Method for producing material for transplantation

Abstract

The purpose of the present invention is to provide a method for easily producing, as a material for transplantation, a structure in which tissue that is improved and suitable for practical use is constructed.　A method for producing a material for transplantation according to the present invention is characterized by comprising: an introduction step for introducing cells into a container; and an incubation step for incubating the introduced cells in the container, wherein the incubation step includes a first incubation step and a second incubation step, the first incubation step is for performing incubation under hypoxic and unpressurized conditions, and the second incubation step is for performing incubation under hypoxic and pressurized conditions. It is preferable to repeat the introduction step and the incubation step.

Description

Method for manufacturing transplant materials

[0001] This invention relates to a method for producing transplant materials using cells for use in living organisms.

[0002] In the field of regenerative medicine, cell sheets for transplantation have recently attracted attention. One reported method for manufacturing these cell sheets involves culturing cells in a single layer to prepare a single-layer sheet, and then stacking multiple single-layer sheets to form a three-dimensional laminate (Patent Document 1). However, with such a method, it is difficult to form laminates with complex shapes, for example.

[0003] On the other hand, in the medical field, there is a need for a simple method to manufacture implant materials that have complex shapes and require sufficient strength, such as vascular grafts.

[0004] International Publication No. 01 / 068799

[0005] Therefore, the present invention aims to provide a simple method for producing a structure suitable for practical application using cells.

[0006] The present invention provides a method for producing a transplantable material, comprising an introduction step of introducing cells into a container, and an incubation step of incubating the introduced cells in the container, wherein the incubation step comprises a first incubation step and a second incubation step, the first incubation step being a step of incubation under low-oxygen and non-pressurized conditions, and the second incubation step being a step of incubation under low-oxygen and pressurized conditions.

[0007] According to the manufacturing method of the present invention, by incubating cells introduced into the container under low-oxygen and non-pressurized conditions, and then further incubating them under low-oxygen and pressurized conditions, a structure suitable for practical use, including an extracellular matrix structure that contributes to strength, can be obtained. The formation of the extracellular matrix structure improves strength, for example, and since the structure can be obtained by the simple two-stage incubation process, the manufacturing method of the present invention is useful, for example, as a method for producing transplantable materials such as vascular grafts.

[0008] Figure 1A is a photograph showing the staining results of the cell stacking sheet (untreated with decellation) of Example 2. Figure 1B is a photograph showing the staining results of the cell stacking sheet (untreated with decellation) of Comparative Example 2. Figure 2 is a scanning electron microscope image of the decellation sheet of Example 1. Figure 3A is a photograph showing the staining results of the cell stacking sheet (untreated with decellation) of Example 2. Figure 3B is a photograph showing the staining results of the cell stacking sheet (untreated with decellation) of Comparative Example 2. Figure 4 is a graph showing the amount of DNA (ng) per dry weight (mg) of the decellation sheet of Example 1. Figure 5 is a graph showing the change in size of the decellation sheet of Example 1 after transplantation. Figure 6A is a photograph showing the state after transplantation of the decellation sheet of Example 1. Figure 6B is a photograph showing the state 12 weeks after transplantation of the decellation sheet of Example 1. Figure 7 is a photograph of the Elastica staining of excised blood vessels after transplantation of the decellation sheet of Example 1. Figure 8 is a graph showing the stress-strain curve of the decellatized sheet of Example 4.

[0009] The present invention includes, for example, the following embodiments: [1] A method for producing a transplantable material, comprising an introduction step of introducing cells into a container, and an incubation step of incubating the introduced cells in the container, wherein the incubation step comprises a first incubation step and a second incubation step, the first incubation step being a step of incubation under low-oxygen and non-pressurized conditions, and the second incubation step being a step of incubation under low-oxygen and pressurized conditions. [2] The method according to [1], wherein the introduction step is performed under atmospheric conditions. [3] The method according to [1] or [2], wherein in the first incubation step, the low-oxygen condition is an oxygen partial pressure of 10 mmHg or more to 160 mmHg. [4] The method according to any one of [1] to [3], wherein in the second incubation step, the low-oxygen condition is an oxygen partial pressure of 20 mmHg or more to less than 160 mmHg, and the pressurized condition is greater than atmospheric pressure and 200 kPa or less. [5] The manufacturing method according to any one of [1] to [4], wherein in the second incubation step, pressure is applied to the cells in the container, and the application of pressure is continuous or intermittent. [6] The manufacturing method according to [5], wherein in the second incubation step, pressure is applied as continuous application of periodic pressure. [7] The manufacturing method according to [6], wherein the periodic pressure has a frequency of 0.0005 to 0.01 Hz. [8] The manufacturing method according to [6] or [7], wherein in the periodic pressure, the high pressure value is 150 to 200 kPa and the low pressure value is greater than atmospheric pressure to 115 kPa. [9] The manufacturing method according to any one of [1] to [8], wherein between the introduction step and the first incubation step, there is further a standing step, and the standing step is a step of standing the cells introduced into the container under atmospheric conditions.

[10] The manufacturing method according to any one of [1] to [9], wherein in the introduction step, the cells are introduced into the container in which scaffolding material is arranged inside.

[11] The method of production according to any one of [1] to

[10] , wherein the cells are fibroblasts or smooth muscle cells.

[12] The method of production according to

[11] , wherein the cells are umbilical cord-derived fibroblasts or smooth muscle cells.

[13] A manufacturing method according to any one of [1] to

[12] , wherein the introduction step and the incubation step are repeated to form a laminate in which cells are stacked.

[14] A manufacturing method according to

[13] , wherein the number of layers of the laminate formed by repeating the introduction step and the incubation step is 2 to 100 layers.

[15] A manufacturing method according to

[13] or

[14] , further comprising a decellation step, wherein the decellation step is a step of removing cells from the laminate to prepare a decellation structure.

[16] A manufacturing method according to any one of [1] to

[15] , wherein the transplant material is an artificial blood vessel material.

[17] A transplant material obtained by a manufacturing method for a transplant material according to any one of [1] to

[16] .

[0010] In this specification, hypoxia means that the partial pressure of oxygen is lower than the partial pressure of oxygen in the atmosphere. Generally, the proportion of oxygen in the atmosphere is about 21%, and the partial pressure of oxygen in the atmosphere is 160 mmHg. Therefore, hypoxia is defined as a partial pressure of oxygen that is, for example, less than 160 mmHg.

[0011] When no artificial pressure is applied to an object, it is normally subjected to atmospheric pressure. In this specification, pressurization means applying a pressure exceeding atmospheric pressure to an object, and non-pressurization means not applying a pressure exceeding atmospheric pressure to an object. Non-pressurization is, for example, atmospheric pressure. Generally, atmospheric pressure is 101 kPa. Therefore, a pressure exceeding atmospheric pressure is, for example, a pressure exceeding 101 kPa.

[0012] In this specification, "extracellular matrix" refers to an insoluble substance present outside a cell. Examples of the extracellular matrix include collagen (e.g., collagen I, III, etc.), mucopolysaccharides (e.g., acidic mucopolysaccharides), elastic fibers, glycoproteins, etc., and these are collectively referred to as the extracellular matrix group. In this specification, "extracellular matrix structure" means a structure formed by the bonding of the extracellular matrix group to one another, and "extracellular matrix structure" means a structure having the extracellular matrix structure. In the present invention, cell adhesion means that adjacent cells adhere to each other via adhesion proteins.

[0013] [1. Method for Manufacturing Transplant Materials] The method for manufacturing transplant materials of the present invention, as described above, includes an introduction step of introducing cells into a container and an incubation step of incubating the introduced cells in the container, wherein the incubation step includes a first incubation step and a second incubation step, the first incubation step being a step of incubation under low-oxygen and non-pressurized conditions and the second incubation step being a step of incubation under low-oxygen and pressurized conditions.

[0014] (Cells) In the manufacturing method of the present invention, the type of cells is not particularly limited, and for example, cells that can become constituent cells of a material for transplantation into a living organism can be used. Examples of such cells include fibroblasts or smooth muscle cells, and the tissue from which the cells are derived is, for example, the umbilical cord, and a specific example is, for example, the umbilical artery, and the cells are, for example, umbilical cord-derived fibroblasts or umbilical cord-derived smooth muscle cells.

[0015] The cells are, for example, derived from an animal, and the animal may be human or a non-human animal. Non-human animals are, for example, mammals, and specific examples include mice, rats, hamsters, guinea pigs, rabbits, ferrets, cows, pigs, sheep, goats, dogs, cats, monkeys, chimpanzees, gorillas, horses, bears, rhinos, elephants, giraffes, okapis, pandas, dolphins, whales, orcas, etc. The animal from which the cells are derived may be the same as or different from the type of organism to which the transplant material is to be transplanted, but it is preferable that they be the same. For example, when the transplant material is used for human transplantation, human-derived cells are preferred.

[0016] The cells may be collected from a living organism, for example, or a cell line may be used. When collected from a living organism, for example, the cells may be those of the subject to whom the transplant material obtained by the present invention will be transplanted (autologous cells), or cells from a living organism other than the subject (allogeneic cells), or both may be used. The cells used in the manufacturing method of the present invention may be used after being grown by culture in advance, for example. When used after being grown by culture, for example, the cells may be cultured in a culture vessel, detached from the culture vessel by treatment with an enzyme such as trypsin, dispersed in a liquid medium, and recovered by centrifugation for use in the present invention. For example, monolayer culture is preferred for the culture.

[0017] In the manufacturing method of the present invention, for example, one type of cell may be used, or two or more types of cells may be used in combination. Furthermore, for example, different cells may be used in each of the introduction steps described later, or the ratio of two or more types of cells may be changed.

[0018] (Introduction Process) First, the introduction process is the process of introducing cells into a container. In this specification, the introduction of cells is also called cell seeding, and the introduction process is also called the cell seeding process.

[0019] The introduction process is carried out under atmospheric conditions. Therefore, in the introduction process, the pressure conditions inside the container at the time of cell introduction are, for example, unpressurized, i.e., atmospheric pressure, and the oxygen conditions are, for example, the partial pressure of oxygen in the atmosphere.

[0020] The temperature at which the cells are introduced is not particularly limited and can be appropriately determined depending on the cells used, for example. The temperature may be, for example, the temperature of the container, the temperature inside the container, the temperature of the clean bench, or the set temperature of the incubator that incubates the container. The temperature may be the same as or different from the incubation temperature in the incubation step described later or the standing temperature in any standing step, for example, 30-40°C, 35-38°C, etc. The temperature may also be, for example, room temperature, or a low temperature (around 15±3°C) that does not cause the cells to freeze. The temperature in the introduction step may be constant or varied, for example.

[0021] The cells may be introduced into the container as a cell suspension, for example, by first introducing the liquid medium into the container and then introducing the cells to form a cell suspension, or by introducing the cells into the container first and then introducing the liquid medium to form a cell suspension. It is more preferable to introduce the cell suspension into the container in the introduction step. The cells contained in the cell suspension will be referred to as a cell group below. The introduction step below will be illustrated using the introduction as a cell suspension, but is not limited to this and can be interpreted as referring to the other examples described above.

[0022] The type of liquid medium is not particularly limited, and any medium capable of maintaining cell survival is acceptable. Examples of the medium include water, buffer solution, physiological saline, buffered physiological saline, and culture medium, with culture medium being preferred. The medium can be appropriately set according to the type of cell. Examples of the buffer solution include phosphate buffer and HEPES (4-(2-HydroxyEthyl)-1-Piperadine Ethane Sulfonic Acid) buffer. Examples of the buffered physiological saline include phosphate-buffered physiological saline. Examples of the culture medium include DMEM medium and DMEM-F12 medium. The liquid medium may also have additives added. Examples of the additives include serum and growth factors such as Basic Fibroblast Growth Factor, and examples of serum include bovine serum such as fetal bovine serum.

[0023] The amount of cells to be introduced into the container is not particularly limited and can be determined, for example, based on the area of ​​the bottom surface inside the container. The bottom surface inside the container is, for example, the bottom surface inside the container in the direction of gravity when the container is in the state of the container during the incubation step or any standing step described later. The direction of gravity is also called, for example, the direction of cell stacking or thickness. In the introduction step, it is preferable to introduce an amount of cells such that they come into contact with each other in the direction perpendicular to the direction of gravity, and it is more preferable to introduce an amount greater than that.

[0024] The amount of cells to be introduced can be expressed, for example, as the number of cells per unit area of ​​the bottom surface (also called the cell density per unit area). The cell density can be appropriately determined, for example, depending on the type of cells, the shape of the container, etc. The cell density, i.e., 1 cm ２ Specific examples of the number of cells per unit are that the lower limit is, for example, 1,000 or more, 100,000 or more, or 250,000 or more, while the upper limit is not particularly restricted, for example, 10 million or less.

[0025] When introducing cells as a cell suspension into the container, the cell concentration of the cell suspension is not particularly limited, and is adjusted to a concentration that satisfies the number of cells per unit area as described above. Examples of cell concentrations in the cell suspension include 1 million cells / mL to 10 million cells / mL.

[0026] (Settling Step) In the manufacturing method of the present invention, the following incubation step may be started immediately after the introduction step, for example, or after a period of time. In the latter case, a further settling step may be optionally included between the introduction step and the incubation step. The settling step is a step of setting the cells introduced into the container under atmospheric conditions.

[0027] In the standing step, the cells can be allowed to settle (also called precipitation) by allowing the cell suspension to stand in the container. The standing step is preferably carried out until the cells settle to the bottom of the cell suspension, and more specifically, until the cells settle to the bottom surface inside the container. Furthermore, as will be described later, when the introduction step and the incubation step are repeated, for the second set and beyond, it is preferable to carry out the step until the cells that were introduced in the introduction step settle to the bottom of the cell suspension, and more specifically, until the cells settle to the surface of the layer after the previous incubation step.

[0028] The duration of the standing process is not particularly limited and can be set appropriately depending on, for example, the type and quantity of cells, the type and quantity of the liquid medium used in the cell suspension, etc. The standing time may be extended, for example, until the cells settle visually. Furthermore, as a specific example, the lower limit of the standing time is not particularly limited and can be, for example, 30 minutes or more, 1 hour or more, 6 hours or more, 12 hours or more, or 20 hours or more, and the upper limit is also not particularly limited and can be, for example, a few days or less, or 72 hours or less.

[0029] The aforementioned standing process is carried out, for example, under atmospheric conditions. Therefore, in the standing process, the pressure conditions inside the container are, for example, unpressurized, i.e., atmospheric pressure, and the oxygen conditions are, for example, the partial pressure of oxygen in the atmosphere.

[0030] The manufacturing method of the present invention may or may not include the standing step between the introduction step and the incubation step, and preferably the former.

[0031] (Incubation Step) Next, after the introduction step, or after any of the standing steps, an incubation step is performed. The incubation step is a step of incubating the cells introduced into the container, and includes a first incubation step and a second incubation step. The first incubation step is a step of incubation under low-oxygen and non-pressurized conditions, and the second incubation step is a step of incubation under low-oxygen and pressurized conditions following the first incubation step.

[0032] As described above, the manufacturing method of the present invention may or may not include the settling step between the introduction step and the incubation step. If the manufacturing method of the present invention does not include the settling step, the cells may be allowed to settle in the first incubation step. In this case, the first incubation step can be said to be, for example, a step of allowing the cells introduced into the container to settle and incubate under low-oxygen and non-pressurized conditions. Also, if the manufacturing method of the present invention includes the settling step, for example, the cells introduced into the container may also be allowed to settle and settle in the first incubation step.

[0033] The incubation step is a step of forming an extracellular matrix structure, that is, a step of forming a monolayer of cells containing an extracellular matrix structure from the introduced cell population. In this step, for example, the cells may or may not proliferate. The incubation step is also called, for example, an extracellular matrix structure formation step or a culture step.

[0034] The first incubation step is carried out under low-oxygen and non-pressurized conditions, as described above.

[0035] In the first incubation step, the hypoxic conditions are such that the upper limit of the oxygen partial pressure is, for example, less than 160 mmHg, 100 mmHg or less, 80 mmHg, 60 mmHg or less, 40 mmHg or less, and the lower limit of the oxygen partial pressure is not particularly limited and is, for example, 10 mmHg or more, 20 mmHg or more, 30 mmHg or more. The range can be exemplified by, for example, 10 mmHg or more to less than 160 mmHg, 10 - 100 mmHg, 10 - 80 mmHg, 20 - 100 mmHg, 20 - 80 mmHg, 20 - 60 mmHg, 20 - 40 mmHg, 30 - 80 mmHg, 30 - 60 mmHg, 30 - 40 mmHg, etc.

[0036] The pressure condition of the first incubation step is non - pressurized, that is, atmospheric pressure.

[0037] The temperature of the first incubation step is not particularly limited and can be appropriately determined depending on, for example, the cells to be used. The temperature may be, for example, the temperature of the container, the temperature inside the container, or the set temperature of the incubator for incubating the container. Examples of the temperature include 30 - 40 °C, 35 - 38 °C, etc. The temperature in the first incubation step may be, for example, constant or variable, and preferably a constant temperature.

[0038] The time of the first incubation step is not particularly limited and can be appropriately set according to, for example, the type and amount of cells, the type and amount of the liquid medium used in the cell suspension, etc. The lower limit of the standing time is, for example, 30 minutes or more, 2 hours or more, 6 hours or more, 24 hours or more, and it may be left standing, and the upper limit is also not particularly limited and is, for example, 48 hours or less, 72 hours or less.

[0039] The second incubation step following the first incubation step is carried out under hypoxic and pressurized conditions as described above.

[0040] In the second incubation step described above, the low-oxygen conditions are such that the upper limit of the partial pressure of oxygen is, for example, less than 160 mmHg, 100 mmHg or less, 90 mmHg or less, 80 mmHg, or 75 mmHg or less, and the lower limit of the partial pressure of oxygen is not particularly limited, for example, 20 mmHg or more, 30 mmHg or more, or 40 mmHg or more, and the range can be exemplified by, for example, 20 mmHg or more to less than 160 mmHg, 20 to 100 mmHg, 20 to 90 mmHg, 20 to 80 mmHg, 20 to 75 mmHg, 30 to 100 mmHg, 30 to 90 mmHg, 30 to 80 mmHg, 30 to 75 mmHg, etc.

[0041] Both the first incubation step and the second incubation step can be carried out under low-oxygen conditions, and the low-oxygen conditions of the first incubation step and the low-oxygen conditions of the second incubation step may be the same or different. In the latter case, for example, the partial oxygen pressure (B) of the second incubation step may be higher than the partial oxygen pressure (A) of the first incubation step (A < B), or the partial oxygen pressure (B) of the second incubation step may be lower (A > B).

[0042] Incubating under pressurized conditions means, for example, incubating cells while applying pressure to them. Since the cells exist as a cell suspension in the container, as described above, applying pressure to the cells can also be said to mean applying pressure to the cell suspension. Furthermore, it is preferable that the same pressure, i.e., hydrostatic pressure, is applied inside the container. The pressures exemplified below can be interpreted as hydrostatic pressure, for example.

[0043] In the second incubation step, the pressure under pressurization conditions is, as described above, a pressure exceeding atmospheric pressure (101 kPa). The lower limit of the pressure is, for example, 102 kPa or more, 105 kPa or more, or 110 kPa or more, and the upper limit of the pressure is, for example, 200 kPa or less, 190 kPa or less, or 150 kPa or less, and the range is, for example, above atmospheric pressure and 200 kPa or less, above atmospheric pressure and 190 kPa or less, 105 to 190 kPa, 105 to 150 kPa, or 110 to 150 kPa.

[0044] In the second incubation step, the pressure may be applied continuously or intermittently. Continuous application is a method of continuously applying pressure to the object. Intermittent application is a method of applying pressure to the object intermittently, that is, a method of repeating pressurization and non-pressurization, and the pressure may be applied at regular intervals or at random intervals.

[0045] In the second incubation step, continuous application of pressure is preferred. Continuous application may be, for example, continuous application of a constant pressure (aperiodic pressure) without changing the pressure, or continuous application of a periodic pressure with pressure variation, and the latter is preferred.

[0046] In the case of continuous application of periodic pressure, for example, the pressure may be changed and applied every few seconds to several hundred seconds, and as a specific example, every 60 to 300 seconds. The types of pressure to be changed may be, for example, two types or three or more types. As a specific example, the periodic pressure can be exemplified by repeating high pressure and low pressure. The high pressure and the low pressure are conditions where the former is relatively higher than the latter and the latter is relatively lower than the former. The high pressure and the low pressure may each be, for example, the same pressure value each time or different pressure values.

[0047] The lower limit of the high pressure value (H) is, for example, 150 kPa or more, 160 kPa or more, the upper limit is, for example, 200 kPa or less, 190 Pa or less, and the range is, for example, 150 to 200 kPa, 160 to 190 kPa. The lower limit of the low pressure value (L) is, for example, exceeding atmospheric pressure, 101 kPa or more, 105 kPa or more, the upper limit is, for example, 115 kPa or less, and the range is, for example, exceeding atmospheric pressure and 115 kPa or less, 101 kPa to 115 kPa, 105 to 115 kPa. The difference (H - L) between the high pressure value (H) and the low pressure value (L) is not particularly limited and is, for example, 30 to 50 kPa, 60 to 80 kPa, 90 to 99 kPa. The periodic pressure may start from the high pressure value or the low pressure value.

[0048] In periodic pressure, the combination of high pressure (H) and low pressure (L) is not particularly limited. For example, combinations such as a high pressure (H) of 150-200 kPa and a low pressure (L) above atmospheric pressure but 115 kPa or less, or a high pressure (H) of 160-190 kPa and a low pressure (L) above atmospheric pressure but 115 kPa can be exemplified. In periodic pressure, if one application of high pressure (H) and one application of low pressure (L) are considered as one set, the application time (T) of the high pressure (H) in one set is... Ｈ ) and the application time (T) of the low pressure value (L) Ｌ ) means, for example, the same time or (T Ｈ = T Ｌ ), or different times, but the former is preferred.

[0049] When periodic pressure is applied, the period can be expressed, for example, by frequency. Preferably, the pressure period is, for example, an extremely long period. Specific examples of the frequency include a lower limit of, for example, 0.0005 Hz or more and 0.001 Hz or more, an upper limit of, for example, 0.01 Hz or less and 0.004 Hz or less, and a range of, for example, 0.0005 to 0.01 Hz and 0.001 Hz to 0.004 Hz. 0.0005 Hz is, for example, a 2000-second period of 1000 seconds of high pressure and 1000 seconds of low pressure; 0.001 Hz is, for example, a 1000-second period of 500 seconds of high pressure and 500 seconds of low pressure; 0.004 Hz is, for example, a 250-second period of 125 seconds of high pressure and 125 seconds of low pressure; and 0.01 Hz is, for example, a 100-second period of 50 seconds of high pressure and 50 seconds of low pressure.

[0050] Furthermore, in the case of intermittent application as described above, for example, in the repeated cycle of pressurization and depressurization, each pressurization step may be a pressurization step with a different pressure value. Specific examples include repeated cycles of high pressure application, depressurization, low pressure application, and depressurization. The pressure conditions in intermittent application can be based on the examples used in continuous application, for example.

[0051] The temperature of the second incubation step is not particularly limited and can be appropriately determined depending on the cells used, for example. The temperature may be, for example, the temperature of the container, the temperature inside the container, or the set temperature of the incubation device that incubates the container. Examples of such temperatures include 30-40°C, 35-38°C, etc. The temperature in the incubation step may be constant or variable, and is preferably constant. The temperature of the second incubation step may be the same as or different from that of the first incubation step, and is preferably the former.

[0052] The incubation time for the second incubation is not particularly limited and can be appropriately determined depending on, for example, the type of cells used, the amount of cells introduced in the introduction step, the type of liquid medium, etc. The duration of each second incubation step has a lower limit of, for example, 30 minutes or more, 90 minutes or more, 3 hours or more, or 5 hours or more, and a higher limit of, not particularly limited, for example, 72 hours or less, 48 ​​hours or less, or 24 hours or less. As mentioned above, the incubation step aims to form an extracellular matrix structure by the extracellular matrix group, so the duration of each incubation step can be, for example, 1 to 2 months.

[0053] (Repetition of the process) In the manufacturing method of the present invention, a cell monolayer containing an extracellular matrix structure composed of an extracellular matrix group can be formed by performing the introduction step and the incubation step (the first incubation step and the second incubation step). In this specification, the monolayer is, for example, a cell layer formed by one introduction step and one incubation step. In the monolayer, the number of cells in the thickness direction may be, for example, one or two or more.

[0054] In the manufacturing method of the present invention, it is preferable to repeat the introduction step and the incubation step. One introduction step and one incubation step together are hereinafter referred to as one set of single-layer formation treatment.

[0055] In the manufacturing method of the present invention, by repeating the monolayer formation process two or more times, a laminate in which cells are stacked, specifically a three-dimensional laminate in which monolayers of cells are stacked, can be obtained. As described above, according to the incubation process, for example, a monolayer containing an extracellular matrix structure in which extracellular matrix groups are bound to each other is formed from the cell group introduced in the introduction process. Therefore, when the monolayer formation process is repeated n times (where n is an integer of 2 or more), for example, in each incubation process, an extracellular matrix structure is formed in the same set of cell group introduced in the introduction process, and binding of extracellular matrix groups occurs between the extracellular matrix structure formed in the (n-1)th set and the extracellular matrix structure formed in the (n)th set. As a result, the extracellular matrix structures are bound in both the vertical direction of the monolayer and the stacking direction in which the monolayers are stacked, and a three-dimensional laminate in which multiple layers are bound together by extracellular matrix structures is obtained. The laminate can also be called, for example, a cell aggregate.

[0056] According to this embodiment, for example, by repeatedly performing the single-layer formation process, it is possible to form a laminate of a desired thickness. The number of sets (n) of repetitions is not particularly limited and can be appropriately determined by, for example, the desired number of single-layer layers, the desired thickness of the final transplant material, etc.

[0057] The number of layers (n) of the laminate formed by repeating the introduction step and the incubation step is not particularly limited, with a lower limit of, for example, 2, 4, 6, or 8 layers, and an upper limit of, for example, 100, 50, 20, or 10 layers, and the range can be exemplified by, for example, 2 to 100 layers, 2 to 50 layers, 2 to 20 layers, 2 to 10 layers, 10 ± 5 layers, or 10 ± 4 layers. The number of sets (n) of repeating the introduction step and the incubation step can be, for example, by referring to the example of the number of layers (n), and the number of layers can be read as the number of sets.

[0058] When the aforementioned monolayer formation process is repeated, the conditions of the introduction step in each set may be the same or different. For example, the amount and type of cells introduced, the processing temperature, etc., may be the same or different. Also, the conditions of the incubation step in each set may be the same or different. For example, the low-oxygen conditions, processing temperature, processing time, the pressurization conditions in the second incubation step, etc., may be the same or different.

[0059] After the completion of the repeated monolayer formation process, the resulting laminate may be subjected to further incubation steps, for example, in order to strengthen the binding of the extracellular matrix groups.

[0060] As described above, the monolayer formation process may optionally include the standing step between the introduction step and the incubation step. Furthermore, if the manufacturing method of the present invention involves repeating the monolayer formation process two or more times, it may consist only of sets including the standing step, only of sets without the standing step, or both sets. In the sets without the standing step, for example, as described above, the cells may be allowed to settle in the first incubation step.

[0061] In the manufacturing method of the present invention, for example, the liquid medium in the cell suspension may or may not be replaced. From the viewpoint of maintaining a favorable environment for the cells, for example, the liquid medium may be replaced as appropriate. The timing of replacing the liquid medium is not particularly limited. For example, if the monolayer formation process is performed two or more times, the liquid medium can be replaced by using a new liquid medium during the new introduction step, or the replacement may be performed between the first incubation step and the second incubation step. When replacing the liquid medium, the pressure conditions and oxygen conditions are not particularly limited, for example, atmospheric conditions.

[0062] (Decellularization process) The obtained laminate may, for example, be used as a transplant material as is, or the decellularized structure obtained by removing cells from the laminate may be used as a transplant material. In the latter case, the manufacturing method of the present invention further includes a decellularization process, the decellularization process being a process of removing cells from the laminate to prepare a decellularized structure. As described above, the laminate is a laminate of cells having a three-dimensional extracellular matrix structure formed from an extracellular matrix group. Therefore, by performing the decellularization process, a three-dimensional extracellular matrix structure composed of an extracellular matrix group can be obtained.

[0063] The method for the decellularization treatment is not particularly limited, and for example, a method that does not destroy the three-dimensional structure of the extracellular matrix structure is preferred. Examples of the decellularization treatment methods include freeze-thaw treatment, treatment with surfactants, treatment with chemicals, high-pressure treatment, and electroporation treatment. Examples of the chemicals include surfactants, acids or bases, hypertonic solutions, alcohols, acetone, and tributyl phosphate. The surfactant is not particularly limited, and nonionic surfactants such as polyoxyethylene (10) octylphenyl ether (Tritoon® X-100); ionic surfactants such as sodium dodecyl sulfate, sodium deoxycholate, and sodium 2-[2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethanesulfonate (Tritoon® X-200); and amphoteric surfactants such as 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-(decyldimethylammonio)propane-1-sulfonate (sulfobetaine-10), and 3-[dimethyl(palmityl)ammonio]propane-1-sulfonate (sulfobetaine-16). In addition, other methods can also be used, such as those reported in the paper (Peter M. Crapo, Thomas W. Gilbert, and Stephen F. Badylak. Biomaterials 32 (2011) 3233-3243).

[0064] When treating with the aforementioned drug, for example, a decellularized structure can be obtained by immersing the laminate in a solution containing the drug and then washing it with a washing solution after immersion. The immersion time is not particularly limited, and is, for example, 30 minutes to 24 hours. The washing solution is not particularly limited, and can be, for example, water, buffer solution, saline solution, or other aqueous solvents. The washing can be performed, for example, by immersing the laminate in the liquid solvent for washing and then replacing it with a new liquid solvent, and the washing time is, for example, 1 to 7 days.

[0065] The manufacturing method of the present invention will be further explained with specific examples. However, the manufacturing method of the present invention is not limited to these examples.

[0066] [Embodiment 1] The use of the transplant material produced by the manufacturing method of the present invention is not particularly limited, but for example, vascular grafts are preferred. Vascular grafts are used, for example, for the replacement of damaged blood vessels and for the reconstruction of blood vessels. The laminate and the decellularized structure obtained by the manufacturing method of the present invention both contain, for example, sufficient extracellular matrix and have excellent strength, and are therefore suitable for use as vascular grafts. For this reason, the manufacturing method of the present invention is particularly suitable for producing transplant material as vascular grafts.

[0067] In the production of vascular grafts, it is preferable that the subject and the animal species from which the cells are derived are of the same species, for example, from the viewpoint of reducing the immune response. Specifically, when transplanting into a human, it is preferable to use human-derived cells. Furthermore, the cells used may be autologous or allogeneic, but autologous cells are preferred from the viewpoint of further reducing the immune response.

[0068] In the production of vascular grafts, the cells are preferably, for example, fibroblasts and smooth muscle cells as described above, more preferably smooth muscle cells, and particularly preferably umbilical artery smooth muscle cells, from the viewpoint of the ratio of vascular components. The liquid solvent for the cells is preferably, for example, DMEM medium, and serum may or may not be added.

[0069] When the transplant material is used as a vascular graft, as described above, for example, the laminate having an extracellular matrix structure may be used as is, or a decellularized structure (i.e., a three-dimensional extracellular matrix structure) obtained by removing cells from the laminate may be used. The vascular graft is preferably transplanted into a living organism, and after new blood vessels are regenerated using the vascular graft as a scaffold, it is ultimately degraded in the living organism. In this case, for example, it is preferable to use the decellularized structure as the vascular graft. Hereinafter, the term "transplant material" includes, for example, both the laminate of cells and the decellularized structure.

[0070] The blood vessel is a hollow tubular body, but the shape of the transplant material is not particularly limited; for example, it may be in the form of a sheet or a hollow tubular body (also called a tube shape). In the case of the sheet-shaped transplant material, for example, it can be manufactured by repeatedly performing the introduction step and the incubation step using a tray-shaped container such as a petri dish.

[0071] When manufacturing the tubular implant material, the container is preferably a cylindrical container such as a syringe, and it is preferable to form the tubular laminate against the inner wall of the cylindrical container. After forming the laminate inside the cylindrical container, the tubular laminate may be peeled off from the cylindrical container and used as an implant material as is, or it may be subjected to decellularization treatment and used as a tubular decellularized structure.

[0072] When using the cylindrical container and using the cylindrical laminate for the inner wall of the cylindrical container, for example, each step can be performed as follows: With the opening at one end of the cylindrical container closed, the cell suspension is introduced into the cylindrical container. Then, with the cylindrical container lying on its side, the incubation step is performed. The cell suspension introduced into the cylindrical container preferably has a cell density such that it does not spill out even when the container is lying on its side. Furthermore, it is preferable that the cell suspension fills approximately the lower half of the cylindrical container when it is lying on its side. With this method, for example, the incubation step allows a single layer to be formed on the lower half of the inner wall of the cylindrical container when it is lying on its side. Then, by introducing the cell suspension into the cylindrical container in the further introduction step, and laying the container on its side so that the inner wall that was on the lower side in the previous incubation step is now on the upper side, the incubation step is performed, and another single layer can be formed on the remaining half of the inner wall. In this way, a single layer that is connected in the circumferential direction can be formed on the inner wall of the cylindrical container. Thereafter, by repeating the introduction step and the incubation step, a cylindrical laminate can be formed on the inner wall of the cylindrical container in the same manner.

[0073] The shape and size of the transplant material are preferably similar to those of the blood vessels at the transplant site. The shape and size of the transplant material can be adjusted, for example, by the shape of the container.

[0074] The thickness of the transplant material can be appropriately set according to, for example, the required strength of the target transplant site. The thickness of the transplant material can also be adjusted by, for example, the cell concentration of the cell suspension used in the introduction step, the amount of cells per area in one introduction step, and the number of sets of repeated introduction and incubation steps.

[0075] When the aforementioned transplant material is used as a vascular fume hood, the following conditions are examples. Note that the following examples are merely illustrative, and the present invention is not limited thereto.

[0076] (Size and shape) Shape: Sheet Thickness: 0.05-2 mm Total number of layers: 5-30

[0077] (Conditions for the introduction process) Temperature: 36.5–37.5°C Cell suspension concentration: 500,000–3,500,000 cells / mL Number of introduced cells per area: 20,000–150,000 cells / cm² ２ Oxygen partial pressure: 160 mmHg (partial pressure of oxygen under atmospheric conditions) Pressure: Atmospheric pressure

[0078] (Conditions for the standing process) Temperature: 36.5–37.5°C Time: 0–24 hours Oxygen partial pressure: 160 mmHg (Oxygen partial pressure under atmospheric conditions) Pressure: Atmospheric pressure

[0079] (Conditions for the first incubation process) Temperature: 36.5–37.5°C Time: 12–72 hours Oxygen partial pressure: 20–60 mmHg Pressure: Atmospheric pressure (Conditions for the second incubation process) Temperature: 36.5–37.5°C Time: 12–36 hours Oxygen partial pressure: 30–80 mmHg Applied conditions: Continuous pressurization of periodic pressure Period: 0.001–0.004 Hz Pressure: 105–190 kPa High pressure value: 160–190 kPa Low pressure value: 105–115 kPa

[0080] (Repeating the introduction process, the standing process, and the incubation process) Number of sets: 5 to 30

[0081] (Deoxygenation treatment) Deoxygenation treatment: Types: Freeze-thaw and immersion in surfactant

[0082] When the aforementioned transplant material is used as a vascular draft, the type and location of the target blood vessel are not particularly limited. The blood vessel may be, for example, a vein or an artery. The target animal is not particularly limited and may be a human or a non-human animal, and non-human animals include, for example, the mammals mentioned above.

[0083] [Embodiment 2] In the manufacturing method of the present invention, the container in which cells are introduced in the introduction step (hereinafter also referred to as the cell container) and the apparatus for performing the incubation step from the introduction step are not particularly limited.

[0084] (Cell container) The cell container is made of a material that has little effect on the growth and survival of the cells, as cells are introduced into it. Furthermore, since pressure is applied to the cells inside the cell container during the second incubation step, it is preferable that the container be made of a material that is not easily affected by changes in shape due to pressurization. Examples of such materials include glass, metal, and plastic.

[0085] The size and shape of the cell container are not particularly limited and can be selected, for example, according to the shape and size of the target transplant material. The shape of the cell container is not particularly limited and can be, for example, a tray-like container such as a petri dish, a cylindrical container such as a syringe, a flask-like container, etc.

[0086] In the cell container, for example, the properties of its inner surface (also called the inner wall), particularly the surface in contact with the introduced cells, are not particularly limited. For example, from the viewpoint of monolayer cell culture, the surface may be treated to facilitate cell adhesion. Examples of such surface treatments include fibronectin coating and polylysine coating. On the other hand, as another example, from the viewpoint of ease of peeling (recovering) the laminate from the cell container, the surface may be left untreated. If the laminate is thin, or has a complex shape such as a cylinder, leaving the surface untreated allows for easy recovery of the laminate from the cell container.

[0087] Furthermore, the cell container may or may not include, for example, a scaffold material for the cells inside. The scaffold material may be recovered from the cell container together with the laminate after the formation of the laminate, or it may be left inside the cell container and only the laminate may be removed. The scaffold material is not particularly limited, and for example, a collagen membrane such as an atelocollagen membrane can be used. When using the scaffold material, for example, a so-called membrane culture method can be used, in which cells are incubated on the scaffold material. In this case, for example, a culture medium container such as a petri dish for holding the culture medium and a frame on which the scaffold material is installed can be used, and the frame can be positioned so that the scaffold material installed on the frame is in contact with the culture medium in the culture medium container, and incubation can be performed.

[0088] When using the scaffolding material, the liquid culture medium can be replaced, for example, by replacing the culture medium in the culture medium container with a new liquid culture medium during the introduction step, or by replacing the culture medium in the culture medium container with a new liquid culture medium between the first incubation step and the second incubation step. The liquid culture medium in the culture medium container may also be replaced by replacing the entire culture medium container, for example.

[0089] (Apparatus) Commercially available equipment can be used for the incubation process, and can be combined with, for example, equipment for supplying low-oxygen gas, equipment for adjusting pressure, equipment for adjusting temperature, etc. Below, an example of a method for performing the first incubation process and the second incubation process by combining various devices is shown, but the present invention is not limited to these examples.

[0090] The first incubation step is a step of incubation under low-oxygen and non-pressurized conditions. For example, a multi-gas incubator can be used for the first incubation step. The multi-gas incubator can, for example, control the composition of the gas and introduce the gas into the chamber to bring the chamber to a desired oxygen concentration under atmospheric pressure. The oxygen concentration can be adjusted, for example, by introducing nitrogen gas. The multi-gas incubator can also, for example, adjust the gas with the adjusted oxygen concentration to a desired temperature and introduce it into the chamber, thereby controlling the chamber to a desired incubation temperature.

[0091] The second incubation step is a step of incubation under low-oxygen and pressurized conditions. The second incubation step can be performed by combining, for example, the multi-gas incubator, an air compressor for compressing gas, an air tank for storing the compressed gas, an air compressor, and a regulator for adjusting the amount of gas introduced. In the second incubation step, for example, the cell container containing the cells is placed in a pressure-resistant container. Then, for example, gas with an adjusted oxygen concentration in the multi-incubator is drawn in and compressed by the air compressor, the compressed gas is stored in the air tank, and the compressed gas is introduced from the air tank into the pressure-resistant container. The introduction of the compressed gas can be controlled by the regulator. By introducing the compressed gas, the inside of the pressure-resistant container can be made low-oxygen, and at the same time, pressure can be applied to the cells inside the pressure-resistant container. Furthermore, since the multi-gas incubator can be adjusted to a desired temperature, if the pressure-resistant container is placed inside the multi-gas incubator, the inside of the pressure-resistant container can be controlled to a desired incubation temperature. The pressure-resistant container may, for example, be placed inside the chamber of the multi-gas incubator, or it may be placed outside the chamber of the multi-gas incubator.

[0092] [2. Transplant Material] The transplant material of the present invention is obtained by the method for producing the transplant material of the present invention as described above. According to the method for producing the transplant material of the present invention, for example, a structure including an extracellular matrix structure in which an extracellular matrix group is bound can be obtained. The structure is useful as a transplant material, for example, as a vascular graft, because it has excellent strength.

[0093] The transplant material may be, for example, the laminate described above, or a decellularized structure that has undergone decellularization treatment. The transplant material may include, for example, an extracellular matrix structure formed from a group of 5 to 100 types (approximately 40 types as a specific example) of extracellular matrix components. The main components of the extracellular matrix structure are, for example, collagen (collagen fibers) such as collagen I and collagen III, and mucopolysaccharides such as acidic mucopolysaccharides.

[0094] The aforementioned decellularized structure can, for example, be observed with the naked eye to reveal features of a membrane-like structure.

[0095] The present invention will be described in detail below with reference to examples, etc., but the present invention is not limited to these examples.

[0096] [Example 1] Using smooth muscle cells derived from the human umbilical artery, a transplant material was prepared by the manufacturing method of the present invention.

[0097] (1) Preparation of smooth muscle cells Smooth muscle cells were prepared by the Explant method, which involves collecting cells that have migrated out, as shown below. First, the umbilical artery obtained during a cesarean section at birth was acclimatized by immersion in physiological saline (PSS, Physiological Salt Solution) for 24 hours. Then, 10 cm of the fetal portion was cut from the umbilical artery and further divided into tissue pieces measuring 1 cm square. The tissue pieces were placed in a cell culture dish, and a mesh for the Explant method (product name Cell Amigo®, Tsubakimoto Chain Co., Ltd.) was placed on top of the tissue pieces as a weight. The cells were cultured in DMEM medium containing 10% FBS, and the umbilical artery smooth muscle cells that migrated and proliferated were isolated. The culture conditions for the Explant method were atmospheric pressure, 37°C, and 5% CO2. ２ The suggested duration was 2 to 8 weeks.

[0098] The isolated smooth muscle cells were suspended in a medium to a concentration of 600,000 cells / mL to prepare a cell suspension. The medium used was DMEM medium containing 10% FBS.

[0099] (2) Preparation of cell laminate As a scaffold material for membrane culture, a commercially available permeable collagen membrane (circular membrane of atelocollagen derived from cowhide, for 6-well plate, volume 2,860 mm ３ , area 530 mm ２ , manufactured by Koken Co., Ltd.) was used. A container in which the collagen membrane was set in a dedicated cylindrical frame made of polystyrene resin was used as the cell container.

[0100] First, a 6-cm diameter petri dish was placed in a clean bench under atmospheric conditions, and 7 mL of DMEM medium containing 10% FBS was placed in the petri dish. The cell container was placed in the petri dish. Then, 1 mL of the cell suspension (600,000 cells / mL) was put into the cell container (introduction step). Then, the cell container together with the petri dish was placed in an incubator under atmospheric conditions, and left standing at 37 °C for 3 hours under non-pressurized (atmospheric pressure) conditions to sediment the cells in the cell suspension (standing step). After confirming the sedimentation of the cells, the cell container together with the petri dish was taken out of the incubator.

[0101] Next, the incubation steps (the first incubation step and the second incubation step) were performed as follows using the multi-gas incubator (trade name: direct heat type multi-gas incubator, manufactured by Astec Co., Ltd.) exemplified in Embodiment 2.

[0102] The cell container together with the petri dish was accommodated inside the multi-gas incubator. Then, the temperature of the multi-gas incubator was set to 37 °C, the pressure was set to non-pressurized (atmospheric pressure), and the set value of the oxygen concentration was set to 1%. The incubator was started, and the first incubation step was performed on the cells in the cell container for 21 hours. When the set value of the oxygen concentration was 1%, the actually measured value of the oxygen partial pressure under atmospheric pressure was 30 - 40 mmHg, specifically 38 mmHg.

[0103] After the first incubation step, the cell container was removed from the multi-gas incubator along with the petri dish. The petri dish was replaced with a new petri dish containing fresh culture medium, and the cell container was placed in it to perform a culture medium exchange. The conditions for the culture medium exchange were, for example, atmospheric conditions, i.e., unpressurized (atmospheric pressure) and atmospheric oxygen partial pressure.

[0104] The cell container, along with the new petri dish, was placed in a pressure-resistant container, and the pressure-resistant container was placed in the multi-gas incubator. The temperature of the multi-gas incubator was set to 37°C and the oxygen concentration to 1%, and the incubator was started. The gas, whose oxygen concentration and temperature had been adjusted in the multi-gas incubator, was drawn into an air compressor and compressed, and the compressed gas was stored in an air tank. The compressed gas stored in the air tank was then introduced into the pressure-resistant container, and the cells in the cell container were incubated for 24 hours (second incubation step). The introduction of the compressed gas was controlled by the regulator. The regulator was set to the following conditions. When the oxygen concentration was set to 1%, the measured value of the partial pressure of oxygen under the following pressurized conditions was 70-80 mmHg, specifically 73 mmHg. (Pressurization conditions) Continuous application of periodic pressure switching between high and low pressure High pressure value: 180 kPa Low pressure value: 110 kPa Frequency: 0.002 Hz (500-second cycle, with 250 seconds of high pressure followed by 250 seconds of low pressure as one set)

[0105] After the second incubation step, the pressure-resistant container was removed from the multi-gas incubator, and the cell container was removed from the pressure-resistant container along with the petri dish. Then, the introduction step, the standing step, and the incubation step (first incubation step and second incubation step) were considered as one set, and a total of 10 sets were repeated under the same conditions. In the introduction step, the cell container was removed from the petri dish used in the previous set, the cell container was placed in a new petri dish containing new culture medium, and the cell suspension for the next set was introduced into the cell container. By the above method, a cell layer sheet was formed on the scaffold material, consisting of 10 layers of single-layer cell sheets formed in one set.

[0106] After the 10 sets were completed, the petri dish used in the 10th set was replaced with a new petri dish containing ascorbic acid-supplemented medium, and the culture medium in the cell container was also replaced with the new ascorbic acid medium. The cells in the cell container were then incubated in the multi-gas incubator for 7 days (first post-treatment step). The ascorbic acid-supplemented medium was prepared by adding ascorbic acid (product name: 2-O-alpha-D-Glucopyranosyl-L-ascorbic acid, Tokyo Chemical Industry Co., Ltd.) to 10% FBS-containing DMEM to a concentration of 50 μg / ml. The conditions for the first post-treatment step were non-pressurized (atmospheric pressure), atmospheric oxygen partial pressure, 37°C, and 5% CO2. ２ The ascorbic acid medium in the petri dish and the cell container was replaced with fresh ascorbic acid medium every 1-3 days.

[0107] The cell container was removed from the multi-gas incubator along with the petri dish, and the cell sheet was peeled off the scaffold material in the cell container. The cell sheet was then incubated for another 7 days using the ascorbic acid-supplemented culture medium under the same conditions as the first post-treatment step (second post-treatment step).

[0108] The aforementioned cell sheet is a cell-layered sheet in which 10 single-layer cell sheets, each formed as a single set, are stacked. Visual and microscopic observation confirmed that there is no physical separation between the layers, and that the entire sheet is integrated.

[0109] (3) Preparation of Decellularized Sheet Next, the cell-laminated sheet was subjected to decellularization treatment. Decellularization treatment was carried out using a freeze-thaw method and a treatment method using a surfactant. First, the cell-laminated sheet was frozen by treating it at -80°C for 24 hours or more, and the frozen cell-laminated sheet was thawed by treating it at 4°C for 4 hours or more. The cell-laminated sheet was washed with distilled water before and after freezing and thawing.

[0110] Next, the freeze-thawed sheets were immersed in a mixture containing 0.5% sodium dodecyl sulfate and 0.5% sodium deoxycholate (solvent: distilled water) and treated at room temperature for 6 hours. Furthermore, the treated sheets were immersed in PBS (product name: phosphate-buffered physiological saline (pH 7.4), Nacalai Tesque Co., Ltd.) and treated at 4°C for 7 days. The PBS was replaced with fresh PBS every 1-2 days. This resulted in decellularized sheets from which cells had been removed.

[0111] [Example 2] (1) Detection of collagen by staining A cell stacking sheet (untreated with decellation treatment) was prepared in the same manner as in Example 1, except that decellation treatment was not performed. This was designated as the cell stacking sheet (untreated with decellation treatment) of Example 2. The cell stacking sheet (untreated with decellation treatment) was stained with Sirius Red to visualize collagen and detect it. Sirius Red staining was performed using a commercially available reagent (product name Picrosirius Red Stain Kit, Cosmo Bio Co., Ltd.) according to its instructions. According to Sirius Red staining, collagen in general is stained red, and further observation with a polarizing lens reveals that type 1 collagen is detected as yellow to orange, and type 3 collagen as green.

[0112] Furthermore, the cell-laminated sheet (untreated with decellation) of Comparative Example 2 was prepared by the following method. Specifically, the cell-laminated sheet (untreated with decellation) was prepared in the same manner as in Example 1, except that the first incubation step of the incubation process was performed under an oxygen concentration of 21% and atmospheric conditions, and the second incubation step was performed under a low oxygen concentration and pressurized conditions, and no decellation treatment was performed. The low oxygen concentration in the second incubation step was the same as in Example 1, at 1%, and the pressurized conditions were also the same as in Example 1, with continuous pressurization.

[0113] These results are shown in Figure 1. Figure 1A is a photograph showing the staining results of the cell layer sheet (untreated with decells) of Example 2, and Figure 1B is a photograph showing the staining results of the cell layer sheet (untreated with decells) of Comparative Example 2. In each figure, the scale bar is 100 μm.

[0114] Comparing Figure 1A of Example 2 with Figure 1B of Comparative Example 2, the cell-layered sheet of Example 2 showed a larger red area stained with Sirius Red than the cell-layered sheet of Comparative Example 2. Furthermore, detection results through a polarizing lens (not shown) showed that the cell-layered sheet of Example 2 had a larger orange area indicating type I collagen compared to the cell-layered sheet of Comparative Example 2. From these results, it was found that the cell-layered sheet of Example 2 (untreated with decellation) had a higher collagen content than the cell-layered sheet of Comparative Example 1 (untreated with decellation).

[0115] (2) Evaluation of the fibrous structure The fibrous structure of the decellularized sheet of Example 1 was confirmed using a scanning electron microscope. The results are shown in Figure 2. Figure 2 is a scanning microscope image of the decellularized sheet of Example 1, and the scale bar is 1 μm. As shown in Figure 2, a fibrous structure in which very fine fibers are interwoven was confirmed in the decellularized sheet.

[0116] (3) Detection of residual cells and acidic mucopolysaccharides by staining The cell stack sheets (untreated with decellation) from Example 2 and Comparative Example 2 (untreated with decellation) were stained with Alcian blue. According to the Alcian blue staining, acidic mucopolysaccharides are stained light blue, and cell nuclei are stained red. Therefore, the presence of acidic mucopolysaccharides and the presence of residual cells can be evaluated from the staining results. Alcian blue staining was performed using a commercially available reagent (product name Alcian Blue Staining Solution for Cytological Use (Yamamoto Method), Muto Chemical Co., Ltd.) according to its standard procedure.

[0117] These results are shown in Figure 3. Figure 3A is a photograph showing the staining results of the cell stacking sheet (untreated with decellation) of Example 2, and Figure 3B is a photograph showing the staining results of the cell stacking sheet (untreated with decellation) of Comparative Example 2. Acidic mucopolysaccharides were detected over a wide area in the decellation sheet of Example 2, and in Figure 3A, the remaining cells in the decellation sheet (the scattered gray areas in the figure) were significantly reduced compared to the cell stacking sheet of Comparative Example 2 in Figure 3B. From these results, it was found that the cell stacking sheet of Example 2 was easier to decellate by decellation treatment compared to the cell stacking sheet of Comparative Example 2.

[0118] (4) Detection of residual DNA DNA was detected in the decellation sheet of Example 1 by the following method. In order to evaluate the degree of DNA removal by the decellation treatment (amount of residual DNA), a cell-laminate sheet (untreated with decellation treatment) was prepared by the same method as in Example 1, except that the decellation treatment was not performed, and DNA was detected in the same manner.

[0119] The decellation sheet and the cell-layered sheet (without decellation treatment) were both freeze-dried, and then DNA extraction was performed using a commercially available DNA extraction kit (NucleoSpin Tissue XS, Takara Bio Inc.). The amount of DNA in the extracted DNA samples was then measured using a commercially available DNA measurement kit (QuantiFluor dsDNA system, Promega Inc.). In addition, the decellation sheet and the cell-layered sheet (without decellation treatment) that were not freeze-dried were subjected to nuclear staining using DAPI, and the number of remaining cells was evaluated.

[0120] These results are shown in Figure 4. Figure 4 is a graph showing the amount of DNA (ng) per dry weight (mg) of the sheet. These results are expressed as the mean ± SEM of n=5-6 independent experiments. As shown in Figure 4, compared to the cell-laminate sheet (untreated with decellation), the decellation sheet showed a significant reduction in the amount of remaining DNA, confirming that decellation was sufficiently performed. Furthermore, the amount of remaining DNA in the decellation sheet was sufficiently lower than the standard for general decellated products (50 ng / mg dry body). In addition, no positivity was confirmed in the decellation sheet as a result of nuclear staining. From these results, it was found that decellation was sufficiently performed in the decellation sheet of Example 1.

[0121] [Example 3] The decellation sheet from Example 1 was used as a vascular graft and evaluated by in vivo transplantation.

[0122] Two rats (Wistar, 5 weeks old) had their abdomens opened, and the blood vessels (abdominal aorta) were incised along their long axis. The incised areas were covered with the aforementioned blood vessel grafts, and the grafts were sutured to the blood vessels in a patch transplantation procedure. The rats were then observed for 12 weeks.

[0123] (1) Changes in size The major axis, minor axis, and area of ​​the transplanted vascular grafts were measured immediately after transplantation and 12 weeks after transplantation (n=2). The direction of blood flow in the vessel is called the major axis, and the direction perpendicular to it is called the minor axis. The length in the major axis is called the major axis, and the length in the minor axis is called the minor axis. The major axis and minor axis were measured using the sutures used to sew the graft as markers. The results for the major axis, minor axis, and area are shown in Figure 5. Figure 5 is a graph showing the changes in the size of the vascular graft, with the major axis on the left, the minor axis in the middle, and the area on the right. In addition, photographs of the vascular graft immediately after transplantation and 12 weeks later are shown in Figure 6. Figure 6A is a photograph immediately after transplantation, and Figure 6B is a photograph 12 weeks later. In Figure 6A, the distance between the upper and lower seams in the long axis direction is the major axis (L), and the distance between the right and left seams in the short axis direction is the minor axis (M). The area enclosed by the dotted line indicates the area of ​​the sutured vascular graft. In Figure 6B, the distance between the upper and lower seams in the long axis direction is the major axis (L), and the distance between the right and left seams in the short axis direction is the minor axis (M). The area enclosed by the dotted line indicates the area where the sutured vascular graft has expanded and where the regenerated blood vessels have expanded (grown).

[0124] The rats' body weight increased from 100g (at the time of transplantation) to 300g (after 12 weeks), and their blood vessels also expanded. As shown in Figures 5 and 6, the transplanted blood vessel grafts also expanded in response.

[0125] (2) Size variation Twelve weeks after transplantation, the blood vessels including the transplantation site of the blood vessel graft were excised. The excised blood vessels were stained with Elastica stain and the tissue was examined. Elastica staining was performed using commercially available reagents and according to the standard procedure. Elastica staining is a staining method used to detect elastic fibers.

[0126] These results are shown in Figure 7. Figure 7 is a photograph of the excised blood vessel stained with Elastica. Specifically, the stained blood vessel was cross-sectioned at approximately the center of the site where the blood vessel graft was transplanted, and the photograph was taken from the cross-section side. The straight line in the figure indicates the boundary position between the blood vessel graft and the blood vessel at the time of transplantation, and the area below the straight line in the figure is the site where the blood vessel graft was transplanted. The scale bar in the figure is 500 μm.

[0127] As shown in Figure 7, the area enclosed by the dotted circle is the adventitia side of the blood vessel, where the remaining vascular graft was confirmed. Above the remaining vascular graft, i.e., the area enclosed by the solid circle, is the luminal side of the blood vessel, where regenerated medial vascular tissue was confirmed. These results confirm that transplanting the decellularized sheet leads to the infiltration of rat-derived cells into the transplantation site and the regeneration of elastic fibers.

[0128] [Example 4] The mechanical properties of the cell-free sheet from Example 1 were confirmed.

[0129] The decellularized sheets (n=4) were wrapped around the outer circumference of a glass rod (inner diameter 1.1 mm, outer diameter 1.68 mm) so as not to slacken. One end of the decellularized sheet wrapped around the glass rod was secured with a 10-0 needle-attached suture to prevent it from unraveling, and then it was removed from the glass rod. The removed decellularized sheet, still in its tubular form, was then subjected to stress and strain measurements using a high-precision tensile strength meter (product name DMT560 tissue puller, Danish MyoTechonolody, Denmark), and a stress-strain curve was generated. The stress measurement was based on the paper (Kojima T et al., Acta Biomater. 171: 209-222. 2023).

[0130] Figure 8 shows the results of the stress-strain curve (n=4). The stress-strain curve is a graph that shows the relationship between strain and stress. The final point on the right side of each plot (n=4) represents the stress and strain at which the decellularized sheet fractured.

[0131] When considering the transplantation of cell sheets into the human pulmonary artery, it has been reported that the pulmonary artery diameter doubles in patients aged 1 to 18 years. In this case, it is desirable that the strain of the transplanted cell sheet exceeds, for example, 200%. As shown in Figure 8, the decellularized sheets in this embodiment all exhibited a strain of well over 200% when fractured. From this, it was found that the decellularized sheets can be transplanted into living organisms as vascular grafts.

[0132] Furthermore, it is known that vascular grafts clinically applied to human saphenous veins have a fracture stress of approximately 1600 mmHg. In this embodiment, as shown in Figure 8, the decellularized sheets all exhibited a fracture stress of 2199 ± 175 mmHg, which significantly exceeded 1600 mmHg. From this, it was found that the decellularized sheets can be transplanted into the body as vascular grafts, and specifically, transplantation into the saphenous vein is possible.

[0133] Although the present invention has been described above with reference to embodiments, the present invention is not limited to the above embodiments. Various modifications to the structure and details of the present invention can be made, as can be understood by those skilled in the art within the scope of the present invention.

[0134] This application claims priority based on Japanese Patent Application No. 2025-9175, filed on 22 January 2025, and incorporates all of its disclosures herein.

[0135] According to the manufacturing method of the present invention, a structure suitable for practical use, including an extracellular matrix structure, can be obtained by incubating cells introduced into the container under low-oxygen and non-pressurized conditions, and then further incubating them under low-oxygen and pressurized conditions. The extracellular matrix structure can, for example, improve strength, and since the structure can be obtained by the simple two-step incubation process, the manufacturing method of the present invention is useful, for example, as a method for producing transplantable materials such as vascular grafts.

Claims

1. A method for producing a transplantable material, comprising: an introduction step of introducing cells into a container; and an incubation step of incubating the introduced cells in the container, wherein the incubation step comprises a first incubation step and a second incubation step, the first incubation step being a step of incubation under low-oxygen and non-pressurized conditions, and the second incubation step being a step of incubation under low-oxygen and pressurized conditions.

2. The manufacturing method according to claim 1, wherein the introduction step is performed under atmospheric conditions.

3. The manufacturing method according to claim 1 or 2, wherein in the first incubation step, the low-oxygen condition is an oxygen partial pressure of 10 mmHg or more and less than 160 mmHg, and the non-pressurized condition is atmospheric pressure.

4. The manufacturing method according to any one of claims 1 to 3, wherein in the second incubation step, the low-oxygen conditions are such that the oxygen partial pressure is 20 mmHg or more and less than 160 mmHg, and the pressurization conditions are such that the pressure is greater than atmospheric pressure and 200 kPa or less.

5. The manufacturing method according to any one of claims 1 to 4, wherein in the second incubation step, pressure is applied to the cells in the container, and the pressure is applied continuously or intermittently.

6. The manufacturing method according to claim 5, wherein in the second incubation step, the application of pressure is a continuous application of periodic pressure.

7. The manufacturing method according to claim 6, wherein the periodic pressure has a frequency of 0.0005 to 0.01 Hz.

8. The manufacturing method according to claim 6 or 7, wherein, in the periodic pressure, the high pressure value is 150 to 200 kPa and the low pressure value is greater than atmospheric pressure to 115 kPa.

9. The manufacturing method according to any one of claims 1 to 8, further comprising a standing step between the introduction step and the first incubation step, wherein the standing step is a step of standing the cells introduced into the container under atmospheric conditions.

10. The manufacturing method according to any one of claims 1 to 9, wherein in the introduction step, the cells are introduced into the container in which scaffolding material is arranged inside.

11. The manufacturing method according to any one of claims 1 to 10, wherein the cells are fibroblasts or smooth muscle cells.

12. The manufacturing method according to claim 11, wherein the cells are umbilical cord-derived fibroblasts or smooth muscle cells.

13. The manufacturing method according to any one of claims 1 to 12, comprising repeating the introduction step and the incubation step to form a laminate in which cells are stacked.

14. The manufacturing method according to claim 13, wherein the number of layers of the laminate formed by repeating the introduction step and the incubation step is 2 to 100 layers.

15. The manufacturing method according to claim 13 or 14, further comprising a decellation step, wherein the decellation step is a step of removing cells from the laminate to prepare a decellation structure.

16. The manufacturing method according to any one of claims 1 to 15, wherein the implantation material is an artificial blood vessel material.

17. A transplant material obtained by a method for manufacturing a transplant material according to any one of claims 1 to 16.

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