阅读说明：本技术 用于制造脱细胞化的组织的方法、脱细胞化的组织、和用于制造脱细胞化的组织的设备 (Method for producing decellularized tissue, and apparatus for producing decellularized tissue ) 是由 筱原悟史 铃木章悟 鸟井昭吾 于 2018-03-29 设计创作，主要内容包括：一种用于制造脱细胞化的组织的方法,其包括如下步骤：使用包含液化气体的液体将生物组织的细胞裂解,和使用溶核酶将所述生物组织的裂解的细胞中包含的核酸组分降解。(A method for producing decellularized tissue comprising the steps of: lysing cells of a biological tissue using a liquid comprising a liquefied gas, and degrading nucleic acid components contained in the lysed cells of the biological tissue using a nucleolytic enzyme.)

1. A method for making decellularized tissue, the method comprising:

lysing cells of the biological tissue using a liquid comprising a liquefied gas; and

degrading nucleic acid components contained in the lysed cells of the biological tissue using a nucleolytic enzyme.

2. The method for making a decellularized tissue of claim 1, wherein said liquid further comprises a solvent.

3. The method for producing a decellularized tissue according to claim 2, wherein said solvent is at least one of water and ethanol.

4. The method for producing decellularized tissue of claim 1, wherein

The liquefied gas is at a temperature in the range of 1 ℃ to 40 ℃.

5. The method for producing decellularized tissue of claim 1, wherein

The liquefied gas is at a pressure in the range of 0.2MPa to 5 MPa.

6. The method for making decellularized tissue of claim 1, further comprising:

washing the biological tissue with degraded nucleic acid components.

7. The method for producing decellularized tissue of claim 6, wherein

Washing the biological tissue having degraded nucleic acid components with at least one of water, a physiological saline solution, an aqueous ethanol solution, and the liquid comprising liquefied gas.

8. The method for producing decellularized tissue of claim 7, wherein

The biological tissue having degraded nucleic acid components is washed with an aqueous ethanol solution or the liquid containing liquefied gas and then washed with water or a physiological saline solution.

9. A decellularized tissue comprising DNA in an amount less than 50ng/mg on a dry weight basis.

10. An apparatus for producing decellularized tissue, the apparatus comprising:

lysis means for lysing cells of the biological tissue using a liquid containing a liquefied gas; and

degradation means for degrading nucleic acid components contained in the lysed cells of the biological tissue using a nucleolytic enzyme.

Technical Field

The present invention relates to a method for producing a decellularized tissue, and an apparatus for producing a decellularized tissue.

Background

In the field of regenerative medicine, decellularized tissue produced by removing cellular components such as cytoplasmic, cytoskeletal and cell membrane components from biological tissue of a human or some other mammal is used as, for example, scaffold tissue implantation for regenerating failing (diseased, damaged) organs of a patient. Decellularized tissues are composed primarily of extracellular matrix components such as elastin, collagen (type I, type IV) and laminin.

A conventional method for producing a decellularized tissue includes decellularizing a biological tissue using a treatment liquid containing a surfactant (see, for example, patent documents 1 to 3). Specifically, the biological tissue is immersed in the treatment liquid containing the surfactant for several days while being agitated. However, decellularized tissue may be damaged because the surfactant causes degradation of proteins constituting components of the extracellular matrix. Moreover, decellularization using the above method takes time and residual surfactant may remain in the decellularized tissue.

In this regard, a method of decellularizing a biological tissue using supercritical carbon dioxide is known (see, for example, patent document 4).

However, in this method, a large amount of DNA remains in the decellularized tissue.

It is to be noted that the amount of DNA of the decellularized tissue is desirably less than 50ng/mg on a dry weight basis (see, for example, non-patent document 1).

CITATION LIST

Patent document

[ PTL 1] Japanese translation of PCT International application publication No. JP-T-2005-once 514971

[ PTL 2] Japanese translation of PCT International application publication No. JP-T-2006-507851

[ PTL 3] Japanese translation of PCT International application publication No. JP-T-2005-531355

[ PTL 4] Japanese unexamined patent application publication No.2007- & 105081

Non-patent document

[ NPL 1] biomaterials 2011, 4 months, 32(12): 3233-3243-

Disclosure of Invention

Technical problem

An object of the present invention is to provide a method for producing a decellularized tissue, which comprises: it is substantially free from damage and has an amount of DNA of less than 50ng/mg on a dry weight basis.

Solution to the problem

According to one aspect of the invention, a method for producing decellularized tissue comprises the steps of: lysing cells of a biological tissue (cellysis) using a liquid containing a liquefied gas, and degrading nucleic acid components contained in the lysed cells of the biological tissue using a nucleolytic enzyme.

Advantageous effects of the invention

According to one aspect of the present invention, there can be provided a method for producing a decellularized tissue as follows: it is substantially free from damage and has an amount of DNA of less than 50ng/mg on a dry weight basis.

Drawings

FIG. 1 is a flow diagram illustrating an exemplary method for producing decellularized tissue according to one embodiment of the invention.

FIG. 2 is a graph showing a saturated vapor pressure curve of dimethyl ether.

FIG. 3 is a schematic diagram illustrating an exemplary decellularization pretreatment apparatus according to an embodiment of the invention.

FIG. 4 is a schematic diagram illustrating another exemplary decellularization pretreatment apparatus according to an embodiment of the invention.

FIG. 5 is an optical micrograph of decellularized tissue stained with hematoxylin and eosin of example 1.

FIG. 6 is an optical micrograph of decellularized tissue stained with hematoxylin and eosin of example 2.

FIG. 7 is an optical micrograph of decellularized tissue stained with hematoxylin and eosin of example 3.

FIG. 8 is an optical micrograph of untreated biological tissue stained with hematoxylin and eosin.

Detailed Description

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings.

(method for producing decellularized tissue)

FIG. 1 illustrates an exemplary method for producing decellularized tissue according to one embodiment of the invention.

The method for producing decellularized tissue includes a step of lysing (destroying) cells of the biological tissue using a liquid containing a liquefied gas (step S1), and a step of degrading nucleic acid components contained in the lysed cells using a nucleolytic enzyme (nuclease) (step S2).

In step S1, for example, the biological tissue may be contacted with a liquid containing a liquefied gas to lyse (destroy) cells of the biological tissue and expose the nucleic acid component to the outside of the cells. In this way, the decellularized tissue can be substantially free of damage, and liquefied gas is less likely to remain in the decellularized tissue. In this embodiment, the liquid comprising liquefied gas causes the cell membrane components to dissolve and thus lyse (destroy) the cells.

In the present specification, a liquefied gas refers to a liquid form of a substance constituting a gas at a standard temperature and a standard pressure (0 ℃, 1atm (0.101325 MPa)).

Although the liquefied gas used in the present embodiment is not particularly limited as long as it can lyse cells of a biological tissue, for example, dimethyl ether, methyl ethyl ether, formaldehyde, ketene, acetaldehyde, propane, butane, liquefied petroleum gas, or a combination of two or more of the above may be used. Of the above, methylethyl ether and dimethyl ether may be suitably used in view of the fact that they can be liquefied at relatively low temperature and low pressure. In particular, dimethyl ether may be preferably used.

Dimethyl ether can be liquefied at about 1 ℃ to 40 ℃ and about 0.2MPa to 5MPa (see fig. 2), and therefore, relatively low-cost equipment can be used for production equipment for producing decellularized tissue. Moreover, since liquefied dimethyl ether is easily vaporized at standard temperature and standard pressure, it is less likely to remain in decellularized tissue.

In order to maintain the liquid state of the liquefied gas, for example, the process of step S1 may be performed in an airtight extraction tank under an environment of saturated vapor pressure or higher.

Although the method for bringing the biological tissue into contact with the liquid containing the liquefied gas is not particularly limited, for example, a suitable method may be selected based on the nature and properties of the biological tissue, such as a method of mixing and stirring the liquid containing the liquefied gas and the biological tissue, a method of immersing the biological tissue in the liquid containing the liquefied gas, or a method of circulating the liquid containing the liquefied gas and bringing it into contact with the biological tissue.

The liquid comprising liquefied gas may further comprise a solvent (entrainer).

Although the solvent used is not particularly limited, for example, ethanol, water, a physiological saline solution, PBS (phosphate buffered physiological saline solution), or a combination of two or more of the above may be used.

The solvent is preferably added in an amount adjusted to be less than or equal to its solubility in the liquefied gas. In this way, homogeneity of the liquid containing the liquefied gas can be achieved.

The temperature of the liquefied gas is preferably in the range of 1 ℃ to 40 ℃, and more preferably in the range of 10 ℃ to 30 ℃. When the temperature of the liquefied gas is in the range of 1 ℃ to 40 ℃, the cost of the decellularization pretreatment apparatus as described below can be reduced.

The pressure of the liquefied gas is preferably in the range of 0.2 to 5MPa, and more preferably in the range of 0.3 to 0.7 MPa. When the pressure of the liquefied gas is in the range of 0.2MPa to 5MPa, the cost of the decellularization pretreatment apparatus as described below can be reduced.

Although the biological tissue is not particularly limited, for example, the biological tissue may be: soft tissue obtained from a human or some other mammal, such as skin, blood vessels, heart valves, cornea, amnion, dura mater, or a portion thereof; an organ obtained from a human or some other mammal, such as the heart, kidney, liver, pancreas, brain or a part thereof; or connective tissue obtained from a human or some other mammal, such as bone, cartilage, tendon, or a portion thereof.

After the biological tissue is brought into contact with the liquid containing the liquefied gas and then the temperature and pressure are adjusted back to the standard temperature and standard pressure, the liquefied gas is vaporized and removed.

Note that, when cell lysis is insufficiently performed by performing the process (procedure) of step S1 only once, the process of step S1 may be repeated a plurality of times.

In step S2, for example, the biological tissue that has undergone the cell lysis process of step S1 may be contacted with a solution containing a nucleolytic enzyme to cause degradation of nucleic acid components exposed to the outside of the cell. As a result, the amount of DNA of the decellularized tissue on a dry weight basis may be less than 50 ng/mg.

Although the nucleolytic enzyme used in the present embodiment is not particularly limited as long as it can degrade DNA, for example, a deoxyribonuclease (e.g., deoxyribonuclease I) can be used.

Although a method for contacting the lysed cells with the solution containing the nucleolytic enzyme is not particularly limited, exemplary methods that can be used include a method of mixing and stirring a solution containing the nucleolytic enzyme and a biological tissue that has been subjected to a cell lysis process, a method of immersing a biological tissue that has been subjected to a cell lysis process in a solution containing the nucleolytic enzyme, and a method of circulating a solution containing the nucleolytic enzyme and contacting it with a biological tissue that has been subjected to a cell lysis process.

The method for contacting the lysed cells with the solution containing the nucleolytic enzyme may be appropriately selected based on the nature and properties of the biological tissue that has undergone the cell lysis process.

It is noted that in some embodiments, the process of step S2 may be included in the process of step S1. That is, for example, cells of a biological tissue can be lysed and nucleic acid components included in the cells can be degraded using a liquid containing a liquefied gas and a nucleolytic enzyme. In this case, for example, the solution containing the nucleolytic enzyme may be introduced while the biological tissue is brought into contact with the liquid containing the liquefied gas.

In a preferred embodiment, the method for producing a decellularized tissue further comprises a step of washing (washing) the biological tissue that has been subjected to the nucleic acid component degradation process (step S3).

In step S3, for example, the biological tissue that has undergone the nucleic acid component degradation process of step S2 may be contacted with a washing solution to be washed.

Examples of wash solutions include water, physiologically compatible liquids, aqueous solutions of physiologically acceptable organic solvents, liquids comprising liquefied gases, and the like.

Although the physiologically compatible liquid is not particularly limited, examples include a physiological saline solution, PBS (phosphate buffered physiological saline solution), and a combination of two or more of the above. In particular, for example, a physiological saline solution can be preferably used.

Although the physiologically acceptable organic solvent is not particularly limited, for example, ethanol or the like can be used.

The liquid containing liquefied gas may be the same as or different from the liquid containing liquefied gas used in step S1.

It is to be noted that in some embodiments, the process of step S3 may include, for example, washing the biological tissue that has undergone the nucleic acid component degradation process with an aqueous solution of a physiologically acceptable organic solvent or a liquid comprising a liquefied gas prior to washing the biological tissue with water or a physiologically compatible liquid.

Although a method for contacting the biological tissue that has been subjected to the nucleic acid component degradation process with the washing solution is not particularly limited, exemplary methods that can be used include a method of mixing and stirring the washing solution and the biological tissue that has been subjected to the nucleic acid component degradation process, a method of immersing the biological tissue that has been subjected to the nucleic acid component degradation process in the washing solution, and a method of circulating and contacting the washing solution with the biological tissue that has been subjected to the nucleic acid component degradation process.

The method of contacting the biological tissue having undergone the nucleic acid component degradation process with the washing solution may be appropriately selected based on the nature and properties of the biological tissue having undergone the nucleic acid component degradation process.

It is to be noted that the biological tissue which has been subjected to the degradation process of the nucleic acid component is preferably washed with a washing solution at a temperature of 4 ℃ to 40 ℃.

In the case of washing a biological tissue that has undergone a nucleic acid component degradation process with a liquid containing a liquefied gas, for example, in order to maintain the liquid state of the liquefied gas, the washing process is preferably carried out in an environment at least at a saturated vapor pressure, for example, in an airtight extraction tank.

It is to be noted that the treatment time of the process of washing the biological tissue having undergone the nucleic acid component degradation process with the washing solution is not particularly limited as long as the washing is performed for a sufficient period of time for sufficiently removing the enzyme used in step S2 and the cell components exposed to the outside of the cells in step S1.

It is noted that, in some embodiments, when a biological tissue that has undergone a nucleic acid component degradation process is washed with a washing solution, for example, the washing solution may be replaced and the washing process may be repeatedly performed.

By carrying out the method for producing a decellularized tissue according to the present embodiment, a decellularized tissue substantially free from damage and having an amount of DNA of less than 50ng/mg on a dry weight basis can be obtained. When the amount of DNA of the decellularized tissue is less than 50ng/mg on a dry weight basis, an immune response when the decellularized tissue is transplanted into a living body can be prevented.

(decellularization pretreatment apparatus)

The decellularization pretreatment apparatus used in the present embodiment is not particularly limited as long as it can lyse cells of a biological tissue using a liquid containing a liquefied gas.

Hereinafter, an exemplary case in which liquefied dimethyl ether is used as the liquid containing the liquefied gas will be described.

The decellularization pretreatment apparatus can lyse cells of the biological tissue by contacting the biological tissue with liquefied dimethyl ether, which is produced, for example, by pressurizing dimethyl ether to at least its saturated vapor pressure in an extraction tank. Also, the decellularization pretreatment apparatus may vaporize liquefied dimethyl ether by reducing the pressure to less than the saturated vapor pressure to remove the liquefied dimethyl ether from the biological tissue that has undergone the cell lysis process.

Specifically, the decellularization pretreatment apparatus comprises liquid transfer means (a) for transferring liquefied dimethyl ether from storage means (g) to contact means (b), contact means (b) for contacting the biological tissue with the liquefied dimethyl ether, and lead-out means (c) for leading out the liquefied dimethyl ether that has been in contact with the biological tissue from the contact means (b). Also, the decellularization pretreatment apparatus includes a separation means (d) which may be a separation tank for separating dimethyl ether by adjusting temperature and pressure or a membrane separation tank for separating dimethyl ether by membrane separation, and a condensation means (e) for condensing dimethyl ether by adjusting temperature and pressure. Further, the decellularization pretreatment apparatus includes gasification means (f) for gasifying liquefied dimethyl ether by adjusting temperature and pressure, storage means (g) for storing liquefied dimethyl ether, supply means (h) for supplying (replenishing) liquefied dimethyl ether, and detection means (i) for detecting temperature and pressure.

Although the liquid transport means (a) is not limited to a specific configuration as long as it can adjust the flow rate of the liquefied dimethyl ether, for example, an infusion pump or a heat drive mechanism may be used.

Hereinafter, a decellularization pretreatment apparatus suitable for carrying out the process of step S1 will be described.

FIG. 3 illustrates an exemplary configuration of a decellularization pretreatment apparatus 100 according to an embodiment of the invention.

It is to be noted that FIG. 3 is only a schematic illustration for facilitating understanding of the overall shape, size and arrangement of the constituent elements of the decellularization pretreatment apparatus according to the present embodiment. The present invention is not limited to the following description in any way, and constituent elements of the decellularization pretreatment apparatus may be appropriately modified or changed within the scope of the present invention.

The decellularization pretreatment apparatus 100 includes a storage tank 1 for storing liquefied dimethyl ether 2, an extraction tank 6 for bringing biological tissue 7 into contact with the liquefied dimethyl ether 2, a separation tank 11 for separating liquid led out from the extraction tank 6, and a pump 3 for transporting the liquefied dimethyl ether 2 from the storage tank 1 to the extraction tank 6. Further, the decellularization pretreatment apparatus 100 includes conduits 5, 10, 12, 14, 16, 19, and 20 for leading out or introducing (liquefied) dimethyl ether, and valves 4, 9, 13, 15, 18, and 21 for adjusting air pressure in each tank and controlling leading out and introducing of (liquefied) dimethyl ether. The pressure in the extraction tank 6 and the separation tank 11 can be adjusted to maintain the liquefied dimethyl ether in a liquid state.

In the decellularization pretreatment apparatus 100, the pump 3, the valve 4 and the conduit 5 for introducing the liquefied dimethyl ether 2 from the storage tank 1 to the extraction tank 6 serve as the liquid transport means (a). The extraction groove 6 serves as a contact means (b). A conduit 10 and a valve 9 for leading the liquefied dimethyl ether 2 out of the extraction tank 6 serve as the leading means (c). Further, the separation tank 11 serves as a separation means (d). The condenser 17 serves as the condensing means (e). The conduit 12 and the valve 13 connected to the separation tank 11 serve as the vaporization means (f). The storage tank 1 serves as a storage means (g). The conduits 19 and 20 serve as the supply means (h).

The decellularization pretreatment apparatus 100 may include additional constituent elements such as a thermometer and a manometer for detecting the temperature and pressure in each tank, a stirrer for stirring the contents of each tank, and a device for circulating an inert gas (e.g., nitrogen) to, for example, pump a reactive gas (e.g., oxygen) into the tank and the conduit.

Hereinafter, a method of implementing the process of step S1 using the decellularization pretreatment apparatus 100 will be described.

First, the biological tissue 7 is introduced into the extraction tank 6 having the filters 8 arranged at the upstream side and the downstream side thereof. At this time, the valves 4, 9, 13, 15, 18, 21, 22 are closed. It is to be noted that when a sufficient amount of liquefied dimethyl ether 2 is not stored in the storage tank 1, the valve 21 is opened and the liquefied dimethyl ether 2 is supplied to the storage tank 1 via the conduit 20, and thereafter the valve 21 is closed. At this time, the valve 18 may be opened and closed together with the valve 21. It is noted that liquefied dimethyl ether is produced by pressurizing dimethyl ether to at least its saturated vapor pressure (see fig. 2).

Then, the valve 4 is opened, and the liquefied dimethyl ether 2 in the storage tank 1 is taken out (pumped) by the pump 3, introduced into the extraction tank 6 via the conduit 5, and brought into contact with the biological tissue 7, after which the valve 4 is closed. As a result, phospholipids, which are main components of the cell membrane of the biological tissue 7, are dissolved, and the cells 7 of the biological tissue are lysed.

Then, the valves 4 and 9 are opened, and liquefied dimethyl ether is taken out from the extraction tank 6 by the pump 3 and introduced into the separation tank 11 via the conduit 10. As a result, the liquefied dimethyl ether having the phospholipids dissolved therein in the extraction tank 6 is introduced into the separation tank 11 via the conduit 10. At this time, since the filters 8 are arranged at the upstream side and the downstream side of the extraction tank 6, the biological tissue 7 that has undergone cell lysis remains in the extraction tank 6.

The timing of opening the valves 4 and 9 is controlled so that a predetermined period of time elapses from the time of introducing liquefied dimethyl ether into the extraction tank 6 to enable the liquefied dimethyl ether to come into contact with the biological tissue 7. It is to be noted that the liquefied dimethyl ether and the biological tissue 7 may be left standing for a predetermined period of time, or may be stirred, for example, while the liquefied dimethyl ether is in contact with the biological tissue 7.

Then, valve 4 is closed, valves 9, 13 and 22 are opened and the pressure is reduced to less than the saturated vapour pressure of dimethyl ether so that the liquefied dimethyl ether present between valve 4 and valve 13 is vaporised and discharged from conduit 23 via conduit 14. It is to be noted that, for discharging dimethyl ether, a pump may be used, for example, as required. As a result, the biological tissue 7 that has undergone cell lysis remains in the extraction tank 6, and the phospholipid remains in the separation tank 11.

It is noted that vaporized dimethyl ether is introduced into condenser 17 via conduit 16 when valve 22 is closed and valve 15 is open. As a result, liquefied dimethyl ether produced by the condensation of dimethyl ether can be reused.

Although the above has described the exemplary case where the liquefied dimethyl ether 2 in the storage tank 1 is discontinuously withdrawn, in other examples, the liquefied dimethyl ether 2 in the storage tank 1 may be continuously withdrawn.

Specifically, the valves 4 and 9 may be opened so that the liquefied dimethyl ether 2 in the storage tank 1 may be continuously introduced into the extraction tank 6 from the conduit 5, and the liquefied dimethyl ether having the phospholipid dissolved therein in the extraction tank 6 may be continuously withdrawn from the extraction tank 6 to the conduit 10. In this case, the internal structure of the extraction tank 6 is preferably configured such that the liquefied dimethyl ether is in contact with the biological tissue 7.

Also, it is noted that, in some embodiments, the decellularization pretreatment apparatus can be configured to: to liquefy (or vaporize) dimethyl ether, the temperature is adjusted instead of the pressure.

(nucleic acid component degradation apparatus)

The nucleic acid component degradation device used in the present embodiment is not particularly limited as long as it can degrade a nucleic acid component contained in a cell lysed by the decellularization pretreatment device using a ribozyme. For example, a known agitator may be used.

It is noted that in some embodiments, the nucleic acid component degradation device may be included in a decellularization pretreatment device. In this case, for example, while the biological tissue 7 is contacted with liquefied dimethyl ether in the extraction tank 6, a solution containing a nucleolytic enzyme may be introduced into the extraction tank 6 using, for example, a known method.

(washing apparatus)

The washing apparatus used in the present embodiment is not particularly limited as long as it can wash biological tissue that has undergone a nucleic acid component degradation process by the nucleic acid component degradation apparatus. For example, a known agitator may be used.

It is to be noted that, in the case where the biological tissue that has undergone degradation of the nucleic acid component is washed with liquefied dimethyl ether, for example, the decellularization pretreatment apparatus 100 may be used.