] [Patent documents]

[0069] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-21984 [Non-patent literature]

[0070] [Non-Patent Document 1] Long et al., Preparation and characteristics of gelatin sponges crosslinked by microbial transglutaminase, PeerJ 5: e3665 (2017)

Claims

1. A method for producing a porous structure, comprising the following steps (a) to (e): (a) forming a hydrogel; (b) freezing the hydrogel; (c) drying the freeze-drying device; (d) placing the frozen hydrogel after step (b) in the freeze-drying apparatus previously dried in step (c); and (e) freeze-drying the hydrogel after step (d).

2. The method according to claim 1, further comprising the following steps (a-1) and (a-2) after step (a) and before step (b): (a-1) cooling the hydrogel to a refrigeration temperature; and (a-2) A step of exposing the hydrogel cooled in step (a-1) to the outside air at room temperature.

3. The method according to claim 1, wherein the step (c) is a step of drying the freeze-drying apparatus under a reduced pressure of 100 Pa or less.

4. 2. The method according to claim 1, wherein the relative humidity in the freeze-drying apparatus immediately after installation in step (d) is 50% or less.

5. The method of claim 1, wherein the average pore size of the porous structure is from 5 μm to 1000 μm.

6. A porous structure produced by the method according to any one of claims 1 to 5.

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