JP5526573B2 - Method for preparing decellularized biological tissue - Google Patents

Description

  The present invention relates to a method for preparing a decellularized biological tissue for decellularizing a biological tissue derived from an animal, a washing solution used in the method for preparing the decellularized biological tissue, and a graft including the decellularized biological tissue.

  When transplanting a graft derived from a living tissue of another person, rejection of the graft by the recipient side tissue is a problem. Therefore, development of an artificial tissue is awaited as a means for solving such a problem.

  Various synthetic polymers have been tried as materials for artificial tissues. However, since the compatibility between these materials and the living tissue is low, dropout or infection may occur at the joint between the graft and the living tissue.

  Thus, in order to improve compatibility with living tissue, a technique has recently been developed in which decellularized living tissue, which is a supporting tissue remaining after removing cells from living tissue, is used as a graft. Removal of cellular components, that is, decellularization, first destroys cells by treating living tissue with a treatment solution containing a surfactant, or applying ultrahigh static pressure to the living tissue, Next, it is performed by removing the destroyed cells from the living tissue (see Patent Documents 1, 2, and 3). Conventionally, as a washing solution for removing cells, a buffer solution containing phosphate ions such as PBS (phosphate buffered saline) has been used (see Patent Document 3).

JP 2005-514971 A JP 2006-507851 gazette JP-T-2005-53355

  However, there is a problem that a living tissue cleaned using a conventional cleaning solution is likely to undergo calcification with time after transplantation into a living body for some reason.

  The present invention has been made in view of the above circumstances, and a method for preparing a decellularized biological tissue capable of suppressing the occurrence of calcification, a washing liquid used for the method for preparing the decellularized biological tissue, and the decellularization thereof It aims at providing the graft | grafting provided with a biological tissue.

  In the method for preparing a decellularized biological tissue including a procedure for destroying cells in a biological tissue derived from an animal, the present inventors have a phosphate ion content of 2.0 mmol / L after the procedure for destroying cells. Decellularized with a buffer containing at least one compound selected from the group consisting of citric acid, ethylenediaminetetraacetic acid (hereinafter also referred to as EDTA), and physiologically acceptable salts thereof. It has been found that the calcification can be suppressed with high efficiency by further washing the living tissue, and the present invention has been completed. Specifically, the present invention provides the following.

(1) A method for preparing a decellularized biological tissue in which an animal-derived biological tissue is decellularized,
A disruption procedure that destroys cells in the isolated biological tissue;
One or more kinds of the destroyed cells having a phosphate ion content of 2.0 mmol / L or less and selected from the group consisting of citric acid, ethylenediaminetetraacetic acid, and physiologically acceptable salts thereof A method for preparing a decellularized biological tissue, comprising a step of removing with a buffer containing the compound.

  (2) The decellularized organism according to (1), wherein the buffer solution contains one or more compounds selected from the group consisting of citric acid and physiologically acceptable salts of citric acid. Tissue preparation method.

  (3) The method for preparing a decellularized biological tissue according to any one of (1) and (2), wherein the buffer further contains sodium chloride and D-glucose.

  (4) The method for preparing a decellularized biological tissue according to any one of (1) to (3), wherein a buffer that does not substantially contain a surfactant is used as the buffer solution.

  (5) The method for preparing a decellularized biological tissue according to any one of (1) to (4), wherein the destruction procedure includes a procedure of applying an ultrahigh hydrostatic pressure to the biological tissue.

  (6) The method for preparing a decellularized living tissue according to (5), wherein the ultrahigh hydrostatic pressure is 1000 atm or more.

  (7) The decellularization according to any one of (1) to (6), including a step of bringing the living tissue into contact with an organic solvent or an aqueous solution of an organic solvent between the destruction procedure and the removal procedure. Tissue preparation method.

  (8) The method for preparing a decellularized tissue according to (7), wherein the organic solvent contains a lower alcohol.

  (9) The method for preparing a decellularized tissue according to any one of (1) to (6), which does not include a step of bringing the living tissue into contact with an organic solvent or an aqueous solution of an organic solvent.

(10) A washing solution used for removing broken cells in the isolated biological tissue,
A cleaning solution having a phosphate ion content of 2.0 mmol / L or less and containing at least one compound selected from the group consisting of citric acid, ethylenediaminetetraacetic acid, and physiologically acceptable salts thereof.

  (11) The cleaning solution according to (10), wherein the cleaning solution contains at least one compound selected from the group consisting of citric acid and physiologically acceptable salts of citric acid.

  (12) The cleaning liquid according to (10) or (11), wherein the cleaning liquid further contains sodium chloride and D-glucose.

(13) A graft transplanted into an animal,
A graft comprising a decellularized biological tissue prepared by the preparation method according to any one of (1) to (9).

  ADVANTAGE OF THE INVENTION According to this invention, when decellularizing the inside of a biological tissue, it becomes possible to prepare the decellularized biological tissue which can suppress generation | occurrence | production of calcification. Furthermore, the decellularized living tissue obtained by the method of the present invention is also useful as a graft.

It is a figure which shows the observation result of the calcification of the decellularized biological tissue obtained by Example 1 and 2 of this invention. It is a figure which shows the observation result of the calcification of the decellularized biological tissue obtained by Example 3 and 4 of this invention. It is a figure which shows the observation result of the calcification of the decellularized biological tissue obtained by Example 5 and 6 of this invention. It is a figure which shows the observation result of the calcification of the decellularized biological tissue obtained by Example 7 and 8 of this invention. It is a figure which shows the observation result of the calcification of the decellularized biological tissue obtained by the comparative examples 1 and 2 of this invention. It is a figure which shows the observation result of the calcification of the decellularized biological tissue obtained by the comparative examples 3 and 4 of this invention.

  Hereinafter, although embodiment of this invention is described, it is not intending limiting this invention.

<Preparation method>
The method for preparing a decellularized biological tissue of the present invention includes a disruption procedure for destroying cells in the biological tissue, a phosphate ion content of 2.0 mmol / L or less, citric acid, ethylenediaminetetraacetic acid, and these And removing with a buffer containing one or more compounds selected from the group consisting of the physiologically acceptable salts.

[Destruction procedure]
The procedure for destroying the cells in the living tissue is not particularly limited as long as the cells are favorably destroyed, and may be performed by a conventionally known method.

  Examples of animal-derived biological tissue subjected to the cell destruction procedure include cornea, cartilage, trachea, pericardium, amniotic membrane, soft tissue such as skin, bulky tissue such as myocardium, and hard tissue such as bone.

As a specific example of a suitable procedure for destroying cells in living tissue,
1) A biological tissue derived from an animal is washed with a washing solution containing a surfactant selected from artificial compounds such as SDS, Triton X-100 (registered trademark), PEG and PEO, and biologically derived compounds such as sodium cholate. Examples thereof include a method of washing a living tissue, or 2) a method of applying an ultrahigh hydrostatic pressure to an animal-derived living tissue.

  Among these methods, because of the fact that cells and viruses in living tissue can be destroyed depending on conditions, and there is no problem of residual surfactant or the like in living tissue, 2) It is more preferable to use a method of applying a hydrostatic pressure.

The method of applying the ultrahigh hydrostatic pressure of 2) will be described more specifically.
The ultrahigh hydrostatic pressure at the time of destroying the cells in the living tissue is preferably 1000 atm or more, more preferably 4000 atm or more in terms of being able to destroy the bacteria in the living tissue, and the virus can be destroyed. More preferably, the pressure is 6000 atm or more.

  The application of ultrahigh hydrostatic pressure is performed in a medium, and water or an aqueous solution of a water-soluble polysaccharide can be used as the medium. When the animal-derived biological tissue is, for example, soft tissues such as cornea, cartilage, trachea, pericardium, amniotic membrane, and skin, it is preferable to use an aqueous solution of a water-soluble polysaccharide as a medium. This is because by using an aqueous solution of a water-soluble polysaccharide as a medium, swelling of soft tissue after application of ultrahigh hydrostatic pressure can be significantly suppressed.

  Preferable examples of the water-soluble polysaccharide used in the aqueous solution of the water-soluble polysaccharide used as a medium include dextran, alginic acid, hyaluronic acid, trehalose, 2-methacryloyloxyethyl phosphorylcholine, and polyvinylpyrrolidone. These water-soluble polysaccharides may be used alone or in combination of two or more.

  The concentration of the water-soluble polysaccharide in the case of using an aqueous solution of the water-soluble polysaccharide as a medium is appropriately determined so that the degree of swelling can be limited within an allowable range depending on the applied ultrahigh hydrostatic pressure value, the type of soft tissue used, etc. May be set.

  As a specific procedure for applying an ultra-high hydrostatic pressure, for example, a medium of a water-impermeable film is first filled with a medium, and animal-derived biological tissue is moistened in the medium. Then, the bag is tightly sealed while taking care not to leave any gas inside. This bag may be installed in a chamber of an ultrahigh hydrostatic pressure processing apparatus (for example, “Dr. CHEF (model)” (manufactured by Kobe Steel)) and the apparatus may be operated. The application time of the ultrahigh hydrostatic pressure is not particularly limited as long as cells are destroyed well, and is usually about 10 to 30 minutes, and the medium temperature at the time of application is usually 25 to 40 ° C, preferably around 30 ° C.

[Removal procedure]
After the cells in the living tissue are destroyed by the above method, prior to performing the removal procedure, an organic solvent that does not affect the living tissue or an aqueous solution of the organic solvent, and a method such as immersing the living tissue in which the cells are destroyed You may process by making it contact. This is because, when washing with a buffer solution described later, it is easy to remove the broken cells. The organic solvent is preferably a lower alcohol such as ethanol or isopropanol, glycerin, or an aqueous solution thereof, and more preferably ethanol or an aqueous solution thereof because it can be easily removed from a living tissue after contact.

  In addition, an embodiment that does not include a procedure of bringing an organic solvent or an aqueous solution of an organic solvent into contact with a living tissue in which cells have been destroyed is also preferable as a method for preparing a decellularized tissue. When ethanol or the like is used for washing a living tissue, it is necessary to completely remove the ethanol or the like from the washed living tissue by a method such as freeze drying, and the amount of ethanol remaining in the living tissue is analyzed. This is because an expensive device such as a freeze-drying device or a residual solvent analyzer is required, and the amount of work for preparing a decellularized tissue increases.

  In the destruction procedure, the cells in the destroyed biological tissue are removed from the biological tissue by the removal procedure. In the removal procedure, the phosphate ion content is 2.0 mmol / L or less, and contains at least one compound selected from the group consisting of citric acid, EDTA, and physiologically acceptable salts thereof. The broken cells in the living tissue are washed with a buffer solution. More preferably, the buffer solution does not substantially contain phosphate ions. “Containing substantially no phosphate ion” includes a case where a small amount of phosphate ion is inevitably mixed or a case where a small amount is contained for detour purposes, and the phosphate ion content is 1.0 mmol. / L or less, more specifically 0.7 mmol / L or less, and more specifically 0 mmol / L.

  The phosphate ion content in the buffer solution used for washing the destroyed cells in the living tissue should be measured by a known method such as ion chromatography, capillary electrophoresis, atomic absorption method, molybdenum blue method, etc. Can do.

  In the present invention, the biological tissue in which the cells are destroyed has a very low phosphate ion content of 2.0 mmol / L or less, and citric acid, EDTA, and physiologically acceptable salts thereof, etc. By washing with a buffer solution containing a compound having an effect of chelating metal ions such as calcium, calcification of decellularized living tissue can be remarkably suppressed.

  Examples of physiologically acceptable salts include alkali metal salts such as sodium and potassium, amino acid salts such as arginine, lysine, histidine, and ornithine, ammonium salts, and salts of various organic bases. Among them, alkali metal salts such as sodium and potassium are more preferable because they are easily available and have less problems when remaining in decellularized living tissue.

  The concentration of one or more compounds selected from the group consisting of citric acid, EDTA, and physiologically acceptable salts thereof in the buffer is the concentration of the solution normally used as the buffer, Although it will not restrict | limit especially if the effect of invention is not impaired, Usually, 0.1-100 mmol / L, More preferably, it is 0.1-70 mmol / L.

  The compound contained in the buffer is one selected from the group consisting of citric acid and physiologically acceptable salts of citric acid because there are fewer problems when remaining in the decellularized living tissue. It is more preferable to use the above compound.

  More preferably, the buffer solution contains sodium chloride and D-glucose in addition to one or more compounds selected from the group consisting of citrate, EDTA, and physiologically acceptable salts thereof. When the buffer solution contains sodium chloride and D-glucose, the concentration of sodium chloride in the buffer solution is preferably 0.1 to 10 g / L, more preferably 0.5 to 5 g / L. -The concentration of glucose is preferably 1 to 50 g / L, more preferably 5 to 30 g / L. Among these buffers, those particularly preferably used include Alsever's solution, which is a buffer solution containing a specific amount of citric acid, citrate, sodium chloride, and D-glucose.

  As described above, the buffer used for washing the decellularized living tissue in the present invention is substantially free of a surfactant in order to make it difficult to cause calcification of the obtained decellularized living tissue. It is preferable to use it.

  Removal of the destroyed cells in the biological tissue is performed by immersing the biological tissue in which the cells are destroyed in the buffer solution. The treatment temperature immersed in the buffer solution is usually 25 to 40 ° C, more preferably around 37 ° C. The treatment time immersed in the buffer is not particularly limited as long as the destroyed cells can be removed well, but is usually 1 to 7 days, more preferably about 3 days.

  The preservation method of the decellularized biological tissue thus prepared is not particularly limited as long as it is in a sterilized state, and may be in a frozen state, a wet state in a liquid, or a dry state.

<Graft>
The decellularized living tissue thus prepared is useful as a component of a graft to be transplanted into an animal. That is, the graft of the present invention includes the aforementioned decellularized living tissue.

<Examples 1-8 and Comparative Examples 1-2>
[Destruction procedure]
The porcine aorta was purchased from an edible pig farm and transported at 4 ° C. The aorta was cut into 1 cm pieces and wetted in a polyethylene film bag filled with 5 ml of Alsever's solution. The bag was placed in a chamber of “Dr. CHEF” (manufactured by Kobe Steel), and a hydrostatic pressure of 10,000 atm was applied for 10 minutes while maintaining the temperature at 30 ° C. During this time, “Dr. CHEF” was controlled such that the pressure increase and decrease rates were 666 atm / min. The aorta (living tissue) after application was removed by a clean operation.

[Removal procedure]
The biological tissue obtained by the disruption procedure was immersed for 14 days at 37 ° C. in about 100 mL of EBM-2 with 500 μL of DNase (4 mg / ml, Roche Diagnostics Co., Ltd.). Washing was performed.

  Next, if desired, the biological tissue was immersed in about 100 mL of an 80% by weight ethanol aqueous solution (prepared by diluting ethanol using Alsever's solution) at 37 ° C. for 3 days, and then washed. Immersion treatment was performed for 3 days at 37 ° C. in about 100 mL of the buffer solution shown.

[Observation of calcification]
The decellularized biological tissue obtained through the disruption procedure and the removal procedure is immersed in FBS (Invitrogen Corp.) for 10 days, and then Kossa staining is performed. Observed black spots and compared the degree of calcification.

  The treatment conditions after the EBM immersion treatment in Examples 1 to 8 and Comparative Examples 1 to 4 and the observation results of calcification are shown in Table 1.

* 1: Alsever's solution (Sigma-Aldrich, composition: sodium chloride 4
. 2 g / L, trisodium citrate dihydrate 8.0 g / L, citric acid monohydrate 0.55 g / L, D-glucose 20.5 g / L. )
* 2: PBS / Alsever's mixed solution (PBS: Alsever's solution mixed at 1: 9 (volume ratio). Phosphate ion concentration 0.66 mmol / L)
* 3: Saline + citric acid (citric acid concentration 41.6 mmol / L)
* 4: Saline + EDTA (disodium salt) (EDTA concentration 5.9 mmol / L)
* 5: PBS (composition: sodium chloride 9000 mg / L, sodium monohydrogen phosphate (anhydrous) 800 mg / L, potassium dihydrogen phosphate (anhydrous) 144 mg / L, phosphate ion concentration 6.65)

  As shown in Table 1 and FIGS. 5 to 6, PBS containing 6.65 mmol / L and a large amount of phosphate ions, and physiological saline that does not contain phosphate ions but does not contain citric acid, EDTA, etc. It can be seen that severe decalcification occurs in the decellularized biological tissue obtained by Comparative Examples 1 to 4 used as the above.

  On the other hand, as shown in Table 1 and FIGS. 1 to 4, an implementation using a buffer containing a specific amount of a compound selected from citric acid, EDTA, and salts thereof, containing a small amount of phosphate ion or not at all. It can be seen that in the decellularized living tissue obtained by Examples 1 to 8, calcification does not occur or is slight.

Claims (10)

A method for preparing a decellularized biological tissue in which an animal-derived biological tissue is decellularized,
A disruption procedure that destroys cells in the isolated biological tissue;
One or more kinds of the destroyed cells having a phosphate ion content of 2.0 mmol / L or less and selected from the group consisting of citric acid, ethylenediaminetetraacetic acid, and physiologically acceptable salts thereof a removal procedure of removing the buffer containing compounds, only including,
A method for preparing a decellularized biological tissue, wherein the buffer further contains sodium chloride and D-glucose .   The preparation of decellularized biological tissue according to claim 1, wherein the buffer solution contains one or more compounds selected from the group consisting of citric acid and physiologically acceptable salts of citric acid. Method. The method for preparing a decellularized biological tissue according to claim 1 or 2, wherein a buffer that does not substantially contain a surfactant is used as the buffer. The method for preparing a decellularized biological tissue according to any one of claims 1 to 3 , wherein the destruction procedure includes a procedure of applying an ultrahigh hydrostatic pressure to the biological tissue. The method for preparing a decellularized living tissue according to claim 4 , wherein the ultrahigh hydrostatic pressure is 1000 atm or more. The method for preparing a decellularized tissue according to any one of claims 1 to 5 , further comprising a step of bringing the living tissue into contact with an organic solvent or an aqueous solution of an organic solvent between the destruction procedure and the removal procedure. The method for preparing a decellularized tissue according to claim 6 , wherein the organic solvent contains a lower alcohol. The method for preparing a decellularized tissue according to any one of claims 1 to 5 , which does not include a step of bringing the living tissue into contact with an organic solvent or an aqueous solution of an organic solvent. A washing solution used to remove broken cells in isolated biological tissue,
The phosphate ion content is 2.0 mmol / L or less, and contains at least one compound selected from the group consisting of citric acid, ethylenediaminetetraacetic acid, and physiologically acceptable salts thereof ,
The cleaning liquid further contains sodium chloride and D-glucose . The cleaning liquid according to claim 9 , wherein the cleaning liquid contains at least one compound selected from the group consisting of citric acid and physiologically acceptable salts of citric acid.