WO2009025398A1

WO2009025398A1 - Decellularizing solution, method of preparing decellularized tissue, graft and culture member

Info

Publication number: WO2009025398A1
Application number: PCT/JP2008/065475
Authority: WO
Inventors: Akio Kishida; Toshiya Fujisato; Tsuyoshi Kimura; Seiichi Funamoto
Original assignee: National University Corporation, Tokyo Medical And Dental University; Josho Gakuen Educational Foundation
Priority date: 2007-08-23
Filing date: 2008-08-22
Publication date: 2009-02-26
Also published as: JP2009050297A

Abstract

It is intended to provide a decellularizing solution, whereby decellularization can be sufficiently conducted while improving safety and production efficiency, and so on. A decellularizing solution for decellularizing a starting tissue originating in an animal which comprises an animal-origin serum or a serum derivative as the active ingredient. As the serum derivative, an immobilizing serum or one having been treated under ultrahigh hydrostatic pressure in a liquid is preferred. A decellularized tissue is prepared by immersing a starting tissue in the decellularizing solution as described above.

Description

Specification

Decellularized treatment solution, decellularized tissue preparation method, graft, and culture member

[0001] The present invention relates to a decellularized treatment solution for decellularizing an original tissue derived from an animal, a method for preparing a decellularized tissue using the decellularized treatment solution, and a graft and a culture comprising the decellularized tissue. It relates to members.

Background art

[0002] When transplanting a graft derived from another person's living tissue, rejection of the graft by the recipient tissue is a problem. Therefore, the development of artificial tissue is awaited as a solution to such problems.

[0003] Various synthetic polymers have been tried as materials for artificial tissues. However, since the compatibility between these materials and living tissue is low, dropout and infection may occur at the joint between the graft and living tissue.

[0004] Therefore, a technique has recently been developed in which a decellularized tissue, which is a supporting tissue that remains after removing cells from a living tissue that improves compatibility with the living tissue, is used as a graft.

Removal of cellular components, that is, decellularization is generally performed by washing the original tissue with a treatment solution containing a surfactant.

[0005] Conventionally, as surfactants, artificial compounds such as SDS, Triton X-100 (registered trademark), PEG, and PEO, and biological compounds such as sodium cholate have been used (for example, patents (Ref. 1, 2).

Patent Document 1: Japanese Translation of Special Publication 2005—514971

Patent Document 2: Japanese Translation of Special Publication 2006—507851

Disclosure of the invention

Problems to be solved by the invention

However, since many of the aforementioned artificial compounds have been shown to have dose-dependent cytotoxicity, it is desirable to remove them when used in the human body. As a result, the production efficiency of the graft is reduced. Also compatible with in-vivo substances such as collagen that make up living tissue Because sex is unknown, there is also concern that unexpected symptoms may occur after transplantation.

[0007] On the other hand, since a compound derived from a living body has low solubility in a solvent such as water, the content of the compound in the treatment liquid becomes small. For this reason, the penetration of the compound into the raw tissue in contact with the treatment solution becomes insufficient, and decellularization becomes insufficient. Alternatively, a long time is required for the penetration of the compound into the original tissue, and the production efficiency of the graft is reduced.

[0008] The present invention has been made in view of the above circumstances, and a decellularization treatment solution that can improve safety and production efficiency and can be sufficiently decellularized, and decellularization using the decellularization treatment solution. It is an object of the present invention to provide a tissue preparation method, and a graft and a culture member provided with the decellularized tissue.

Means for solving the problem

[0009] The present inventors have found that serum or serum derivatives have excellent permeability and decellularization ability, and have completed the present invention. Specifically, the present invention provides the following.

[0010] (1) A decellularization treatment solution for decellularizing an animal-derived raw tissue,

A decellularized treatment solution containing animal-derived serum or serum derivative as an active ingredient.

[0011] According to the invention of (1), since serum or serum derivative is used as an active ingredient, the original tissue is rapidly and sufficiently decellularized. In addition, since serum or serum derivatives are derived from animals, they are more biocompatible than human compounds. For this reason, even if it is not necessary to wash serum or serum derivatives, it takes a short time.

Therefore, the production efficiency and safety of the graft can be greatly improved and sufficient decellularization can be achieved.

[0012] It should be noted that “serum or serum derivative as an active ingredient” means any ingredient other than serum or serum derivative, as long as safety, production efficiency, and decellularization ability are not inhibited beyond an acceptable range. It may be contained. In addition, “serum derivative” refers to a serum that has been treated so that safety, production efficiency, and decellularization ability are not inhibited beyond acceptable limits.

[0013] (2) The decellularized treatment solution according to (1), wherein the serum derivative is one obtained by inactivating serum. [0014] Complement, which is a major component responsible for humoral immunity, exists in serum. If the amount of complement in the treatment solution is excessive, a considerable amount of complement remains in the decellularized tissue, and there is a concern that inflammation may occur after transplantation.

Therefore, according to the invention of (2), since serum has been inactivated, complement in serum is inactivated. Therefore, since inflammation after transplantation is suppressed, safety can be further improved.

[0015] (3) The decellularized treatment solution according to (1) or (2), wherein the serum derivative is a serum in which an ultrahigh hydrostatic pressure is applied.

[0016] Various foreign substances are considered to exist in the serum. If the amount of foreign matter in the treatment solution is excessive, a considerable amount of foreign matter remains in the decellularized tissue, and there is a concern that some trouble may occur after transplantation. On the other hand, if an artificial compound or the like is used to remove foreign substances, the safety of the serum derivative itself is impaired, and there is a concern that the safety of the decellularized tissue may be reduced.

Therefore, according to the invention of (3), since the ultrahigh hydrostatic pressure is applied to the serum in a liquid, the safety can be further improved.

[0017] (4) The decellularized treatment solution according to (3), wherein the ultrahigh hydrostatic pressure is 1000 atm or more.

[0018] When the pressure applied to the original tissue is insufficient, the destruction of the resident bacteria present in the original tissue becomes insufficient, and the resident bacteria may remain in the prepared decellularized soft tissue.

Therefore, according to the invention of (4), since the ultrahigh hydrostatic pressure is set to 1000 atmospheres or more, the resident bacteria are sufficiently destroyed, and the resident bacteria remain in the decellularized tissue. Therefore, safety can be further improved.

[0019] (5) A method for preparing a decellularized tissue obtained by decellularizing an original tissue derived from an animal, wherein the original tissue is a decellularized treatment solution according to any one of (1) to (4) A preparation method with a procedure for impregnation in it.

[6] (6) A graft transplanted into an animal,

(5) A graft comprising a decellularized tissue prepared by the preparation method described above.

[0021] (7) The graft according to (6), further comprising an adjacent tissue located on the decellularized tissue and adjacent to the original tissue in the animal.

[0022] (8) A decellularized tissue prepared by the preparation method according to (5), comprising: A culture member used for culturing cells of an adjacent tissue adjacent to the original tissue. The invention's effect

[0023] According to the present invention, since serum or serum derivative is used as an active ingredient, the original tissue is rapidly and sufficiently decellularized. In addition, since serum or serum derivatives are derived from animals, they have higher biocompatibility than artificial compounds. For this reason, even if it is not necessary to wash the serum or serum derivative, it takes a short time. Therefore, the production efficiency and safety of the graft can be greatly improved and sufficient decellularization can be achieved.

Brief Description of Drawings

FIG. 1 is a view showing a usage state of a decellularized treatment solution according to an example of the present invention.

FIG. 2 is a view showing a state of decellularization of the original tissue when a decellularization treatment solution according to an example of the present invention is used.

FIG. 3 is a view showing a state of decellularization of another original tissue when the decellularization treatment solution according to the example of the present invention is used.

BEST MODE FOR CARRYING OUT THE INVENTION

[0025] Embodiments of the present invention will be described below, but are not intended to limit the present invention.

[0026] <Decellularized treatment solution>

The decellularized treatment solution contains animal-derived serum or serum derivative as an active ingredient.

[0027] [Serum]

The animal from which the serum is derived is not particularly limited, and examples thereof include humans, rabbits, and pigs. In order to produce a graft for humans, human-derived serum is preferable in that an inflammatory reaction caused by complement or the like can be suppressed.

[0028] [Serum derivatives]

As described above, a serum derivative refers to a product obtained by subjecting serum to a treatment that does not inhibit the safety, production efficiency, and decellularization ability beyond an acceptable range. Examples of such treatment include, but are not limited to, deactivation, application of ultrahigh hydrostatic pressure, dilution, and the like. In addition, high-temperature and high-pressure sterilization such as autoclave and irradiation with 0-ray for a long time are not applicable to the above treatment. [0029] (Deactivation)

Inactivation can be achieved by heating to inactivate complement by heating, usually at about 56 ° C for about 30 minutes. By deactivating, inflammation after transplantation is suppressed, so safety can be further improved.

[0030] (Application of ultra-high hydrostatic pressure)

The application of ultra-high hydrostatic pressure is a process that destroys cells, resident bacteria, and viruses in the original tissue by applying ultra-high hydrostatic pressure to serum in a liquid. The intensive treatment ensures a very high level of safety, so that serum after an inappropriate period of time can be used for transfusion applications. Since such serum can be obtained at low cost, the manufacturing cost of the graft can be reduced while maintaining safety.

[0031] The ultra-high hydrostatic pressure refers to a hydrostatic pressure that can destroy normal bacteria present in serum, and specifically, it is preferably 1000 atm or higher. In addition, the ultrahigh hydrostatic pressure is 4,000 atmospheres or more, for example, 10,000 atmospheres, in that it can sufficiently destroy viruses more preferably 4000 atmospheres or more in terms of sufficiently destroying bacteria present in serum. Is more preferable.

[0032] As a specific procedure, for example, first, serum is filled in a bag of a water-impermeable film. Then, the bag is tightly sealed while taking care not to leave any gas inside. This bag is placed in the liquid in the chamber of the ultra-high hydrostatic pressure treatment device (eg “Dr. CHEF (model)” (Kobe Steel)) and the device is operated.

[0033] The application time is not particularly limited as long as a desired cell destructive property can be obtained, but it may usually be about 10 to 30 minutes.

[0034] At this time, it is preferable to keep the temperature of the serum at or above the melting point of the serum at an ultrahigh hydrostatic pressure. As a result, even under conditions where the pressure has been increased to an ultrahigh hydrostatic pressure, coagulation of serum can be suppressed, and deterioration of the characteristics of the prepared serum can be suppressed.

[0035] As a specific procedure, first, the melting point of serum at a preset ultrahigh hydrostatic pressure value is calculated. Then, the ultra-high hydrostatic pressure processing device may be controlled so that the temperature in the chamber is equal to or higher than the calculated melting point. The serum temperature may be fixed at a fixed value, or may be varied within the range above the melting point of the serum.

[0036] Further, in a procedure for limiting the applied pressure applied to the serum to be equal to or higher than the melting pressure of the serum. is there. When the applied pressure is increased or decreased, the serum temperature may change suddenly and the characteristics of the serum may be greatly impaired. Therefore, the serum melting pressure is calculated in advance, and in particular, the rapid change in serum temperature is suppressed by reducing the applied pressure increase and κ or pressure decrease rate to a predetermined value or less. As a result, coagulation of serum during the process of pressurization and hypotension is suppressed, and a decrease in serum characteristics can be further suppressed.

[0037] Since the inactivation and application of ultra-high hydrostatic pressure have different purposes for removal, the combination of both may improve safety and decellularization ability.

[0038] (Dilution)

Dilution is performed with water or buffer. Since serum (especially human serum) is expensive, the cost of producing a graft can be reduced by using a serum derivative in which serum is diluted.

[0039] The buffer solution is not particularly limited, and a conventionally known buffer solution such as a PBS aqueous solution, a HEPES buffer solution, or a MES buffer solution can be used.

[0040] The dilution ratio may be appropriately set so that the cell can be decellularized within an allowable time in accordance with the thickness, characteristics, and the like of the original tissue to be decellularized. Specifically, when decellularizing soft tissues such as pericardium, amniotic membrane, and skin, it is sufficient to dilute so that the serum concentration is 0.1 to 2% by mass. When decellularizing soft tissue, the serum concentration should be from 2 to 10% by mass. When decellularizing bulky tissue such as myocardium and hard tissue such as bone, the serum concentration is 20%. What is necessary is just to dilute so that it may become the mass%.

[0041] The mechanism by which the above serum or serum derivative has excellent permeability and decellularization ability is presumed as follows. Serum plays a role in removing unnecessary cells in tissues as appropriate in vivo, and as a result of various ingredients being balanced, it contains a large amount of known active ingredients such as albumin and sodium cholate. To do. Indeed, it is difficult to achieve serum concentrations of albumin and sodium cholate under artificial conditions. However, serum contains not only the above known ingredients but also unknown active ingredients. It is presumed that the exquisite synergism of these unknown components and known components provides excellent permeability and decellularization ability.

[0042] Preparation Method for Decellularized Tissue> The above decellularized treatment solution is used for the preparation of decellularized tissue. Specifically, the original tissue is impregnated in a decellularization treatment solution. The decellularized solution rapidly penetrates into the original tissue, destroys cells in the original tissue, and dissolves cell components. By convection or circulation of the decellularized treatment solution, removal of cell debris is promoted, and an immune reaction caused by the remaining debris can be suppressed. Further, a step of washing with beak water or a buffer solution that promotes removal of cell debris may be further provided.

[0043] As described above, the original tissue is not particularly limited, and examples thereof include soft tissues such as pericardium, amniotic membrane, and skin, moderate soft tissues such as blood vessels and cartilage, bulky tissues such as myocardium, and bones. Hard tissue. The impregnation time may be set as appropriate according to the combination of the raw tissue and the decellularization solution used.

[0044] The raw tissue may or may not be an ultrahigh hydrostatic pressure applied in a liquid. Although depending on the type of the original tissue, the original tissue to which no ultra-high hydrostatic pressure is applied may be sufficiently decellularized using the decellularization treatment solution of the present invention. However, the application of ultra-high hydrostatic pressure is thought to be effective in removing abnormal prions and retroviruses mixed in the original tissue.

[0045] The preservation method of the decellularized tissue thus prepared is not particularly limited as long as it is sterilized, and may be frozen, wet in liquid, or dried. It is an advantage of the decellularized tissue of the present invention that the preservation method is not limited.

[0046] <Graft>

The decellularized tissue thus prepared is useful as a component of a graft to be transplanted into an animal. That is, the graft of the present invention comprises the aforementioned decellularized tissue. In addition, the graft may include an adjacent tissue on the decellularized tissue in which the original tissue from which the decellularized tissue is derived is adjacent in the animal body. For example, when the original tissue from which the decellularized tissue is derived is the cornea, the adjacent tissue is the corneal epithelium or corneal endothelium.

<0047> Culture member

The decellularized tissue of the present invention is also useful as a culture material used for culturing cells of adjacent tissues. That is, the culture member of the present invention comprises the decellularized tissue described above, and cells derived from adjacent tissues are placed on the decellularized tissue under appropriate conditions. By culturing, cell culture can be performed without using a special apparatus and suppressing infection.

Example

[0048] Pig-derived aorta was purchased from an edible pig farm and transported at 4 ° C. The aorta was cut into lcm pieces and wetted in a polyethylene film bag filled with PBS solution. This bag was placed in a chamber of “Dr. CHEF” (manufactured by Tohko), and a hydrostatic pressure of 6000 atmospheres was applied for 10 minutes while maintaining the temperature at 25 ° C. During this time, "Dr. CHEFJ was controlled so that the pressure increase and pressure decrease speeds were 2000 atm. Z. The aorta (original tissue) after application was removed by a clean operation, and the decellularization solution shown in A to D below. (Refer to Fig. 1.) As shown in Fig. 1, only treatment liquid D was cloudy.

Treatment liquid A: 100% FBS

Treatment liquid B: Supernatant obtained by applying ultrahigh hydrostatic pressure of 10,000 atm at 10 ° C for 10 minutes to 100% FBS and then centrifuging at 3500 rpm for 10 minutes

Treatment solution C: Supernatant obtained by heating 100% FBS at 56 ° C for 30 minutes and then centrifuging at 3500 rpm for 10 minutes

Treatment solution D: Supernatant obtained by sterilizing 100% FBS at 121 ° C and 2 atm with high-temperature steam and centrifuging at 3500i: pm for 10 minutes

[0049] The impregnation was continued for 11 days while changing the treatment solution every two days. Thereafter, the major artery was collected, and the section was stained with hematoxylin and eosin according to a conventional method. Figure 2 shows the results. In the figure, cells appearing as spots are cells. Note that the control in FIG. 2 is the aorta before impregnation with the treatment liquids A to D.

[0050] As shown in FIG. 2, in the aorta impregnated with the treatment solutions A to C, cells started to decrease after 7 days, and most of the cells disappeared after 11 days. As a result, it was confirmed that by using a treatment solution containing animal-derived serum or serum derivative, decellularization can be sufficiently performed in a relatively short period of 11 days.

[0051] On the other hand, in the aorta impregnated with the treatment solution D, the cells were not decreased much even after 11 days. From this, it was confirmed that high-temperature and high-pressure sterilization treatment of serum may impair the decellularization effect of serum. [0052] The aorta, to which no hydrostatic pressure of 6000 atmospheres was applied, was impregnated with the treatment liquid C under the same conditions as described above. The aorta after impregnation was collected, and the section was stained with hematoxylin.eosin according to a conventional method. The results are shown in Fig. 3.

[0053] As shown in Fig. 3, most of the cells in the aorta disappeared 5 days after the impregnation. Compared with Fig. 2, the 5th day was much shorter than the 11th day in the aorta, which was applied with a hydrostatic pressure of 6000 atmospheres. Therefore, it was confirmed that ultra-high hydrostatic pressure is applied, and the force of the raw tissue and the original tissue may be sufficiently decellularized using the decellularization treatment solution of the present invention.

Claims

- The scope of the claims

[1] A decellularization treatment solution for decellularizing an animal-derived raw tissue,

[2] The decellularization treatment solution according to [1], wherein the serum derivative is one obtained by inactivating serum.

[3] The decellularized treatment solution according to claim 1 or 2, wherein the serum derivative is a serum applied with an ultrahigh hydrostatic pressure in a liquid.

4. The decellularized treatment solution according to claim 3, wherein the ultrahigh hydrostatic pressure is 1000 atm or more.

[5] A method for preparing a decellularized tissue in which an original tissue derived from an animal is decellularized,

A preparation method comprising a step of impregnating the raw tissue in the decellularized treatment solution according to any one of claims 1 to 4.

[6] A graft to be transplanted into an animal,

A graft comprising the decellularized tissue prepared by the preparation method according to claim 5.

7. The graft according to claim 6, further comprising an adjacent tissue located on the decellularized tissue and adjacent to the original tissue in the animal.

8. A culture member comprising the decellularized tissue prepared by the preparation method according to claim 5, and used for culturing cells of an adjacent tissue adjacent to the original tissue in the animal.