JP2019136011A - Method for producing decellularized carrier, decellularized carrier, cell filling method, cell sheet production method, and decellularized solution kit - Google Patents

Abstract

To provide decellularized carrier production methods capable of producing a decellularized carrier with high performance.SOLUTION: A decellularized carrier production method of the present invention comprises a cryopreservation step, a thawing step, and a decellularization step. The cryopreservation step cryopreserves a removed organ or tissue. The thawing step thaws the cryopreserved organ or tissue in the cryopreservation step. The decellularization step decellularizes the thawed organ or tissue in the thawing step. Here, the decellularization step comprises a poorly water soluble surfactant perfusion step and a hydrophilic water soluble surfactant perfusion step. The poorly water soluble surfactant perfusion step perfuses the thawed organ or tissue with a poorly water soluble surfactant-containing solution. The hydrophilic water soluble surfactant perfusion step perfuses the organ or tissue perfused with a poorly water soluble surfactant-containing solution, with a hydrophilic water soluble surfactant-containing solution.SELECTED DRAWING: Figure 10

Description

  The present invention particularly relates to a method for producing a decellularized carrier, a decellularized carrier, a cell filling method, a cell sheet preparation method, and a decellularized solution kit.

  Conventionally, techniques relating to decellularization have been developed to remove cells from organs and tissues and to produce decellularized carriers made of extracellular matrix. Such a decellularized carrier is used as a scaffold for growing cells (scaffold) for transplantation into a living body, or used as a biomaterial for research.

Here, referring to Patent Document 1, as a conventional decellularization carrier, an extracellular matrix includes an outer surface, and the extracellular matrix including a vascular tree substantially forms the form of the extracellular matrix before decellularization. Decellularized mammalian organs are described that are retained and the outer surface is substantially intact.
The decellularized carrier of Patent Document 1 includes a step of creating a cannulated organ by inserting a cannula into the organ in one or a plurality of cavities, blood vessels, and / or tubes; and a cannula Perfusing a first cell disruption medium through one or more cannula insertions into the organ into which is inserted. The cell disruption medium includes at least one surfactant. The surfactant is selected from the group consisting of SDS, PEG, or Triton X.

JP2015-164549A

  However, the conventional decellularized carrier according to Patent Document 1 has a weak cytoskeleton and extracellular matrix, and the decellularized carrier is atrophic or deflated due to the influence of in vivo environment such as movement by muscle and abdominal pressure. It was damaged. Therefore, application to actual medical treatment has been very difficult. In addition, conventional decellularized carriers have broken structures of microvasculature such as capillaries that are running in organs or tissues, and it is difficult to restore organs or tissues after cell filling. It was. That is, the conventional decellularized carrier has low performance.

  The present invention has been made in view of such circumstances, and it is an object of the present invention to provide a method for producing a high-performance decellularized carrier that eliminates the above-described problems and maintains hardness and flexibility. To do.

The method for producing a decellularized carrier of the present invention includes a cryopreservation step of cryopreserving an extracted organ or tissue, a thawing step of thawing the organ or tissue cryopreserved in the cryopreservation step, and the thawing A decellularization step of decellularizing the organ or tissue thawed in the step, wherein the decellularization step perfuses the thawed organ or tissue with a poorly water-soluble surfactant-containing solution. A poorly water-soluble surfactant perfusion step, and a hydrophilic water-soluble surfactant perfusion step of perfusing the organ or the tissue perfused with the poorly water-soluble surfactant-containing solution with the hydrophilic water-soluble surfactant-containing solution. It is characterized by that.
In the method for producing a decellularized carrier of the present invention, in the cryopreservation step, blood removal is performed in a freezing blood removal step in which the organ or the tissue is perfused with physiological saline to remove blood, and in the freezing blood removal step. A cryopreservation solution perfusion step of perfusing the organ or tissue that has been perfused with a cryopreservation solution, and a swelling that fills and expands the organ or tissue perfused in the cryopreservation solution perfusion step A step, a surplus solution removing step of removing surplus cryopreservation solution adhering to the swollen organ or tissue, and the organ having the surplus cryopreservation solution removed in the surplus solution removal step or the organ And a freezing step of freezing the tissue at −20 ° C. or lower.
The method for producing a decellularized carrier according to the present invention is characterized in that the cryopreservation step further includes a period storage step for storing the organ or the tissue before the decellularization step for a specific period after the freezing step.
In the method for producing a decellularized carrier of the present invention, the cryopreservation solution is a physiological saline containing 0.01 to 0.3% hydrogen peroxide solution, and the physiological saline and / or The cryopreservation solution may contain 500 to 4000 U heparin.
In the method for producing a decellularized carrier of the present invention, the thawing step includes the cryopreserved organ or the tissue in a low temperature refrigerated state in which the thawing step is thawed in a low temperature refrigerated state, and the thawed in the low temperature refrigerated thawing step. Or, the tissue is perfused with a physiological saline containing a proteolysis inhibitor, and the blood is removed by thawing, and the organ or the tissue that has been removed by the blood removal in the thawing is checked for damage. And a connection step of connecting the organ or the tissue confirmed in the confirmation step to a perfusion container.
The method for producing a decellularized carrier of the present invention is characterized in that the thawing blood removal step includes a thrombus removal step of removing a microthrombus contained in the organ or the tissue.
In the method for producing a decellularized carrier of the present invention, in the decellularization step, the poorly water-soluble surfactant contained in the poorly water-soluble surfactant-containing solution is NP-40, and the hydrophilic water-soluble surfactant The hydrophilic water-soluble surfactant contained in the containing solution is SDS.
In the method for producing a decellularized carrier of the present invention, the decellularization step includes a stabilization step of stabilizing after washing the organ or the tissue perfused in the hydrophilic water-soluble surfactant perfusion step. Features.
The method for producing a decellularized carrier of the present invention is characterized in that in the stabilization step, the organ or the tissue is perfused with a 0.1-3% formaldehyde solution.
In the method for producing a decellularized carrier according to the present invention, the organ includes a liver, a kidney, a spleen, a heart, a lung, a small intestine, and a testis, and the tissue includes a skin, a ureter, and a blood vessel. .
The decellularized carrier of the present invention is produced by the method for producing a decellularized carrier.
The decellularized carrier of the present invention is characterized in that 30% sucrose solution is immersed and cryopreserved.
The cell filling method of the present invention includes a physiological saline perfusion step in which the decellularized carrier is perfused with physiological saline, and the decellularized carrier perfused in the physiological saline perfusion step is perfused with a culture solution to replace it. A replacement step, a cell injection step of injecting cultured cells from the decellularized carrier artery perfused with the culture medium in the replacement step, and stopping the perfusion after the cultured cells are injected in the cell injection step A cell adhesion step for adhering the cells to the decellularized carrier, and a perfusion culture step for culturing by resuming the perfusion of the culture solution to the decellularized carrier to which the cells are adhered by the cell adhesion step. It is characterized by including.
The cell filling method of the present invention is characterized in that when the organ or the tissue is a small intestine, perfusion is performed by connecting an artery and a portal vein.
The cell sheet preparation method of the present invention includes a thin-layer sectioning step in which the decellularized carrier is thin-sectioned into decellularized tissue fragments, and the decellularization in which the thin-layer sectioning is performed by the thin-layer sectioning step. The method includes a seeding step of washing a tissue fragment and seeding a cultured cell, and a culture step of culturing the decellularized tissue fragment seeded with the cultured cell by the seeding step.
The decellularization solution kit of the present invention is a decellularization solution kit for producing a decellularization carrier from an organ or tissue, and contains a poorly water-soluble surfactant that perfuses the thawed organ or tissue. And a hydrophilic water-soluble surfactant-containing solution for perfusing the organ or the tissue perfused with the poorly water-soluble surfactant-containing solution.

  According to the present invention, the extracted organ or tissue is cryopreserved, the cryopreserved organ or tissue is thawed, the thawed organ or tissue is perfused with a poorly water-soluble surfactant-containing solution, and then the hydrophilic By perfusing with a soluble surfactant-containing solution, a method for producing a high-performance decellularized carrier capable of maintaining the hardness and flexibility of the cytoskeleton and extracellular matrix can be provided.

It is a photograph of the poorly water-soluble surfactant perfusion process of the liver which concerns on the Example of this invention. It is a photograph of the hydrophilic water-soluble surfactant perfusion process of the liver based on the Example of this invention. It is a photograph of the hydrophilic water-soluble surfactant perfusion process of the kidney which concerns on the Example of this invention. It is a photograph of the blood removal process at the time of thawing | decompression and the hydrophilic water-soluble surfactant perfusion process which concern on the Example of this invention. It is a photograph of the hydrophilic water-soluble surfactant perfusion process of the heart based on the Example of this invention. It is a photograph of the hydrophilic water-soluble surfactant perfusion process of the lung which concerns on the Example of this invention. It is the photograph which confirmed the cell engraftment property of the decellularized tissue fragment of the heart based on the Example of this invention. It is the photograph which confirmed the cell viability of the decellularized tissue fragment of the kidney based on the Example of this invention. It is the photograph which confirmed the cell engraftment property of the decellularized tissue fragment of the lung which concerns on the Example of this invention. It is the photograph which confirmed cell engraftment about the decellularized tissue fragment of the liver concerning the example of the present invention. It is an electron micrograph about the decellularization support | carrier of the kidney which concerns on the Example of this invention. It is an electron micrograph about the decellularization support | carrier of the heart which concerns on the Example of this invention, and a lung. It is an electron micrograph about the decellularization support | carrier of the liver which concerns on the Example of this invention, and a small intestine. It is a photograph of the analysis (immunostaining) regarding the extracellular matrix which concerns on the Example of this invention. It is a photograph of a transplantation experiment of a decellularized carrier (liver) according to an example of the present invention.

<Embodiment>
In recent years, technical reports on decellularized carriers have been developed, and various technologies have been developed at home and abroad. However, in the conventional method for producing a decellularized carrier, it took 24 to 72 hours to complete decellularization, for example, in rats. In addition, when the washing step is included, it takes 3 to 7 days. For this reason, there was a high possibility that the extracellular matrix was broken or rotted during the decellularization period. That is, in the conventional method for producing a decellularized carrier, the cytoskeleton and the extracellular matrix are fragile. Therefore, the blood vessel anastomosis process performed at the time of transplantation has become very difficult.
In contrast, the present inventors have conducted intensive experiments and tests, and found that it becomes easy to produce a high-quality decellularized carrier by cryopreserving the removed organ or tissue. Furthermore, when trial and error were repeated, the cryopreserved organ or tissue was thawed, and the thawed organ or tissue was perfused with a sparingly water-soluble surfactant-containing solution, and then the hydrophilic water-soluble surfactant-containing solution was used. We found that it would be better to perfuse, and came to complete the invention. By the method for producing a decellularized carrier of the present embodiment, a high-performance decellularized carrier that facilitates vascular anastomosis while maintaining high hardness and flexibility of the cytoskeleton and extracellular matrix can be produced.

  Hereinafter, the configuration and effects of the method for producing a decellularized carrier according to the embodiment of the present invention will be described specifically and in detail.

A method for producing a decellularized carrier according to an embodiment of the present invention includes a cryopreservation step of cryopreserving an extracted organ or tissue, a thawing step of thawing an organ or tissue cryopreserved in the cryopreservation step, A decellularization step of decellularizing the organ or tissue thawed in the thawing step.
By comprising in this way, a decellularization support | carrier can be manufactured in a shorter time than a prior art. In addition, the hardness and flexibility of the cytoskeleton and extracellular matrix of the decellularized carrier can be maintained at a high level. Therefore, a high-quality decellularized carrier can be produced. The produced decellularized carrier can be used for advanced experiments using organ structures. In addition, the produced decellularized carrier can be easily subjected to vascular anastomosis, and can be clinically applied as a medical member such as skin, small intestine, blood vessel, and heart valve. In addition, the produced decellularized carrier can be used as a regenerative medical material and a scaffold material.
Furthermore, the decellularized carrier produced by the method for producing a decellularized carrier according to the present embodiment is capable of engrafting functional cells induced to differentiate from iPS cells, ES cells, etc. Can be possible. Therefore, application in the field of regenerative medicine can be expected.

In addition, the decellularized carrier produced by the decellularized carrier producing method of the present embodiment minimizes the loss of the extracellular matrix. As a result, as shown in the immunostaining and electron microscopic images of Examples described later, a decellularized carrier having a highly maintained detailed structure is obtained.
In addition, the decellularized carrier prepared in the present invention has almost no antigenicity because the double-stranded DNA (hereinafter referred to as “dsDNA”) and the sugar chain structure disappear. For this reason, living body application becomes easy.

Further, since the conventional method for producing a decellularized carrier is “organ-specific”, it is necessary to match the protocol and decellularized solution to the organ or tissue to be decellularized.
In contrast, in the decellularized carrier manufacturing method according to the embodiment of the present invention, the organ includes the liver, kidney, spleen, heart, lung, small intestine, and testis, and the tissue includes the skin, ureter, and It includes blood vessels.
That is, in the decellularized carrier manufacturing method of this embodiment, the liver, kidney, spleen, heart, lung, small intestine, and testis, and tissues such as skin, ureter, and blood vessel, which are swept out, are the same. It can be decellularized with the composition solution and the same protocol, and can be processed in a short time.

In the method for producing a decellularized carrier according to the embodiment of the present invention, the cryopreservation step includes a blood removal step during freezing by perfusion of an organ or tissue with physiological saline and a blood removal step during freezing. A cryopreservation solution perfusion step of perfusing an organ or tissue removed from the blood with a cryopreservation solution; and a swelling step of filling and swelling the organ or tissue perfused in the cryopreservation solution perfusion step with a cryopreservation solution; A surplus solution removing step for removing surplus cryopreservation solution adhering to the swollen organ or tissue, and freezing for freezing the organ or tissue from which the surplus cryopreservation solution has been removed in the surplus solution removing step at −20 ° C. or lower And a process.
Constructed in this way, in the cryopreservation solution perfusion step of perfusing with the cryopreservation solution, blood is removed, perfusion with the cryopreservation solution is expanded, and then the organ or tissue is frozen and then thawed in the subsequent thawing step Thus, a fine hole can be opened in a cell membrane constituting an organ or tissue. For this reason, the permeability of the decellularized solution into the cytoplasm is increased, and the decellularization time can be shortened.
Specifically, in the method for producing a decellularized carrier of the present embodiment, the time required for decellularization is shortened to about 20% as compared with the conventional technique described in Patent Document 1. Further, as described above, the treatment can be performed with the same composition liquid without depending on the target organ.

Further, in the conventional method for producing a decellularized carrier, decellularization is performed immediately after the organ is removed. In other words, there was a limit to the preservation period of the extracted organ.
In contrast, the method for producing a decellularized carrier of the present embodiment is characterized in that the cryopreservation step further includes a period storage step of storing the organ or tissue after the freezing step and before the decellularization step for a specific period. To do.
Thus, since the decellularization organ is once frozen, organ or tissue stock becomes possible. The specific period can be arbitrarily set in units of several hours to several years, and can be stored without substantial deterioration in a commercial medical freezer that does not perform defrosting heating. That is, a decellularization carrier can be produced by thawing the decellularization organ as necessary.

In the method for producing a decellularized carrier according to the embodiment of the present invention, the cryopreservation solution is a physiological saline containing 0.01 to 0.3% by weight of hydrogen peroxide and 500 to 4000 U heparin. It is characterized by.
Constructed in this way, as a cryopreservation solution, by using a physiological saline containing 0.01 to 0.3% by weight of hydrogen peroxide solution and 500 to 4000 U heparin, damage to the cytoskeleton and extracellular matrix is prevented. While minimizing the permeability, it is possible to increase the permeability of the surfactant and to open a fine hole optimal for cytoplasm removal. In other words, by using physiological saline containing hydrogen peroxide in the decellularization process and making minute holes in the cell membrane, the inflow of each surfactant is improved, and cytoplasmic substances such as nuclei and various structures Removability is improved. For this reason, as described above, in the subsequent decellularization step, the time during which the cytoskeleton and the extracellular matrix are in contact with the decellularized solution is shortened. Therefore, the production time of the decellularized carrier can be shortened. Moreover, it becomes possible to suppress degradation and denaturation of amino acids.

In the method for producing a decellularized carrier according to the embodiment of the present invention, the thawing step includes a low-temperature refrigeration thawing step in which a cryopreserved organ or tissue is thawed in a low-temperature refrigerated state, and an organ thawed in the low-temperature refrigerated thawing step. Alternatively, a blood removal process at the time of thawing in which the tissue is perfused with a physiological saline containing a proteolysis inhibitor to remove blood, and a confirmation process for confirming whether or not the organ or tissue removed by the blood removal process at the time of thawing is damaged. And a connecting step of connecting the organ or tissue confirmed by the confirmation step to the perfusion container.
Here, the low temperature refrigerated state may be about 2 ° C to 10 ° C.
By such a thawing step after the cryopreservation step, a “drip” is appropriately generated from a minute hole in a cell membrane constituting an organ or tissue, and the cytoplasm can be easily removed. Thereby, the permeability of the decellularized solution into the cytoplasm is increased, and the decellularization time can be shortened. Therefore, as described above, the decellularization time can be shortened, and degradation of amino acids in the cytoskeleton or extracellular matrix, suppression of denaturation, etc. can be made possible.

Further, in the conventional method for producing a decellularized carrier, there was no means for removing the thrombus. For this reason, it was necessary to remove the organ in a state of pulsation with blood flow.
In contrast, the method for producing a decellularized carrier according to the embodiment of the present invention is characterized by including a thrombus removal step of removing microthrombus contained in an organ or tissue in the blood removal step upon thawing.
With this configuration, decellularization is possible even if a microthrombus is formed. That is, in the method for producing a decellularized carrier of the present embodiment, decellularization is possible even in the presence of a microthrombus by performing blood removal by sufficient perfusion at the time of freeze-thawing in which the thawing step is performed after the freezing step. . More specifically, the method for producing a decellularized carrier according to the present embodiment makes it possible to produce a decellularized carrier as long as the target organ or tissue is not spoiled.

Further, in the method for producing a decellularized carrier of the present embodiment, the decellularization step includes a poorly water-soluble surfactant perfusion step of perfusing an organ or tissue thawed with a poorly water-soluble surfactant-containing solution, And a hydrophilic water-soluble surfactant perfusion step of perfusing an organ or tissue perfused with the hydrophilic surfactant-containing solution with a hydrophilic water-soluble surfactant-containing solution.
Here, as the poorly water-soluble surfactant contained in the poorly water-soluble surfactant-containing solution of the present embodiment, a surfactant having a hydrophilic group that does not ionize when dissolved in water, the influence of water hardness and electrolyte It is preferable to use a nonionic surfactant that is not easily affected. As this nonionic surfactant, polyoxyethylene phenyl ether surfactants such as Triton X-100 and NP-40, DDM, digitonin, Twin 20, Twin 80 and the like can be used. In the present embodiment, it is particularly preferable to use NP-40. At this time, the NP-40 is not necessarily NP-40 (octyl-phenoxy-polyethyl-ethanol) itself, but various NP-40 substitutes, Nonidet (TM) P-40, and the like can be used.
By configuring in this way, in the decellularization step, the concentration change due to the substance in the cytoplasm is less likely to occur, and it is possible to first remove the substance in the cytoplasm after leaving the extracellular matrix etc. Become.

In addition, the hydrophilic water-soluble surfactant contained in the liquid for containing a hydrophilic water-soluble surfactant of the present embodiment includes an anionic surface activity in which a portion having a hydrophobic group is ionized to a negative (anion) ion when dissolved in water. An agent (an anionic surfactant) can be preferably used. In this embodiment, alkyl sulfate ester salts may be used. As this alkyl sulfate ester salt, it is particularly preferable to use SDS (Sodium Dodecyl Sulfate).
By comprising in this way, in a decellularization process, after processing with a poorly water-soluble surfactant (NP-40) containing solution, after removing this with a hydrophilic surfactant (SDS) containing solution, The water washing time can be shortened to half of the conventional one. That is, in the prior art described in Patent Document 1, only water is used to remove the surfactant, but another surfactant is used to remove the surfactant as in this embodiment. As a result, the cleaning time can be reduced by at least 50%.

The decellularization solution kit according to the embodiment of the present invention is a decellularization solution for producing a decellularization carrier from an organ or tissue, and is poorly water-soluble to perfuse a thawed organ or tissue. A surfactant-containing solution and a hydrophilic water-soluble surfactant-containing solution for perfusing an organ or tissue perfused with a poorly water-soluble surfactant-containing solution.
With such a configuration, the slightly water-soluble surfactant and the hydrophilic water-soluble surfactant-containing solution may be prepared as a decellularization solution kit. The slightly water-soluble surfactant and the hydrophilic water-soluble surfactant-containing solution may be prepared at the time of use.

In the method for producing a decellularized carrier according to the embodiment of the present invention, the decellularization step includes a stabilization step of stabilizing after washing the organ or tissue perfused in the hydrophilic water-soluble surfactant perfusion step. It is characterized by.
In addition, this stabilization step is characterized in that the organ or tissue perfused in the hydrophilic water-soluble surfactant perfusion step is perfused with a 0.1-3% formaldehyde solution. At this time, the formaldehyde solution used in the stabilization process of the present embodiment is particularly preferably 0.5 to 1%.
Constituting in this way and maintaining the properties of the cytoskeleton and extracellular matrix by perfusing a 0.1-3% formaldehyde solution after perfusion in the hydrophilic water-soluble surfactant perfusion step for 0.5-2 hours, The hardness of the produced decellularized carrier can be ensured. This facilitates blood vessel anastomosis during transplantation. As a result, as will be described below, it becomes easy to apply to a scaffold treatment in which a decellularized carrier is used as a scaffold material for filling cells or a regenerative medical material.
Note that physiological saline containing hydrogen peroxide may be used in the decellularization step. Also in this case, by making minute holes in the cell membrane, the inflow property of each surfactant and the removability of cytoplasmic substances are improved, and the decellularized carrier preparation time can be shortened.

In addition, the cell filling method according to the embodiment of the present invention replaces the decellularized carrier perfused by the perfusion step with a physiological saline perfusion step in which the decellularized carrier is perfused with physiological saline and then perfused with a culture solution. A replacement step, a cell injection step of injecting cultured cells from the decellularized carrier artery perfused with the culture medium in the replacement step, and the perfusion is stopped after the cultured cells are injected in the cell injection step to remove the cells. A cell adhesion step for adhering to a cellized carrier; and a perfusion culture step for culturing by resuming the perfusion of the culture solution on the decellularized carrier to which cells are adhered by the cell adhesion step.
By configuring in this way, cultured cells such as functional cells and primary passage cells derived from iPS cells, ES cells, or other stem cells (hereinafter referred to as “iPS cells”) are used as a decellularization carrier. Can be filled. Moreover, as shown in the Example mentioned later, the cell-filled decellularized carrier can be engrafted even after transplanted cultured cells are transplanted. As described above, this is considered to be because the decellularized carrier of the present embodiment has an antigenicity of “0” as much as possible and there is little disappearance of the extracellular matrix. For this reason, the decellularization support | carrier filled with the cell of this embodiment can be used in order to solve the shortage of the organs for transplantation considered chronically a problem.
In addition, since the hardness of the decellularized carrier is ensured by the above-described stabilization step, it becomes easier to function as an organ in which the filled cultured cells are engrafted.

  Moreover, the cell filling method according to the embodiment of the present invention is mainly intended for vertebrates. This vertebrate is not particularly limited, and includes, for example, humans, domestic animal species, and wild animals. Here, the cell filling method of this embodiment has succeeded in decellularization of at least each organ of pig, rat, and mouse experimentally. For this reason, the decellularized carrier manufacturing method according to the embodiment of the present invention can be used for animal treatment, livestock treatment, xenotransplantation, etc. in addition to human treatment. It can also be applied in the pet industry.

In addition, the cell filling method according to the embodiment of the present invention is characterized in that when an organ or tissue is a small intestine, perfusion is performed by connecting an artery and a portal vein.
By comprising in this way, the decellularization support | carrier filled with the cell can be used as a functioning small intestine, and the possibility of transplanting can be increased. This is because the decellularized carrier of this embodiment, as shown in the examples described later, retains the structure of the villi portion and muscle layer portion of the small intestine at a high level. It is because is filled.
For this reason, it is possible to perfuse a mixture of cells differentiated into the villi portion and muscle layer portion by a technique that induces differentiation from iPS cells and the like to regenerate various organs, and to fix them separately in the villi portion and muscle layer portion. I can expect.

In addition, the cell sheet preparation method according to the embodiment of the present invention includes a thin-layer sectioning step of thinning a decellularized carrier into a decellularized tissue fragment, and a thin-layer sectioning step. And a seeding step of washing the decellularized tissue fragment and seeding the cultured cell, and a culture step of culturing the decellularized tissue fragment seeded with the cultured cell by the seeding step.
Here, the thickness of the thin-layer slice is preferably about 0.1 to 5 mm. Further, the cultured cells may be seeded by repeating about 0.5 to 10 × 10 ⁵ cells a plurality of times.
As a result of seeding cells on a 1 mm-thick thin-layer slice of the decellularized carrier produced in this embodiment, the cell adhesion was 30 minutes to 1 hour from the start of culture. It has been confirmed through preliminary experiments. This is because, as a result of shortening the decellularization processing time, it is possible to suppress degradation and degeneration of amino acids in the cytoskeleton and extracellular matrix, and as a result, the cells seeded on the obtained decellularized carrier sheet are shorter than before. It is thought that the cells adhered with time.
This cell-attached decellularized carrier can be used as a cell sheet. In other words, it is possible to create a correct adhesion of a functional cell derived from iPS cell or ES cell and a functionally expressed organ.
In addition, after seed | inoculating a cultured cell, you may use these cell sheets, after incubating overnight (overnight).

In addition, the decellularized carrier according to the embodiment of the present invention is manufactured by a method for producing a decellularized carrier.
As described above, the decellularized carrier thus configured is excellent in cell adhesion, and the decellularized organ filled with cells can engraft the filled cells even after transplantation. That is, it is high quality as a decellularization carrier.

Further, the decellularized carrier of the present embodiment is characterized in that a 30% sucrose solution is immersed and stored frozen.
By comprising in this way, the decellularization support | carrier of this embodiment can be cryopreserved easily. Moreover, it can be made into a usable state, without using a special method, maintaining the quality of the decellularization support | carrier of this embodiment. This facilitates the application of the decellularized carrier.

  Each of the above-described steps may include a step that can be executed in parallel, and some of the steps before and after the step may be changed. Optimization within the scope of the present invention by a person skilled in the art is possible. May be performed.

  In addition, the method for producing a decellularized carrier according to the embodiment of the present invention can be used in combination with a process using another composition.

  In the embodiment of the present invention, when a decellularized carrier is used for treatment, the period during which the disease is improved or alleviated is not particularly limited, but may be temporary improvement or alleviation. It may be an improvement or reduction of the period.

  Hereinafter, the method for producing a decellularized carrier according to the embodiment of the present invention will be described more specifically as examples based on experiments. However, this embodiment is only an example, and the present invention is not limited to this.

〔reagent〕
The reagents and manufacturers used in this example are listed below:
NP-40 Substitute (manufactured by Wako Pure Chemical Industries, Ltd.), 10% SDS solution (manufactured by Wako Pure Chemical Industries, Ltd.), physiological saline (manufactured by Otsuka Pharmaceutical Factory Co., Ltd., hereinafter referred to as “raw food”), heparin sodium (Manufactured by Nipro Co., Ltd.), hydrogen peroxide (manufactured by Wako Pure Chemical Industries, Ltd.), water for injection (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), antibiotic-antifungal mixed solution (Antibiotic-Antimycotic, 100X, manufactured by Gibco) , MEMalpha (Gibco), FBS (Fetal Bovine Serum, Gibco), PBS (TaKaRaBio), protein degradation inhibitor (ProteoGuard EDTA-Free Protease Inhibitor Cocktail, Clontech) Lilium injection solution (manufactured by Astellas Pharma Inc.), sucrose (manufactured by Wako Pure Chemical Industries, Ltd.), 10% neutral buffered formalin or formaldehyde (manufactured by Wako Pure Chemical Industries, Ltd.).

[Equipment]
Gauze, stainless steel clips, mosquito forceps, levers and scissors were used as surgical instruments. These surgical instruments were sterilized at 121 ° C. for 15 minutes in an autoclave before use.
The tapper (for treatment) and the equipment of the sealed pack were EOG sterilized before use.

  The following instruments used were sterilized purchased items: general-purpose inhalation catheter, aspirator, connecting tube, polypropylene disposable syringe, injection needle (26G), 6-well plate.

(Preparation of reagents)
In this example, NP-40 was used as the poorly water-soluble surfactant in the poorly water-soluble surfactant perfusion step in the decellularization step. In this example, the poorly water-soluble surfactant-containing solution using NP-40 is hereinafter referred to as “solution A”. Specifically, in this example, 20 mL of NP-40 substrate was added to 1980 mL of water for injection or purified water, and dissolved so as not to foam, and used as solution A.
Moreover, SDS was used as the hydrophilic water-soluble surfactant in the hydrophilic water-soluble surfactant perfusion step in the decellularization step. In this example, the water-soluble surfactant-containing solution using SDS is hereinafter referred to as “Liquid B”. Specifically, in this example, 200 mL of 10% SDS solution was added to 1800 mL of water for injection or purified water, and dissolved so as not to be foamed to become a 1% SDS solution, and used as B solution.
Other solutions were prepared according to the volumes described in the reagent manual.

<Example of processing for production of decellularized carrier>
Hereafter, the detail of the process in each process of the decellularization support | carrier manufacturing method based on a present Example is demonstrated.

[Freeze preservation process]
First, a cryopreservation process for cryopreserving an extracted organ or tissue in the present example will be described.
In the cryopreservation process of this example, as shown in the following examples, a blood removal process during freezing, a cryopreservation solution perfusion process, a swelling process, a surplus solution removal process, and a freezing process were performed.

(Bleeding process when frozen)
In the blood removal step at the time of freezing in this example, a catheter for perfusion fixation is placed, and the organ or tissue is perfused with heparinized physiological saline (2000 Unit (U) / 500 mL, hereinafter referred to as “heparin saline”) to remove it. Blooded.

For this reason, first, a perfusion fixation catheter was placed in an organ or tissue. Specifically, the blood vessel of an organ or tissue was exposed and peeled, and the blood vessel outer diameter was measured with a measure. Next, a catheter suitable for the outer diameter of the blood vessel was selected. As this catheter, an extension tube for blood transfusion or an IVH catheter was selected corresponding to the outer shape of the blood vessel. The vessel outer diameter, the catheter used, and the suture were selected as follows:

Vascular outer diameter Catheter Suture> 4.0 mm, left atrial appendage Transfusion extension tube 3-0 silk thread 3.0-4.0 mm Transfusion extension tube 3-0 silk thread 2.0-2.9 mm 14 GIVH catheter 4-0 nylon thread 1.5-2.0mm 16GIVH catheter 4-0 nylon thread 1.0-1.4mm 18GIVH catheter 5-0 nylon thread

  Next, a three-way stopcock and a 20 mL syringe (Terumo Corporation) were attached to the selected catheter, filled with heparinized saline, and the organ or tissue was vented. Next, the catheter was cut at a position of 40 cm from the three-way stopcock connecting portion. Next, the vascular center side was ligated and sutured. Next, an incision was made with a metzen on the blood vessel center side, and a catheter was inserted. Next, the blood vessel and the catheter were simply fixed with bulldog forceps. Then, ligation and fixation were performed at two places of the catheter insertion site and one place of the catheter.

Next, perfusion for blood removal to an organ or tissue was actually performed.
Specifically, about 1 cm of a catheter was inserted and placed in the artery and vein of each organ or tissue. Then, 200 mL or more of heparin saline was pumped and injected from the artery side catheter, and it was confirmed that blood was discharged from the vein side catheter. It was confirmed that white color was missing.

(Cryopreservation solution perfusion process)
Next, in the cryopreservation solution perfusion step, the organ or tissue that was blood-depleted in the above-described blood removal step during freezing was perfused with the cryopreservation solution.
As the cryopreservation solution of this example, a solution adjusted to 0.1% hydrogen peroxide in physiological saline was used. Further, the three-way stopcock on the venous side was closed, and a cryopreservation solution was injected from the arterial side.

(Expansion process)
In the swelling step, the organ or tissue perfused in the cryopreservation solution perfusion step perfused with the cryopreservation solution was filled with the cryopreservation solution and swollen.
Specifically, it was confirmed that the cryopreservation solution was injected and the internal organ or tissue was swollen (as a whole), and the arterial three-way stopcock was closed.

(Excess solution removal step)
In the surplus solution removing step, surplus cryopreservation solution adhering to the swollen organ or tissue was removed.
Specifically, excess water adhering to the surface of the organ or tissue was removed with a paper towel.

(Freezing process)
In the freezing step, the organ or tissue from which the excess cryopreservation solution was removed in the surplus solution removing step was frozen at −20 ° C. or lower.
Specifically, the organ or tissue was transferred into a container such as Ziplock and stored frozen at -20 ° C or -80 ° C.

[Thawing step and decellularization step]
Here, an example of performing the thawing step and the decellularization step through the cryopreservation step described above for each of the liver, kidney, heart, spleen, and lung extracted from a laboratory pig weighing about 10 kg will be described below. To do.
In addition, each perfusion time of the following organ is a standard, and priority was given to visual confirmation.

[liver]
(Low temperature refrigeration thawing process)
The cryopreserved organ was thawed in a low-temperature refrigerated state. Specifically, frozen organs were thawed at 4 ° C. for 24 ± 1 hours. It was confirmed that the organ was completely thawed.

(Through blood removal process, confirmation process, connection process)
Next, the organs thawed in the low-temperature refrigerated thawing step were perfused with physiological saline containing a proteolysis inhibitor to remove blood.
Specifically, first, the liver was slaughtered to remove blood clots and the like that had adhered and remained.
Next, a raw meal (50 mL × 5 each) was injected from the portal vein catheter and the hepatic artery catheter using a disposable syringe, and it was confirmed that there was no resistance or leakage, and that it was discharged from the hepatic vein. I felt resistance in the first pumping, but I was careful because if I push it forcibly at this point, the blood vessels and the membrane may burst. It was confirmed that the resistance gradually disappeared and the raw food could be pumped smoothly.
Next, an infusion tube of an automatic perfusion device was connected to the three-way stopcock of the portal vein catheter, and the three-way stopcock of the portal vein catheter and the three-way stopcock of the hepatic artery catheter were connected by a connector.
A raw food containing a proteolysis inhibitor in a volume described in the manual (hereinafter referred to as “proteolysis-containing inhibitor raw food”) was prepared, 2000 mL was placed in a sterile pack, and a sterile gauze was attached to the top. The liver was placed on a gauze so that the liver was immersed in about 1 cm. At this time, if the organ is immersed in the liquid level too much, it will float and move in the perfusate. Also, care was taken that the whole organ was on the gauze. In addition, it was confirmed that both the hepatic artery and the portal vein were not bent because the blood vessels came out of the hepatic portal portion.
Next, the liver was moved to a 10 ° C. refrigerator or a 10 ° C. environment. Here, when it set to 4 degreeC, since the perfusate may precipitate in a next process, it was careful. In addition, at room temperature, the construction damage of the decellularized carrier may occur due to the production of proteolytic enzyme by autolysis.
Next, the three-way stopcock of the hepatic artery was closed, the three-way stopcock was opened only in the portal vein, and a protein-containing degradation inhibitor raw diet was perfused at 60-80 mL / min for 15 minutes using an automatic perfusion apparatus.
Next, the three-way stopcock of the portal vein was closed, the three-way stopcock was opened only in the hepatic artery, and a protein-containing degradation inhibitor raw diet was perfused at 60-80 mL / min for 15 minutes using an automatic perfusion device.
Next, the three-way stopcocks of the portal vein and hepatic artery were both opened, and a protein-containing degradation inhibitor saline was perfused twice at 80 mL / min for 30 minutes using an automatic perfusion apparatus. When the raw food became red and cloudy, it was perfused again for 15 minutes.
That is, by irrigating until the raw food becomes red and cloudy in this way, it was possible to remove microthrombus from an organ or tissue as a thrombus removal step in the blood removal step upon thawing. The same applies to the other organs described below.

As a final confirmation step, the presence or absence of damage to the liver that had been blood-removed in the blood removal step during thawing was confirmed. At this time, organs or tissues with cracks or perforations were excluded from the decellularized carrier production.
In addition, this confirmation process performed the same process also with another organ.

(Slightly water-soluble surfactant perfusion process)
Here, the automatic perfusion apparatus was stopped and replaced with solution A. Then, using an automatic perfusion apparatus, solution A was perfused in the order of hepatic artery and portal vein at 60 mL / min for 15 to 30 minutes, and then both were opened, and perfusion was performed at 80 to 120 mL / min for 20 minutes × 3 times. At this time, immediately after the replacement with the liquid A, it was perfused while observing the state at 20 mL / min, and it was confirmed that the perfusate smoothly flowed and gradually increased to 60 mL / min.
According to FIGS. 1 (a) and 1 (b), the circumference of the blood vessel changed to a translucent state after a while after perfusion. Here, FIG. 1A shows a photograph immediately after perfusion, and FIG. 1B shows a photograph of the third perfusion.
According to FIG.1 (c), it confirmed that the edge part of the liver also changed a little translucent after that.

(Water-soluble surfactant perfusion process)
Then, the automatic perfusion apparatus was stopped and replaced with solution B. Then, using an automatic perfusion apparatus, solution B was perfused in the order of hepatic artery and portal vein at 60 mL / min for 15 to 30 minutes, and then both were opened and perfused 20 to 6 times at 80 to 120 mL / min. At this time, immediately after exchanging with the liquid B, the perfusion was perfused while observing the state at 20 mL / min as in the case of the poorly water-soluble surfactant perfusion step, and it was confirmed that the perfusate smoothly flowed and gradually increased to 60 mL / min. I went.
According to FIGS. 2 (a) and 2 (b), in the first perfusion of the B liquid, the liver becomes dramatically transparent, so that the liquid becomes cloudy immediately after the perfusion. FIG. 2A shows a photograph before the first perfusion, and FIG. 2B shows a photograph after the first perfusion.
According to FIG. 2 (c), it was confirmed that the liver was completely translucent decellularized tissue after the completion of the perfusion.

Next, the perfusion apparatus was temporarily removed, and perfusion was performed 500 mL × 3 times from the hepatic artery catheter and the portal vein with B liquid separately prepared. A part of the third perfusate was collected and dsDNA was measured using an absorptiometer to confirm that it was 0.5 ng / μL or less. When this value was exceeded, the B liquid was additionally executed at 80 to 120 mL / min for 30 minutes using an automatic perfusion apparatus, and the same concentration measurement was performed again. In addition, this concentration measurement was excluded when the total perfusion time of solution B exceeded 6 hours.
Next, all the B liquid was sucked and replaced with purified water (water for injection). Specifically, using an automatic perfusion device, purified water is perfused in the order of hepatic artery and portal vein at 60 mL / min for 15 to 30 minutes, and then both are opened, then 80 to 120 mL / min for 15 minutes × 8 times, 30 Perfusion was performed for 6 minutes, then (120 minutes × 4 times / day) × 6 days. At this time, since the central part of the decellularized organ has a fine network structure, the washing perfusion took not only the number of times but also a sufficient time.
Thereafter, the final perfusate (purified water) is collected, dsDNA is measured using an absorptiometer, and it is confirmed that the dsDNA is 0.0 ng / μL (below the detection limit). confirmed.

[kidney]
(Low temperature refrigeration thawing process)
The cryopreserved organ was thawed at 4 ° C. for 24 ± 1 hour. After confirming that the organ was completely thawed, the following treatment was performed.

(Through blood removal process, confirmation process, connection process)
Next, a raw meal was applied to the kidneys to wash off any adhering or remaining blood clots.
Next, a raw meal (50 mL × 3) was injected from the renal artery catheter using a disposable syringe, and it was confirmed that there was no resistance or leakage and that it was discharged from the hepatic vein. I felt resistance in the first pumping, but I was careful because if I push it forcibly at this point, the blood vessels and the membrane may burst. It was confirmed that the resistance gradually disappeared and the raw food could be pumped smoothly.
Next, the injection tube of the automatic perfusion device was connected to the three-way stopcock of the renal artery catheter.
Next, 2000 mL of a protein-containing degradation inhibitor raw food was placed in a sterilized pack, and a sterilized gauze was attached to the top. I put it on the gauze so that the kidney was immersed in about 1 cm of raw food. Be careful not to immerse it too much in the liquid surface as it will float and move in the perfusate. Care was taken that the whole organ was on the gauze. It was confirmed that the blood vessels of the renal arteries and veins exited from the hilar part and were not bent.
The kidneys were then moved to a 10 ° C. refrigerator or 10 ° C. environment. Here, when it set to 4 degreeC, since the perfusate may precipitate in a next process, it was careful. In addition, it was avoided at room temperature because there is a possibility that the cell carrier is damaged.
Next, the three-way stopcock of the renal artery was opened, and the protein-containing degradation inhibitor raw diet was perfused twice for 15 minutes × 40-60 mL / min using an automatic perfusion apparatus. If the raw food became red and cloudy, it was perfused again for 15 minutes.

(Slightly water-soluble surfactant perfusion process)
After the final confirmation step, the automatic perfusion apparatus was stopped and replaced with solution A. And A liquid was perfused 20 times * 3 times by 40-60 mL / min using the automatic perfusion apparatus. Immediately after replacement with solution A, the solution was perfused while observing the state at 20 mL / min, and it was confirmed that the perfusate smoothly flowed, and was gradually increased to 60 mL / min. After a while after perfusion, the circumference of the blood vessel changed to a translucent state. After that, it was confirmed that the whole kidney was slightly translucent.

(Water-soluble surfactant perfusion process)
Next, the automatic perfusion apparatus was stopped and replaced with solution B. And B liquid was perfused 20 minutes x 6 times at 60 mL / min using the automatic perfusion apparatus. Immediately after the replacement with the B liquid, it was perfused while observing the state at 20 mL / min, and it was confirmed that the perfusate smoothly flowed and gradually increased to 60 mL / min.
According to FIG. 3 (a), in the first perfusion of the B liquid, the kidney became dramatically transparent, so that the liquid became cloudy immediately after the perfusion.
According to FIG. 3B, it was confirmed that the kidney was completely translucent decellularized tissue after 6 perfusions.

Next, the perfusion apparatus was temporarily removed, and perfusion was performed 50 mL × 3 times from the renal artery catheter with a separately prepared B solution. A part of the third perfusate was collected and dsDNA was measured using an absorptiometer to confirm that it was 0.5 ng / μL or less. When this value was exceeded, the liquid B was additionally perfused at 40-60 mL / min for 15 minutes using an automatic perfusion apparatus, and the same concentration measurement was performed again. Further, when the total perfusion time of the liquid B exceeded 6 hours, this concentration determination was not performed.
Next, all the B liquid was sucked and replaced with purified water (water for injection). Then, using an automatic perfusion apparatus, purified water was perfused at 40 to 60 mL / min for 15 minutes × 8 times, 30 minutes × 6 times, and then (120 minutes × 4 times / day) × 6 days. At this time, since the central part of the decellularized organ has a fine network structure, the washing perfusion took not only the number of times but also a sufficient time.
Thereafter, the final perfusate (purified water) is collected, dsDNA is measured using an absorptiometer, and it is confirmed that the dsDNA is 0.0 ng / μL (below the detection limit). confirmed.

[spleen]
(Low temperature refrigeration thawing process)
Frozen organs were thawed at 4 ° C. for 24 ± 1 hours. After confirming that the organ was completely thawed, the following treatment was performed.

(Through blood removal process, confirmation process, connection process)
Next, the spleen was eaten raw, and the clots and the like that adhered and remained were washed away.
Next, a raw meal (50 mL × 6) was injected from the splenic artery catheter using a disposable syringe, and it was confirmed that there was no resistance or leakage and the splenic vein was discharged. At first pumping, you feel resistance, but be careful because if you force it in at this point, the blood vessels and skin may burst. In particular, the splenic artery catheter may be thinner than other organs, so care was taken to prevent the catheter from being pulled out by pressure. It was confirmed that the resistance gradually disappeared and the raw food could be pumped smoothly.
Next, the injection tube of the automatic perfusion device was connected to the three-way stopcock of the splenic artery catheter.
Next, 2000 mL of the protein-containing degradation inhibitor raw food was placed in a sterile pack, and a sterile gauze was attached to the top. The spleen was placed on a gauze so that it was immersed in about 1 cm of raw food. Be careful not to immerse it too much in the liquid surface as it will float and move in the perfusate. Care was taken that the whole organ was on the gauze. In both splenic arteries and veins, it was confirmed that the blood vessels exited from the splenic hilum and were not bent.
Next, the spleen was moved to a 10 ° C. refrigerator or a 10 ° C. environment. Here, when it set to 4 degreeC, since the perfusate may precipitate in a next process, it was careful. In addition, the construction damage of the decellularized carrier may occur at room temperature, so it was avoided.
Next, the three-way stopcock of the splenic artery was opened, and the protein-containing degradation inhibitor saline was perfused 15 times × 5 times at 40 to 60 mL / min using an automatic perfusion apparatus. If the raw food became red and cloudy, it was perfused again for 15 minutes. Blood tended to remain at the margin of the spleen. When the omission was poor, there was a tendency for blood removal to occur with a light hand. When proceeding to the next step in a situation where blood removal was not possible, the perfusion time would be long and the fine structure might not be maintained.
According to FIG. 4 (a), the whole spleen was thoroughly perfused with saline until it was bleeded and turned pink.

(Slightly water-soluble surfactant perfusion process)
After the final confirmation step, the automatic perfusion apparatus was stopped and replaced with solution A. And A liquid was perfused 20 times * 3 times by 40-60 mL / min using the automatic perfusion apparatus. Immediately after replacement with solution A, the solution was perfused while observing the state at 20 mL / min, and it was confirmed that the perfusate smoothly flowed, and was gradually increased to 60 mL / min. Some time after the perfusion, the entire spleen changed to translucent. After that, it was confirmed that the whole spleen changed slightly translucent.

(Water-soluble surfactant perfusion process)
Next, the automatic perfusion apparatus was stopped and replaced with solution B. And B liquid was perfused 20 minutes x 6 times at 60 mL / min using the automatic perfusion apparatus. Immediately after the replacement with the B liquid, it was perfused while observing the state at 20 mL / min, and it was confirmed that the perfusate smoothly flowed and gradually increased to 60 mL / min.
According to FIG.4 (b), in the first perfusion of B liquid, since the kidney became dramatically transparent, the liquid became cloudy immediately after perfusion.
According to FIG. 4 (c), it was confirmed that the spleen was completely translucent decellularized tissue after 6 perfusions.

Next, the perfusion apparatus was temporarily removed, and perfusion was performed 500 mL × 3 times from the splenic artery catheter with a separately prepared B solution. A part of the third perfusate was collected and dsDNA was measured using an absorptiometer to confirm that it was 0.5 ng / μL or less. When this value was exceeded, the liquid B was additionally perfused at 40-60 mL / min for 15 minutes using an automatic perfusion apparatus, and the same concentration measurement was performed again. When the total perfusion time of B liquid exceeded 6 hours, this concentration determination was not performed.
Next, all the B liquid was sucked and replaced with purified water (water for injection). Then, using an automatic perfusion apparatus, purified water was perfused at 40 to 60 mL / min for 15 minutes × 8 times, 30 minutes × 6 times, and then (120 minutes × 4 times / day) × 6 days. At this time, since the central part of the decellularized organ has a fine network structure, the washing perfusion took not only the number of times but also a sufficient time.
Thereafter, the final perfusate (purified water) is collected, dsDNA is measured using an absorptiometer, and it is confirmed that the dsDNA is 0.0 ng / μL (below the detection limit). confirmed.

[heart]
(Low temperature refrigeration thawing process)
Frozen organs were thawed at 4 ° C. for 24 ± 1 hours. After confirming that the organ was completely thawed, the following treatment was performed.

(Through blood removal process, confirmation process, connection process)
Next, the heart was eaten raw, and the clots and the like remaining on the heart were washed away.
Next, a raw meal (50 mL × 6) was injected from the aortic catheter using a disposable syringe, and it was confirmed that there was no resistance or leakage and the pulmonary artery was discharged. I felt resistance at the first pumping, but I was careful because the auricle could rupture if I push it forcibly at this point. Both left and right atrial appendages were inflated by pumping. At this time, it was confirmed that blood in the coronary artery was flowing and the entire heart was perfused.
Next, the injection tube of the automatic perfusion device was connected to the three-way stopcock of the aortic catheter.
Next, 2000 mL of the protein-containing degradation inhibitor raw food was placed in a sterile pack, and a sterile gauze was attached to the top. I put it on the gauze so that the heart was immersed in raw food about 1 cm. Be careful not to immerse it too much in the liquid surface as it will float and move in the perfusate. Also, care was taken that the whole organ was on the gauze.
Next, the heart was moved to a 10 ° C. refrigerator or a 10 ° C. environment. When set to 4 ° C., it was noted that the perfusate may precipitate in a later step. Also, room temperature was avoided because there is a possibility of destructive carrier construction damage.
Next, the three-way stopcock of the aorta was opened, and the protein-containing degradation inhibitor saline was perfused 15 times × 5 times at 40 to 60 mL / min using an automatic perfusion apparatus. If the raw food became red and cloudy, it was perfused again for 15 minutes.

(Slightly water-soluble surfactant perfusion process)
After the final confirmation step, the automatic perfusion apparatus was stopped and replaced with solution A. And A liquid was perfused 20 times * 3 times by 40-60 mL / min using the automatic perfusion apparatus. Immediately after replacement with solution A, the solution was perfused while observing the state at 20 mL / min, and it was confirmed that the perfusate smoothly flowed, and was gradually increased to 60 mL / min. After a while after perfusion, the circumference of the blood vessel changed to a translucent state. After that, it was confirmed that the whole heart changed slightly translucent.

(Water-soluble surfactant perfusion process)
Next, the automatic perfusion apparatus was stopped and replaced with solution B. And B liquid was perfused 20 minutes x 6 times at 60 mL / min using the automatic perfusion apparatus. Immediately after the replacement with the B liquid, it was perfused while observing the state at 20 mL / min, and it was confirmed that the perfusate smoothly flowed and gradually increased to 60 mL / min.
According to FIG. 5 (a), in the first perfusion of the B liquid, the heart became dramatically transparent, so that the liquid became cloudy immediately after the perfusion.
According to FIG. 5 (b), it was confirmed that the heart was completely translucent decellularized tissue after 6 perfusions.

Next, the perfusion apparatus was temporarily removed, and perfusion was performed 50 mL × 3 times from the aortic catheter with a separately prepared B solution. A part of the third perfusate was collected and dsDNA was measured using an absorptiometer to confirm that it was 0.5 ng / μL or less. When this value was exceeded, the liquid B was additionally perfused at 40-60 mL / min for 15 minutes using an automatic perfusion apparatus, and the same concentration measurement was performed again. When the total perfusion time of B liquid exceeded 6 hours, this concentration determination was not performed.
Next, all the B liquid was sucked and replaced with purified water (water for injection). Then, using an automatic perfusion apparatus, purified water was perfused at 40 to 60 mL / min for 15 minutes × 8 times, 30 minutes × 6 times, and then (120 minutes × 4 times / day) × 6 days. At this time, since the central part of the decellularized organ has a fine network structure, the washing perfusion took not only the number of times but also a sufficient time.
Thereafter, the final perfusate (purified water) is collected, dsDNA is measured using an absorptiometer, and it is confirmed that the dsDNA is 0.0 ng / μL (below the detection limit). confirmed.

[lung]
(Low temperature refrigeration thawing process)
Frozen organs were thawed at 4 ° C. for 24 ± 1 hours. After confirming that the organ was completely thawed, the following treatment was performed. In addition, the edge of the trachea was picked with forceps and completely closed.

(Through blood removal process, confirmation process, connection process)
Next, the lungs were subjected to a raw diet to wash away any clots that had adhered and remained.
Next, a raw meal (50 mL × 10 to 20) was injected from the pulmonary artery catheter using a disposable syringe, and it was confirmed that there was no resistance or leakage and that it was discharged from the pulmonary vein. I felt resistance at the first pumping, but I was careful because if I forced it at this point, the alveoli could rupture. In addition, it was confirmed that the whole lung could be exsanguinated and perfused pink.
Next, the injection tube of the automatic perfusion device was connected to the three-way stopcock of the pulmonary artery catheter.
Next, 2000 mL of the protein-containing degradation inhibitor raw food was placed in a sterile pack, and a sterile gauze was attached to the top. It was put on the gauze so that the lung could be immersed in the raw food about 1 cm. Be careful not to immerse it too much in the liquid surface as it will float and move in the perfusate. Care was taken that the whole organ was on the gauze.
The lungs were then moved to a 10 ° C refrigerator or 10 ° C environment. When set to 4 ° C., it was noted that the perfusate may precipitate in a later step. Also, room temperature was avoided because there is a possibility of destructive carrier construction damage.
Next, the three-way stopcock of the pulmonary artery was opened, and the protein-containing degradation inhibitor saline was perfused 15 times × 5 times at 40 to 60 mL / min using an automatic perfusion apparatus. If the raw food became red and cloudy, it was perfused again for 15 minutes.

(Slightly water-soluble surfactant perfusion process)
After the final confirmation step, the automatic perfusion apparatus was stopped and replaced with solution A. And A liquid was perfused 20 times * 3 times by 40-60 mL / min using the automatic perfusion apparatus. Immediately after replacement with solution A, the solution was perfused while observing the state at 20 mL / min, and it was confirmed that the perfusate smoothly flowed, and was gradually increased to 60 mL / min. After a while after perfusion, it was confirmed that the whole lung changed to a translucent state.

(Water-soluble surfactant perfusion process)
Next, the automatic perfusion apparatus was stopped and replaced with solution B. Using an automatic perfusion apparatus, solution B was perfused at 60 mL / min for 20 minutes × 6 times. Immediately after the replacement with the B liquid, it was perfused while observing the state at 20 mL / min, and it was confirmed that the perfusate smoothly flowed and gradually increased to 60 mL / min.
According to FIG. 6 (a), in the first perfusion of the B liquid, the lung becomes dramatically transparent, so that the liquid becomes cloudy immediately after the perfusion.
According to FIG. 6 (b), it was confirmed that the heart was completely translucent decellularized tissue after 6 perfusions.

Next, the perfusion apparatus was temporarily removed, and perfusion was performed 500 mL × 3 times from the pulmonary artery catheter with a separately prepared B solution. A part of the third perfusate was collected and dsDNA was measured using an absorptiometer to confirm that it was 0.5 ng / μL or less. When this value was exceeded, the liquid B was additionally perfused at 40-60 mL / min for 15 minutes using an automatic perfusion apparatus, and the same concentration measurement was performed again. When the total perfusion time of B liquid exceeded 6 hours, this concentration determination was not performed.
Next, all the B liquid was sucked and replaced with purified water (water for injection). Then, using an automatic perfusion apparatus, purified water was perfused at 40 to 60 mL / min for 15 minutes × 8 times, 30 minutes × 6 times, and then (120 minutes × 4 times / day) × 6 days. Since the central part of the decellularized organ has a fine network structure, the washing perfusion took not only the number of times but also a sufficient time.
Thereafter, the final perfusate (purified water) is collected, dsDNA is measured using an absorptiometer, and it is confirmed that the dsDNA is 0.0 ng / μL (below the detection limit). confirmed.

<Method of cryopreserving decellularized carrier>
Here, a method for cryopreserving an organ or tissue converted into a decellularized carrier will be described.
First, a 30% sucrose solution (PBS) was prepared.
And the automatic perfusion apparatus connected to the decellularization support | carrier manufactured with the above-mentioned decellularization support | carrier manufacturing method was stopped, all the purified water was attracted | sucked, and it replaced with 30% sucrose solution. Using an automatic perfusion apparatus, 30% sucrose solution was perfused at 60-100 mL / min for 15 minutes × 3 times.
Next, the automatic perfusion device was stopped and the three-way stopcock was closed. The organs were not too tight. Then, the organ was moved to a sterile pack filled with 30% sucrose solution. Gauze was placed on the liquid surface so that the organ was completely immersed in the sucrose solution.
Next, it was immersed in a storage (set temperature: 4 ° C.) for 3 hours. Thereafter, the decellularized organ was placed in a sealed pack and stored frozen in a freezer (set temperature: −80 ° C.).

<Thawing of cryopreserved decellularized carrier, treatment method before use>
Here, a process when the decellularized organ or tissue cryopreserved by the above-described cryopreservation method for a decellularized carrier is thawed and used will be described.
First, it thawed at 4 ° C. for 24 ± 1 hour. It was confirmed that the organ was free from cracks due to freezing. And the above-mentioned automatic perfusion apparatus was reconnected. Then, the sucrose solution was replaced with purified water. Purified water was perfused 15 times × 10 times at 60 to 100 mL / min using an automatic perfusion apparatus. Next, the purified water was replaced with raw food. The raw food was perfused 15 times × 5 times at 60-100 mL / min using an automatic perfusion apparatus. Depending on the purpose, 20 mL of an antibiotic-antimycotic mixed solution was added to 1980 mL of a final replacement solution such as a culture solution, and a preparation solution was further added with 1 ampule of cephamedin. Finally, the purified water was replaced with the preparation solution, and perfusion was performed twice at 80 mL / min for 30 minutes.

<Stabilization method for stabilizing the decellularized carrier without cryopreservation>
(Stabilization process)
Here, in this example, a specific example of a stabilization process for stabilizing an organ or tissue converted to a decellularized carrier will be described. In this example, a formaldehyde fixed storage method in which a decellularized carrier is fixed and stored with a 0.5% formaldehyde solution will be described. In this example, an example of refrigerated storage without freezing storage will be described.
First, a 0.5% formaldehyde solution (formalin) was prepared. Then, the automatic perfusion apparatus connected to the decellularized carrier produced by the above-described decellularized carrier production method was stopped, and all purified water was sucked and replaced with a 0.5% formaldehyde solution.
Next, a 0.5% formaldehyde solution was perfused at 60 to 100 mL / min for 15 minutes × 3 times using an automatic perfusion apparatus. Then, the automatic perfusion device was stopped and the three-way stopcock was closed. The organs were not too tight. Here, if the treatment is performed in a state where the organ is concave or too tight, the shape will be fixed as it is.
The organs were then transferred to a sterile pack filled with 0.5% formaldehyde solution. Gauze was placed on the liquid surface so that the organ was completely immersed in a 0.5% formaldehyde solution.
Next, the decellularized organ was placed in a sealed pack and stored in a refrigerated storage (set temperature: 4 ° C.).

<Measurement of cell residue of decellularized carrier>
The following items were confirmed for the quality confirmation of organs or tissues converted into decellularized carriers.
This confirmation was carried out in accordance with the guidelines for ensuring the quality and safety of human (self) -derived cells (primary passage cells), tissue-processed pharmaceuticals and the like. dsDNA: Confirmed to be 50 ng or less per gram of lyophilized weight. DNA fragment length: confirmed to be 200 bp or less. HE or DAP I: Confirmed by micro that there is no nuclear staining and no remaining cells. Ultra-fine structure, electron microscope: Confirm that the fine structure is maintained and that no cells remain. Microbiological examination (sterility test): A part of the decellularized organ was collected and confirmed to be sterile. Mycoplasma negative test: A portion of the decellularized organ was collected and confirmed to be free of mycoplasma. Endotoxin test: A portion of the decellularized organ was collected and confirmed to contain no endotoxin.

<Experimental Example 1 for Confirming Cell Engraftment by Cell Loading Method of Decellularized Carrier>
The cell-freeness of the decellularized carrier preserved by the cryopreservation method of the above-mentioned decellularized carrier or preserved by the formaldehyde fixed preservation method was filled in vitro to confirm cell engraftment. The process of Experimental Example 1 will be described below.

(Thin layer sectioning process)
A decellularized carrier was prepared, and purified water was perfused 20 times × 10 times at 40 to 60 mL / min using an automatic perfusion apparatus.
At this point, cells to be filled were prepared. In this example, 1-5 × 10 ⁵ HepG2 cells were prepared.
In addition, the decellularized carrier was cut into 10 mm × 10 mm × 2 mm block pieces (decellularized tissue fragments).

(Seeding process)
The decellularized tissue fragment that had been sliced in the thin-layer sectioning step was washed again with purified water for 5 minutes × 6 times.
Next, the antibiotic-antimycotic mixed solution was washed with 180 mL of sterilized PBS with 20 mL (normal 5-fold concentration) for 10 minutes × 4 times.
In addition, decellularized tissue fragments were placed in the medium.
Moreover, a 2 to 5 times concentrated antibiotic-antifungal mixed solution + 10% FBS-added MEM (hereinafter referred to as “added MEM”) was prepared.
The corner of the decellularized tissue fragment was picked with a sterilized insulator, and 1 mL of 1 to 5 × 10 ⁵ / mL of cells was injected from all directions using a disposable syringe equipped with a 26 G injection needle.

(Culture process)
2 mL of additional MEM was injected into the decellularized tissue fragment seeded with cultured cells.
Then, it incubated at 37 degreeC overnight (Overnight).
The cell engraftment was confirmed under a stereomicroscope.
The medium was washed and replaced once a day.
In addition, after 72 hours of culture, cell viability was confirmed under a stereomicroscope.

FIG. 7A shows an example of confirming engraftment of a decellularized tissue fragment of the heart under a stereomicroscope. According to this result, swelling of the seeded cells was observed corresponding to the structure of the decellularized carrier of the heart.
FIG. 7B shows an example of confirming engraftment of a decellularized tissue fragment of kidney under a stereomicroscope. According to this result, the seeded cells were somewhat small, corresponding to the structure of the decellularized carrier of the kidney.
FIG. 7C shows an example of confirming engraftment of a decellularized tissue fragment of lung under a stereomicroscope. According to this result, in accordance with the structure of the decellularized carrier of the lung, the seeded cells were fixed in a flat layer shape.

<Experimental Example 2 for Confirming Cell Engraftment by Cell Loading Method of Decellularized Carrier>
Moreover, about the decellularization support | carrier, depending on the kind of organ, the cell viability was confirmed in vitro (in vitro) by dripping the cell fluid on the decellularization support. The process of Experimental Example 2 will be described below.

(Thin layer sectioning process)
First, the decellularized carrier was cut into 20 mm × 20 mm × 1 mm decellularized tissue fragments.
The decellularized tissue fragment was then placed in a well of a 6-well plate.

(Seeding process)
Next, 1 mL of 5 × 10 ⁵ / mL cell solution was gradually dropped into each well, and the cell solution leaked to the side was aspirated and dropped again three times.
The 6-well plate was then incubated at 37 degrees for 15 minutes.
These seeding processes were repeated three times.

(Culture process)
2 mL of added MEM was added dropwise to the decellularized tissue fragment in which the cultured cells were seeded by the seeding process.
Thereafter, incubation was performed at Overnight at 37 ° C.
The cell engraftment was confirmed under a stereomicroscope.
The medium was washed and replaced once a day.
In addition, after 72 hours of culture, cell viability was confirmed under a stereomicroscope.

  FIG. 7D shows an example of confirming engraftment of a decellularized tissue fragment of liver under a stereomicroscope. According to this result, cells engrafted in a regular arrangement, such as liver lobule, were found in some cases corresponding to the structure of the decellularized carrier of the liver.

Here, in Experimental Example 2 described above, the culture solution was then collected, and the decellularized tissue fragment was lightly washed with a saline diet.
Next, the decellularized tissue fragment was immersed in a 4% PFA solution or a 10% neutral buffered formalin solution for 5 minutes × 2 times.
Next, a histopathological examination of the decellularized tissue fragment was performed, and finally cell engraftment was confirmed by HE staining and anti-HLA immunohistochemical staining.

<Electron microscope analysis>
Each decellularized carrier was sent to a contractor to obtain an electron microscope image.
FIG. 8A is an electron microscope image of the kidney and spleen, FIG. 8B is the heart and lung, and FIG. 8C is the liver and small intestine. In each figure, in the electron microscopic images of the kidney, spleen, heart lung, and liver, an enlarged image of the left figure is shown in the right figure. In the small intestine, the left figure shows an electron microscopic image of the villus part, and the right figure shows an electron microscopic image of the muscle layer part.
Each of these electron micrographs showed that the extracellular matrix along the organ construction including the cytoskeleton remained very highly.

<Analysis of extracellular matrix (immunostaining)>
Next, each decellularized carrier was made into a thin-layer section, and DAB (3,3′-diaminobenzidine tetrahydrochloride) staining was performed using each commercially available antibody and kit (manufactured by Takara Bio Inc.).
According to FIG. 9, it was confirmed from the image of H & E (double staining of hematoxylin and eosin) that the cell nucleus disappeared. Further, Sirius red and Collagen IV staining confirmed the disappearance of the cytoplasm and the remaining cell membrane. In addition, as a result of examining each extracellular matrix remaining on the cell membrane by staining with Fibronectin and Laminin, the density of DAB staining was confirmed according to the constituent factors of the extracellular matrix.

<Transplantation experiment of decellularized carrier (liver)>
The liver was transplanted with a decellularized carrier (unfilled with cells) that had been treated by the decellularization method of the present example for the liver, and it was confirmed that blood was filled inside the carrier. The program was conducted at the Advanced Medical Technology Development Center in Jichi Medical University.

According to FIGS. 10 (a) to 10 (c), an angiographic agent is administered from the portal vein to a decellularized liver transplanted by the APOLT method under a heart beat, and contrasted by a C-arm (radiological imaging apparatus). I got an image. 10A is a photograph of the treatment, and FIGS. 10B and 10C are contrast photographs. As a result, it was found that the vascular structure was maintained in detail even with the carrier not filled with cells. This indicates that the decellularized carrier treated by the decellularization method of the present example maintains a high cytoskeleton etc. even in a fragile structure such as a capillary. It was.
10D and 10E, the transplanted decellularized liver was sectioned to confirm the location of red blood cells. According to this, the liver lobule structure, which is a characteristic structure of the liver, was shown only by blood (erythrocytes). This fact also indicates that the decellularized carrier of this example maintains a high cytoskeleton and the like.

  It should be noted that the configuration and operation of the above-described embodiment are examples, and it is needless to say that the configuration and operation can be appropriately changed and executed without departing from the gist of the present invention.

  According to the present invention, since a decellularized carrier applicable to various experiments and advanced medicine can be produced, it can be used industrially.

Claims (16)

A cryopreservation step of cryopreserving the removed organ or tissue;
A thawing step of thawing the organ or tissue cryopreserved in the cryopreservation step;
A decellularization step of decellularizing the organ or tissue thawed in the thawing step,
The decellularization step includes
A poorly water-soluble surfactant perfusion step of perfusing the thawed organ or tissue with a poorly water-soluble surfactant-containing solution;
A method of producing a decellularized carrier, comprising: a hydrophilic water-soluble surfactant perfusion step of perfusing the organ or the tissue perfused with the poorly water-soluble surfactant-containing solution with the hydrophilic water-soluble surfactant-containing solution. . The cryopreservation step includes
A blood removal step upon freezing by perfusion of the organ or the tissue with physiological saline,
A cryopreservation solution perfusion step of perfusing the organ or the tissue that has been exsanguinated in the blood removal step during freezing with a cryopreservation solution;
A swelling step of filling and swelling the organ or tissue perfused in the cryopreservation solution perfusion step with the cryopreservation solution;
A surplus solution removing step of removing surplus cryopreservation solution adhering to the swollen organ or tissue;
The decellularization carrier according to claim 1, further comprising a freezing step of freezing the organ or the tissue from which the surplus cryopreservation solution has been removed in the surplus solution removing step at -20 ° C or lower. Production method. The cryopreservation step includes
The method for producing a decellularized carrier according to claim 2, further comprising a period storage step of storing the organ or the tissue before the decellularization step after the freezing step for a specific period. The cryopreservation solution is a physiological saline solution containing 0.01 to 0.3% hydrogen peroxide solution,
The method for producing a decellularized carrier according to claim 2 or 3, wherein the physiological saline and / or the cryopreservation solution in the blood removal step upon freezing contain 500 to 4000 U heparin. The thawing step includes
A low-temperature refrigeration thawing step for thawing the organ or tissue that has been cryopreserved in a low-temperature refrigerated state;
A blood removal step during thawing in which the organ or tissue thawed in the low temperature refrigeration and thawing step is perfused with a physiological saline containing a proteolysis inhibitor to remove blood;
A confirmation step for confirming the presence or absence of damage to the organ or the tissue that has been removed by the blood removal step at the time of thawing;
The method for producing a decellularized carrier according to any one of claims 1 to 4, further comprising a connection step of connecting the organ or the tissue confirmed in the confirmation step to a perfusion container. The thawing blood removal step includes
The method for producing a decellularized carrier according to claim 5, further comprising a thrombus removal step of removing a microthrombus contained in the organ or the tissue. The decellularization step includes
The poorly water-soluble surfactant contained in the poorly water-soluble surfactant-containing solution is NP-40,
The method for producing a decellularized carrier according to any one of claims 1 to 6, wherein the water-soluble surfactant contained in the solution containing the water-soluble surfactant is SDS. The decellularization step includes
The decellularization according to any one of claims 1 to 7, further comprising a stabilization step of stabilizing the organ or the tissue perfused in the hydrophilic water-soluble surfactant perfusion step after washing. A method for producing a carrier The stabilization step includes
Perfuse the organ or tissue with a 0.1-3% formaldehyde solution
The method for producing a decellularized carrier according to claim 8. Said organs include liver, kidney, spleen, heart, lung, small intestine, and testis,
The method for producing a decellularized carrier according to any one of claims 1 to 9, wherein the tissue includes skin, ureters, and blood vessels. A decellularized carrier produced by the method for producing a decellularized carrier according to any one of claims 1 to 10. The decellularized carrier according to claim 11, wherein 30% sucrose solution is immersed and cryopreserved. A physiological saline perfusion step of perfusing the decellularized carrier according to claim 11 or 12 with physiological saline;
A replacement step of replacing the decellularized carrier perfused in the physiological saline perfusion step by perfusion with a culture solution;
A cell injection step of injecting cultured cells from the decellularized carrier artery perfused with the culture medium in the replacement step;
A cell adhesion step in which the perfusion is stopped after the cultured cells are injected by the cell injection step and the cells are adhered to the decellularized carrier;
A perfusion culture step of culturing by resuming perfusion of the culture solution on the decellularized carrier to which the cells are adhered by the cell adhesion step. When the organ or tissue is the small intestine,
The cell filling method according to claim 13, wherein perfusion is performed by connecting an artery and a portal vein. A thin-layer sectioning step of thin-sectioning the decellularized carrier according to claim 11 or 12 into a decellularized tissue fragment,
Washing the decellularized tissue fragment that has been sliced by the thin-layer sectioning step, and seeding the cultured cells; and
And a culture step of culturing the decellularized tissue fragment in which the cultured cells are seeded by the seeding step. A decellularization solution kit for producing a decellularized carrier from an organ or tissue,
A poorly water-soluble surfactant-containing solution for perfusing the thawed organ or tissue;
A decellularized solution kit comprising a hydrophilic water-soluble surfactant-containing solution for perfusing the organ or the tissue perfused with the poorly water-soluble surfactant-containing solution.

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