JP7174984B2 - Decellularized carrier production method, decellularized carrier storage method, cell filling method, cell sheet preparation method, and decellularized solution kit - Google Patents

Description

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

Conventionally, techniques related to decellularization have been developed for removing cells from organs and tissues and producing decellularized carriers composed of extracellular matrices. Such decellularized carriers are used for transplantation into living organisms as scaffolds for cell growth, or as biomaterials for research.

Here, referring to Patent Document 1, as a conventional decellularization carrier, an extracellular matrix includes an outer surface, and the extracellular matrix containing a vascular tree substantially reproduces the morphology of the extracellular matrix before decellularization. Described is a decellularized mammalian organ which retains its morphology and whose outer surface is substantially intact.
The decellularized carrier of Patent Document 1 is prepared by inserting a cannula into the organ at one or more cavities, blood vessels, and/or ducts to create a cannulated organ; perfusing the organ into which is inserted a first cell disruption medium via one or more cannulations. The cell disruption medium includes at least one surfactant. Also, the surfactant is selected from the group consisting of SDS, PEG, or Triton X.

JP 2015-164549 A

However, in the conventional decellularized carrier according to Patent Document 1, the cytoskeleton and extracellular matrix are fragile, and the decellularized carrier undergoes atrophy or shrinkage due to the effects of the in vivo environment such as muscle movement and intra-abdominal pressure. was damaged. Therefore, it was very difficult to apply it to actual medical treatment. In addition, with conventional decellularized carriers, the structure of each vascular system running in an organ or tissue, particularly microvessels such as capillaries, is broken, making it difficult to restore the organ or tissue after cell filling. rice field. In other words, conventional decellularized carriers had low performance.

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

The decellularized carrier production method of the present invention comprises a cryopreservation step of cryopreserving an excised organ or tissue, a thawing step of thawing the organ or tissue cryopreserved in the cryopreservation step, and the thawing step. and a decellularization step of decellularizing the organ or the tissue thawed in the step, wherein the decellularization step includes exfoliating the thawed organ or tissue with a poorly water-soluble surfactant-containing solution. a poorly water-soluble surfactant perfusion step of perfusing the poorly water-soluble surfactant-containing solution within 180 minutes; and a water-soluble surfactant perfusion step of removing the poorly water-soluble surfactant contained in the poorly water-soluble surfactant-containing solution, wherein the organ or tissue excludes the placenta, and the poorly water-soluble surfactant is , the thawing step, the sparingly water-soluble surfactant perfusion step, and the hydrophilic surfactant perfusion step are not repeated and are completed within 48 hours.
In the decellularized carrier production method of the present invention, the cryopreservation step includes a freezing blood removal step of perfusing the organ or tissue with physiological saline to remove blood, and blood removal in the freezing blood removal step. and a cryopreservation solution perfusion step of perfusing the organ or tissue that has been treated with a cryopreservation solution, wherein the cryopreservation solution is physiological saline containing 0.01 to 0.3% hydrogen peroxide. , the physiological saline and/or the cryopreservation solution in the freezing blood removal step may contain 500 to 4000 U of heparin.
In the method for producing a decellularized carrier of the present invention, the cryopreservation step includes a swelling step of filling the organ or tissue perfused in the cryopreservation solution perfusion step with the cryopreservation solution to swell it, and a surplus solution removing step of removing the surplus cryopreservation solution adhering to the organ or tissue; and a freezing step of freezing below.
The decellularized carrier production method of the present invention is characterized in that the cryopreservation step further includes a period preservation step of preserving the organ or tissue for a specific period of time after the freezing step and before the decellularization step.
In the decellularized carrier production method of the present invention, the thawing step includes a low-temperature refrigeration-thawing step of thawing the cryopreserved organ or tissue in a low-temperature refrigeration state, and the organ thawed in the low-temperature refrigeration-thawing step. Alternatively, a thaw-time blood removal step of perfusing the tissue with a physiological saline solution containing a proteolysis inhibitor to remove blood, and confirming whether or not the organ or tissue that has been blood-bleeded in the thaw-time blood removal step is damaged. and a connecting step of connecting the organ or tissue confirmed by the confirming step to a perfusion container.
The decellularized carrier manufacturing method of the present invention is characterized in that the blood removal step during thawing includes a thrombus removal step of removing microthrombi contained in the organ or tissue.
In the method for producing a decellularized carrier of the present invention, in the decellularization step, the sparingly water-soluble surfactant contained in the sparingly water-soluble surfactant-containing solution is NP-40, and the hydrophilic surfactant is The hydrophilic water-soluble surfactant contained in the containing solution is characterized by being SDS.
In the decellularized carrier production method of the present invention, the decellularization step includes a stabilization step of washing and then stabilizing the organ or tissue perfused in the water-soluble surfactant perfusion step. Characterized by
The decellularized carrier production method of the present invention is characterized in that the stabilizing step includes perfusing the organ or tissue with a 0.1 to 3% formaldehyde solution and filling the organ or tissue with the formaldehyde solution. do.
The decellularized carrier production method of the present invention is characterized in that the organs include liver, kidney, spleen, heart, lung, small intestine, and testis, and the tissues include skin, ureter, and blood vessels. .
The method for producing a decellularized carrier of the present invention is characterized by eliminating double-stranded DNA and sugar chain structures.
The method for preserving decellularized carriers of the present invention is characterized in that the decellularized carriers produced by the method for producing decellularized carriers are immersed in a 30% sucrose solution and cryopreserved.
The cell filling method of the present invention comprises a physiological saline perfusion step of perfusing the decellularized carriers produced by the decellularized carrier production method or stored by the decellularized carrier storage method with saline; a replacement step of perfusing and replacing the decellularized carrier perfused in the physiological saline perfusion step with a culture medium; and removing cultured cells from an artery of the decellularized carrier perfused with the culture medium in the replacement step. a cell-injection step of injecting, a cell-adhesion step of stopping perfusion after the cultured cells are injected in the cell-injection step to adhere the cells to the decellularization carrier, and the cell-adhesion step to adhere the cells. and a perfusion culture step of resuming the perfusion of the culture solution to the decellularized carrier for culturing.
The cell filling method of the present invention is characterized in that perfusion is performed by connecting an artery and a portal vein when the organ or tissue is the small intestine.
The method for producing a cell sheet of the present invention comprises thinly sectioning the decellularized carriers produced by the method for producing decellularized carriers or stored by the method for storing decellularized carriers into decellularized tissue fragments. A seeding step of washing the decellularized tissue fragment thinly sliced by the layer sectioning step and seeding the cultured cells, and the cultured cells seeded by the seeding step and a culturing step of culturing the decellularized tissue fragment.
The decellularization solution kit of the present invention is a decellularization solution kit for producing a decellularized carrier from an organ or tissue, wherein the thawed organ or tissue is perfused for less than 120 minutes. Perfusing a surfactant-containing solution and the organ or tissue perfused with the poorly water-soluble surfactant-containing solution within 180 minutes, and obtaining the poorly water-soluble surfactant contained in the poorly water-soluble surfactant-containing solution. and a solution containing a hydrophilic surfactant that removes an agent, said organ or said tissue except for the placenta, said sparingly water-soluble surfactant being lipophilic, and instructions for practicing the method described above. It is characterized by including a book .

According to the present invention, the excised 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 hydrophilic. It is possible to provide a method for producing a high-performance decellularized carrier that can maintain the hardness and flexibility of the cytoskeleton and extracellular matrix by perfusion with a solution containing a soluble surfactant.

FIG. 4 is a photograph of a liver perfusion process with a sparingly water-soluble surfactant according to an example of the present invention. FIG. 4 is a photograph of a liver perfusion process with a hydrophilic surfactant according to an example of the present invention. 4 is a photograph of a kidney perfusion process with a hydrophilic surfactant according to an example of the present invention. FIG. 10 is a photograph of a spleen thawed blood removal step and a water-soluble surfactant perfusion step according to an example of the present invention. FIG. FIG. 4 is a photograph of a heart perfusion process with a hydrophilic surfactant according to an example of the present invention; FIG. 4 is a photograph of a lung perfusion process with a hydrophilic surfactant according to an example of the present invention. Fig. 4 is a photograph confirming the cell engraftment of the heart decellularized tissue fragment according to the example of the present invention. Fig. 4 is a photograph confirming the cell engraftment of the decellularized tissue fragment of the kidney according to the example of the present invention. Fig. 3 is a photograph confirming the cell engraftment of the decellularized lung tissue fragment according to the example of the present invention. Fig. 10 is a photograph confirming the cell engraftment of the decellularized tissue fragment of the liver according to the example of the present invention. FIG. 2 is electron micrographs of kidney and spleen decellularized carriers according to examples of the present invention. FIG. FIG. 2 is electron micrographs of heart and lung decellularized carriers according to examples of the present invention. FIG. Fig. 2 is electron micrographs of liver and small intestine decellularized carriers according to Examples of the present invention. FIG. 10 is a photograph of an analysis (immunostaining) of an extracellular matrix according to an example of the present invention; FIG. 4 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 techniques have been developed both at home and abroad. However, in the conventional decellularized carrier production method, it takes 24 to 72 hours, for example, for rats to complete decellularization. Including the washing process, an additional 3 to 7 days were required. For this reason, there was a high possibility that the extracellular matrix would break down, putrefy, or the like during the period of decellularization. In other words, the cytoskeleton and extracellular matrix become fragile in conventional methods for producing decellularized carriers. Therefore, the process of vascular anastomosis performed at the time of transplantation has become very difficult.
On the other hand, the present inventors conducted extensive experiments and tests and found that cryopreservation of excised organs or tissues facilitates production of high-quality decellularized carriers. Furthermore, as a result of repeated trial and error, the cryopreserved organ or tissue is thawed, the thawed organ or tissue is perfused with a poorly water-soluble surfactant-containing solution, and then with a water-soluble surfactant-containing solution. I found that it is good to perfuse, and came to complete the invention. According to the method for producing decellularized carriers of the present embodiment, it is possible to produce high-performance decellularized carriers that facilitate vascular anastomosis while maintaining a high degree of hardness and flexibility of the cytoskeleton and extracellular matrix.

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

A decellularized carrier production method according to an embodiment of the present invention comprises a cryopreservation step of cryopreserving an excised organ or tissue, a thawing step of thawing the cryopreserved organ or tissue in the cryopreservation step, and a decellularization step of decellularizing the thawed organ or tissue in the thawing step.
By configuring in this way, it becomes possible to produce the decellularized carrier in a shorter period of time than in the conventional technique. In addition, it becomes possible to maintain the hardness and flexibility of the cytoskeleton and extracellular matrix of the decellularized carrier at a high level. Therefore, a high-quality decellularized carrier can be produced. The manufactured decellularized carrier can be used for advanced experiments using organ structures. In addition, the manufactured decellularized carrier facilitates 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 carriers can be used as materials for regenerative medicine and scaffolds.
Furthermore, the decellularized carrier produced by the method for producing a decellularized carrier of the present embodiment, as described later, is capable of engraftment of functional cells differentiated from iPS cells, ES cells, etc. and production of functionally expressed organs. can be made possible. Therefore, application in the field of regenerative medicine can be expected.

In addition, loss of extracellular matrix is minimized in the decellularized carrier produced by the decellularized carrier producing method of the present embodiment. As a result, as shown in the immunostaining and electron microscopic images in Examples described later, the decellularized carrier maintains a high degree of detailed structure.
In addition, the decellularized carrier produced by the present invention has almost no antigenicity due to the loss of double stranded DNA (hereinafter referred to as "dsDNA") and sugar chain structure. Therefore, bioapplication is facilitated.

In addition, since the conventional decellularized carrier manufacturing method is "organ-specific", it was necessary to adapt the protocol and decellularization solution to the organ or tissue to be decellularized.
In contrast, in the decellularized carrier production method according to the embodiment of the present invention, organs include liver, kidney, spleen, heart, lung, small intestine, and testis, and tissues include skin, ureter, and It is characterized by containing blood vessels.
That is, in the method for producing a decellularized carrier of the present embodiment, the liver, kidney, spleen, heart, lung, small intestine, and testis, which are excised organs, and the skin, ureter, and blood vessels, which are tissues, are the same. Decellularization is possible with the composition liquid and the same protocol, and the treatment can be performed in a short time.

Further, in the decellularized carrier production method according to the embodiment of the present invention, the cryopreservation step includes a freezing blood removal step of perfusing the organ or tissue with physiological saline to remove blood, and a freezing blood removal step. a cryopreservation solution perfusion step of perfusing the organ or tissue exsanguinated with a cryopreservation solution with a cryopreservation solution; A surplus solution removing step of removing surplus cryopreservation solution adhering to the swollen organ or tissue; and a step.
In the cryopreservation solution perfusion step of perfusing with the cryopreservation solution, the organ or tissue is frozen after blood removal, perfusion with the cryopreservation solution, swelling, and then thawing in the subsequent thawing step. By doing so, it is possible to open fine holes in the cell membranes constituting the organ or tissue. As a result, the permeability of the decellularization solution into the cytoplasm is increased, and the decellularization time can be shortened.
Specifically, in the decellularized carrier production method of the present embodiment, the time required for decellularization is shortened by about 20% compared to the conventional technique described in Patent Document 1. In addition, as described above, the same liquid composition can be used regardless of the target organ.

In addition, in the conventional decellularized carrier production method, decellularization was performed immediately after the organ was extracted. In other words, there was a limit to the storage period of the excised organs.
On the other hand, in the decellularized carrier production method of the present embodiment, the cryopreservation step further includes a period preservation step of preserving the organ or tissue after the freezing step and before the decellularization step for a specific period of time. do.
In this way, once the decellularized organ is frozen, it becomes possible to stock the organ or tissue. This specific period can be arbitrarily set in units of several hours to several years, and in a commercial medical freezer that does not perform defrosting heating, it is possible to store the product without deterioration. That is, the decellularized organ can be thawed as necessary to produce a decellularized carrier.

Further, in the decellularized carrier production method according to the embodiment of the present invention, the cryopreservation solution is physiological saline containing 0.01 to 0.3% by weight of hydrogen peroxide and 500 to 4000 U of heparin. characterized by
By using a physiological saline containing 0.01 to 0.3% by weight hydrogen peroxide and 500 to 4000 U heparin as a cryopreservation solution, damage to the cytoskeleton and extracellular matrix is minimized. While minimizing, it increases the permeability of surfactants and allows the opening of optimal micropores for cytoplasmic removal. In other words, by using a physiological saline solution containing hydrogen peroxide in the decellularization process and making microscopic holes in the cell membrane, the influx of each surfactant is improved, and intracytoplasmic substances such as the nucleus and various structures are removed. improves the removability of Therefore, as described above, during the subsequent decellularization step, the cytoskeleton and extracellular matrix are exposed to the decellularization solution for less time. Therefore, it is possible to shorten the production time of the decellularized carrier. In addition, it becomes possible to suppress deterioration and denaturation of amino acids.

In the decellularized carrier production method according to the embodiment of the present invention, the thawing step includes a low-temperature refrigeration thawing step of thawing the cryopreserved organ or tissue in a low-temperature refrigeration state, and a low-temperature refrigeration thawing step, and Alternatively, a thaw-time blood removal step of perfusing the tissue with a physiological saline solution containing a proteolysis inhibitor to remove blood, and a confirmation step of confirming whether or not the organ or tissue that has been blood-bleeded in the thaw-time blood removal step is damaged. and a connecting step of connecting the organ or tissue confirmed by the confirming step to the perfusion container.
Here, the low-temperature refrigeration state may be about 2°C to 10°C.
The thawing step after such a cryopreservation step allows the cytoplasm to be easily removed by appropriately causing "drip" through fine holes in the cell membranes constituting the organ or tissue. This increases the permeability of the decellularization fluid into the cytoplasm and shortens the decellularization time. Therefore, as described above, the decellularization time can be shortened, and the deterioration and denaturation of amino acids in the cytoskeleton and extracellular matrix can be suppressed.

Moreover, in the conventional decellularized carrier manufacturing method, there was no means for removing thrombi. For this reason, it was necessary to extract the organ under a pulsating state with blood flow.
In contrast, the decellularized carrier manufacturing method according to the embodiment of the present invention is characterized by including a thrombus removal step of removing microthrombi contained in the organ or tissue in the thawing blood removal step.
By configuring in this way, decellularization becomes possible even when microthrombi are formed. That is, in the decellularized carrier production method of the present embodiment, decellularization is possible even in the presence of microthrombi by performing blood removal by sufficient perfusion during freezing and thawing, in which the thawing step is performed after the freezing step. . More specifically, the method for producing decellularized carriers of the present embodiment enables the production of decellularized carriers as long as the target organ or tissue is not decomposed.

Further, in the decellularized carrier production method of the present embodiment, 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; and a hydrophilic surfactant perfusion step of perfusing the organ or tissue perfused with the hydrophilic surfactant-containing solution with the hydrophilic surfactant-containing solution.
Here, the sparingly water-soluble surfactant contained in the sparingly water-soluble surfactant-containing solution of the present embodiment is a surfactant having a hydrophilic group that does not ionize when dissolved in water. It is preferred to use nonionic surfactants that are less susceptible to . As the nonionic surfactant, polyoxyethylene phenyl ether surfactants such as Triton X-100 and NP-40, DDM, digitonin, Tween 20, Tween 80 and the like can be used. In this embodiment, it is particularly preferable to use NP-40. In this case, NP-40 is not necessarily NP-40 (octyl-phenoxy-polyethoxy-ethanol) itself, but various NP-40 substitutes, Nonidet (TM) P-40, etc. can be used.
By configuring in this way, in the decellularization step, concentration changes due to substances in the cytoplasm are less likely to occur, and it is possible to first remove the substances in the cytoplasm after leaving the extracellular matrix and the like. Become.

In addition, as the hydrophilic surfactant contained in the hydrophilic surfactant-containing liquid of the present embodiment, an anionic surfactant that ionizes the portion with a hydrophobic group into a negative (anion) ion when dissolved in water. Agents (anionic surfactants) can be preferably used. In this embodiment, alkyl sulfate ester salts may be used. SDS (Sodium Dodecyl Sulfate) is particularly suitable as the alkyl sulfate.
With this configuration, in the decellularization step, after treatment with a poorly water-soluble surfactant (NP-40)-containing solution, this is removed with a hydrophilic surfactant (SDS)-containing solution. The water cleaning time can be shortened to half of the conventional time. In other words, in the prior art described in Patent Document 1, etc., only water is used to remove the surfactant, but in order to remove the surfactant as in the present embodiment, another surfactant is used. This makes it possible to reduce the cleaning time by at least 50%.

Further, a decellularization solution kit according to an embodiment of the present invention is a decellularization solution for producing a decellularized carrier from an organ or tissue, which is a poorly water-soluble decellularization solution for perfusing the thawed organ or tissue. It is characterized by comprising a surfactant-containing solution and a hydrophilic water-soluble surfactant-containing solution that perfuses an organ or tissue perfused with the sparingly water-soluble surfactant-containing solution.
With such a configuration, the poorly water-soluble surfactant and the solution containing the hydrophilic surfactant may be prepared as a decellularization solution kit. Moreover, the poorly water-soluble surfactant and the solution containing the hydrophilic surfactant may be prepared at the time of use.

In the decellularized carrier production method 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 water-soluble surfactant perfusion step. characterized by
Also, this stabilization step is characterized by perfusing the organ or tissue perfused in the water-soluble surfactant perfusion step with a 0.1-3% formaldehyde solution. At this time, it is particularly preferable that the formaldehyde solution used in the stabilization step of the present embodiment has a concentration of 0.5 to 1%.
By constructing in this way and perfusing a 0.1 to 3% formaldehyde solution for 0.5 to 2 hours after perfusion in the water-soluble surfactant perfusion step, while maintaining the properties of the cytoskeleton and extracellular matrix, The hardness of the manufactured decellularized carrier can be ensured. This facilitates vascular anastomosis at the time of transplantation. As a result, as described below, it becomes easier to apply the decellularized carrier to scaffold therapy, such as using the decellularized carrier as a scaffold material for cell filling or as a material for regenerative medicine.
A physiological saline containing hydrogen peroxide may be used in the decellularization step. In this case as well, by forming minute holes in the cell membrane, the influx of each surfactant and the ability to remove cytoplasmic substances are improved, and the time required for preparing the decellularized carrier can be shortened.

In addition, the cell filling method according to the embodiment of the present invention includes a physiological saline perfusion step of perfusing the decellularized carrier with a physiological saline and then perfusing it with a culture solution, and replacing the perfused decellularized carrier in the perfusion step. A replacement step, a cell injection step of injecting cultured cells from the artery of the decellularized carrier perfused with the culture medium in the replacement step, and a cell injection step in which the perfusion is stopped after the cultured cells are injected to decellularize the cells. and a perfusion culture step of resuming the perfusion of the culture solution to the decellularized carrier to which the cells have been adhered by the cell adhesion step and culturing the decellularized carrier.
By configuring in this way, cultured cells such as functional cells and primary passage cells differentiated from iPS cells, ES cells, or other stem cells (hereinafter referred to as "iPS cells, etc.") can be used as decellularization carriers. can be filled. In addition, as shown in Examples described later, decellularized carriers filled with cells allow the filled cultured cells to survive after transplantation. This is probably because, as described above, the decellularized carrier of the present embodiment has infinitely "0" antigenicity and little loss of extracellular matrix. Therefore, the decellularized carrier filled with cells of the present embodiment can be used to solve the chronic shortage of organs for transplantation.
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 to which the cultured cells that have been filled are engrafted.

Moreover, the cell filling method according to the embodiment of the present invention is mainly intended for vertebrate animals. The vertebrates are not particularly limited and include, for example, humans, domestic animal species, and wild animals. Here, the cell filling method of the present embodiment has experimentally succeeded in decellularizing at least the organs of pigs, rats, and mice. Therefore, the method for producing a decellularized carrier according to the embodiment of the present invention can be used not only for human treatment, but also for animal treatment, livestock treatment, xenotransplantation, and the like. It is also applicable in the pet industry.

Moreover, the cell filling method according to the embodiment of the present invention is characterized in that perfusion is performed by connecting an artery and a portal vein when the organ or tissue is the small intestine.
By configuring in this way, the cell-filled decellularized carrier can be used as a functioning small intestine, and the possibility of transplantation can be increased. This is because the decellularized carrier of the present embodiment retains a high degree of structure of the villus portion and muscle layer portion of the small intestine, as will be shown in Examples described later. is filled.
For this reason, it is possible to perfuse a mixture of cells differentiated into the villus and muscle layers by perfusing a mixture of cells that have differentiated into the villus and muscle layers using techniques for inducing differentiation from iPS cells and the like to regenerate various organs, and allowing them to settle separately in the villus and muscle layers. I can expect it.

Further, the method for producing a cell sheet according to the embodiment of the present invention includes a thin layer sectioning step of thinly sectioning the decellularized carrier into decellularized tissue fragments, and a thin layer sectioning step. A seeding step of washing the decellularized tissue fragment and seeding the cultured cells, and a culturing step of culturing the decellularized tissue fragment seeded with the cultured cells in the seeding step.
Here, it is preferable that the thickness of this thin slice is about 0.1 to 5 mm. In addition, cultured cells may be seeded several times with about 0.5 to 10×10 ⁵ cells.
As a result of seeding cells on a 1 mm-thick thin slice of the decellularized carrier produced in this embodiment, the cells adhered in 30 minutes to 1 hour from the start of culture, which is much shorter than the conventional technology. Preliminary experiments have confirmed that This is because the shortening of the decellularization treatment time has made it possible to suppress deterioration and denaturation of amino acids in the cytoskeleton and extracellular matrix. It is thought that the cells adhered over time.
This cell-adhered decellularized carrier can be used as a cell sheet. In other words, it is possible to create an organ in which functional cells that are induced to differentiate from iPS cells or ES cells are allowed to adhere and functions are expressed.
These cell sheets may be used after seeding cultured cells and incubating overnight.

Moreover, the decellularized carrier according to the embodiment of the present invention is characterized by being manufactured by a decellularized carrier manufacturing method.
As described above, the decellularized carrier constructed in this manner is excellent in cell adhesion, and the decellularized organ filled with cells can be engrafted with the filled cells even after transplantation. That is, it is of high quality as a decellularized carrier.

Further, the decellularized carrier of the present embodiment is characterized by being immersed in a 30% sucrose solution and cryopreserved.
By configuring in this way, the decellularized carrier of the present embodiment can be easily cryopreserved. In addition, the quality of the decellularized carrier of the present embodiment can be maintained, and it can be put into a usable state without using a special method. This facilitates application of the decellularized carrier.

In addition, each of the above-described steps may include steps that can be executed in parallel, and the steps before and after the steps may be partially changed, and optimization within the scope of the present invention by those skilled in the art may be performed.

Moreover, the method for producing a decellularized carrier according to the embodiment of the present invention can be used in combination with steps using other compositions.

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

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

〔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 "saline"), heparin sodium (manufactured by Nipro Corporation), hydrogen peroxide (manufactured by Wako Pure Chemical Industries, Ltd.), water for injection (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.), antibiotic-antimycotic mixed solution (Antibiotic-Antimycotic, 100X, manufactured by Gibco) , MEMalpha (manufactured by Gibco), FBS (Fetal Bovine Serum, manufactured by Gibco), PBS (manufactured by TakaRaBio), protein degradation inhibitor (ProteoGuard EDTA-Free Protease Inhibitor Coctail, manufactured by Clontech), Cefamedin sodium injection (Astellas Pharmaceutical Co., Ltd.), 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, forceps, and scissors were used as surgical instruments. These surgical instruments and the like were sterilized in an autoclave at 121° C. for 15 minutes before use.
Tupperware (for procedures) and sealed pack instruments were EOG sterilized prior to use.

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

[Preparation of reagent]
In this example, NP-40 was used as a sparingly water-soluble surfactant in the sparingly 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 "A solution". Specifically, in this example, 20 mL of NP-40 Substitute was added to 1,980 mL of water for injection or purified water, and dissolved without foaming to obtain liquid A.
In addition, SDS was used as a hydrophilic surfactant in the hydrophilic surfactant perfusion step in the decellularization step. In this example, the hydrophilic surfactant-containing solution using this SDS is hereinafter referred to as "B solution". Specifically, in this example, 200 mL of 10% SDS solution was added to 1800 mL of water for injection or purified water, and dissolved to form a 1% SDS solution without foaming, which was used as solution B.
Other solutions were prepared according to the volumes described in the reagent manuals.

<Example of treatment for manufacturing decellularized carriers>
The details of the treatment in each step of the decellularized carrier production method according to the present example will be described below.

[Cryopreservation step]
First, the cryopreservation step of cryopreserving the excised organ or tissue in this embodiment will be described.
In the cryopreservation process of this example, as shown in the following examples, a cryopreservation blood removal process, a cryopreservation solution perfusion process, a swelling process, an excess solution removal process, and a freezing process were performed.

(Freezing blood removal process)
In the frozen blood removal step of this embodiment, 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 "heparinized saline") to remove blood. I got blood

Therefore, first, a catheter for perfusion fixation was indwelled in the organ or tissue. Specifically, the blood vessel of the organ or tissue was exposed and peeled, and the outer diameter of the blood vessel was measured with a tape measure. A catheter suitable for the outer diameter of the vessel was then selected. As this catheter, an extension tube for blood transfusion or an IVH catheter was selected according to the vessel shape. The outer diameter of the vessel and the catheters and sutures used were selected as follows:

Outer diameter of blood vessel Catheter suture > 4.0 mm, left atrial appendage Extension tube for blood transfusion 3-0 silk thread 3.0-4.0 mm Extension tube for blood transfusion 3-0 silk thread 2.0-2.9 mm 14GIVH catheter 4-0 nylon thread 1.5-2.0 mm 16GIVH catheter 4-0 nylon thread 1.0-1.4 mm 18GIVH catheter 5-0 nylon thread

Next, the selected catheter was fitted with a three-way stopcock and a 20 mL syringe (Terumo Corporation), filled with heparinized saline, and the organ or tissue was evacuated. The catheter was then cut 40 cm from the three-way stopcock connection. Next, the central side of the blood vessel was ligated and sutured. Next, an incision was made on the central side of the blood vessel with a Metzen, and a catheter was inserted. Then, the blood vessel and the catheter were simply fixed with bulldog forceps. Two catheter insertion sites and one catheter site were ligated and fixed.

Next, the organ or tissue was actually perfused for blood removal.
Specifically, catheters were inserted and indwelled in arteries and veins of each organ or tissue by about 1 cm. After this, 200 mL or more of heparinized saline was pumped and injected from the arterial catheter, and it was confirmed that blood was discharged from the venous catheter. It was confirmed that the color had faded to white.

(Cryopreservation solution perfusion step)
Next, in the cryopreservation solution perfusion step, the organ or tissue exsanguinated in the cryopreservation step was perfused with the cryopreservation solution.
The cryopreservation solution used in this example was a physiological saline solution adjusted to 0.1% hydrogen peroxide. Also, the three-way stopcock on the venous side was closed, and the cryopreservation solution was injected from the arterial side.

(Expansion process)
In the expansion step, the organ or tissue perfused with the cryopreservation solution perfused in the cryopreservation solution perfusion step was filled with the cryopreservation solution and expanded.
Specifically, it was confirmed that the cryopreservation solution was injected and the inside of the organ or tissue was swollen (totally stretched), and the three-way stopcock on the artery side was closed.

(Excess solution removal step)
In the surplus solution removing step, the 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 excess solution removal step was frozen at -20°C or lower.
Specifically, the organ or tissue was transferred to a container such as a ziplock and stored frozen at -20°C or -80°C.

[Thawing step and decellularization step]
Here, an example in which the liver, kidney, heart, spleen, and lungs extracted from experimental pigs weighing about 10 kg are subjected to the above-described cryopreservation process, followed by the thawing process and the decellularization process will be described below. do.
The perfusion time for each organ below is a guideline, and priority was given to visual confirmation.

[liver]
(Low-temperature refrigeration thawing process)
Cryopreserved organs were thawed under cryogenic conditions. Specifically, frozen organs were thawed at 4° C. for 24±1 hours. Make sure the organs are completely thawed.

(Bleeding process at the time of thawing, confirmation process, connection process)
Next, the organ thawed in the low-temperature refrigeration thawing step was perfused with a protein degradation inhibitor-containing physiological saline solution to remove blood.
Specifically, first, raw food was applied to the liver to wash off adhering and remaining blood clots and the like.
Next, saline (50 mL each x 5) was injected from the portal vein catheter and the hepatic artery catheter using a disposable syringe, and it was confirmed that the saline was discharged from the hepatic vein without resistance or leakage. Although I felt some resistance during the first pumping, I was careful at this point because if I forced it in, the blood vessels and membranes could rupture. The resistance gradually disappeared, and it was confirmed that the raw food could be pumped and injected smoothly.
Next, the injection tube of the 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 with a connector.
Saline containing a protein degradation inhibitor in the amount described in the manual (hereinafter referred to as "proteolysis inhibitor-containing saline") was prepared, 2000 mL was placed in a sterilized pack, and sterilized gauze was attached to the top. The liver was put on a gauze so that about 1 cm of the liver was immersed in the saline. At this time, if the organ was too immersed in the liquid surface, it would float and move in the perfusate. Also, care was taken that the entire organ rested on the gauze. In addition, it was confirmed that the hepatic artery and portal vein were not crooked and protruded from the porta hepatis.
The liver was then transferred to a 10°C refrigerator or 10°C environment. Note that if the temperature is set to 4° C., the perfusate may precipitate in subsequent steps. In addition, room temperature was avoided because proteolytic enzyme production due to autolysis may cause damage to the assembly of the decellularized carrier.
Next, the three-way stopcock of the hepatic artery was closed, and only the three-way stopcock of the portal vein was opened.
Next, the three-way stopcock of the portal vein was closed, and only the three-way stopcock of the hepatic artery was opened, and a protein-degradation inhibitor-containing saline was perfused for 15 minutes at 60 to 80 mL/min using an automatic perfusion apparatus.
Next, the three-way stopcocks of the portal vein and hepatic artery were both opened, and normal saline containing a protein degradation inhibitor was perfused twice at 80 mL/min for 30 minutes using an automatic perfusion device. If the saline turned red and cloudy, it was perfused again for 15 minutes.
In other words, it was possible to remove microthrombi from an organ or tissue as a thrombus removal step in the blood removal step during thawing by perfusing the saline until it no longer turned red and cloudy. This is the same for the following other organs.

As a final confirmation step, the presence or absence of damage to the liver that had been exsanguinated in the exsanguination step during thawing was confirmed. At this time, organs or tissues with cracks or perforations were excluded from the decellularized carrier production.
This confirmation step was performed in the same way for other organs as well.

(Poorly water-soluble surfactant perfusion step)
At this point, the automatic perfusion apparatus was stopped and replaced with A solution. Then, using an automatic perfusion apparatus, liquid A was perfused in order through the hepatic artery and portal vein at 60 mL/min for 15 to 30 minutes, and then both were opened and perfused at 80 to 120 mL/min for 20 minutes×3 times. At this time, immediately after the replacement with solution A, perfusion was performed at 20 mL/min while observing the situation, and after confirming that the perfusate flowed smoothly, the rate was gradually increased to 60 mL/min.
According to FIGS. 1(a) and 1(b), after a while after perfusion, the perivascular area changed to a translucent state. Here, FIG. 1(a) shows a photograph immediately after perfusion, and FIG. 1(b) shows a photograph during the third perfusion.
According to FIG. 1(c), it was confirmed that the peripheral portion of the liver also changed to a slightly translucent state after that.

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

Next, the perfusion device was temporarily removed, and 500 mL×3 times perfusion was performed with separately prepared B solution from the hepatic artery catheter and portal vein. A portion of the third perfusate was collected, and dsDNA was measured using an absorbance meter and confirmed to be 0.5 ng/μL or less. When this value was exceeded, solution B was added at 80 to 120 mL/min for 30 minutes using an automatic perfusion apparatus, and the concentration was measured again in the same manner. Also, if the total perfusion time of solution B exceeded 6 hours, this concentration measurement was excluded.
Next, all the liquid B was aspirated and replaced with purified water (water for injection). Specifically, using an automatic perfusion device, purified water was perfused in order of 60 mL/min through the hepatic artery and portal vein for 15 to 30 minutes, and then both were opened, and 80 to 120 mL/min for 15 minutes x 8 times, 30 times. min x 6 times, then (120 min x 4 times/day) x 6 days. At this time, since the central portion of the decellularized organ has a fine network structure, the washing perfusion was performed not only several times but also for a sufficient time.
After that, the final perfusate (purified water) was collected, and dsDNA was measured using an absorbance meter to confirm that dsDNA was 0.0 ng/μL (below the detection limit). confirmed.

[kidney]
(Low-temperature refrigeration thawing process)
Cryopreserved organs were thawed at 4°C for 24±1 hours. After confirming that the organs were completely thawed, the following treatments were performed.

(Bleeding process at the time of thawing, confirmation process, connection process)
Next, a saline solution was applied to the kidney to wash off adhering and remaining blood clots and the like.
Next, saline (50 mL×3) was injected from the renal artery catheter using a disposable syringe, and it was confirmed that the saline was discharged from the hepatic vein without resistance or leakage. Although I felt some resistance during the first pumping, I was careful at this point because if I forced it in, the blood vessels and membranes could rupture. The resistance gradually disappeared, and it was confirmed that the raw food could be pumped and injected smoothly.
Next, the infusion tube of the automatic perfusion apparatus was connected to the three-way stopcock of the renal artery catheter.
Next, 2000 mL of proteolytic inhibitor-containing saline was placed in a sterile pack and sterile gauze was attached on top. The kidney was put on gauze so that it was immersed in the saline by about 1 cm. Be careful not to immerse it in the liquid surface too much, as it will float and move in the perfusate. Care was taken to ensure that the entire organ rested on the gauze. It was confirmed that the blood vessels of both the renal artery and vein exited the renal hilum and were not bent.
Next, the kidney was transferred to a 10°C refrigerator or a 10°C environment. Note that if the temperature is set to 4° C., the perfusate may precipitate in later steps. In addition, room temperature was avoided because it may cause damage to the cellular carrier assembly.
Next, the three-way stopcock of the renal artery was opened, and normal saline containing a protein degradation inhibitor was perfused twice at 40 to 60 mL/min for 15 minutes using an automatic perfusion apparatus. If the saline became red and cloudy, it was perfused again for 15 minutes.

(Poorly water-soluble surfactant perfusion step)
After the final confirmation step, the autoperfusion apparatus was stopped and replaced with solution A. Then, liquid A was perfused 3 times for 20 minutes at 40 to 60 mL/min using an automatic perfusion device. Immediately after replacement with solution A, perfusion was performed at 20 mL/min while monitoring the situation, and after confirming that the perfusate flowed smoothly, the rate was gradually increased to 60 mL/min. Shortly after the perfusion, the perivascular area changed to a translucent state. After that, it was confirmed that the whole kidney changed to a slightly translucent state.

(Water-soluble surfactant perfusion step)
Next, the automatic perfusion apparatus was stopped and replaced with B solution. Then, liquid B was perfused 6 times for 20 minutes at 60 mL/min using an automatic perfusion device. Immediately after replacement with solution B, perfusion was performed at 20 mL/min while observing the situation, and after confirming that the perfusate flowed smoothly, the rate was gradually increased to 60 mL/min.
According to FIG. 3(a), the initial perfusion of liquid B made the kidneys dramatically transparent, and the liquid immediately became cloudy after the perfusion.
According to FIG. 3(b), it was confirmed that the kidney was completely translucent decellularized tissue after 6 perfusions.

Next, the perfusion device was temporarily removed, and 50 mL×3 times perfusion was performed with the separately prepared B solution through the renal artery catheter. A portion of the third perfusate was collected, and dsDNA was measured using an absorbance meter and confirmed to be 0.5 ng/μL or less. When this value was exceeded, solution B was additionally perfused for 15 minutes at 40 to 60 mL/min using an automatic perfusion device, and the concentration was measured again in the same manner. Further, when the total perfusion time of solution B exceeded 6 hours, this concentration determination was not performed.
Next, all the liquid B was aspirated and replaced with purified water (water for injection). Using an automatic perfusion apparatus, purified water was perfused at 40 to 60 mL/min for 15 minutes x 8 times, 30 minutes x 6 times, and thereafter (120 minutes x 4 times/day) x 6 days. At this time, since the central portion of the decellularized organ has a fine network structure, the washing perfusion was performed not only several times but also for a sufficient time.
After that, the final perfusate (purified water) was collected, and dsDNA was measured using an absorbance meter to confirm that dsDNA was 0.0 ng/μL (below the detection limit). confirmed.

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

(Bleeding process at the time of thawing, confirmation process, connection process)
Next, the spleens were soaked with saline to wash off adhering and remaining blood clots and the like.
Next, saline (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 that the saline was discharged from the splenic vein. I felt some resistance during the first pumping, but I was careful at this point because if I forced it in, the blood vessels and membranes could rupture. In particular, the splenic artery catheter may be thinner than other organs, so care was taken not to pull the catheter out due to pressure. The resistance gradually disappeared, and it was confirmed that the raw food could be pumped and injected smoothly.
Next, the infusion tube of the automatic perfusion apparatus was connected to the three-way stopcock of the splenic artery catheter.
Next, 2000 mL of the proteolytic inhibitor-containing saline was placed in a sterile pack, and sterile gauze was attached to the top. The spleen was placed on gauze so that it was immersed in saline by about 1 cm. Be careful not to immerse it in the liquid surface too much, as it will float and move in the perfusate. Care was taken to ensure that the entire organ rested on the gauze. It was confirmed that the blood vessels of both the splenic artery and vein exited the splenic hilum and were not bent.
The spleens were then transferred to a 10°C refrigerator or 10°C environment. Note that if the temperature is set to 4° C., the perfusate may precipitate in later steps. In addition, room temperature was avoided because it may cause structural damage to the decellularized carrier.
Next, the three-way stopcock of the splenic artery was opened, and normal saline containing a protein degradation inhibitor was perfused at 40 to 60 mL/min for 15 minutes×5 times using an automatic perfusion device. If the saline became red and cloudy, it was perfused again for 15 minutes. Blood tended to remain in the margin of the spleen. When it was difficult to remove blood, it tended to be easy to remove blood by rubbing it lightly with the hand. It was noted that proceeding to the next step in a situation where the blood could not be removed would result in a long perfusion time and the possibility that the fine structure could not be maintained.
According to FIG. 4(a), the entire spleen was exsanguinated and thoroughly perfused with saline until it became pink.

(Poorly water-soluble surfactant perfusion step)
After the final confirmation step, the autoperfusion apparatus was stopped and replaced with solution A. Then, liquid A was perfused 3 times for 20 minutes at 40 to 60 mL/min using an automatic perfusion device. Immediately after replacement with solution A, perfusion was performed at 20 mL/min while monitoring the situation, and after confirming that the perfusate flowed smoothly, the rate was gradually increased to 60 mL/min. Shortly after the perfusion, the entire spleen became translucent. After that, it was confirmed that the whole spleen changed to a slightly translucent state.

(Water-soluble surfactant perfusion step)
Next, the automatic perfusion apparatus was stopped and replaced with B solution. Then, liquid B was perfused 6 times for 20 minutes at 60 mL/min using an automatic perfusion device. Immediately after replacement with solution B, perfusion was performed at 20 mL/min while observing the situation, and after confirming that the perfusate flowed smoothly, the rate was gradually increased to 60 mL/min.
According to FIG. 4(b), the initial perfusion of liquid B made the kidneys dramatically transparent, and the liquid immediately became cloudy after the perfusion.
According to FIG. 4(c), it was confirmed that the spleen had completely turned into a translucent decellularized tissue after six perfusions.

Next, the perfusion device was temporarily removed, and 500 mL×3 times of perfusion was performed with the separately prepared B solution through the splenic artery catheter. A portion of the third perfusate was collected, and dsDNA was measured using an absorbance meter and confirmed to be 0.5 ng/μL or less. When this value was exceeded, solution B was additionally perfused for 15 minutes at 40 to 60 mL/min using an automatic perfusion device, and the concentration was measured again in the same manner. This concentration determination was not performed when the total perfusion time of solution B exceeded 6 hours.
Next, all the liquid B was aspirated and replaced with purified water (water for injection). Using an automatic perfusion apparatus, purified water was perfused at 40 to 60 mL/min for 15 minutes x 8 times, 30 minutes x 6 times, and thereafter (120 minutes x 4 times/day) x 6 days. At this time, since the central portion of the decellularized organ has a fine network structure, the washing perfusion was performed not only several times but also for a sufficient time.
After that, the final perfusate (purified water) was collected, and dsDNA was measured using an absorbance meter to confirm that dsDNA was 0.0 ng/μL (below the detection limit). confirmed.

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

(Bleeding process at the time of thawing, confirmation process, connection process)
Next, the heart was sprinkled with saline to wash off adhering and remaining blood clots and the like.
Next, saline (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 that the saline was discharged from the pulmonary artery. I felt some resistance during the first pumping, but I was careful at this point because if I forced it in, the auricle might rupture. Both the left and right auricles were inflated by pumping. At this time, it was confirmed that the blood in the coronary arteries was flowing and the whole heart was perfused.
Next, the infusion tube of the automatic perfusion apparatus was connected to the three-way stopcock of the aortic catheter.
Next, 2000 mL of the proteolytic inhibitor-containing saline was placed in a sterile pack, and sterile gauze was attached to the top. The heart was placed on gauze so that it was immersed in the saline by about 1 cm. Be careful not to immerse it in the liquid surface too much, as it will float and move in the perfusate. Also, care was taken that the entire organ rested on the gauze.
The heart was then moved to a 10°C refrigerator or a 10°C environment. Note that if the temperature is set to 4°C, the perfusate may precipitate in later steps. Also, room temperature was avoided because it may damage the structure of the decellularized carrier.
Next, the three-way stopcock of the aorta was opened, and normal saline containing a proteolysis inhibitor was perfused at 40 to 60 mL/min for 15 minutes×5 times using an automatic perfusion device. If the saline became red and cloudy, it was perfused again for 15 minutes.

(Poorly water-soluble surfactant perfusion step)
After the final confirmation step, the autoperfusion apparatus was stopped and replaced with solution A. Then, liquid A was perfused 3 times for 20 minutes at 40 to 60 mL/min using an automatic perfusion device. Immediately after replacement with solution A, perfusion was performed at 20 mL/min while monitoring the situation, and after confirming that the perfusate flowed smoothly, the rate was gradually increased to 60 mL/min. Shortly after the perfusion, the perivascular area changed to a translucent state. After that, it was confirmed that the whole heart changed to a slightly translucent state.

(Water-soluble surfactant perfusion step)
Next, the automatic perfusion apparatus was stopped and replaced with B solution. Then, liquid B was perfused 6 times for 20 minutes at 60 mL/min using an automatic perfusion device. Immediately after replacement with solution B, perfusion was performed at 20 mL/min while observing the situation, and after confirming that the perfusate flowed smoothly, the rate was gradually increased to 60 mL/min.
According to FIG. 5(a), the initial perfusion of liquid B made the heart dramatically transparent, and the liquid immediately became turbid after the perfusion.
According to FIG. 5(b), it was confirmed that the heart was completely translucent decellularized tissue after six perfusions.

Next, the perfusion device was temporarily removed, and 50 mL of solution B separately prepared was perfused three times through the aortic catheter. A portion of the third perfusate was collected, and dsDNA was measured using an absorbance meter and confirmed to be 0.5 ng/μL or less. When this value was exceeded, solution B was additionally perfused for 15 minutes at 40 to 60 mL/min using an automatic perfusion device, and the concentration was measured again in the same manner. This concentration determination was not performed when the total perfusion time of solution B exceeded 6 hours.
Next, all the liquid B was aspirated and replaced with purified water (water for injection). Using an automatic perfusion apparatus, purified water was perfused at 40 to 60 mL/min for 15 minutes x 8 times, 30 minutes x 6 times, and thereafter (120 minutes x 4 times/day) x 6 days. At this time, since the central portion of the decellularized organ has a fine network structure, the washing perfusion was performed not only several times but also for a sufficient time.
After that, the final perfusate (purified water) was collected, and dsDNA was measured using an absorbance meter to confirm that dsDNA was 0.0 ng/μL (below the detection limit). confirmed.

[lung]
(Low-temperature refrigeration thawing process)
Frozen organs were thawed for 24±1 hours at 4°C. After confirming that the organs were completely thawed, the following treatments were performed. Also, the margin of the trachea was pinched with forceps and completely closed.

(Bleeding process at the time of thawing, confirmation process, connection process)
Next, saline was applied to the lungs to wash off adhering and remaining clots and the like.
Next, saline (50 mL×10-20) was injected from the pulmonary artery catheter using a disposable syringe, and it was confirmed that there was no resistance or leakage and that the saline was discharged from the pulmonary vein. I felt some resistance during the first pumping, but I was careful at this point because if I forced it in, the alveoli might rupture. In addition, it was confirmed that the whole lung was blood-bleeded and perfused to a pink color.
Next, the infusion tube of the automatic perfusion apparatus was connected to the three-way stopcock of the pulmonary artery catheter.
Next, 2000 mL of the proteolytic inhibitor-containing saline was placed in a sterile pack, and sterile gauze was attached to the top. The lungs were put on gauze so that about 1 cm of the lungs were immersed in the saline. Be careful not to immerse it in the liquid surface too much, as it will float and move in the perfusate. Care was taken to ensure that the entire organ rested on the gauze.
The lungs were then transferred to a 10°C refrigerator or a 10°C environment. Note that if the temperature is set to 4°C, the perfusate may precipitate in later steps. Also, room temperature was avoided because it may damage the structure of the decellularized carrier.
Next, the three-way stopcock of the pulmonary artery was opened, and normal saline containing a protein degradation inhibitor was perfused at 40 to 60 mL/min for 15 minutes×5 times using an automatic perfusion apparatus. If the saline became red and cloudy, it was perfused again for 15 minutes.

(Poorly water-soluble surfactant perfusion step)
After the final confirmation step, the autoperfusion apparatus was stopped and replaced with solution A. Then, liquid A was perfused 3 times for 20 minutes at 40 to 60 mL/min using an automatic perfusion device. Immediately after replacement with solution A, perfusion was performed at 20 mL/min while monitoring the situation, and after confirming that the perfusate flowed smoothly, the rate 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 step)
Next, the automatic perfusion apparatus was stopped and replaced with B solution. Liquid B was perfused 6 times for 20 minutes at 60 mL/min using an automatic perfusion device. Immediately after replacement with solution B, perfusion was performed at 20 mL/min while observing the situation, and after confirming that the perfusate flowed smoothly, the rate was gradually increased to 60 mL/min.
According to FIG. 6(a), the initial perfusion of liquid B makes the lungs dramatically transparent, so the liquid immediately becomes turbid after the perfusion.
According to FIG. 6(b), it was confirmed that the heart had completely become a translucent decellularized tissue after six perfusions.

Next, the perfusion device was temporarily removed, and 500 mL×3 times perfusion was performed with the separately prepared B solution through the pulmonary artery catheter. A portion of the third perfusate was collected, and dsDNA was measured using an absorbance meter and confirmed to be 0.5 ng/μL or less. When this value was exceeded, solution B was additionally perfused for 15 minutes at 40 to 60 mL/min using an automatic perfusion device, and the concentration was measured again in the same manner. This concentration determination was not performed when the total perfusion time of solution B exceeded 6 hours.
Next, all the liquid B was aspirated and replaced with purified water (water for injection). Using an automatic perfusion apparatus, purified water was perfused at 40 to 60 mL/min for 15 minutes x 8 times, 30 minutes x 6 times, and thereafter (120 minutes x 4 times/day) x 6 days. Since the central part of the decellularized organ has a fine network structure, the washing and perfusion was performed not only for the number of times but also for a sufficient time.
After that, the final perfusate (purified water) was collected, and dsDNA was measured using an absorbance meter to confirm that dsDNA was 0.0 ng/μL (below the detection limit). confirmed.

<Method for Cryopreservation of Decellularized Carrier>
Here, a cryopreservation method for the decellularized carrierized organ or tissue will be described.
First, a 30% sucrose solution (PBS) was prepared.
Then, the automatic perfusion device connected to the decellularized carriers produced by the method for producing decellularized carriers described above was stopped, and all the purified water was aspirated and replaced with 30% sucrose solution. A 30% sucrose solution was perfused 3 times for 15 minutes at 60 to 100 mL/min using an automatic perfusion device.
The autoperfusion system was then turned off and the 3-way stopcock was closed. The organs were kept in a state that they were not overly taut. Then, the organs were transferred to a sterilized pack filled with 30% sucrose solution. A gauze was placed on the surface of the sucrose solution so that the organs were completely submerged in the sucrose solution.
Next, it was immersed in a storage (set temperature: 4°C) for 3 hours. After that, the decellularized organ was placed in a sealed pack and stored frozen in a freezer (set temperature: -80°C).

<Thawing of cryopreserved decellularized carriers and treatment method before use>
Here, a process for thawing and using the decellularized organ or tissue cryopreserved by the cryopreservation method for the decellularized carrier described above will be described.
First, it was thawed at 4° C. for 24±1 hours. It was confirmed that there were no cracks due to freezing in the organs. The autoperfusion device described above was then reconnected. Then, the sucrose solution was replaced with purified water. Purified water was perfused at 60 to 100 mL/min for 15 minutes×10 times using an automatic perfusion device. Purified water was then replaced with saline. Saline was perfused five times for 15 minutes at 60-100 mL/min using an automatic perfusion device. Depending on the purpose, a preparation solution was prepared by adding 20 mL of the antibiotic-antimycotic mixed solution to 1980 mL of the final replacement solution such as the culture solution, and further adding 1 ampoule of cefamedine. Finally, the purified water was replaced with the prepared solution, and perfusion was performed twice for 30 minutes at 80 mL/min.

<Stabilization method for stabilizing decellularized carriers without cryopreservation>
(Stabilization step)
Here, in this example, a specific example of the stabilization step for stabilizing the decellularized carrier organ or tissue will be described. In this example, a formaldehyde fixation preservation method for fixing and preserving a decellularized carrier with a 0.5% formaldehyde solution will be described. In this example, an example of refrigerated storage without cryopreservation will be described.
First, a 0.5% formaldehyde solution (formalin) was prepared. Then, the automatic perfusion device connected to the decellularized carriers produced by the method for producing decellularized carriers described above was stopped, and all the purified water was aspirated and replaced with a 0.5% formaldehyde solution.
Next, 0.5% formaldehyde solution was perfused 3 times for 15 minutes at 60-100 mL/min using an automatic perfusion device. Then the automatic perfusion apparatus was turned off and the three-way stopcock was closed. The organs were kept in a state that they were not overly taut. Here, if the organ is dented or stretched too much, it will be fixed as it is, so I was very careful.
Organs were then transferred to sterile packs filled with 0.5% formaldehyde solution. A gauze was placed on the liquid surface so that the organ was completely immersed in the 0.5% formaldehyde solution.
Next, the decellularized organ was placed in a sealed pack and stored in a refrigerator (set temperature: 4°C).

<Measurement of cell residual degree of decellularized carrier>
The following items were confirmed for quality confirmation of decellularized and carrierized organs or tissues.
This confirmation was carried out in accordance with guidelines for ensuring the quality and safety of human (autologous)-derived cells (primary passage cells), tissue-processed pharmaceuticals, and the like. dsDNA; confirmed to be 50 ng or less per 1 g of freeze-dried weight. DNA fragment length; confirmed to be 200 bp or less. HE or DAP I: Confirmed by microscopy that there was no nuclear staining and no residual cells. Ultrastructure, electron microscopy: Confirm that the microstructure is preserved 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 part of the decellularized organ was collected to confirm the absence of mycoplasma. Endotoxin test: A part of the decellularized organ was collected and confirmed to be free of endotoxin.

<Experimental example 1 for confirmation of cell engraftment by cell filling method of decellularized carriers>
Decellularized carriers cryopreserved by the above cryopreservation method for decellularized carriers or preserved by formaldehyde-fixed preservation method were subjected to cell filling in vitro to confirm cell engraftment. The processing of Experimental Example 1 will be described below.

(Thin-layer sectioning process)
A decellularized carrier was prepared, and purified water was perfused at 40 to 60 mL/min for 20 minutes×10 times using an automatic perfusion device.
Also at this point, the cells you want to load are ready. In this example, 1-5×10 ⁵ HepG2 cells were prepared.
Also, the decellularized carrier was cut into blocks (decellularized tissue fragments) of 10 mm×10 mm×2 mm.

(Sowing process)
The decellularized tissue fragments sliced into thin slices by the thin slice sectioning step were again washed with purified water for 5 minutes×6 times.
Next, the cells were washed 4 times for 10 minutes with 20 mL of an antibiotic-antimycotic mixed solution (five times the usual concentration) in 180 mL of sterile PBS.
Also, the decellularized tissue fragment was placed in the medium.
Also, a 2- to 5-fold concentration antibiotic-antimycotic mixed solution + 10% FBS-added MEM (hereinafter referred to as "added MEM") was prepared.
A corner of the decellularized tissue fragment was pinched with sterilized forceps, and a total of 1 mL of cells at 1 to 5×10 ⁵ /mL was injected from all sides using a disposable syringe fitted with a 26G injection needle.

(Culturing process)
An additional 2 mL of MEM was injected into the decellularized tissue fragment seeded with cultured cells.
After that, it was incubated overnight at 37°C.
Then, cell engraftment was confirmed under a stereoscopic microscope.
In addition, the medium was washed and replaced once a day.
In addition, after 72 hours of culture, cell engraftment was confirmed under a stereoscopic microscope.

FIG. 7A shows an example of confirming the engraftment properties of decellularized tissue fragments of the heart under a stereoscopic microscope. According to this result, swelling of the seeded cells was observed corresponding to the structure of the heart decellularized carrier.
FIG. 7B shows an example of engraftment confirmed under a stereoscopic microscope for decellularized tissue fragments of the kidney. The results showed that the seeded cells were somewhat smaller, corresponding to the structure of the renal decellularized carrier.
FIG. 7C shows an example of confirming the engraftment properties of decellularized tissue fragments of the lung under a stereoscopic microscope. According to this result, the seeded cells were settled in a flat layer, corresponding to the structure of the lung decellularized carrier.

<Experimental example 2 for confirming cell engraftment by cell filling method of decellularized carrier>
In addition, regarding the decellularized carrier, depending on the type of organ, the cell solution was added dropwise to the decellularized carrier to confirm in vitro cell engraftment. The processing of Experimental Example 2 will be described below.

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

(Sowing process)
Next, 1 mL of a 5×10 ⁵ /mL cell solution was gradually dropped into each well, and the cell solution that leaked out to the side was sucked and dropped again 3 times.
The 6-well plate was then incubated for 15 minutes at 37 degrees.
These seeding step treatments were repeated three times.

(Culturing process)
2 mL of additional MEM was added dropwise to the decellularized tissue fragments seeded with cultured cells in the seeding step.
After that, it was incubated overnight at 37°C.
Then, cell engraftment was confirmed under a stereoscopic microscope.
In addition, the medium was washed and replaced once a day.
In addition, after 72 hours of culture, cell engraftment was confirmed under a stereoscopic microscope.

FIG. 7D shows an example of confirming engraftment properties of decellularized tissue fragments of liver under a stereoscopic microscope. According to this result, cells engrafted in a regular arrangement like liver lobules were found here and there, corresponding to the structure of the liver decellularized carrier.

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

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

<Analysis of extracellular matrix (immunostaining)>
Next, each decellularized carrier was cut 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 H&E (double staining of hematoxylin and eosin) image that the cell nuclei had disappeared. Sirius red and Collagen IV staining confirmed disappearance of cytoplasm and residual cell membrane. Further, each extracellular matrix remaining on the cell membrane was examined by staining with Fibronectin and Laminin. As a result, the intensity of DAB staining was confirmed according to the constituent factors of the extracellular matrix.

<Transplant experiment of decellularized carrier (liver)>
A decellularized carrier (unfilled with cells) in which the liver was treated by the decellularization method of this embodiment was transplanted into a pig body, and it was confirmed that the inside of the carrier was filled with blood. It was carried out at the Advanced Medical Technology Development Center attached to the Jichi Medical University campus.

According to FIGS. 10(a) to (c), an angiographic agent was administered from the portal vein to the decellularized liver transplanted by the APOLT method while the heart was beating, and contrast was obtained with a C-arm (radiation imaging diagnostic device). got the image. FIG. 10(a) is a photograph of treatment, and FIGS. 10(b) and 10(c) are photographs of contrast enhancement. As a result, it was found that the vascular structure was maintained in detail even in the cell-unfilled carrier. This indicates that the decellularized carrier treated by the decellularization method of the present example maintains the cytoskeleton and the like to a high degree, even if it is a fragile structure such as a capillary. rice field.
In FIGS. 10(d) and (e), the transplanted decellularized liver was sectioned to confirm the locations of red blood cells. According to this, the hepatic lobule structure, which is the characteristic structure of the liver, was shown only in blood (erythrocytes). This fact also indicates that the decellularized carrier of this example maintains the cytoskeleton and the like to a high degree.

It goes without saying that the configuration and operation of the above-described embodiment are examples, and can be modified as appropriate without departing from the scope of the present invention.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to produce a decellularized carrier applicable to various experiments and advanced medical treatments, and thus it is industrially applicable.

Claims (16)

a cryopreservation step of cryopreserving the extracted 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 within 120 minutes;
the organ or tissue perfused with the poorly water-soluble surfactant-containing solution is perfused with the water-soluble surfactant-containing solution within 180 minutes, and the poorly water-soluble interface contained in the poorly water-soluble surfactant-containing solution a hydrophilic surfactant perfusion step to remove the active agent;
said organ or said tissue, except for the placenta,
The poorly water-soluble surfactant is lipophilic,
A method for producing a decellularized carrier, wherein the thawing step, the sparingly water-soluble surfactant perfusion step, and the hydrophilic surfactant perfusion step are completed within 48 hours without being repeated. The cryopreservation step is
a frozen blood removal step of perfusing the organ or the tissue with a physiological saline solution to remove blood;
a cryopreservation solution perfusion step of perfusing the organ or tissue exsanguinated in the cryopreservation step with a cryopreservation solution;
The cryopreservation solution is a physiological saline solution containing 0.01 to 0.3% hydrogen peroxide,
2. The method for producing a decellularized carrier according to claim 1, wherein the physiological saline and/or the cryopreservation solution in the step of removing blood during freezing may contain 500 to 4000 U of heparin. The cryopreservation step is
An expansion step in which the organ or tissue perfused in the cryopreservation solution perfusion step is filled with the cryopreservation solution and expanded to close the vein side and the artery side;
a surplus solution removing step of removing the surplus cryopreservation solution adhering to the swollen organ or tissue;
and 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 is
The decellularized carrier production method according to claim 2 or 3, further comprising a period of storage step of storing the organ or tissue before the decellularization step after the freezing step for a specific period of time. The thawing step includes
a low-temperature refrigeration thawing step of thawing the cryopreserved organ or tissue in a low-temperature refrigeration state without immersion in a solution;
a thaw-time blood removal step of perfusing the organ or tissue thawed in the low-temperature refrigeration and thawing step with a physiological saline solution containing a proteolytic inhibitor to remove blood;
a confirming step of confirming whether or not the organ or tissue exsanguinated in the exsanguination step during thawing is damaged;
The method for producing decellularized carriers according to any one of claims 1 to 4, further comprising a connecting step of connecting the organ or tissue confirmed by the confirming step to a perfusion container. The blood removal step during thawing includes:
6. The decellularized carrier manufacturing method according to claim 5, further comprising a thrombus removal step of removing microthrombi contained in the organ or tissue. The decellularization step includes
The sparingly water-soluble surfactant contained in the sparingly 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 hydrophilic surfactant contained in the hydrophilic surfactant-containing solution is SDS. The decellularization step includes
8. The decellularization according to any one of claims 1 to 7, further comprising a stabilization step of stabilizing the organ or tissue perfused in the hydrophilic surfactant perfusion step after washing. method for producing a carrier. The stabilization step includes
9. The decellularization according to claim 8, wherein the organ or tissue is perfused with a 0.1-3% formaldehyde solution to prevent luminal obstruction, and the organ or tissue is filled with the formaldehyde solution. Carrier manufacturing method. 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, ureter, and blood vessel. The method for producing a decellularized carrier according to any one of claims 1 to 10, wherein the double-stranded DNA and the sugar chain structure are eliminated. A decellularized carrier produced by the method for producing a decellularized carrier according to any one of claims 1 to 10,
A method for preserving a decellularized carrier, characterized in that the decellularized carrier is immersed in a 30% sucrose solution and cryopreserved. Decellularized carriers produced by the method for producing decellularized carriers according to any one of claims 1 to 10 or preserved by the method for preserving decellularized carriers according to claim 12 are washed with physiological saline. a perfusing saline perfusion step;
a replacement step of perfusing and replacing the decellularized carrier perfused in the physiological saline perfusion step with a culture solution;
a cell injection step of injecting cultured cells from an artery of the decellularized carrier perfused with the culture medium in the replacement step;
a cell adhesion step of stopping perfusion after the cultured cells are injected in the cell injection step to adhere the cells to the decellularized carrier;
and a perfusion culture step of resuming perfusion of the culture medium to the decellularized carrier to which the cells have adhered by the cell adhesion step and culturing the cells. when the organ or tissue is the small intestine,
14. The cell filling method according to claim 13, wherein perfusion is performed by connecting an artery and a portal vein. Decellularized tissue decellularized carriers produced by the method for producing decellularized carriers according to any one of claims 1 to 10 or preserved by the method for preserving decellularized carriers according to claim 12 . a thin sectioning step of thinly sectioning into pieces;
A seeding step of washing the decellularized tissue fragment thinly sectioned by the thin sectioning step and seeding cultured cells;
and a culturing step of culturing the decellularized tissue fragment seeded with the cultured cells in the seeding step. A decellularization solution kit for producing a decellularized carrier from an organ or tissue,
a poorly water-soluble surfactant-containing solution that perfuses the thawed organ or tissue within 120 minutes;
a hydrophilic interface that perfuses the organ or tissue perfused with the poorly water-soluble surfactant-containing solution within 180 minutes to remove the poorly water-soluble surfactant contained in the poorly water-soluble surfactant-containing solution; an active agent-containing solution;
said organ or said tissue, except for the placenta,
The poorly water-soluble surfactant is lipophilic,
including instructions for carrying out the method according to any of claims 1-11
A decellularization solution kit characterized by:

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