JP2020005622A - Method for producing decellularized tissues, and apparatus for producing decellularized tissues - Google Patents

Abstract

To provide methods for producing decellularized tissues, which can efficiently produce decellularized tissues.SOLUTION: A method for producing a decellularized tissue comprises: a step (S1) of degrading DNA contained in living tissue in which cells are destroyed, using a first processing solution; a step (S2) of delipidating the biological tissue in which the DNA has been degraded, using a second processing solution; and a step (S3) of washing the delipidated living tissue, using a third processing solution, at least one of the step (S1), the step (S2), and the step (S3) being carried out by a flow system.SELECTED DRAWING: Figure 1

Description

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

  In regenerative medicine, as a supporting tissue for regenerating a defective organ of a patient, a cellular tissue such as a cytoplasmic component, a cytosolic component, a cytoskeleton, or a cell membrane component is removed from a living tissue of a human or a heterologous mammal. Cellular tissue has been reimplanted. The decellularized tissue has as its main component an extracellular matrix component such as elastin, collagen (type I, type IV, etc.), laminin and the like.

  Patent Literature 1 discloses a process of destroying cells in a living tissue using an aqueous solution of polyethylene glycol, a process of decomposing DNA contained in the living tissue in which the cells have been destroyed by using DNase, and a process of using PBS. A method for producing a decellularized tissue including a step of washing a living tissue in which DNA has been degraded is disclosed (for example, see Patent Document 1).

  However, it takes a long time to degrade DNA contained in living tissue in which cells have been destroyed and to wash the living tissue in which DNA has been degraded using PBS, so that decellularized tissue can be efficiently produced. There is a problem that you can not.

  An object of the present invention is to provide a method for producing a decellularized tissue and an apparatus for producing a decellularized tissue, which can efficiently produce a decellularized tissue.

  One embodiment of the present invention provides a method for producing a decellularized tissue, wherein a first treatment solution is used to degrade DNA contained in living tissue in which cells have been destroyed, and a second treatment A second step of delipidating the living tissue in which the DNA has been decomposed using a liquid, and a third step of washing the delipidated living tissue using a third treatment liquid, And at least one of the second step and the third step is carried out in a flow-through manner.

  Another embodiment of the present invention provides a device for producing a decellularized tissue, wherein the first treatment solution is used to degrade DNA contained in living tissue in which cells have been destroyed, A second means for delipidating the biological tissue in which the DNA has been decomposed using the treatment liquid, and a third means for washing the delipidated biological tissue using the third treatment liquid, At least one of the first means, the second means, and the third means is of a flow type.

  According to one aspect of the present invention, it is possible to provide a method for producing a decellularized tissue and an apparatus for producing the decellularized tissue, which can efficiently produce the decellularized tissue.

It is a flowchart which shows an example of the manufacturing method of the decellularized tissue of this embodiment. It is the schematic which shows an example of the manufacturing apparatus of the decellularized tissue of this embodiment. It is the schematic which shows the cell disruption apparatus of an Example. It is the schematic which shows the manufacturing apparatus of the decellularized tissue of an Example.

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

(Method for producing decellularized tissue)
FIG. 1 shows an example of a method for producing a decellularized tissue according to the present embodiment.

  The method for producing a decellularized tissue includes a step (S1) of decomposing DNA contained in living tissue in which cells have been destroyed using a first treatment liquid, and a step of decomposing DNA using a second treatment liquid. The method includes a step (S2) of delipidating the decomposed living tissue and a step (S3) of washing the delipidated living tissue using the third treatment liquid, and includes the steps (S1), (S2), and ( At least one of S3) is performed in a flow-through manner. For this reason, a decellularized tissue can be efficiently produced.

  In the present specification and the claims, the flow system is a system in which a treatment liquid is subjected to treatment with a treatment liquid on a living tissue in a process in which the treatment liquid flows into and out of the contact tank. In this method, a living tissue is treated with a treatment liquid in the course of flowing through the contact tank.

  In the step (S1), for example, a liquid containing an enzyme or a surfactant is brought into contact with a living tissue in which cells have been destroyed to degrade DNA contained in the living tissue in which cells have been destroyed.

  Examples of the enzyme include, but are not particularly limited to, DNase (for example, DNase I), trypsin, and the like.

  Although it does not specifically limit as a surfactant, An ionic surfactant (for example, sodium dodecyl sulfate (SDS)), a nonionic surfactant (for example, Triton X-100) etc. are mentioned.

  Examples of DNase include DNaseI.

  The liquid is not particularly limited as long as it is physiologically compatible. Examples of the liquid include liquids containing water as a main solvent such as physiological saline and PBS (phosphate-buffered physiological saline). Is also good. Of these, physiological saline is preferred.

  The method of bringing the liquid containing DNase into contact with the living tissue in which the cells are destroyed is not particularly limited, and the method of mixing the living tissue in which the cells are destroyed with the liquid containing DNase and stirring the mixture may be used. A method of flowing a liquid containing DNase through a broken living tissue may be used. Among them, a method of flowing a liquid containing DNase through a living tissue in which cells are destroyed is preferable in that DNA can be efficiently degraded.

  When the living tissue in which cells are destroyed is mixed with a liquid containing DNase and stirred, the liquid containing DNase may be appropriately replaced.

  The temperature of the environment in which the liquid containing DNase is brought into contact with the living tissue in which the cells are destroyed is preferably 4 to 40 ° C. When the temperature of the environment in which the liquid containing DNase is brought into contact with the living tissue in which the cells are destroyed is 4 ° C. or more, damage caused by ice crystals of the extracellular matrix contained in the living tissue can be suppressed, and the temperature of 40 ° C. The following conditions can suppress the denaturation of the protein contained in the living tissue.

  Further, the temperature of the environment in which the liquid containing DNase is brought into contact with the living tissue in which the cells are destroyed is more preferably 25 to 37 ° C. Generally, DNase shows high activity in the range of 25 to 37 ° C, and can efficiently degrade DNA.

  In the step (S2), for example, the living tissue in which the DNA has been decomposed in step (S1) is brought into contact with a liquid containing an organic solvent to defatt the living tissue in which the DNA has been decomposed.

  In the present specification and claims, defatting refers to removing fat-soluble components from living tissue.

  The organic solvent is not particularly limited as long as it is a physiologically acceptable organic solvent capable of defatting the biological tissue in which DNA has been decomposed, and includes ethanol and the like, and two or more kinds may be used in combination. . Among them, ethanol is preferred because it is harmless to living tissues.

  The liquid is not particularly limited as long as it is physiologically compatible. Examples of the liquid include liquids containing water as a main solvent such as physiological saline and PBS (phosphate-buffered physiological saline). Is also good. Of these, physiological saline is preferred.

  The method for bringing the biological tissue in which the DNA is degraded into contact with a liquid containing an organic solvent is not particularly limited, but a method in which the biological tissue in which the DNA is decomposed is mixed with a liquid containing an organic solvent and agitated, A method of flowing a liquid containing an organic solvent through a living tissue may be used. Among these, a method of flowing a liquid containing an organic solvent through a living tissue in which DNA has been degraded is preferable because it can be efficiently defatted.

  When the living tissue in which the DNA is decomposed is mixed with a liquid containing an organic solvent and stirred, the liquid containing the organic solvent may be appropriately replaced.

  The temperature of the environment in which the living tissue in which the DNA has been degraded is brought into contact with a liquid containing an organic solvent is preferably 4 to 40 ° C. When the temperature of the environment in which the living tissue in which the DNA is degraded is brought into contact with a liquid containing an organic solvent is 4 ° C. or higher, damage caused by ice crystals of the extracellular matrix contained in the living tissue can be suppressed, and 40 ° C. or lower. In this case, denaturation of a protein contained in a living tissue can be suppressed.

  Step (S1) and step (S2) may be performed simultaneously, or the order of step (S1) and step (S2) may be reversed.

  In the step (S3), for example, the delipidated living tissue is brought into contact with a liquid in the step (S2) to wash the delipidated living tissue.

  In the present specification and claims, washing refers to removing water-soluble components from living tissue.

  The liquid is not particularly limited as long as it is physiologically compatible. Examples of the liquid include liquids containing water as a main solvent such as physiological saline and PBS (phosphate-buffered physiological saline). Is also good. Of these, physiological saline is preferred.

  The method of bringing the delipidated living tissue into contact with the liquid is not particularly limited, and examples thereof include a method of mixing and stirring the delipidated living tissue with the liquid, a method of flowing the liquid through the delipidated living tissue, and the like. Among these, a method of flowing a liquid through a delipidated living tissue is preferable because it can be efficiently washed.

  When the living tissue in which the DNA has been decomposed is mixed with a liquid and stirred, the liquid may be appropriately replaced.

  The temperature of the environment in which the delipidated living tissue is brought into contact with the liquid is preferably 4 to 40 ° C. When the temperature of the environment in which the delipidated living tissue is brought into contact with the liquid is 4 ° C. or more, damage due to ice crystals of the extracellular matrix contained in the living tissue can be suppressed. Can suppress denaturation of the protein contained in.

  Further, the method for producing a decellularized tissue according to the present embodiment includes dissolving a cell membrane component of the living tissue, and dissolving the cells of the living tissue as a step prior to the step of decomposing DNA contained in the living tissue in which the cells are destroyed. The method may further include a breaking step.

(Method of destroying cells in living tissue)
The method for disrupting cells of a living tissue is not particularly limited, but a method of contacting a living tissue with a liquid containing a liquefied gas, a mechanical method (ultrasonic waves, microbeads, freezing, etc.) (for example, JP-T-2006-523541) ), Chemical methods (acids, alkalis, surfactants, etc.), methods combining mechanical methods and chemical methods, and methods of permeating supercritical carbon dioxide into living tissues (for example, JP-A-6-218036) ) And the like. Among them, a method of contacting a liquid containing a liquefied gas with a living tissue is preferable, since the decellularized tissue is not substantially damaged and the liquefied gas hardly remains.

  Here, the liquid containing the liquefied gas dissolves cell membrane components, and thus can destroy cells of a living tissue.

  In the present specification and claims, the liquefied gas is a liquefied substance that is a gas at normal temperature and normal pressure (0 ° C., 1 atm (0.101325 MPa)).

  The liquefied gas is not particularly limited as long as it can destroy cells of a living tissue, and includes dimethyl ether, ethyl methyl ether, formaldehyde, ketene, acetaldehyde, propane, butane, liquefied petroleum gas, and the like. The above may be used in combination. Among these, ethyl methyl ether and dimethyl ether are preferable, and dimethyl ether is particularly preferable, in that liquefaction is performed at a relatively low temperature and low pressure.

  Since dimethyl ether is liquefied at 1 to 40 ° C. and about 0.2 to 5 MPa, the cost of the apparatus is reduced. In addition, liquefied dimethyl ether is easily vaporized at normal temperature and normal pressure, and therefore hardly remains in the decellularized tissue.

  When a living body tissue is brought into contact with a liquid containing a liquefied gas, the liquid state of the liquefied gas is maintained in an environment having a saturated vapor pressure or higher, such as in an airtight extraction tank, in order to maintain the liquid state.

  The method of bringing the liquid containing the liquefied gas into contact with the living tissue is not particularly limited, and examples thereof include a method of immersing the living tissue in the liquid containing the liquefied gas and a method of flowing the liquid containing the liquefied gas through the living tissue. Among these, a method of flowing a liquid containing a liquefied gas through a living tissue is preferable because cells can be efficiently destroyed.

  The liquid containing the liquefied gas may further contain a solvent.

  Although it does not specifically limit as a solvent, Ethanol, water, physiological saline, PBS (phosphate buffered physiological saline) etc. are mentioned, You may use 2 or more types together.

  It is preferable that the amount of the solvent added be equal to or less than the solubility in the liquefied gas. Thereby, the liquid containing the liquefied gas can be made uniform.

  The temperature of the liquefied gas is preferably 1 to 40C, more preferably 10 to 30C.

  The pressure of the liquefied gas is preferably from 0.2 to 5 MPa, and more preferably from 0.3 to 0.7 MPa.

  When the living tissue is brought into contact with a liquid containing a liquefied gas and then returned to normal temperature and normal pressure, the liquefied gas is vaporized, so that the liquefied gas can be easily removed.

  The step of bringing the living tissue into contact with the liquid containing the liquefied gas may be repeated a plurality of times.

(Living tissue)
Biological tissue means a tissue obtained from any of plants, fungi, archaea, eubacteria, or animals without a cell wall having a cell wall. Although not particularly limited, leaves, branches, trees, petals, stems, roots, pulp, pericarp, and seeds, and human or xenogeneic mammal-derived skin, blood vessels, heart valves, cornea, amniotic membrane, dura mater, and the like, or soft tissues thereof A part thereof includes an organ including a heart, a kidney, a liver, a pancreas, a brain, or a part thereof, and a bone, a cartilage, a tendon, a part thereof, and the like.

(Decellularized tissue manufacturing equipment)
The apparatus for producing a decellularized tissue of the present embodiment uses a first processing solution, a first means for decomposing DNA contained in living tissue in which cells are destroyed, and a second processing solution. And a second means for delipidating the biological tissue in which the DNA has been degraded, and a third means for washing the delipidated biological tissue using a third treatment solution, wherein the first means and the second means There is no particular limitation as long as at least one of the means and the third means is of a flow type.

  The apparatus for producing a decellularized tissue of the present embodiment includes a first storage unit that stores a first processing solution, a second storage unit that stores a second processing solution, and a third storage solution. It is preferable to further include a third storage means. The apparatus for producing a decellularized tissue according to the present embodiment includes a first liquid supply unit that supplies the first processing liquid from the first storage unit to the contact tank, and a second tank from the second storage unit. It is preferable to further include a second liquid sending means for sending the second processing liquid, and a third liquid sending means for sending the third processing liquid from the third storage means to the contact tank.

  Further, the apparatus for producing a decellularized tissue of the present embodiment uses a liquid containing a liquefied gas, a cell destruction means for destroying cells of a living tissue, and a fourth storage means for storing a liquid containing a liquefied gas. Preferably, the apparatus further includes a fourth liquid sending means for sending a liquid containing a liquefied gas from the fourth storage means to the contact tank. In this case, it is preferable that the cell disrupting means is of a flow type.

  Further, the apparatus for producing a decellularized tissue according to the present embodiment is in contact with each of the supplied treatment liquids, the contact means for bringing the living tissue in which the cells are destroyed into contact, and the living tissue in which the cells are destroyed. It is preferable that the apparatus further includes a recovery unit that recovers the processing liquid, a separation unit that removes contaminants mixed in the recovered processing liquid, and a fifth liquid transmission unit that transmits the processing liquid from the recovery unit to the separation unit. .

  Further, it is preferable that the apparatus for producing a decellularized tissue of the present embodiment further includes a temperature detection adjusting unit that detects and adjusts the internal temperature.

  FIG. 2 shows an example of an apparatus for producing a decellularized tissue according to the present embodiment.

  The decellularized tissue manufacturing apparatus 100 includes a tank 1 for storing a first processing liquid, a tank 5 for storing a second processing liquid, a tank 1 ′ for storing a third processing liquid, and a liquefied gas. It has a tank 1 '' for storing the liquid to be contained. Further, the decellularized tissue manufacturing apparatus 100 includes a pressurized pump 3 (for example, a syringe pump) for sending a first treatment liquid and a pump 6 (for example, a plunger pump) for sending a second treatment liquid. ), A pressurized pump 3 ′ (for example, a syringe pump) for sending a third processing liquid, and a pressurized pump 3 ″ (for example, a syringe pump) for sending a liquid containing a liquefied gas.

  In addition, the decellularized tissue manufacturing apparatus 100 was brought into contact with the contact bath 8 for contacting each of the supplied treatment liquids, the living tissue 9 in which the cells were destroyed, and the living tissue 9 in which the cells were destroyed. It has a collection tank 11 for collecting each processing solution.

  The flow rate of the processing liquid flowing through the contact tank 8 may not be uniform between the vicinity of the wall surface of the contact tank 8 and the central portion, and the extraction efficiency may be suppressed. Therefore, a member that promotes uniformity of the velocity distribution generated from the vicinity of the wall surface of the contact tank 8 to the center thereof may be provided on the end face on the upstream side of the contact tank 8.

  A member that promotes uniformity of the velocity distribution generated from the vicinity of the wall surface of the contact tank 8 to the central portion is not particularly limited, but a porous member having micropores is preferable from the viewpoint of the efficiency of uniformity of the velocity distribution.

  Further, the decellularized tissue manufacturing apparatus 100 includes separation means 15, 15 ', and 16 for removing contaminants mixed in each of the collected processing solutions.

  In addition, the decellularized tissue manufacturing apparatus 100 sends the first processing solution from which impurities are removed to the tank 1 and sends the second processing solution from which impurities are removed to the tank 5; It has a pump 12 (for example, a plunger pump) for sending the third processing liquid from which impurities have been removed to the tank 1 'or for transporting the vaporized liquefied gas to the condenser 15' '.

  Further, the decellularized tissue manufacturing apparatus 100 includes a temperature detection adjusting unit 17 that detects and adjusts the internal temperature.

  The decellularized tissue manufacturing apparatus 100 includes valves 2, 2 ′, 2 ″, 4, 4 ′, 4 ″, 7, 10, 13, 14, 14 ′, 14 ″ and a back pressure valve 18. And

  Next, a method for manufacturing a decellularized tissue using the decellularized tissue manufacturing apparatus 100 will be described.

  First, in the step (S1), the living tissue 9 in which cells are destroyed is introduced into the contact tank 8, and the tank 1 is filled with the first treatment liquid. Next, after the valves 2, 4, 7, and 10 are opened and the other valves are closed, the first processing liquid filled in the tank 1 is supplied to the contact tank using the pressurized pump 3. 8 The DNA of the living tissue 9 in which the cells are destroyed is decomposed by the action of DNase, for example. Next, the first treatment liquid containing the decomposed product of DNA is collected in the collection tank 11. At this time, by using a pressurized pump, it is possible to prevent cavitation of the first processing liquid having water as a main solvent into which air bubbles easily enter.

  Next, after the valve 14 is opened and the other valves are closed, the first processing liquid containing the recovered DNA degradation product is sent to the tank 1 using the pump 12. At this time, since the decomposed product of the DNA is removed by the separation means 15, the first processing liquid from which the contaminants have been removed can be collected in the tank 1. The first treatment liquid collected in the tank 1 is again passed through the contact tank 8 as it is, and can be used for DNA degradation of the living tissue 9 in which cells have been destroyed. Therefore, the DNA can be decomposed with a small amount of the first processing solution without replacing or adding the first processing solution.

  In the step (S2), the tank 5 is filled with the second processing liquid. Next, after the valves 7 and 10 are opened and the other valves are opened, the second treatment liquid filled in the tank 5 is circulated to the contact tank 8 using the pump 6, The biological tissue 9 in which is decomposed is degreased, and the second treatment liquid containing the removed lipid is collected in the collection tank 11. Next, after the valve 14 is opened and the other valves are closed, the second processing liquid containing the collected lipid is sent to the tank 5 using the pump 12. At this time, since the lipid is removed by the separation means 16, the second treatment liquid from which the contaminants have been removed can be collected in the tank 5. The second treatment liquid collected in the tank 5 can be again passed through the contact tank 8 as it is, and used for defatting the biological tissue 9 in which the DNA has been decomposed. Therefore, degreasing can be performed with a small amount of the second processing liquid without replacing or adding the second processing liquid.

  In the step (S3), the third processing liquid is filled in the tank 1 '. Next, after the valves 2 ', 4', 7, and 10 are opened and the other valves are opened, the third processing liquid filled in the tank 1 'is discharged using the pressurized pump 3'. Then, the delipidated biological tissue 9 is circulated through the contact tank 8 and washed, and the third treatment liquid containing the removed water-soluble compound is collected in the collection tank 11. At this time, by using a pressurized pump, it is possible to prevent the cavitation of the third processing liquid having water as a main solvent, in which air bubbles easily enter. Next, after the valve 14 'is opened and the other valves are closed, the third processing liquid containing the recovered water-soluble compound is sent to the tank 1' using the pump 12. At this time, since the water-soluble compound is removed by the separation means 15 ', the third treatment liquid from which impurities have been removed can be collected in the tank 1'. The third treatment liquid collected in the tank 1 'can be again passed through the contact tank 8 as it is, and used for washing the delipidized living tissue 9. Therefore, it is possible to wash with a small amount of the third processing liquid without replacing or adding the third processing liquid.

  In addition, the decellularized tissue manufacturing apparatus 100 can destroy cells of a living tissue using a liquid containing a liquefied gas, as described later. When cells are destroyed using a liquid containing a liquefied gas, the liquid containing the liquefied gas is vaporized, which may cause damage to the living tissue due to drying. When sending the liquid containing the liquefied gas, the pressurized pump 3 ″ is used. However, since the pressure can be adjusted by the back pressure valve 18, the vaporization of the liquid containing the liquefied gas can be adjusted and suppressed. In addition, it is possible to reduce damage to living tissues due to drying.

  In addition, cell destruction, DNA degradation, defatting, and washing can be continuously performed in a flow-through manner by the following operations.

  First, the living tissue 9 is introduced into the contact tank 8. Next, the tank 1 ″ is filled with a liquid containing a liquefied gas, the tank 1 is filled with a first processing liquid, the tank 5 is filled with a second processing liquid, and the tank 1 ′ is filled. Is filled with a third processing solution. Next, after the valves 2 ″, 4 ″, 7, and 10 were opened, the back pressure valve 18 was adjusted to a predetermined pressure, and the other valves were closed, the tank 1 ″ was filled. The liquid containing the liquefied gas is sent by the pressurized pump 3 ″. Next, the pressurizing pump 3 ″ is stopped, the valves 2 ″, 4 ″ are closed, and the liquid feeding is terminated. Next, the liquefied gas existing between the valve 4 ″ and the valve 14 ″ is vaporized by setting the valve 19 to the open state and making the pressure lower than the saturated vapor pressure of the liquefied gas. At this time, if necessary, the vaporized dimethyl ether may be discharged using a pump. Instead of opening the valve 19, the valve 14 '' is opened, and the vaporized liquefied gas is transported to the condenser 15 '' using the pump 12, liquefied, and reused. Is also good. Next, after the valves 2, 4, 7, and 10 are opened and the other valves are closed, the first processing liquid filled in the tank 1 is sent by the pressurized pump 3. At this time, by gradually reducing the set pressure of the back pressure valve 18 from the above-mentioned predetermined pressure to the atmospheric pressure, it becomes possible to suppress the vaporization of the liquefied gas remaining in the contact tank 8 and to dry the living tissue 9 by drying. Damage can be reduced. Next, the pressurizing pump 3 is stopped, the valves 2 and 4 are closed, and the liquid feeding is terminated. Next, the second processing liquid filled in the tank 5 is sent by the pump 6. Next, the pump 6 is stopped, and the liquid feeding ends. Next, the valves 2 'and 4' are opened, and the third processing liquid filled in the tank 1 'is sent by the pressurized pump 3'. Next, the pressurizing pump 3 'is stopped, the valves 2', 4 ', 7, and 10 are closed, and the liquid feeding is terminated.

  As described above, the processing time can be shortened by continuously performing cell destruction, DNA degradation, defatting, and washing in a flow system.

  Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the embodiments.

[Example 1]
(Destruction of swine-derived aorta cells)
The cells of the aortic tissue 55 derived from pigs were destroyed using the cell disruption apparatus shown in FIG.

  Specifically, swine-derived aortic tissue 55 was charged into the extraction tank 54. Next, the separation tank 60 was replaced with dimethyl ether in advance, and the valves 52, 53, 56, 58, and 59 were closed. Next, the set pressure of the back pressure valve 57 was set to 0.7 MPa, the valves 52, 53, 56, and 58 were opened, and liquefied dimethyl ether was passed using the pump 51, and the extraction tank 54 was filled with liquefied dimethyl ether. By the way, the valves 53 and 56 were closed, and the aortic tissue 55 derived from pig was immersed in liquefied dimethyl ether to extract cell membrane components such as phospholipids. Next, the valves 53 and 56 were opened, the flow rate of liquefied dimethyl ether was adjusted to 1 mL / min, and the extract was collected in the separation tank 60. Next, the valve 58 was closed, the separation tank 60 was removed from the apparatus, and the liquefied dimethyl ether was volatilized at atmospheric pressure in the fume hood.

  By the above operation, after contacting 60 mL of liquefied dimethyl ether with the aortic tissue 55 derived from pig, the valve 53 is closed, the valves 56, 58, and 59 are opened, and the pressure in the extraction tank 54 is set to atmospheric pressure. The liquefied dimethyl ether in the extraction tank 54 was volatilized and exhausted. As a result, swine-derived aortic tissue in which cells were destroyed was obtained.

(DNA degradation, delipidation and washing of aortic tissue derived from swine from which cells were destroyed)
Using the apparatus for producing a decellularized tissue shown in FIG. 4, DNA degradation, defatting, and washing of the aortic tissue 66 derived from pigs in which cells were destroyed were carried out in a flow-through manner.

Specifically, a swine-derived aortic tissue 66 in which cells were destroyed was charged into the contact tank 65. Next, the tank 61 is filled with a physiological saline solution containing 0.2 mg / mL of DNase I (manufactured by Roche Diagnostics) and 0.05 M of MgCl ₂ (manufactured by Wako Pure Chemical Industries, Ltd.). 62, 64, and 67 were opened, and the valve 71 was closed. Next, a physiological saline solution containing DNase I and MgCl ₂ filled in the tank 61 was circulated through the contact tank 65 at a rate of 1 mL / min by using the syringe pump 63 to pass the swine-derived aortic tissue 66 in which the cells were destroyed. The contained DNA was decomposed, and the decomposed DNA was recovered in the recovery tank 68.

  Next, the tank 69 was filled with physiological saline containing 80% by volume of ethanol, the valves 62 and 64 were closed, and the valves 67 and 71 were opened. Next, the physiological saline containing ethanol filled in the tank 69 is passed through the contact tank 65 at 1 mL / min using the plunger pump 70 to defatte the pig-derived aortic tissue 66 in which the DNA has been degraded, The removed lipid was collected in the collection tank 68.

  Next, after the physiological saline containing ethanol was discharged from the tank 69, the physiological saline was filled into the tank 69. Next, the physiological saline filled in the tank 69 is circulated through the contact tank 65 at 1 mL / min using the plunger pump 70 to wash the defatted swine-derived aortic tissue 66 and remove the removed porcine aortic tissue 66. The material was collected in the collection tank 68. As a result, a decellularized tissue of swine-derived aortic tissue was obtained.

[Comparative Example 1]
The DNA degradation, delipidation and washing of the aortic tissue 66 derived from pigs in which the cells were destroyed were performed in a batch system, that is, except that they were mixed with each treatment solution and shaken in the same manner as in Example 1. Of the aortic tissue was obtained.

  Table 1 shows the amount of DNA per dry mass of the decellularized tissue of the aortic tissue derived from pig and the untreated aortic tissue of the pig.

ここで、ＤＮＡは、フェノール・クロロホルム抽出により、ブタ由来の大動脈組織の脱細胞化組織及び未処理のブタ由来の大動脈組織から抽出した。また、ＤＮＡ量は、Ｑｕａｎｔ−ｉＴ ＰｉｃｏＧｒｅｅｎ ｄｓＤＮＡ Ａｓｓａｙ Ｋｉｔを用いて、２本鎖のＤＮＡ量を定量した。

Here, DNA was extracted from porcine-derived aortic tissue derived from swine-derived aortic tissue and untreated swine-derived aortic tissue by phenol-chloroform extraction. The amount of double-stranded DNA was quantified using Quant-iT PicoGreen dsDNA Assay Kit.

  From Table 1, it can be seen that the decellularized tissue of the aortic tissue derived from pig in Example 1 has a small amount of DNA per dry mass and can efficiently produce the decellularized tissue.

  In contrast, the decellularized tissue of the pig-derived aortic tissue of Comparative Example 1 was dried since the DNA degradation, defatting and washing of the pig-derived aortic tissue 66 in which the cells were destroyed were not carried out in a flow-through manner. Since the amount of DNA per mass is large, decellularized tissue cannot be produced efficiently.

[Example 2]
A decellularized tissue of swine-derived aortic tissue was obtained in the same manner as in Example 1 except that the degreasing and washing were performed in a batch manner.

  Specifically, the pig-derived aortic tissue 66 in which DNA had been degraded was placed in a physiological saline solution containing 80% by volume of ethanol, and then shaken and defatted. The defatted swine-derived aortic tissue 66 was placed in physiological saline, shaken, and washed.

[Example 3]
A decellularized tissue of swine-derived aortic tissue was obtained in the same manner as in Example 1, except that the DNA degradation and washing were performed in a batch system.

Specifically, swine-derived aortic tissue 66 in which cells were destroyed was treated with 0.2 mg / mL of DNase I (manufactured by Roche Diagnostics) and 0.05 M MgCl ₂ (manufactured by Wako Pure Chemical Industries). After that, the cells were shaken to decompose DNA contained in a pig-derived aortic tissue 66 in which cells were destroyed. The defatted swine-derived aortic tissue 66 was placed in physiological saline, shaken, and washed.

[Example 4]
A decellularized tissue of swine-derived aortic tissue was obtained in the same manner as in Example 1, except that DNA degradation and delipidation were performed in a batch system.

Specifically, swine-derived aortic tissue 66 in which cells were destroyed was treated with 0.2 mg / mL of DNase I (manufactured by Roche Diagnostics) and 0.05 M MgCl ₂ (manufactured by Wako Pure Chemical Industries). After that, the cells were shaken to decompose DNA contained in a pig-derived aortic tissue 66 in which cells were destroyed. Further, the aortic tissue 66 derived from pigs in which DNA was degraded was placed in a physiological saline solution containing 80% by volume of ethanol, and then shaken and defatted.

  Table 2 shows the amount of residual DNA, the amount of remaining lipid, and the amount of remaining water-soluble compound in the decellularized tissue of aortic tissue derived from pig.

実施例１のブタ由来の大動脈組織の脱細胞化組織は、ＤＮＡ残存量と同様にして、脱脂残存量及び水溶性化合物残存量も、比較例１のブタ由来の大動脈組織の脱細胞化組織よりも少なくなる。

The decellularized tissue of the swine-derived aortic tissue in Example 1 was also similar to the DNA-remaining amount in that the remaining amount of defatted and the remaining amount of the water-soluble compound were the same as those of the decellularized tissue of the swine-derived aortic tissue of Comparative Example 1. Is also reduced.

  The decellularized tissue of the swine-derived aortic tissue in Example 2 was subjected to DNA degradation in a flow-through manner, and the degreasing and washing were performed in a batch manner. Less than decellularized tissue.

  The decellularized tissue of the swine-derived aortic tissue in Example 3 was subjected to defatting in a flow-through manner, and the DNA degradation and washing were performed in a batch manner. Less than decellularized tissue.

  The decellularized tissue of the swine-derived aortic tissue of Example 4 was washed by a flow-through method, and the DNA degradation and defatting were performed by a batch method. Tissue is less decellularized than tissue.

  From the above, it can be seen that when at least one of the steps of DNA degradation, defatting, and washing is carried out by a flow system, a decellularized tissue can be efficiently produced.

1, 1 ', 1 ", 5 tank 2, 2', 2", 4, 4 ', 4 ", 7, 10, 13, 14, 14, 14', 14", 19 Valve 18 Back pressure valve 3 3 ', 3''pressurized pump 6, 12 pump 8 contact tank 9 living tissue 11 recovery tank 15, 15', 16 separation means 15 '' condenser 17 temperature detection adjustment means 100 decellularized tissue manufacturing apparatus

Japanese Patent Publication No. 2005-53355

Claims (12)

Using a first treatment solution, a first step of decomposing DNA contained in living tissue in which cells are destroyed,
A second step of delipidating the biological tissue in which the DNA has been decomposed, using a second treatment liquid;
A third step of washing the delipidated living tissue using a third treatment liquid,
A method for producing a decellularized tissue, wherein at least one of the first step, the second step, and the third step is performed by a flow system.   The method for producing a decellularized tissue according to claim 1, wherein the first treatment liquid is a liquid containing an enzyme or a surfactant.   The method for producing a decellularized tissue according to claim 1, wherein the second treatment liquid is a liquid containing an organic solvent.   The method for producing a decellularized tissue according to claim 3, wherein the organic solvent is ethanol.   The method for producing a decellularized tissue according to any one of claims 1 to 4, wherein the third treatment liquid is mainly composed of water. Using a liquid containing a liquefied gas, further comprising a fourth step of destroying cells of the living tissue,
The method for producing a decellularized tissue according to any one of claims 1 to 5, wherein the fourth step is performed under a predetermined environment in which the liquefied gas maintains a liquid state.   The method for producing a decellularized tissue according to claim 6, wherein the liquefied gas is liquefied dimethyl ether.   The method for producing a decellularized tissue according to claim 7, wherein the predetermined environment is an environment of 1 to 40C and 0.2 to 5 MPa. At least one of the first step, the second step, the third step and the fourth step is performed in a contact tank,
The method for producing a decellularized tissue according to any one of claims 6 to 8, wherein the contact tank has a porous member on an end surface on an upstream side.   The said 1st process, the said 2nd process, the said 3rd process, or the said 4th process is implemented at 4-40 degreeC, The Claims any one of Claims 6-9 characterized by the above-mentioned. A method for producing a decellularized tissue.   The method according to any one of claims 1 to 10, further comprising a step of removing contaminants from a processing solution used in at least one of the first step, the second step, and the third step. The method for producing a decellularized tissue according to claim 1. First means for decomposing DNA contained in living tissue in which cells have been destroyed, using a first treatment solution;
A second means for delipidating the biological tissue in which the DNA has been decomposed, using a second treatment solution;
Using a third treatment solution, including a third means for washing the delipidated biological tissue,
An apparatus for producing a decellularized tissue, wherein at least one of the first means, the second means, and the third means is of a flow type.

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