阅读说明：本技术 脱细胞化组织制品的制备方法、及具备脱细胞化组织制品的移植片 (Method for producing decellularized tissue product, and graft having decellularized tissue product ) 是由 岸田晶夫 舩本诚一 桥本良秀 根岸淳 桑总一郎 江间崇夫 小林国治 于 2012-09-03 设计创作，主要内容包括：本发明提供能够一边抑制构成脱细胞化组织的支持组织的结构的变化,一边在脱细胞化组织中填充液体的脱细胞化组织制品的制备方法、及具备脱细胞化组织制品的移植片。脱细胞化组织制品的制备方法包括使来自动物的脱细胞化组织材料和液体在减压条件下接触的减压工序及/或在加压条件下接触的加压工序。另外,移植片具有通过该制备方法制备的脱细胞化组织制品。(The invention provides a method for producing a decellularized tissue product, which can fill a liquid into a decellularized tissue while suppressing a change in the structure of a supporting tissue constituting the decellularized tissue, and a graft having the decellularized tissue product. The method for producing a decellularized tissue product comprises a pressure reduction step of bringing a decellularized tissue material derived from an animal into contact with a liquid under reduced pressure and/or a pressure step of bringing the decellularized tissue material into contact under pressurized conditions. In addition, the graft has a decellularized tissue product prepared by the preparation method.)

1. A method for producing a decellularized tissue product, comprising a pressure-reducing step of bringing a decellularized tissue material derived from an animal, which has been freeze-dried and has a collagen structure, into contact with a liquid under reduced pressure and/or a pressure-increasing step of bringing the decellularized tissue material into contact under increased pressure, wherein the decellularized tissue material does not include a bone tissue material,

the pressure reducing step is followed by the pressurizing step,

the pressurizing step is a step of maintaining the atmospheric pressure at 0.1 to 100 atm when the atmospheric pressure is 0 atm.

2. The process according to claim 1, wherein the pressure-reducing step is carried out under a vacuum of 0 to-0.101 MPa.

3. The method according to claim 1 or 2, wherein the decellularized tissue material is a decellularized tissue which is immersed in the treatment solution and then freeze-dried.

4. The production method according to claim 1 or 2, wherein the liquid contains a functionality-imparting agent.

5. A graft to be transplanted into an animal, said graft comprising the decellularized tissue product produced by the production method according to claim 1 or 2.

Technical Field

The present invention relates to a method for producing a decellularized tissue product, and a graft having a decellularized tissue product.

Background

In recent years, in the field of transplantation medicine and the like, artificial materials having excellent compatibility with living tissues have been developed. As the artificial material, for example, a technique of using a decellularized tissue, which is a supporting tissue remaining after removing cells from a living tissue, as a graft is given (for example, patent document 1).

The supporting tissue (collagen, etc.) constituting the living tissue has voids of various sizes therein. Usually, the voids are filled with biomass, moisture, and the like. In recent years, attempts have been made to impart functionality to biological tissues by filling the voids with functional substances such as proteins, polysaccharides, enzymes, and synthetic polymers, and to use the resulting material as a biological tissue material.

As such an attempt, a method of immersing a living tissue in a liquid containing a functional substance or the like is known. However, in the method using immersion, a functional substance or the like is fixed only to the surface layer portion of the living tissue, and it is difficult to fill the whole living tissue up to the inside of the living tissue with a liquid.

Further, a method of filling a liquid into a living tissue by heat, microwave, ultrasonic wave, or the like is also conceivable, but in the case of this method, a supporting tissue constituting the living tissue is modified, destroyed, or the like, and thus it is difficult to use the method as a living tissue material.

Therefore, it is desired to develop a method capable of filling a liquid into a living tissue while suppressing a change in the structure of a supporting tissue constituting the living tissue.

Patent document 1: WO2008/111530 Specification

Disclosure of Invention

The purpose of the present invention is to provide a method for producing a decellularized tissue product, which can fill a liquid into a biological tissue while suppressing a change in the structure of a supporting tissue constituting the biological tissue, and a graft having the decellularized tissue product.

The present inventors have found that a tissue can be filled with a liquid while suppressing a structural change of collagen or the like constituting a biological tissue by a pressure reducing step of bringing a decellularized tissue material derived from an animal into contact with a liquid under a reduced pressure condition and/or a pressure increasing step of bringing a decellularized tissue material derived from an animal into contact with a liquid under a pressurized condition, and have completed the present invention. Specifically, the present invention provides the following.

(1) A method for producing a decellularized tissue product, comprising a pressure-reducing step of bringing a decellularized tissue material derived from an animal into contact with a liquid under reduced pressure and/or a pressure-increasing step of bringing the decellularized tissue material into contact under increased pressure.

(2) The production process according to (1), wherein the pressure-reducing step is carried out in a vacuum of 0 to-0.101 MPa.

(3) The production method according to (1) or (2), further comprising the pressurization step after the depressurization step.

(4) The production method of any one of (1) to (3), wherein the decellularized tissue material is a freeze-dried decellularized tissue.

(5) The method according to (4), wherein the decellularized tissue material is a decellularized tissue which is immersed in the treatment solution and then freeze-dried.

(6) The production method according to any one of (1) to (5), wherein the liquid contains a functionality-imparting agent.

(7) A graft to be transplanted into an animal, the graft comprising the decellularized tissue product produced by the production method according to any one of (1) to (6).

According to the present invention, the tissue can be filled with the liquid while suppressing the change in the structure of the supporting tissue constituting the biological tissue by the pressure reducing step of bringing the decellularized tissue material derived from the animal and the liquid into contact under the reduced pressure condition and/or the pressure step of bringing the decellularized tissue material derived from the animal and the liquid into contact under the pressurized condition.

Drawings

FIG. 1 is a cross-sectional view showing a cornea which has been restored by immersion or impregnation of a freeze-dried decellularized cornea.

FIG. 2(A) is a cross-sectional view showing an aorta after the decellularized aorta which has been freeze-dried is restored by immersion or impregnation. (B) The figure shows the water content of the aorta after the freeze-dried decellularized aorta is restored by immersion or impregnation.

FIG. 3(A) is a cross-sectional view showing a skin which has been restored by immersing or impregnating a freeze-dried decellularized skin. (B) Is a graph showing the water content of the skin after the freeze-dried decellularized skin has been restored by immersion or impregnation.

FIG. 4 is a cross-sectional view showing an aorta after the freeze-dried decellularized aorta has been immersed or impregnated with a heparin solution.

FIG. 5 shows the results of an elution test of heparin, which was performed after immersing or impregnating the freeze-dried decellularized aorta with a heparin solution.

FIG. 6 is a view showing a cross section of a cornea after the freeze-dried decellularized cornea was immersed or impregnated in β -dextran solution.

FIG. 7 is a view showing a cross section of a cornea which was restored by immersion from a freeze-dried decellularized cornea and transplanted into a Japanese white rabbit for one month. (A) Shows the results of staining based on hematoxylin-eosin staining. (B) Shows the results of staining based on Masson trichrome staining (Masson trichrome stain).

FIG. 8 is a cross-sectional view of an aorta in which a decellularized aorta which has been freeze-dried by changing the freezing conditions in the freeze-drying is immersed or impregnated in a rhodamine solution or a rhodamine-labeled PEG solution.

FIG. 9 is a cross-sectional view showing an aorta after the decellularized aorta which has been freeze-dried by changing the freezing conditions in the freeze-drying is impregnated with a heparin solution.

FIG. 10 is a view showing a cross section of a cornea which has been restored by impregnation from a decellularized cornea freeze-dried under a different freezing condition in freeze-drying.

Detailed Description

The following describes embodiments of the present invention, but the present invention is not limited to the embodiments.

< pressure reduction step and/or pressurization step >

The preparation method of the present invention includes a pressure reduction step of bringing a decellularized tissue material derived from an animal into contact with a liquid under reduced pressure and/or a pressure step of bringing the decellularized tissue material and the liquid into contact under pressure. Hereinafter, the state of the present invention in which a decellularized tissue material is filled with a liquid under reduced pressure or increased pressure is referred to as "impregnation".

(pressure reducing step)

In the supporting tissue (collagen or the like) constituting the decellularized tissue material, a large number of voids are present. Inside the void, moisture, biological substances, gas, and the like exist. In the production method of the present invention, when water or the like in the space in the support tissue constituting the decellularized tissue material comes into contact with the introduced liquid, a difference in osmotic pressure can be generated between the inside and the outside of the decellularized tissue material. In this case, under reduced pressure, the water or the like in the voids in the supporting tissue constituting the decellularized tissue material can be physically replaced with the introduced liquid. When the introduced liquid enters the inside of the void in the support tissue, the impregnation of the liquid into the decellularized tissue material is accelerated. In the production method of the present invention, the gas and the like in the space in the support tissue constituting the decellularized tissue material and the introduced liquid may be physically replaced under a reduced pressure. When the air in the decellularized tissue material is exhausted from the tissue under reduced pressure and reduced, the voids in the decellularized tissue material are in a reduced pressure state equivalent to the surrounding of the decellularized tissue material. It is assumed that the introduced liquid subsequently penetrates into the interstices in the decellularized tissue material.

By either or both of the above actions, the liquid can be satisfactorily immersed in the decellularized tissue material. In addition, according to the production method of the present invention, the decellularized tissue material can be uniformly impregnated with the liquid while suppressing the change in the structure of the supporting tissue constituting the decellularized tissue material by the above-described action. In addition, a liquid having low compatibility with the decellularized tissue material can be satisfactorily immersed in the decellularized tissue material. The above-described effect can be promoted by further performing the following pressurization step.

In the pressure reduction step, the pressure reduction may be performed at least once during the process of impregnating the decellularized tissue material with the liquid. For example, the decellularized tissue material may be brought into contact with a liquid by reducing the pressure of the atmosphere of the decellularized tissue material and injecting the liquid or the like while maintaining the reduced pressure; the decellularized tissue material may be brought into contact with a liquid by dipping or the like, and then reduced in pressure.

The pressure reduction condition is not particularly limited as long as it is a pressure lower than atmospheric pressure, and it is sufficient if a vacuum degree of 0 to-0.101 MPa can be maintained. The "degree of vacuum" in the present invention is expressed as gauge pressure. This value represents how close to an ideal vacuum state (absolute vacuum) the atmospheric pressure is zero. The ideal vacuum state in the present invention is a vacuum degree of-0.101 MPa, and the closer to this value, the closer to the ideal vacuum state. In the present invention, "maintaining" the pressure does not necessarily require that the pressure be in the above-mentioned range throughout the entire decompression step, and may be performed so long as the decellularized tissue material can be decompressed in the above-mentioned pressure range at least for a certain time (for example, 0.01 second to 24 hours). When the pressure is reduced in the above-mentioned pressure range, the infiltration of the liquid into the decellularized tissue material becomes uniform.

The pressure range may be appropriately adjusted according to the liquid used for impregnation. For example, when the decellularized tissue material contains moisture or the like, when the moisture or the like comes into contact with the introduced liquid, an osmotic pressure difference may be generated between the inside and the outside of the decellularized tissue material. In this case, when a pressure close to the absolute vacuum is used, there is a possibility that moisture, liquid, or the like vaporizes before the moisture, liquid, or the like is physically replaced with the introduced liquid by the osmotic pressure difference. The vaporized water or the like freezes on the surface of the decellularized tissue material, and may destroy the surface structure of the decellularized tissue material or interfere with impregnation of the liquid into the decellularized tissue material. Therefore, in this case, it is preferable to set the pressure to a pressure close to atmospheric pressure, to cool the liquid in advance, or to mix an additive or the like in advance for the purpose of adjusting the vapor pressure.

In addition, when the decellularized tissue material is a dried material, and when a liquid containing a polymer or the like is immersed in the decellularized tissue material, a pressure close to an absolute vacuum may be used as follows: while the decellularized tissue material rapidly absorbs the liquid, polymers and the like are deposited on the surface of the decellularized tissue material, and as a result, the supporting tissue of the decellularized tissue material is deformed, and impregnation of the liquid into the decellularized tissue material is hindered. In this case, in order to uniformly impregnate the decellularized tissue material with the liquid, it is preferable to set the pressure to a pressure close to atmospheric pressure, or to gradually bring the decellularized tissue material into a high vacuum state after contacting the liquid.

The decompression time is not particularly limited, and may be 1 second to 60 minutes, preferably 5 seconds to 10 minutes. According to the present invention, since the impregnation is performed under reduced pressure, the liquid is impregnated into the inside of the decellularized tissue material even if the contact time between the decellularized tissue material and the liquid is short in comparison with a method such as immersion under atmospheric pressure.

After the decellularized tissue material is brought into contact with the liquid under reduced pressure, the reduced pressure can be released, the pressure is increased to about atmospheric pressure (about 0.1MPa), and the decellularized tissue product is recovered. In this case, the impregnation of the liquid into the decellularized tissue material can be promoted by the increase in pressure.

(pressing step)

In the production method of the present invention, the introduced liquid is allowed to permeate into the voids of the decellularized tissue material under a pressurized condition. It is presumed that the liquid is pushed into the voids of the decellularized tissue material by the pressurization, and the liquid permeates into the voids. Thus, the decellularized tissue material is uniformly impregnated with the liquid while suppressing a change in the structure of the supporting tissue constituting the decellularized tissue material. In addition, even if the decellularized tissue material has low compatibility with the liquid, the liquid can be satisfactorily immersed in the decellularized tissue material.

As the pressurizing step of bringing the decellularized tissue material into contact with the liquid under a pressurized condition, a method of pressurizing at least once in the process of impregnating the decellularized tissue material with the liquid may be used. For example, the decellularized tissue material may be brought into contact with a liquid by pressurizing the atmosphere of the decellularized tissue material and injecting the liquid while maintaining the pressurized state, or the decellularized tissue material may be brought into contact with the liquid by immersion or the like and then pressurized.

The pressurizing condition may be a pressure higher than atmospheric pressure, and when the atmospheric pressure is 0 atm, the pressurizing condition may be a pressure capable of maintaining 0.1 to 10000 atm, preferably 0.1 to 1000 atm, and particularly preferably 0.1 to 100 atm. When the air pressure is 1000 atm or more, the cells of the resident bacteria are sufficiently destroyed, and the resident bacteria remaining in the decellularized tissue material are suppressed. The pressure may be appropriately adjusted according to the components contained in the liquid, the degree of impregnation of the liquid into the decellularized tissue material, and the like. The pressure is maintained as long as the decellularized tissue material can be pressurized in the above-described pressure range at least for a certain period of time (for example, 0.01 second to 24 hours). The pressurization may be performed by using a device such as a first type pressure vessel, a second type pressure vessel, a small-sized pressure vessel, etc., which are prescribed in japanese labor safety and health law, in accordance with the value of the pressure used for pressurization, etc.

The pressing time is not particularly limited, and may be 1 second to 24 hours, preferably 1 minute to 60 minutes. According to the present invention, since the impregnation is performed under a pressurized condition, the liquid is impregnated into the inside of the decellularized tissue material even if the contact time between the decellularized tissue material and the liquid is short as compared with a method such as immersion under atmospheric pressure.

The pressurizing temperature is not particularly limited as long as the decellularized tissue material is not modified, and may be-20 to 30 ℃ and preferably-10 to 25 ℃.

In the production method of the present invention, the pressure reduction step and the pressurization step may be combined. In the vacuum step and the pressurizing step, any step may be performed first, but when the pressurizing step is performed after the vacuum step, since the permeation of the liquid into the decellularized tissue material can be accelerated after the air in the decellularized tissue material is reduced, uniform impregnation can be rapidly achieved in the entire tissue, which is particularly preferable from the viewpoint of the ability to achieve uniform impregnation.

< acellular tissue material >

The decellularized tissue material can be prepared by a conventionally known method (for example, WO 2008/111530).

The decellularized tissue material may be maintained in a prepared state, or may be subjected to a normal preservation treatment (freeze-drying treatment, freezing treatment, drying treatment, or the like). According to the production method of the present invention, impregnation can be promoted by physical replacement of water, gas, or the like inside the voids in the support tissue constituting the decellularized tissue material with the introduced liquid in the reduced-pressure step, regardless of the presence or absence of preservation treatment of the decellularized tissue material. In particular, the decellularized tissue material is preferably a freeze-dried material in view of facilitating impregnation by physical replacement of the introduced liquid with a gas or the like inside the voids in the supporting tissue constituting the decellularized tissue material. In addition, if the decellularized tissue material is a freeze-dried material, even if a polymer or the like is contained in the liquid to be introduced, the polymer or the like is not easily deposited on the surface of the decellularized tissue material, and therefore the liquid can be satisfactorily immersed in the decellularized tissue material. According to the present invention, it is possible to restore a decellularized tissue material subjected to a preservation treatment while suppressing a change in the structure of the decellularized tissue material.

The method for freeze-drying the decellularized tissue material is not particularly limited, and the following methods may be mentioned: a method of freezing the decellularized tissue by dipping in liquid nitrogen, rapid freeze-drying by dry ice, slow freeze-drying at-80 to-4 ℃ using a FREEZER (manufactured by NIHON FREEZER Co., Ltd.), controlled freezing at-10 to-80 ℃ using a program FREEZER (program FREEZER), and the like, and then drying in a vacuum state.

The decellularized tissue material may be a material which is immersed in any solvent generally used for preservation of the decellularized tissue material and then freeze-dried, but a material which is immersed in a treatment solution and then freeze-dried is particularly preferable. Examples of the treatment solution include solutions containing sucrose, trehalose, lactose, maltose, disaccharides such as cellobiose, trisaccharides such as raffinose, melezitose and maltotriose, glycerol, ribitol, galactitol, xylitol, sorbitol, erythritol, mannitol and maltitol, either alone or in combination, or solutions obtained by dissolving them in water, a buffer solution, a physiological saline solution or the like. The treatment solution is capable of suitably removing water from the decellularized tissue material without binding to free water in the decellularized tissue material, causing an increase in osmotic pressure in the decellularized tissue material, and modifying and swelling proteins. This can suppress the change of the support tissue of the decellularized tissue material. By immersing the tissue material in the treatment solution before freeze-drying, the tissue material can be stored for a long period of time while suppressing the change in the original structure of the decellularized tissue material even after freeze-drying.

The impregnation in the solvent may be carried out at 4 to 30 ℃ for 30 minutes to 1 day.

The animal used as a source of the decellularized tissue material in the present invention is not particularly limited, and examples thereof include mammals such as pigs, cows, horses, goats, sheep, rabbits, kangaroos, monkeys, and humans.

The decellularized tissue in the present invention is not particularly limited, and examples thereof include organs such as cornea, heart valve, blood vessel, skin, cartilage, bone, tendon, muscle, bladder, small intestine, heart, liver, lung, trachea, esophagus, crystalloid, vitreous body, retina, nerve, adipose tissue, brain, dura mater, pleura, diaphragm, ureter, kidney, pancreas, gall bladder, gum, periodontal membrane, tooth, placenta, and genitalia.

< liquid >

The liquid to be impregnated into the decellularized tissue material by contacting it is not particularly limited as long as it is a liquid at the time of contacting. A liquid such as a solution, slurry, dispersion, or the like can be used.

The functional agent is not particularly limited, and examples thereof include low-molecular drugs (aspirin and the like), natural drug substances (antibiotics and the like), proteins (growth factors and the like), polymerizable monomers, polysaccharides (heparin, β -dextran and the like), nucleic acids (plasmids and the like), synthetic polymers (polyethylene glycol (PEG and the like), lipids (cholesterol and the like), surfactants and the like, and combinations thereof.

When a plurality of reactive substances (polymerizable monomers and the like) are contained as the functionality-imparting agent, the above reactive substances may be chemically reacted with each other after the acellular tissue material is impregnated with the functional material.

In the production method of the present invention, since the liquid can be satisfactorily impregnated into the decellularized tissue material regardless of the physical properties of the liquid, for example, a substance having a high viscosity (e.g., β -dextran), a substance having poor compatibility with the decellularized tissue material (e.g., a hydrophobic substance such as heparin), or a high molecular substance (e.g., polyethylene glycol) can be satisfactorily impregnated into the decellularized tissue material.

< decellularized tissue preparation >

Whether or not a decellularized tissue product in which a decellularized tissue material is impregnated with a liquid is obtained can be determined by measuring an index corresponding to the introduced liquid for the decellularized tissue product. For example, when an aqueous solution is impregnated into a decellularized tissue material, it is known to measure the water content of a decellularized tissue product or the like. In addition, when a specific substance is impregnated, it is known to stain a decellularized tissue product with a reagent capable of staining the substance.

< transplant & gt

The decellularized tissue product obtained by the production method of the present invention is useful as a structure of a graft to be transplanted into an animal. That is, the graft of the present invention has the above-mentioned decellularized tissue product.