Method for producing decellularized tissue and decellularized tissue

Abstract

To provide a decellularized tissue production process that can produce a decellularized tissue having excellent blood compatibility, such as the ability to hardly induce a blood coagulation reaction, and to provide a decellularized tissue.SOLUTION: A disclosed decellularized tissue production process is an antithrombotic decellularized tissue production process that has a decellularization treatment step of decellularization treating a living tissue, a freeze-drying step of immersing the living tissue after decellularization treatment in a freeze-dryable organic solvent and then freeze-drying, and an alcohol treatment step of immersing the living tissue after freeze-drying step in a treatment liquid containing at least one alcohol selected from alcohol having 2 or more and 4 or fewer carbon atoms, and then drying under reduced pressure.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing decellularized tissue that can be used in cardiovascular medical devices such as artificial blood vessels and artificial valves, and to decellularized tissue produced by said method. [Background technology]

[0002] Development is underway to create artificial extracellular matrices that can function in vivo, replacing the original tissues within the body. Artificial extracellular matrices can be manufactured from synthetic materials or biological tissues. As an artificial extracellular matrix with high biocompatibility and low rejection, decellularized tissue, from which cellular components have been removed by decellularization treatment of biological tissue, is preferred. Decellularized tissue, from which cellular components have been removed, is being applied primarily in Europe and North America to the treatment of soft tissue prosthetics, tendons, breast tissue, dental tissue, urethra, pericardium, ophthalmology, bone, and cardiac tissue, as well as to medical devices (transplant grafts) in vascular surgery.

[0003] In this context, cardiovascular medical devices such as artificial blood vessels and artificial valves (bioprosthetic valves) are used in a blood environment. Therefore, the decellularized tissue used for these cardiovascular medical devices must possess blood compatibility, including properties that make it difficult to induce blood coagulation reactions.

[0004] However, since decellularized tissue is mainly composed of collagen and elastin, which promote blood coagulation, contact with blood induces a blood coagulation reaction through platelet adhesion and protein adsorption, leading to the formation of a thrombus. For this reason, the use of decellularized tissue as a cardiovascular medical device such as an artificial blood vessel or artificial valve is limited. By suppressing this blood coagulation reaction, decellularized tissue that is less prone to thrombus formation can lead to improved treatment outcomes for cardiovascular medical devices such as artificial blood vessels and artificial valves made from decellularized tissue, as well as an improvement in patients' quality of life.

[0005] Patent Document 1 describes an amino acid sequence (POG) that is an endothelial cell adhesion peptide. n -X-(REDV) mBy immobilizing (surface modifying) peptides containing these peptides onto the lumen of artificial blood vessels, thrombus formation is prevented.

[0006] Furthermore, Patent Document 2 describes a method for producing biocompatible transplant scaffolds derived from living organisms, including antithrombotic properties. This method includes the steps of freeze-drying living soft tissue, then heating it under vacuum at a temperature of 100°C to 200°C to partially cross-link and immobilize the proteins contained therein, and incubating the partially cross-linked tissue with elastase to selectively remove elastin, thereby improving the durability of the transplant scaffolds and suppressing calcification. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2014 / 065017 [Patent Document 2] Patent No. 5050197 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] However, Patent Document 1 requires the use of expensive synthetic peptides and the need for uniform and even coating. Therefore, there is a need for a low-cost technology to prevent thrombus formation that does not rely on the method described in Patent Document 1. Furthermore, the transplant scaffold described in Patent Document 2 has the problem that platelet adhesion occurs on the surface of the scaffold, leading to the formation of thrombi.

[0009] This invention has been made in view of the above circumstances, and aims to provide a method for producing decellularized tissue and decellularized tissue that can produce decellularized tissue with excellent blood compatibility, such as being less likely to induce blood coagulation reactions. [Means for solving the problem]

[0010] The inventors of the present invention have found that a decellularized tissue excellent in blood compatibility such as a performance that hardly induces a blood coagulation reaction can be obtained by a production method having a decellularization treatment step of decellularizing a biological tissue, a lyophilization step of immersing the biological tissue after the decellularization treatment step in a lyophilizable organic solvent and then lyophilizing it, and an alcohol treatment step of immersing the biological tissue after the lyophilization step in a treatment liquid containing at least one alcohol selected from alcohols having 2 to 4 carbon atoms and then drying it under reduced pressure, and have completed the present invention. Specifically, the present invention provides the following.

[0011] (1) A method for producing a decellularized tissue, comprising: a decellularization treatment step of decellularizing a biological tissue; a lyophilization step of immersing the biological tissue after the decellularization treatment step in a lyophilizable organic solvent and then lyophilizing it; and an alcohol treatment step of immersing the biological tissue after the lyophilization step in a treatment liquid containing at least one alcohol selected from alcohols having 2 to 4 carbon atoms and then drying it under reduced pressure.

[0012] (2) The method for producing a decellularized tissue according to (1), wherein the decellularization treatment includes a decellularization treatment by pressurization.

[0013] (3) The method for producing a decellularized tissue according to (1) or (2), wherein the concentration of the alcohol in the treatment liquid is 50% by mass or more.

[0014] (4) The method for producing a decellularized tissue according to any one of (1) to (3), wherein the biological tissue is a blood vessel or a valve.

[0015] (5) The method for producing a decellularized tissue according to any one of (1) to (4), further comprising an immersion step of immersing in a treatment liquid containing ethanol and then in a phosphate buffer after the alcohol treatment step.

[0016] (6) Decellularized tissue prepared by subjecting a living tissue to the following in this order: decellularization treatment, freeze-drying after immersion in a freeze-dryable organic solvent, and vacuum drying after immersion in at least one alcohol selected from alcohols having 2 to 4 carbon atoms. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide a method for producing decellularized tissue and decellularized tissue that can produce decellularized tissue with excellent blood compatibility, such as being less likely to induce blood coagulation reactions. [Brief explanation of the drawing]

[0018] [Figure 1] This figure shows the quantitative results of platelet adhesion behavior (platelet adhesion range). [Modes for carrying out the invention]

[0019] The present invention will be described below based on preferred embodiments.

[0020] Method for producing decellularized tissue, and decellularized tissue itself. A method for producing decellularized tissue comprises a decellularization step of decellularizing biological tissue, a freeze-drying step of immersing the biological tissue after the decellularization step in a freeze-dryable organic solvent and then freeze-drying it, and an alcohol treatment step of immersing the biological tissue after the freeze-drying step in a treatment solution containing at least one alcohol selected from alcohols having 2 to 4 carbon atoms, and then drying it under reduced pressure. The alcohol treatment step may also include an immersion step of immersing the tissue in a treatment solution containing ethanol and then in a phosphate buffer. The following describes each step in the method for producing decellularized tissue.

[0021] <Decellation process> In the decellularization process, living tissue is decellularized. Decellularization removes cellular components from the living tissue. By removing cellular components, the foreign body reaction after transplantation can be reduced, and the biocompatibility of the resulting decellularized tissue can be increased, thus lowering the risk of rejection.

[0022] Examples of biological tissue include blood vessels and valves derived from the body. The blood vessels can be arteries or veins, but arteries are preferred from the standpoint of strength. By using blood vessels and valves as biological tissues, artificial blood vessels, artificial valves (bioprosthetic valves), and artificial myocardial patches can be manufactured as antithrombotic decellularized tissues. Living tissue can be obtained by surgically removing it from a living organism. It can also be obtained by purchasing commercially available, already removed living tissue. Examples of living organisms include birds such as ratites and non-human animals such as mammals. However, the living organism may also be a human. Examples of mammals include mice, rats, rabbits, goats, sheep, monkeys, pigs, cows, and horses. Examples of flightless birds include emus, kiwis, ostriches, cassowaries, and rheas. When the biological tissue is blood vessels, among the organisms mentioned above, ratites are preferred. Ratatodes generally have long necks, and the blood vessels in these necks, such as the carotid arteries, are thin, long, and have few branches. Therefore, by using the blood vessels of ratites to create artificial blood vessels, it is possible to create artificial blood vessels with a smaller, longer, and less branched lumen cross-section. In addition, ratites and similar organisms are easy to breed, so a large amount of biological tissue can be supplied stably.

[0023] The size and shape of the biological tissue should be appropriately selected according to the desired decellularized tissue. When the biological tissue is a blood vessel, the inner diameter is, for example, 5 mm or less, but it may also be 4 mm or less or 3 mm or less. Furthermore, the inner diameter of the blood vessel is, for example, 1 mm or more. If the biological tissue is a blood vessel, the area of ​​the lumen in its cross-section is, for example, π × 2 2 mm 2as follows, π×1.5 2 mm 2 hereinafter, π×1 2 mm 2 hereinafter, π×0.75 2 mm 2 hereinafter, or π×0.5 2 mm 2 may be used. In this case, the shape of the lumen in the cross-section of the blood vessel is not limited to a perfect circle. When the biological tissue is a blood vessel, the length may be, for example, 10 cm or more, 20 cm or more, 30 cm or more, 40 cm or more, 50 cm or more, 60 cm or more, 70 cm or more, or 80 cm or more. The decellularized tissue of the present embodiment is excellent in blood compatibility (e.g., antithrombotic property) such as the performance of being difficult to induce a blood coagulation reaction. Therefore, as biological tissues, blood vessels with a small inner diameter and cross-sectional area, or long blood vessels may be used, and artificial blood vessels with a small inner diameter and cross-sectional area, or long artificial blood vessels may be formed. Note that the inner diameter, lumen area, length, etc. of the decellularized tissue produced in the present embodiment are substantially the same as those of the biological tissue used.

[0024] The method of decellularization treatment is not particularly limited as long as cell components (cells and components of cells) are removed from the biological tissue. Examples of decellularization treatment include decellularization treatment by pressurization, decellularization treatment with a surfactant (e.g., sodium dodecyl sulfate, Triton X-100 (registered trademark), sorbitan), and decellularization treatment with a solvent having a high salt concentration. Decellularization treatment by pressurization is preferred.

[0025] The specific method of decellularization treatment by pressurization is not particularly limited as long as pressure can be applied to the biological tissue to the extent that cell components (cells and components of cells) are removed from the biological tissue, and a well-known pressurization device can be used.

[0026] The magnitude of the pressure applied to the biological tissue is not particularly limited, but for example, it is preferably 200 MPa or more and 1000 MPa or less, more preferably 300 MPa or more and 1000 MPa or less, and even more preferably 500 MPa or more and 1000 MPa or less.

[0027] The specific method for applying pressure to biological tissue is not particularly limited, but for example, pressure can be applied to biological tissue in a liquid. As a liquid, for example, water, physiological saline, phosphate buffer, PBS (phosphate buffered saline), etc., can be used.

[0028] The pressurized temperature is not particularly limited, but for example, the temperature of the biological tissue may be controlled to be between 25°C and 100°C. The pressurization time is not particularly limited, but for example, it is between 5 and 20 minutes.

[0029] After the decellation treatment by pressurization described above, DNase treatment may be performed. DNase treatment allows for more efficient destruction and removal of remaining cells and their components (specifically, DNA).

[0030] DNase treatment can be performed by adding DNase (e.g., various commercially available DNases) and biological tissue to a liquid in which DNase can function. As a liquid, for example, a DNase-treated solution can be used, which is made by adding antibiotics such as penicillin or streptomycin, or a source of divalent ions such as MgCl2 and / or CaCl2, as needed, to PBS (phosphate-buffered saline) or physiological saline.

[0031] The concentration of DNase in the DNase-treated solution is not particularly limited, but for example, it may be between 1 U / mL and 1000 U / mL, between 10 U / mL and 500 U / mL, or between 40 U / mL and 100 U / mL.

[0032] Since DNase requires divalent ions (e.g., magnesium ions and / or calcium ions) to exhibit its activity, it is preferable that the DNase-treated solution contains a source of divalent ions such as MgCl2 and / or CaCl2.

[0033] The concentrations of MgCl2 and CaCl2 in the DNase-treated solution are not particularly limited and can be appropriately set according to the characteristics of the DNase used. For example, the concentrations of MgCl2 and CaCl2 in the DNase-treated solution may be between 10 mM and 50 mM, or between 20 mM and 40 mM.

[0034] The temperature for DNase treatment is not particularly limited, but is preferably, for example, 35°C to 40°C.

[0035] The processing time for DNase treatment is not particularly limited, but is, for example, 6 days or less, preferably 5 days or less, more preferably 4 days or less, and even more preferably 3 days or less. The processing time for DNase treatment is, for example, 0.5 days or more, and may be 1 day or more.

[0036] The processing time for DNase treatment can also be set according to the processing time for the washing treatment described later. For example, it is preferable that the sum of the processing time for DNase treatment and the processing time for washing treatment be 6 days or less.

[0037] If the amount of cellular components (cells and cellular components) remaining in the biological tissue before DNase treatment is taken as 100 (100%), it is preferable to reduce this amount to 90 (90%) or less after DNase treatment, and more preferably to 80 (80%) or less, 70 (70%) or less, 60 (60%) or less, 50 (50%) or less, 40 (40%) or less, 30 (30%) or less, 20 (20%) or less, 10 (10%) or less, 5 (5%) or less, or 1 (1%) or less.

[0038] The biological tissue may be subjected to decellularization treatment by pressurization, etc., followed by washing. Washing allows for more efficient removal of remaining cells and their components (specifically, DNA).

[0039] The cleaning process can be carried out by washing the biological tissue with a cleaning solution. More specifically, the biological tissue can be washed by immersing it in the cleaning solution and, if necessary, shaking it.

[0040] For washing, for example, PBS or physiological saline can be used. The washing solution may contain antibiotics such as penicillin or streptomycin. Furthermore, it is preferable that the washing solution contains EDTA (ethylenediaminetetraacetic acid). EDTA can chelate divalent ions (e.g., magnesium ions), thereby suppressing the activity of enzymes such as DNase (e.g., DNase derived from biological tissue or DNase treatment used in DNase treatment). By suppressing the activity of enzymes such as DNase, it is possible to achieve more strongly decellularized tissue.

[0041] The concentration of EDTA in the washing solution is not particularly limited, but for example, it may be between 1 mg / L and 1 g / L, between 10 mg / L and 1 g / L, or between 100 mg / L and 500 mg / L.

[0042] The processing time for the cleaning treatment is not particularly limited, but is preferably 6 days or less, more preferably 5 days or less, more preferably 4 days or less, and even more preferably 3 days or less. The processing time for the cleaning treatment may be, for example, 0.5 days or more, and may be 1 day or more.

[0043] The temperature of the washing process is not particularly limited, but for example, it is between 4°C and 37°C.

[0044] The timing of the washing treatment is not particularly limited and may be performed before the DNase treatment, but it is preferable to perform it after the DNase treatment.

[0045] If the amount of cellular components (cells and cellular components) remaining in the biological tissue before DNase treatment and washing is taken as 100 (100%), it is preferable to reduce this amount to 90 (90%) or less after DNase treatment, and more preferably to 80 (80%) or less, 70 (70%) or less, 60 (60%) or less, 50 (50%) or less, 40 (40%) or less, 30 (30%) or less, 20 (20%) or less, 10 (10%) or less, 5 (5%) or less, or 1 (1%) or less.

[0046] <Lyophilization process> In the freeze-drying process, the biological tissue, after the decellation process, is immersed in a freeze-dryable organic solvent and then freeze-dried.

[0047] It is preferable to dehydrate the biological tissue after the decellation process before immersing it in a freeze-dryable organic solvent. For example, the biological tissue is dehydrated by immersing it in a solution containing ethanol.

[0048] A solution containing ethanol may also contain water in addition to ethanol.

[0049] When immersing biological tissue in an ethanol-containing solution, it is preferable to first immerse it in a solution with a low ethanol concentration (e.g., 50% by mass), and then in a solution with a high ethanol concentration (e.g., substantially 100% by mass). This allows for sufficient replacement of the water in the biological tissue with ethanol. A substantially 100% by mass ethanol concentration means, for example, an ethanol concentration of 98.0% by mass or higher, preferably 99.0% by mass or higher, and more preferably 99.5% by mass or higher.

[0050] The immersion time of biological tissue in an ethanol-containing solution is not particularly limited, but it is preferably 10 minutes to 30 minutes for each concentration of ethanol-containing solution.

[0051] The immersion temperature of biological tissue in an ethanol-containing solution is not particularly limited, but it is preferably at room temperature (for example, 10°C to 35°C).

[0052] Preferably, the freeze-dryable organic solvents used to immerse dehydrated biological tissue after the decellularization process include tert-butyl alcohol, 1,4-dioxane, dimethyl carbonate, benzene, DMSO (dimethyl sulfoxide), 1,1,1,3,3,3-hexafluoro-2-propanol, 2-xylene (o-xylene), 4-xylene (p-xylene), tert-amyl alcohol, or mixtures thereof. It is preferable to use only tert-butyl alcohol as the freeze-dryable organic solvent.

[0053] The tert-butyl alcohol used to immerse the biological tissue after the decellation process is preferably tert-butyl alcohol with a concentration of substantially 100% by mass. A tert-butyl alcohol concentration of substantially 100% by mass means, for example, that the tert-butyl alcohol concentration is 98.0% by mass or higher, and preferably 99.0% by mass or higher. By immersing the biological tissue in tert-butyl alcohol with a concentration of substantially 100% by mass and cooling it (for example, to 5°C or below) to freeze the tert-butyl alcohol, the biological tissue after the decellation process can be sufficiently freeze-dried.

[0054] The immersion time of biological tissue in a freeze-dryable organic solvent is not particularly limited, but is preferably between 10 minutes and 30 minutes.

[0055] The immersion temperature of the biological tissue in a freeze-dryable organic solvent is not particularly limited, but it is preferably at room temperature (for example, 10°C to 35°C, preferably 25°C or higher).

[0056] Freeze-drying is carried out under reduced pressure. The freeze-drying pressure is, for example, 50 Pa or less, and preferably 30 Pa or less.

[0057] The freeze-drying time is not particularly limited, as long as it is sufficient for the biological tissue to dry completely, but is preferably between 5 and 20 hours, and between 8 and 15 hours.

[0058] <Alcohol treatment process> In the alcohol treatment process, the biological tissue after the freeze-drying process is immersed in a treatment solution containing at least one alcohol selected from alcohols with 2 to 4 carbon atoms, and then dried under reduced pressure.

[0059] Examples of alcohols containing 2 to 4 carbon atoms in the alcohol-containing treatment solution include ethanol, n-propanol, isopropyl alcohol, n-butanol, isobutanol, sec-butanol, and tert-butanol. The alcohol-containing treatment solution preferably contains ethanol.

[0060] The alcohol concentration in the alcohol-containing processing solution is preferably 50% by mass or more, more preferably 60% by mass or more, even more preferably 70% by mass or more, and particularly preferably substantially 100% by mass, from the viewpoint of having particularly excellent blood compatibility, such as being less likely to induce blood coagulation reactions. A substantially 100% by mass alcohol concentration means, for example, an alcohol concentration of 98.0% by mass or more, preferably 99.0% by mass or more, and more preferably 99.5% by mass or more.

[0061] The immersion time of the biological tissue in the alcohol-containing treatment solution is not particularly limited, but is preferably between 3 minutes and 30 minutes.

[0062] The immersion temperature of the biological tissue in the alcohol-containing treatment solution is not particularly limited, but room temperature (for example, 10°C to 35°C) is preferred.

[0063] The process involves immersing the tissue in an alcohol-containing treatment solution to allow the alcohol to penetrate the tissue after freeze-drying, followed by vacuum drying. The pressure for reduced-pressure drying is, for example, 50 Pa or less, and preferably 30 Pa or less.

[0064] The reduced-pressure drying time is not particularly limited, as long as it is sufficient for the biological tissue to dry completely, but is preferably between 3 minutes and 24 hours, and between 30 minutes and 12 hours.

[0065] The reduced-pressure drying temperature is not particularly limited, but room temperature (for example, 10°C to 35°C) is preferred.

[0066] Thus, decellularized tissue, produced by subjecting biological tissue to decellularization treatment, freeze-drying after immersion in a freeze-dryable organic solvent, and vacuum drying after immersion in at least one alcohol selected from alcohols with 2 to 4 carbon atoms, exhibits excellent blood compatibility, such as being less likely to induce blood coagulation and less likely to trigger blood coagulation reactions, as shown in the examples described later.

[0067] To create artificial tissues with high biocompatibility and low rejection rates, biological tissues are decellularized. However, this process can induce blood coagulation and increase the likelihood of thrombus formation. This is because decellularization exposes the collagen in the biological tissue, and platelets adhere to this exposed collagen. However, the decellularized tissue produced by the manufacturing method of this embodiment undergoes freeze-drying after decellularization, followed by immersion in alcohol with 2 to 4 carbon atoms and vacuum drying. As a result, platelet adhesion is suppressed, and blood compatibility, such as the ability to less induce blood coagulation reactions, can be improved. In the alcohol treatment process, it is hypothesized that the biotissue after freeze-drying is denatured by alcohol with 2 to 4 carbon atoms and vacuum drying, resulting in the suppression of platelet adhesion to collagen.

[0068] The decellularized tissue obtained by the above manufacturing method is used to prepare a concentrated platelet solution (PRP (platelet-rich plasma) solution, 8.0 × 10⁶) from arterial blood collected from miniature pigs, as shown in the <Quantification of Platelet Adhesion Behavior (Platelet Adhesion Range)> section of the examples described later. 7When 0.25 mL of (platelets / ml) was dropped onto the luminal surface of decellularized tissue (graft) hydrated with ethanol and phosphate buffer, and allowed to stand at 37°C for 1 hour, after washing with physiological saline and rhodamine staining, the attached platelets were observed at three locations using a confocal laser microscope, and the average area of ​​attached platelets at each observation site (platelet adhesion area) was, for example, 70,000 μm. 2 / mm 2 The following is true: 60,000 μm 2 / mm 2 Below 50,000 μm 2 / mm 2 Below 40,000 μm 2 / mm 2 Below 30,000 μm 2 / mm 2 or less than 25,000 μm 2 / mm 2 The following is also possible. Note that this "platelet adhesion area" is, as the unit suggests, 1 mm. 2 This represents the area of ​​attached platelets.

[0069] Furthermore, the above manufacturing method is a simple method in which the biological tissue after the decellularization process is immersed in a freeze-dryable organic solvent, freeze-dried, immersed in a treatment solution containing at least one alcohol selected from alcohols with 2 to 4 carbon atoms, and then dried under reduced pressure. Therefore, decellularized tissue can be easily produced.

[0070] Furthermore, according to the above manufacturing method, it is not necessary to use expensive chemical treatment agents such as synthetic peptides. Therefore, expensive chemical treatment agents and the washing process for such chemical treatment agents are unnecessary. In addition, uniform coating of the chemical treatment agent onto the biological tissue is unnecessary.

[0071] Furthermore, Patent Document 2 describes a process for freeze-drying biological tissue. However, the freeze-drying in Patent Document 2 is performed as a pre-treatment for heating, which involves dehydrating and condensing collagen to create crosslinks. Patent Document 2 does not consider improving blood compatibility (e.g., antithrombotic properties), such as making the tissue less susceptible to inducing blood coagulation reactions, by immersing the tissue in a treatment solution containing at least one alcohol selected from alcohols with 2 to 4 carbon atoms after freeze-drying, followed by vacuum drying.

[0072] Furthermore, the decellularized tissue after the alcohol treatment process is in a dry state. Therefore, it is possible to provide decellularized tissue in a lightweight form (dry state) with excellent storage stability, which facilitates product distribution and enables the realization of cardiovascular medical devices with excellent therapeutic outcomes, such as artificial blood vessels and artificial valve grafts, and has a very large ripple effect on the medical industry.

[0073] <Soaking process> The method for producing decellularized tissue according to this embodiment may include an immersion step, in which, after the alcohol treatment step, the decellularized tissue that has undergone the alcohol treatment step is immersed in a treatment solution containing ethanol, and then further immersed in a phosphate buffer. This immersion process hydrates the decellularized tissue after the alcohol treatment process, making it ready for transplantation. Note that this immersion step is optional.

[0074] The ethanol-containing treatment solution used in the immersion process may also contain water in addition to ethanol. It is preferable to immerse the ethanol-containing treatment solution in a treatment solution with a high ethanol concentration (e.g., substantially 100% by mass) followed by immersion in a treatment solution with a low ethanol concentration (e.g., 50% by mass). This allows for sufficient hydration after subsequent immersion in phosphate buffer. The pH of the phosphate buffer used in the immersion process is preferably 6.0 to 8.0, and more preferably 6.8 to 7.6.

[0075] After immersion in phosphate buffer, the transplant may be immersed in other transplant-suitable solutions such as physiological saline or heparinized saline, if necessary.

[0076] ≪How to use decellularized tissue≫ The decellularized tissue obtained by the above manufacturing method exhibits excellent blood compatibility, such as being less likely to induce blood coagulation reactions, making it useful as a transplant graft for animals such as humans, for example, as a cardiovascular medical device such as an artificial blood vessel or an artificial valve. The transplant may be performed on an organism different from the organism from which the tissue was sourced (e.g., a ratite), or on an organism of the same species as the organism from which the tissue was sourced.

[0077] Decellularized tissue produced without an immersion step after the alcohol treatment step is rehydrated by performing the same treatment as the immersion step when using the decellularized tissue, and then used by immersing the decellularized tissue in a solution suitable for transplantation.

[0078] Decellularized tissue produced without an immersion step after the alcohol treatment step may be provided as decellularized tissue produced without an immersion step after the alcohol treatment step, or it may be provided as a decellularized tissue transplantation kit that includes decellularized tissue produced without an immersion step after the alcohol treatment step, a treatment solution containing ethanol used in the immersion step, and a phosphate buffer used in the immersion step.

[0079] Decellularized tissue produced by performing an immersion process after an alcohol treatment process can be used directly for transplantation. [Examples]

[0080] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0081] <Graft fabrication> (Comparative Example 1) Carotid artery taken from the neck of an ostrich (inner diameter: 2-4 mm, lumen area in cross-section: 3.14-12.56 mm) 2 After washing the 80cm length specimens with physiological saline, they were packed in a plastic bag with physiological saline. The resulting packs were then subjected to decellation under pressure using a Kobelco ultra-high pressure processing device at 980 MPa, 30°C, and for 10 minutes. Blood vessels (i.e., the extracellular matrix constituting the carotid artery) after pressurized decellularization were immersed in physiological saline containing DNase 40U / ml, Penicillin 100 units / ml, Streptomycin 0.1 mg / ml, and MgCl2 20 mM at 37°C for 72 hours to decompose the cellular components remaining in the extracellular matrix. Next, the blood vessels were rinsed with a saline solution containing EDTA 500 mg / ml, Penicillin 100 units / ml, and Streptomycin 0.1 mg / ml. A graft (decellularized graft) of Comparative Example 1 was prepared by immersing the grafts in a saline solution of the same composition as the one used for rinsing at 37°C for 72 hours.

[0082] (Comparative Example 2) Decellularized grafts prepared in the same manner as in Comparative Example 1 were dehydrated by immersing them in 50% by mass ethanol aqueous solution, 65% by mass ethanol aqueous solution, 80% by mass ethanol aqueous solution, 90% by mass ethanol aqueous solution, 95% by mass ethanol aqueous solution, and substantially 100% by mass ethanol in that order, for 20 minutes each at room temperature (25°C). For substantially 100% by mass ethanol, ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, 99.5%) was used, and this was diluted with water to prepare 50% by mass ethanol aqueous solution, 65% by mass ethanol aqueous solution, 80% by mass ethanol aqueous solution, 90% by mass ethanol aqueous solution, and 95% by mass ethanol aqueous solution, respectively. Next, the decellatinated grafts were immersed in tert-butanol at a concentration of substantially 100% by mass for 20 minutes at room temperature, then replaced with fresh tert-butanol at a concentration of substantially 100% by mass, immersed for another 20 minutes, and then cooled to 4°C to freeze the tert-butanol. As the substantially 100% by mass tert-butanol, t-butanol (manufactured by Fujifilm Wako Pure Chemical Industries, 99.0%) was used. Subsequently, the graft for Comparative Example 2 (freeze-dried graft) was prepared by freeze-drying under a vacuum of 20 Pa overnight (12 hours). The freeze-dried grafts prepared were used in the <Quantification of Platelet Adhesion Behavior (Platelet Adhesion Range)> described later, after being immersed in ethanol and then rehydrated with phosphate buffer (pH 7.2-7.4). For this ethanol immersion, the grafts were immersed in substantially 100% by mass ethanol, 95% by mass ethanol aqueous solution, 90% by mass ethanol aqueous solution, 80% by mass ethanol aqueous solution, 65% by mass ethanol aqueous solution, and 50% by mass ethanol aqueous solution in this order, for 20 minutes each at room temperature (25°C). For substantially 100% by mass ethanol, ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, 99.5%) was used, and this was diluted with water to prepare 50% by mass ethanol aqueous solution, 65% by mass ethanol aqueous solution, 80% by mass ethanol aqueous solution, 90% by mass ethanol aqueous solution, and 95% by mass ethanol aqueous solution, respectively.

[0083] (Comparative Example 3) A carotid artery taken from the neck of an ostrich was washed with physiological saline solution and used as the graft for Comparative Example 3.

[0084] (Example 1) An ethanol aqueous solution was infiltrated into the lumen of a freeze-dried graft prepared in the same manner as in Comparative Example 2, until the entire freeze-dried graft was immersed in the ethanol aqueous solution. The immersion time for the entire freeze-dried graft in the ethanol aqueous solution was 20 minutes, and the immersion temperature (temperature of the ethanol aqueous solution) was 25°C. The concentration of the ethanol aqueous solution at this time was 75% by mass. The ethanol aqueous solution was prepared by diluting ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, 99.5%) with water. Grafts permeated with an ethanol aqueous solution were immersed in it, and then subjected to reduced-pressure drying at a vacuum of 20 Pa for 12 hours to produce the grafts of Example 1 (ethanol-treated grafts). The ethanol-treated grafts prepared were, as in Comparative Example 2, immersed in ethanol and then rehydrated with phosphate buffer for the purpose of determining platelet adhesion behavior (platelet adhesion range) described later.

[0085] (Example 2) An ethanol-treated graft (the graft of Example 2) was prepared in the same manner as in Example 1, except that the ethanol aqueous solution was changed to ethanol that was substantially free of water (manufactured by Fujifilm Wako Pure Chemical Industries, 99.5%; substantially 100% by mass ethanol). Furthermore, the ethanol-treated grafts prepared were used in the <Quantification of Platelet Adhesion Behavior (Platelet Adhesion Range)> described later, in the same manner as in Example 1, by immersing them in ethanol and then rehydrating them with phosphate buffer.

[0086] <Quantitative analysis of platelet adhesion behavior (platelet adhesion range)> The platelet adhesion behavior (platelet adhesion range) to the grafts of Comparative Examples 1-3 and Examples 1-2 was measured. Specifically, arterial blood was first collected from miniature pigs and a concentrated platelet solution (PRP (platelet-rich plasma) solution, 8.0 × 10⁴) was prepared. 7 A solution of platelets (particles / ml) was prepared. Next, the concentrated platelet solution was used to prepare the grafts of Comparative Examples 1-2, the luminal surface of Comparative Example 2 and the grafts of Examples 1-2 that had been rehydrated with phosphate buffer (cut into discs with a diameter of 8 mm (area: 50.24 mm²)). 2 0.25 mL was added to the sample and allowed to stand at 37°C for 1 hour. After washing with physiological saline, the sample was stained with rhodamine, and the attached platelets were observed using a confocal laser microscope (magnification: 10x). Three locations (observation field: 1.25 mm × 1.25 mm) were observed, and the area of ​​attached platelets at each observation site was measured. The average value of the three locations was calculated and defined as the platelet adhesion area. The results are shown in Figure 1. The unit of the vertical axis in Figure 1 is μm. 2 / mm 2 The standard deviation is also shown in Figure 1.

[0087] As shown in Figure 1, the grafts of Examples 1 and 2, which were prepared by following the following steps in order—decellularization, freeze-drying after immersion in a freeze-dryable organic solvent, and reduced-pressure drying after immersion in a treatment solution containing an alcohol with 2 to 4 carbon atoms—show significantly less platelet adhesion behavior (platelet adhesion range) than the grafts of Comparative Examples 1 to 3. Therefore, they can be said to have superior anticoagulant effects and are less likely to induce blood coagulation reactions.

Claims

1. A decellularization process for removing cells from biological tissue, The biological tissue after the decellation process is immersed in a freeze-dryable organic solvent, and then freeze-dried; A method for producing decellularized tissue, comprising: an alcohol treatment step of immersing the biological tissue after the freeze-drying step in a treatment solution containing at least one alcohol selected from alcohols having 2 to 4 carbon atoms, and then drying it under reduced pressure.

2. The method for producing decellularized tissue according to claim 1, wherein the decellularization treatment includes decellularization treatment by pressurization.

3. The method for producing decellularized tissue according to claim 1 or 2, wherein the concentration of the alcohol in the processing solution is 50% by mass or more.

4. The method for producing decellularized tissue according to any one of claims 1 to 3, wherein the biological tissue is a blood vessel or a valve.

5. A method for producing decellularized tissue according to any one of claims 1 to 4, further comprising an immersion step of immersing in a treatment solution containing ethanol after the alcohol treatment step, followed by immersion in a phosphate buffer.

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