JP6027300B2 - Cell or organ preservation solution and preservation method - Google Patents

Description

  The present invention relates to a cell or organ preservation solution and a preservation method.

  In the research on Stherniidae's Antheraea yamamai, the present inventors have an amino acid sequence, aspartic acid-isoleucine-leucine-arginine-glycine (DILRG), and the C-terminus is amidated. In addition, a novel peptide having a molecular weight of 570.959 was found, and patents were obtained by clarifying the dormancy control action and cancer cell growth inhibitory action of this peptide (Patent Documents 1 and 2). This peptide is also named “Yamamarin” by the present inventors.

  In addition, the present inventors proceeded with research on peptide derivatives in order to increase the cell permeability and cytostatic activity of the peptide, and named a conjugate with palmitic acid (named “C16-Yamamarin”). ) Has been reported (Non-patent Document 1).

  On the other hand, with the advancement of medical technology, organ transplant surgery has been widely performed. If the organ damage in the patient is very large and recovery by normal treatment cannot be expected, the donor organ is transplanted to the recipient and treated. Transplanted organs removed from organ donors (donors) for transplantation are in a state where blood flow is interrupted and oxygen is not supplied via the blood flow (ischemic state), and it takes several minutes to several hours until transplantation. Saved. At this time, storage conditions such as storage temperature and storage solution may not be appropriately selected, and it may take a long time to transplant. In such a case, when the blood flow in the transplanted organ is restored by transplantation (at the time of reperfusion), a substrate or functional disorder may occur in the transplanted organ. For example, in liver transplantation, graft dysfunction may be observed due to microcirculatory disturbance with increased coagulation activity and thrombus formation after transplantation. Such a disorder is generally called a post-ischemic reperfusion disorder of a transplanted organ.

  Studies have been made on methods for preventing such symptoms and preserving transplanted organs in a physiologically favorable state. The organ preservation methods reported to date are roughly divided into two methods: 1) perfusion method, and 2) simple immersion preservation method.

  The perfusion method performs organ perfusion during the preservation period, supplies the cells with necessary oxygen and nutrients, and removes waste products, thereby maintaining organ metabolism and extending the preservation period. . However, organ preservation by the perfusion method involves various factors such as perfusion temperature, perfusion pressure, perfusion flow rate, and composition of the perfusate, and detailed optimum conditions have not been established.

  On the other hand, the simple immersion preservation method is a method for preventing tissue damage due to oxygen deficiency by keeping organs at a low temperature and suppressing cell metabolism, and is widely used in clinical settings as a simple and effective method. ing. Specifically, a method is generally used in which blood components are washed from the blood vessel bed of the organ using a low-temperature preservation solution from an inflowing blood vessel or the like before or after the removal, and the extracted organ is immersed in the preservation solution.

  Well-known organ preservation solutions include Eurocollins solution containing glucose and various electrolytes, and Wisconsin solution containing impermeable components, colloid osmotic pressure components, energy metabolism promoting components and hormones, respectively. It has been. However, Eurocollins solution is effective for highly viable kidneys, but it is said that the protective effect on tissues and cells is not sufficient for organs other than kidneys, and Wisconsin solution is not suitable as a preparation. It is said to be stable and has the disadvantage of having to be stored at low temperatures after preparation.

  In order to overcome such drawbacks, various organ preservation solutions have also been proposed (for example, Patent Documents 3, 4, and 5). However, these organ preservation solutions also have problems such as difficulty in preparation of the preparation and maintenance of homeostasis and low solubility in water.

  Further, in any of the above organ preservation solutions, there is a fundamental problem that the recovery of the function of the organ after transplantation is hindered because the organ needs to be kept at a low temperature. Furthermore, it has been pointed out as a point to be improved that a cooling device or the like is required and the burden of transporting the organ is large.

Patent No. 3023790 Patent No. 3571971 JP 2000-191401 A JP 2002-60301 A JP 2005-306749 A

Yang et al. A Palmitonyl Conjugate of an Insect Pentapeptide Causes Growth Arrest in Mammalian Cells and Mimics the Action of Diapause Hormone (2007).

  The present invention provides an organ preservation solution that prevents the occurrence of post-ischemic reperfusion injury in a transplanted organ from the above background, and further exhibits an effective preservation effect even if the preservation temperature of the organ is not low. It is an issue.

  In order to preserve organs, preservation at the cellular level is indispensable. Therefore, in order to solve the above-mentioned problems, the present invention provides the following cell or organ preservation solution and preservation method.

  First, a cell or organ preservation solution comprising a compound represented by the following formula as an active ingredient is provided.

(Wherein R represents an acyl group)

  Secondly, a preservation solution of the first cell or organ in which the acyl group has 6 to 28 carbon atoms is provided, and thirdly, the concentration of the active ingredient is 10 μM to 1000 μM. A cell or organ preservation solution is provided.

  Fourth, the present invention provides a method for preserving cells or organs, which comprises immersing the cells or organs in a cell or organ preservation solution containing a compound represented by the following formula as an active ingredient.

(Wherein R represents an acyl group)

  Fifth, the compound contained as an active ingredient in the preservation solution provides the fourth cell or organ preservation method, wherein the acyl group has 6 to 28 carbon atoms, Provides the method for preserving the fourth or fifth cell or organ, wherein the concentration of the active ingredient is 10 μM to 1000 μM, and seventh, the temperature of the preservation solution is 5 to 37 ° C. 6. A method for preserving 6 cells or organs is provided.

  According to the cell or organ preservation solution of the present invention, the oxygen consumption of the cell can be reversibly suppressed, which is effective for preservation of the cell or organ. Furthermore, in organ transplantation, tissue damage to the organ due to oxygen deficiency can be prevented without maintaining the transplanted organ at a low temperature, and functional recovery of the organ after transplantation can be performed smoothly.

DMSO, palmitic acid, a diagram showing the results of measurement of oxygen consumption when the N-terminal acylated DILRG-NH ₂ was added at each concentration. Symbols * and ** on the graph indicate significance levels, * indicates p <0.005, and ** indicates p <0.001. The medium, C16-DILRG-NH _2, was collected after the addition of palmitic acid and DMSO, the substrate is added, a diagram showing the results of measurement of the oxygen consumption rate of the recovery cell. _C16-DILRG-NH 2 (C16 Yamamarin) due to chronic myeloid leukemia cell preservation effect is a diagram showing a (antiproliferative effect). _C16-DILRG-NH 2 (C16 Yamamarin) in accordance HUVEC storage effect is a diagram showing a (antiproliferative effect). _C16-DILRG-NH 2 (C16 Yamamarin) in accordance NHDF saving effect of a diagram showing a (antiproliferative effect).

The present invention is a preservation solution for enabling preservation of cells or organs for a long period of time by suppressing oxygen consumption of cells. (Hereinafter referred to as “the preservation solution of the present invention”).
The preservation solution of the present invention contains the following compounds as active ingredients.

In the compound, R in the formula represents an acyl group (hereinafter referred to as “N-terminal acylated DILRG-NH ₂ ”), and this N-terminal acylated DILRG-NH ₂ has been conventionally studied by the inventors. An acyl group was introduced at the N-terminus of “a peptide having aspartic acid-isoleucine-leucine-arginine-glycine and amidated at the C-terminus” (hereinafter referred to as “DILRG-NH ₂ ”). Can be synthesized. DILRG-NH _2, for example, larvae from the isolation of wild silkworm (Antheraea yamamai), can either be used after purification, it is also possible to use those produced by known peptide synthesis methods. Although it can be obtained by other methods, taking into consideration economic efficiency, mass productivity, etc., acquisition by a peptide synthesis method is preferable.

The acyl group can be introduced by a known method, and the carbon number of the acyl group in N-terminal acylated DILRG-NH ₂ is 6 to 6 in terms of cell permeability and cell oxygen consumption suppression effect. 28 is preferable, and a palmitoyl group having 16 carbon atoms is particularly preferable.

The preservation solution of the present invention is based on the novel finding that N-terminal acylated DILRG-NH ₂ can reversibly suppress oxygen consumption in cells and organs. No organ preservation solution using such insect-derived peptides has been known at all.

  The reversible effect of suppressing oxygen consumption means that it is useful as a preservative for cells and organs.

That is, the preservation solution of the present invention is prepared by preparing N-terminal acylated DILRG-NH ₂ as a solution. For example, when storing a transplanted organ, the organ is immersed in the preservation solution during the preservation period. By suppressing the oxygen consumption and removing the organ from the preservation solution before transplantation, the oxygen consumption of the organ can be returned to normal again. Therefore, organ damage caused by oxygen deficiency can be prevented.

Furthermore, since N-terminal acylated DILRG-NH ₂ has a small number of 5 amino acids as the skeleton of the chemical structure and is an extremely short small molecule, for example, it is easy to prepare a stock solution and maintain homeostasis. . In addition, it can be stored at room temperature, has excellent storability and handleability, and can be stored for a long time.

Moreover, even if the preservation solution of the present invention is in the form of N-terminal acylated DILRG-NH ₂ alone, N-terminal acylated DILRG-NH ₂ and other, for example, glucose, maltose, sucrose, lactose, raffinose , Carbohydrates such as trehalose, mannitol, hydroxyethyl starch, pullulan, organic acids such as gluconic acid, lactic acid, acetic acid, propionic acid, β-hydroxybutyric acid, citric acid, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, phosphoric acid Sodium dihydrogen, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate and other electrolytes, L-ascorbic acid, vitamin E and other vitamins, glycine, glutamic acid , Amino acids such as lysine Anti-diuretic hormone, hormones such as insulin, anticoagulants such as citric acid, citrate, heparin, sodium edetate, anti-coagulants such as calcium antagonists, adrenergic β receptor antagonists, angiotensin converting enzyme inhibitors, adenosine Nucleobase such as acid phosphate, anti-freezing agent such as anti-freezing protein, active oxygen scavenger, cell activator, antibiotic, antiplatelet factor, liver injury inhibitor, excipient, binder, disintegrant, dispersant, Usually used in organ preservation solutions such as viscosity agents, resorption accelerators, surfactants, solubilizers, preservatives, preservatives, emulsifiers, tonicity agents, stabilizers, buffers, pH adjusters, etc. It may be in the form of a composition with one or more components.

  In addition, when the preservation solution of the present invention is used in a known organ preservation solution such as Eurocollins solution or Wisconsin solution, the organ preservation ability can be improved.

Furthermore, when cells and organs are preserved with the preservative of the present invention, the concentration of N-terminal acylated DILRG-NH _{2 in} the preservation solution is, for example, in the range of 10 μM to 1000 μM, preferably 10 μM to 100 μM. Is preferred.

  And preferable application temperature as conditions on which the preservation | save liquid of this invention is used is 5-37 degreeC, Most preferably, it is 25-37 degreeC. Since the preservation solution of the present invention does not necessarily need to preserve an organ at a low temperature as in the prior art, the function of the organ after transplantation is smoothly restored. Of course, there is a problem of functional recovery in organ transplantation, but the temperature of the preservation solution can be lowered (for example, 5 ° C. or lower), and in this case, cells and organs can be preserved for a longer period of time. .

  The cells that are the target of the preservation solution of the present invention include, for example, stem cells, skin cells, mucosal cells, hepatocytes, islet cells, nerve cells, chondrocytes, endothelial cells isolated from human or non-human animal tissues, It includes epithelial cells, bone cells, and muscle cells, and further includes sperm, eggs or fertilized eggs of animals such as livestock and fish, insect cells, plant cells, and the like.

  Further, organs that are the target of the preservation solution of the present invention include skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, pancreas, and the like.

  Examples of the present invention will be described below. The present invention is not limited to the following examples.

<1> Synthesis of N-terminal acylated DILRG-NH ₂ A peptide, aspartic acid-isoleucine-leucine-arginine-glycine-NH was synthesized by a conventional method using a peptide synthesizer (PSSM-8, manufactured by Shimadzu Corporation). ₂ (DILRG-NH ₂₎ were synthesized. Subsequently, after palmitoylation on the resin, it was subjected to a cleaving reaction to obtain C16-DILRG-NH ₂ having the following chemical structure.

  The purification was carried out by connecting a reverse phase column Develosil-ODS HG-5 (20 mm × 250 mm, manufactured by Nomura Chemical Co., Ltd.) to an HPLC system (Gulliver Corporation JASCO). Elution is carried out at a flow rate of 4 ml / min using an acetonitrile concentration gradient (0 to 100% from 0 to 120 minutes) in the presence of 0.1% trifluoroacetic acid (TFA) to elute the active fraction. It was. Absorbance was measured at 220 nm. The peptide was mixed with an equal amount of matrix (saturated with 40% acetonitrile / 0.1% TFAα-CHCA) on a sample plate, dried, and MALDI-TOF MS (Discovery, manufactured by Shimadzu Corporation) The structure was confirmed by

As a method other than the above, after obtaining free DILRG-NH ₂ , palmitoyl group having 16 carbon atoms is bonded to the N-terminus thereof to introduce a palmitoyl group (acylation), and C16- DILRG-NH ₂ can also be synthesized.

Hereinafter, C16-DILRG-NH ₂ will be described as an example, but the carbon number of the acyl group in the active ingredient of the preservation solution of the present invention may be 6 to 28, and is limited to the example of C16-DILRG-NH _2. Is not to be done.

<2> Activity Test of C16-DILRG-NH ₂ (1) Cell Line Schneider S2 cells, which are Drosophila embryo-derived cultured cell lines, were collected by centrifugation.

(2) Preparation of permeabilized cells Digitonin, a permeabilizing agent, was added to the cells collected by centrifugation to prepare permeabilized cells. The treatment with digitonin can make the cell membrane permeable to the substrate, so that the mitochondrial oxygen consumption can be measured without separating the mitochondria.

(3) Measurement of oxygen consumption (i) Measurement method 5 × 10 ⁷ permeabilized cells were added to the assay medium, and the concentration of C16-DILRG-NH ₂ in the medium was 0 μM (no addition), 50 μM. , 100 μM. The substrate used was 5 mM succinic acid.

  For comparison, DMSO and palmitic acid as solvents were prepared, and the concentrations in the medium were adjusted to 0 μM (no addition), 50 μM, and 100 μM, respectively. The substrate used was 5 mM succinic acid.

Oxygen consumption is determined by measuring the amount of dissolved oxygen in the culture medium using a Clark-type cuvette electrode. By calculating the slope from the amount of dissolved oxygen per hour, the oxygen consumption rate (nmol O ₂ / min / 5 × 10 ⁷ cells). The temperature during measurement was 27 ° C.
(Ii) Results The results are shown in FIG. The oxygen consumption of the DMSO control group was 13.61 at 0 μM (no addition), 15.34 at 50 μM, and 16.67 at 100 μM. The oxygen consumption of the palmitic acid control group was 11.32 at 0 μM (no addition), 11.92 at 50 μM, and 10.58 at 100 μM. When C16-DILRG-NH ₂ was not added (0 μM), the oxygen consumption was 13.76, the C16-DILRG-NH ₂ concentration was 12.35 at 50 μM, and 4.76 at 100 μM. .

In addition, when the respiratory inhibitor (KCN) was added, the oxygen consumption was zero even when DMSO, palmitic acid and C16-DILRG-NH ₂ were added.

From the above results, it was found that the C16-DILRG-NH ₂ increases the oxygen consumption suppression effect as the concentration increases. In particular, at 100 μM, the oxygen consumption suppression effect was significant. It was also shown that there was a sufficient oxygen consumption suppression effect even at room temperature.

In addition, palmitic acid and DMSO used for comparison did not show an effect of suppressing oxygen consumption, and a significant difference was confirmed with C16-DILRG-NH ₂ . In addition, the symbol * and symbol ** on the graph in FIG. 1 indicate significance levels, * indicates p <0.005, and ** indicates p <0.001.

(4) Reversibility test (i) Test method Since C16-DILRG-NH ₂ was found to have an effect of suppressing oxygen consumption, the reversibility of this effect was examined.

C16-DILRG-NH ₂ , palmitic acid and DMSO were added to the assay medium containing the permeabilized cells so as to have a concentration of 100 μM. That is, as shown in the case of 100 μM in FIG. 1, the oxygen consumption rate of the cells was lowered by the addition of C16-DILRG-NH ₂ .

Thereafter, permeabilized cells were centrifuged at 1,000 g for 5 min, the precipitate was collected, 5 mM succinic acid was added as a substrate, and the oxygen consumption rate (nmol O ₂ / min / 5 × 10 ⁷ cells) was measured again. .

(Ii) Results As shown in FIG. 2, the oxygen consumption rate of the recovered cells was 7.44 for DMSO, 6.44 for palmitic acid, and 4.52 for C16-DILRG-NH ₂ . That is, there was almost no difference in oxygen consumption rate between C16-DILRG-NH ₂ and palmitic acid. Therefore, it was found that the oxygen consumption suppression activity by C16-DILRG-NH ₂ is reversible.

In addition, compared with the said oxygen consumption measurement (in the case of 100 micromol), the oxygen consumption rate of the collect | recovered cell has fallen because the cell which could not be collect | recovered by centrifugation exists. As described above, the significant difference in the oxygen consumption rate confirmed in the above oxygen consumption measurement (in the case of 100 μM) is not confirmed between the C16-DILRG-NH ₂ added group and the palmitic acid control group in the recovered cells as described above. Therefore, it can be determined that the oxygen consumption suppression activity by C16-DILRG-NH ₂ is reversible.

As described above, since the oxygen consumption suppression effect of C16-DILRG-NH ₂ is reversible, using C16-DILRG-NH ₂ as an active ingredient can be an effective use as a preservation solution for cells and organs. It was revealed.

<3> Cell preservation effect confirmation test (1) K562 (CML, chronic myelogenous leukemia)
K562 cells were seed-cultured in RPMI medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% FCS (fetal calf serum) at 37 ° C. and CO ₂ concentration of 5%. Thereafter, seed cells were dispensed into 96-well culture plates at 100 μL per well of 2.5 × 10 ⁴ cells / mL cells. Furthermore, a test substance was added to the culture plate into which the cells were dispensed under the following three conditions, and the culture was performed at 37 ° C. under a CO ₂ concentration of 5%.

Hereinafter, in this example, “C16-DILRG-NH ₂ ” is referred to as “C16-Yamamarin” for convenience.
1) Add 1 μL of PBS per well (no addition of C16-Yamamarin)
2) Add 0.5 μL per well of 250 μM C16-Yamamarin dissolved in DMSO (final concentration 12.5 μM)
3) Add 1.0 μL per well of 250 μM C16-Yamamarin dissolved in DMSO (final concentration 25 μM)
The number of cells was measured on the 0th, 1st, 2nd, 3rd, and 7th days in a proliferation confirmation test using a WST-1 assay (Premix WST-1 manufactured by Takara Bio Inc.). That is, 10 μL of Premix WST-1 was added per well of the target microplate, incubated at 37 ° C. under a CO ₂ concentration of 5% for 1 to 4 hours, and then the absorbance was measured at 450 nm. The number of cells by WST-1 assay was determined from a calibration curve prepared in advance.

  The results are shown in FIG. By adding C-16 yamamarin at the start of the culture, it was possible to culture for 1 week without subculture. Similarly, it was found that C-16 yamamarin suppresses the number of cells depending on the addition concentration. That is, it can be determined that cell proliferation is also suppressed by suppressing the oxygen consumption of the cells. In addition, in the additive-free system, cell proliferation was rapid, and dead cells increased on the third day.

  Thus, it is possible to culture cells without subculture for a long period of time by adding C-16 yamamarin, and by adding and culturing C-16 yamamarin to the culture solution, the cells can be subcultured. The culture effort can be significantly reduced.

(2) HUVEC (normal human umbilical vein endothelial cells)
HUVEC (Kurabo) was thawed and seed-cultured in Hu-Media-EG2 (Kurabo) at 37 ° C. and CO ₂ concentration of 5%. Thereafter, seed cells were dispensed into 96-well culture plates at 100 μL per well of 2.5 × 10 ⁴ cells / mL cells. After culturing the plate for 1 day under conditions of 37 ° C and CO ₂ concentration of 5% and confirming that the cells adhere to the bottom of the well, the test substance was added to the culture plate under the following two conditions. The culture was performed at 37 ° C. under a CO ₂ concentration of 5%.
1) Add 1 μL of PBS per well (no addition of C16-Yamamarin)
2) Add 0.5 μL per well of 250 μM C16-Yamamarin dissolved in DMSO (final concentration 12.5 μM)
After culturing for 7 days with the addition of the test substance, remove the culture supernatant and further culture in fresh Hu-Media-EG2 (without test substance) at 37 ° C and CO ₂ concentration of 5% Continued. The number of cells was determined by the WST-1 assay (Premix WST-1, manufactured by Takara Bio Inc.) on the 0th, 3rd, and 7th days after addition of the test substance and on the 3rd and 7th days after the medium change. Measured. That is, 10 μL of Premix WST-1 was added per well of the target microplate, incubated at 37 ° C. under a CO ₂ concentration of 5% for 1 to 4 hours, and then the absorbance was measured at 450 nm. The number of cells by WST-1 assay was determined from a calibration curve prepared in advance.

  The results are shown in FIG. In the additive-free system, it became confluent on the 7th day, and the death of the cells started. On the other hand, when C16-yamamarin was added at the start of the culture, the proliferation of the cells was remarkably suppressed. It was confirmed that the growth was started by removing C16-yamamarin.

  It was confirmed that the addition of C16-yamamarine suppressed the oxygen consumption of the cells and could be stored in an incubator at 37 ° C. in a state where the cells could be grown.

(3) NHDF (normal human adult dermal fibroblasts)
After thawing NHDF (purchased from Kurabo), medium 106S with LSGS custom growth additive (Kurabo, product number: KE-6350) in medium (Kurabo), 37 ° C, CO ₂ concentration 5% Seed culture was performed under conditions. Thereafter, seed cells were dispensed into 96-well culture plates at 100 μL per well of 2.5 × 10 ⁴ cells / mL cells. After culturing the plate for 1 day under conditions of 37 ° C and CO ₂ concentration of 5% and confirming that the cells adhere to the bottom of the well, the test substance was added to the culture plate under the following two conditions. The culture was performed at 37 ° C. under a CO ₂ concentration of 5%.
1) Add 1 μL of PBS per well (no addition of C16-Yamamarin)
2) Add 0.5 μL per well of 250 μM C16-Yamamarin dissolved in DMSO (final concentration 12.5 μM)
After culturing for 7 days with the addition of the test substance, the culture supernatant is removed, and the medium (excluding the test substance) containing LSGS special growth additive in fresh Medium106S is added at 37 ° C with a CO ₂ concentration of 5 The culture was further continued under% conditions. The number of cells was measured on the 0th, 3rd, and 7th days after addition of the test substance and on the 3rd and 7th days after the medium change in the growth confirmation test using the WST-1 assay (Premix WST-1 manufactured by Takara Bio Inc.). It was measured. That is, 10 μL of Premix WST-1 was added per well of the target microplate, incubated at 37 ° C. under a CO ₂ concentration of 5% for 1 to 4 hours, and then the absorbance was measured at 450 nm. The number of cells by WST-1 assay was determined from a calibration curve prepared in advance.

  The results are shown in FIG. In the additive-free system, it became confluent on the 7th day, and the death of the cells started. On the other hand, when C16-yamamarin was added at the start of the culture, the proliferation of the cells was remarkably suppressed. It was confirmed that the growth was started by removing C16-yamamarin.

  It was confirmed that the addition of C16-yamamarine suppressed the oxygen consumption of the cells and could be stored in an incubator at 37 ° C. in a state where the cells could be grown.

Claims (5)

Organ preservation solution characterized by containing C16-DILRG-NH ₂ represented by the following formula as an active ingredient.
Organ preservation solution according to claim 1, wherein the concentration of active ingredient is 10Myuemu～1000myuM. The stock solution organ you containing C16-DILRG-NH ₂ represented by the following formula as an active ingredient, storage method organ you characterized by immersing the organ.
The storage method organ of claim 3, wherein the concentration of active ingredient of preservative solution is 10Myuemu～1000myuM. The temperature of the preservation solution, storage method organ according to claim 3 or 4, characterized in that it is 5 to 37 ° C..