Method for producing biological tissue or RNA preservatives in biological tissue

Abstract

[Problem] To provide a novel biological sample preservative that enables preservation of a biological sample at room temperature, a production method of a biological sample preservative, a preservation method of a biological sample, and a kit for preserving a biological sample. [Solution] A biological sample preservative that contains 1.0-10.0 mol / L of a substance having a polyatomic monovalent cation represented by the chemical formula NH4+ or a compound composed of the polyatomic monovalent cation. As the substance having a polyatomic monovalent cation represented by the chemical formula NH4+ or the compound composed of the polyatomic monovalent cation, use can be made of, for example, ammonium sulfate or ammonium nitrate.

Description

Technical Field

[0001] The present disclosure relates to RNA in biological tissue or biological tissue a method for manufacturing a preservative In the law .

Background Art

[0002] Excellent preservation techniques for various biological samples such as nucleic acids, cells, and tissues collected from organisms such as humans have been conventionally required in various industrial fields. However, such biological samples generally gradually lose their activity over time. Therefore, a technique for long-term preservation of biological samples has become an extremely important technique. For example, Patent Document 1 describes a cryopreservation solution characterized by containing a modified polyvinyl alcohol having an ethylene oxide group. Specifically, after immersing biological cells or tissues in an equilibration solution, a tubular tool such as a pipette is used to take out the cells or tissues together with a small amount of the equilibration solution and transfer them into the cryopreservation solution. Then, after immersing the cells or tissues in the cryopreservation solution for a predetermined time, they are taken out together with a small amount of the cryopreservation solution, and the cells or tissues are dropped and adhered onto a sheet of a cryopreservation tool together with a small amount of the cryopreservation solution. Thereafter, the sheet on which the cells or tissues are placed is immersed in a cooling medium such as liquid nitrogen and frozen. The preservation technique described in Patent Document 1 preserves biological cells and tissues through the above steps.

[0003] However, in the preservation technique described in Patent Document 1, since a cryopreservation solution is used for the preservation of biological cells and tissues, not only various steps are included before preservation, but it is also necessary to maintain a frozen environment after preservation, and its handling is complicated.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, this disclosure is, RNA in biological tissue or biological tissue A novel method that enables storage at room temperature. RNA in biological tissue or biological tissue Method for manufacturing preservatives Law The challenge is to provide it. [Means for solving the problem]

[0017] Furthermore, in order to solve the above problems, the present inventors have found a method for producing a biological sample preservative that enables storage at room temperature by including a substance having a polyatomic monovalent cation represented by the chemical formula NH4+ in a concentration of 1.0 to 10.0 mol / L or a compound composed of said polyatomic monovalent cation. Accordingly, the first aspect of this disclosure includes (1) a first addition step of adding 1.0 to 10.0 mol / L of ammonium sulfate to a predetermined solvent, a second addition step of adding an excipient to the solution obtained in the first addition step, and a freeze-drying step of freeze-drying the solution obtained in the second addition step, wherein the preservative obtained by dissolving the solid form obtained in the freeze-drying step in a solvent allows biological tissue or RNA in biological tissue to be preserved at 37°C for 24 hours, and the higher the concentration of ammonium sulfate in the 1.0 to 10.0 mol / L range, the higher the preservation effect, and when biological tissue or RNA in biological tissue is preserved at 37°C for 24 hours, a turbidity level is observed that is lower than when preserved in phosphate-buffered saline. Furthermore, by adding the excipient in the second addition step, the explosion of ammonium sulfate in the freeze-drying step is suppressed. The invention relates to a method for producing biological tissue or RNA preservatives in biological tissue.

[0018] A preferred embodiment of the present disclosure is (2) in the second addition step, 0.25 to 30% by weight of the excipient is added to the solution obtained in the first addition step. Furthermore, by adding the excipient in the second addition step, the explosion of ammonium sulfate in the freeze-drying apparatus due to rapidly reaching the freezing point during the freeze-drying step is suppressed. This is the manufacturing method. Another preferred embodiment of the present disclosure is a manufacturing method in which, in the freeze-drying step, the solution obtained in the second addition step is freeze-dried at -10°C to -100°C. A preferred embodiment of the present disclosure is a manufacturing method wherein (4) in the first addition step, 1.7 to 5.0 mol / L of ammonium sulfate is added to the predetermined solvent, and the preservative enables the preservation of biological tissue or RNA in biological tissue at 37°C for 24 hours, and the higher the concentration of ammonium sulfate in the 1.7 to 5.0 mol / L range, the greater the preservation effect. 。 [Effects of the Invention]

[0023] This disclosure is, RNA in biological tissue or biological tissue A novel method that enables storage at room temperature. RNA in biological tissue or biological tissue Method for manufacturing preservatives Law It can be provided. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 shows images of the microtubes containing the undiluted, 1 / 2 diluted, and 1 / 4 diluted solutions of the biological sample preservative from Example 1, as well as images of mouse livers after being stored at 37°C for 24 hours in phosphate-buffered saline prepared as a negative control. [Figure 2] This figure shows images of each microtube after storing mouse livers in the respective biological sample preservative solutions of Examples 1-9 and in phosphate-buffered saline prepared as a negative control at 37°C for 24 hours. [Modes for carrying out the invention]

[0025] The following describes in detail embodiments for implementing the biological sample preservative, the method for producing the biological sample preservative, and the biological sample preservation method of this disclosure. However, the following embodiments are examples for illustrating this disclosure, and this disclosure is not limited to these embodiments.

[0026] <Manufacture of Biological Sample Preservative> In the present disclosure, the biological sample preservative can be manufactured, for example, by adding each component to a predetermined solvent and, if necessary, through a freeze-drying process or a dehydration process. Hereinafter, the manufacturing process will be specifically described.

[0027] Here, in the present disclosure, a biological sample is a sample collected from a living body or used for a living body, and is an organ, tissue, cell (including cultured cells), body fluid, nucleic acid, protein, or a combination thereof. More preferably, nucleic acid, protein, or a combination thereof is used as the biological sample. Note that these biological samples may include samples artificially synthesized for a living body in addition to samples collected from a living body. Also, the invention according to the present disclosure is not naturally limited only to those exemplified herein.

[0028] Also, the manufactured biological sample preservative is preferably provided in the form of a solution when preserving a living body, but may be in any form such as a dry form, solid form, powder form, anhydrous form, granular form, etc. Note that when the biological sample preservative is in a form other than the solution form, it is preferably dissolved in a predetermined solvent to reach a desired concentration before use. Also, the invention according to the present disclosure is not limited only to these forms.

[0029] Furthermore, the manufactured biological sample preservative may be provided as a preservative or a preservative composition having the above form, or in addition to the biological sample preservative, it may be provided as a kit for preserving biological samples including a container for preserving biological samples, a tool for collecting biological samples, etc.

[0030] 1. First addition step In the present disclosure, in the first addition step, first, a predetermined solvent is placed in a necessary amount container and stirred at a normal temperature of 5 to 50 °C, more preferably 10 to 40 °C. As the solvent, typically pure water is used, but any treated water such as filtered water, purified water, distilled water, deionized water, or a combination thereof treated by filtration, distillation, deionization, or a combination thereof, or water containing these treated waters may be used.

[0031] Next, a buffer is added to the agitated solvent at a predetermined concentration. As the buffer to be added, various ones such as phosphate buffer, Tris-HCl buffer, ethylene diamine tetraacetic acid buffer, Tris-ethylene diamine tetraacetic acid buffer, Tris-acetate-ethylene diamine tetraacetic acid buffer, Tris-borate-ethylene diamine tetraacetic acid buffer, SSC buffer, SSPE buffer, sodium citrate buffer, carbonate-bicarbonate buffer, sodium borate buffer, 2-morpholinoethanesulfonic acid buffer, and combinations thereof can be used. Among these, as the buffer, more preferably, an ethylene diamine tetraacetic acid buffer is used. However, the invention according to the present disclosure is not limited to only these buffers. Further, the buffer is added so as to have a concentration of 0.004 to 10.000 mol / L, more preferably 0.010 to 5.000 mol / L, in the finally used biological sample preservative.

[0032] Next, to the solution to which the buffer has been added, each material such as other main components and additive materials is added. As the main components, substances having a polyatomic monovalent cation represented by the chemical formula NH4 , + , ,

[0033] or compounds composed of the polyatomic monovalent cation are included. Such substances or compounds are selected from the group consisting of ammonium sulfate, ammonium nitrate, and combinations thereof. By adding substances having a polyatomic monovalent cation represented by these NH4 + or compounds composed of the polyatomic monovalent cation, that is, substances or compounds containing ammonium ions, it is possible to further enhance the preservation effect and preservation stability of biological samples and the like at room temperature. Note that the invention according to the present disclosure is not limited to only these buffers.

[0033] Substances having a polyatomic monovalent cation represented by the chemical formula NH4 + or compounds composed of the polyatomic monovalent cation are added so as to have a concentration of 1.0 to 10.0 mol / L, more preferably 1.7 to 5.0 mol / L, in the finally used biological sample preservative.

[0034] Other components may include chelating reagents. Various chelating reagents can be used, such as sodium citrate, trisodium citrate monohydrate, sodium borate, sodium fluoride, ethylenediaminetetraacetic acid, or combinations thereof. By adding these chelating reagents, the activity of nucleases can be suppressed, and the storage stability of biological samples such as nucleic acids at room temperature can be further enhanced. However, the invention described herein is not limited to these chelating reagents alone. The chelating reagent is added to the final biological sample preservative at a concentration of 0.004 to 10.000 mol / L, more preferably 0.006 to 0.500 mol / L.

[0035] Other possible components include stabilizers. Such stabilizers include 4-(2-aminoethyl)benzenesulfonyl hydrochloride fluoride, aprotinin, E-64, leupeptin hemisulfate monohydrate, silicon carbide, 4-hydroxybenzoic acid, N,N-dialkylpropanamide, 3-morpholinopropanesulfonic acid, polyethylene glycol, poly(oxyethylene) polymer, poly(oxyethylene) polymer, and monoethylene glycol (1,2-ethanediol). Various stabilizers can be used, such as glycol (1,2-ethanediol), glutathione, lithium salts, chaotropic agents, guanidinium salts, guanidine hydrochloride, urea, polyamines, biuret, arginine, cosmotrope, polyethylene glycol, polyvinylpyrrolidone, arginine ethyl ester, glycine, amino acid alkyl esters, amino acid amides, compounds having a guanidinium group, or combinations thereof. By adding these stabilizers, it is possible to stabilize biological samples such as cells, proteins, and nucleic acids. Furthermore, among these, 4-(2-aminoethyl)benzenesulfonyl hydrochloride fluoride, aprotinin, E-64, leupeptin hemisulfate monohydrate, or combinations thereof can particularly enhance stability by inhibiting protease activity. It should be noted that the inventions described herein are not limited to these stabilizers alone. The amount of these stabilizers added can be adjusted as appropriate depending on the selected stabilizer, but when 4-(2-aminoethyl)benzenesulfonyl hydrochloride fluoride, aprotinin, E-64, leupeptin hemisulfate monohydrate, or a combination thereof is selected, it is possible to add them at a concentration of 0.001 to 1.0% by weight, more preferably 0.01 to 0.10% by weight.

[0036] Other components may include bactericidal and antibacterial agents. Various bactericidal and antibacterial agents can be used, including amoxicillin trihydrate, cefotaxime sodium, clarithromycin, erythromycin, pyrethrin, metofluthrin, transfluthrin, sodium azide, 4-hydroxybenzoic acid, alcohols, surfactants, or combinations thereof. However, the invention described herein is not limited to these bactericidal and antibacterial agents alone. The amount of these bactericidal and antibacterial agents added can be appropriately adjusted depending on the selected stabilizer, but they can be added at a concentration of 0.001 to 1.0% by weight, more preferably 0.01 to 0.10% by weight.

[0037] Although not specifically mentioned elsewhere, other additives such as surfactants, pH adjusters, and colorants may be included as appropriate, in addition to the above-mentioned components.

[0038] 2. Stirring process As described above, the solution to which each component has been added in the first addition step is stirred in a required amount in a container at room temperature of 5 to 50°C, more preferably 10 to 40°C. This stirring is continued until each added component is completely dissolved. By the complete dissolution of each added component, the biological sample preservative according to this disclosure is obtained as a solution. Thus, the chemical formula is NH4 + By producing a biological sample preservative containing a substance having a polyatomic monovalent cation as shown above, or a compound composed of such polyatomic monovalent cation, a biological sample preservative with superior storage stability can be obtained.

[0039] 3.Freeze-drying process As described above, the manufactured biological sample preservative can be further processed into a given solid form, such as a dried form, solid form, powder form, anhydrous form, or granular form, taking into consideration the ease of storage and transport of the biological sample preservative itself. These forms of biological sample preservative can then be obtained by treating the manufactured biological sample preservative (solution) with a freeze-drying process. Note that this freeze-drying process is not a mandatory step and can be performed as desired.

[0040] 3-1. Second Addition Step First, in the freeze-drying process, as a second addition step, an excipient is added to the biological sample preservative (solution) manufactured as described above and dissolved by stirring. Such excipients include polyols, monosaccharides, disaccharides, polysaccharides, amino acids, peptides, proteins, hydrophilic polymers, or combinations thereof. Among these, preferred excipients are lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, talc, titanium dioxide, erythritol, mannitol, sorbitol, trehalose, sucrose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carmellose sodium, pullulan, dextrin, acacia gum, agar, gelatin, tragacanth, sodium alginate, polyvinylpyrrolidone, polyvinyl alcohol, or combinations thereof, with calcium carbonate, dextrin, or combinations thereof being particularly preferred. It should be noted that the invention described herein is not limited to these excipients only.

[0041] The amount of these excipients added can be adjusted as appropriate depending on the selected excipient, but they can be added at a concentration of 0.25 to 30% by weight, more preferably 0.1 to 10.0% by weight.

[0042] Here, the biological sample preservative manufactured as described above has the chemical formula NH4 + This invention relates to substances having polyatomic monovalent cations as shown by , or compounds composed of such polyatomic monovalent cations, typically including ammonium sulfate or ammonium nitrate. Therefore, when frozen after dehydration in freeze-drying, the freezing point is reached rapidly, which may cause ammonium sulfate or the like to explode in the freeze-drying apparatus. In this disclosure, as described above, this explosion can be prevented by adding a predetermined amount of excipient in advance during the freeze-drying process.

[0043] 3-2. Freeze-drying process Once the excipient is dissolved in the biological sample preservative, the solution containing the dissolved excipient is freeze-dried. Various methods can be used for this freeze-drying, but one example is that the solution containing the dissolved excipient is degassed under vacuum conditions for a predetermined time (e.g., 60 minutes), and then left at a predetermined temperature (e.g., -75°C) for a predetermined time (e.g., 60 minutes). After that, the obtained biological sample preservative is slowly returned to room temperature and atmospheric pressure to obtain a freeze-dried biological sample preservative. The freeze-drying process or a tableting process after the freeze-drying process can be used to process the product into the desired form via a granulation process.

[0044] 4.Dehydration process The biological sample preservative produced by the first addition and stirring steps can be further processed into a given solid form, such as a dried form, solid form, powder form, anhydrous form, or granular form, taking into consideration the ease of storage and transport of the biological sample preservative itself. Then, the biological sample preservative (solution) produced as described above can be subjected to a dehydration process to obtain biological sample preservatives in these forms. Note that this dehydration process is not a mandatory step and can be performed as desired.

[0045] The required amount of the biological sample preservative (solution) produced by the above process is placed in a vacuum device and left for a predetermined time (e.g., 24 hours). After that, the obtained biological sample preservative is slowly returned to atmospheric pressure, and a dehydrated biological sample preservative is obtained. After the dehydration process, the tableting process or granulation process can be used to process the product into the desired form.

[0046] As described above, it is possible to produce the desired biological sample preservative through the first addition step and stirring step, and optionally through a freeze-drying step or a dehydration step.

[0047] <Composition of the biological sample preservative> The biological sample preservative produced through the above manufacturing process contains the following components. Specifically, the biological sample preservative contains 1.0 to 10.0 mol / L, more preferably 1.7 to 5.0 mol / L of "chemical formula NH4" in a predetermined solvent.+ The biological sample preservative may further contain, as desired, a "substance having a polyatomic monovalent cation represented by or a compound composed of the said polyatomic monovalent cation".

[0048] Furthermore, although not explained in detail here, the biological sample preservative produced through the above manufacturing process is adjusted to a pH of 4.0 to 10.0, more preferably 6.0 to 8.0, by adding a pH adjuster. This makes it possible to further enhance the preservation stability of the biological sample.

[0049] Although not explained in detail here, in addition to the components listed above, other additives such as surfactants, pH adjusters, and colorants may be included as appropriate.

[0050] <Method for Preserving Biological Samples> The preservation method using the above-described biological preservative is used, for example, to preserve organs, tissues, cells (including cultured cells), body fluids, nucleic acids, proteins, or combinations thereof at room temperature. Specifically, the biological sample preservative produced by the first addition step and stirring step is divided into a predetermined container (e.g., vial, microtube, etc.). Then, the biological sample, such as organs, tissues, cells (including cultured cells), body fluids, nucleic acids, proteins, or combinations thereof collected from a living organism, is immersed in the container and left to stand at room temperature (e.g., 5-50°C, more preferably 10-40°C). This makes it possible to preserve the collected biological sample at room temperature.

[0051] If the biological sample preservative has been processed into a dry form, solid form, powder form, anhydrous form, granular form, etc., by a freeze-drying or dehydration process, it can be used by dissolving each component in a predetermined solvent to the concentration of the biological sample preservative in solution form (the concentration may be adjusted as appropriate depending on the amount of solvent). Typically, pure water is used as the solvent, but any treated water such as filtered water, purified water, distilled water, deionized water, or a combination thereof, or water containing such treated water, is also acceptable.

[0052] As described above, this disclosure provides a novel biological sample preservative that enables the storage of biological samples at room temperature, a method for producing the biological sample preservative, and a biological sample This makes it possible to provide storage methods. [Examples]

[0053] The present disclosure will be further described below with reference to examples, but the disclosure is not limited thereto.

[0054] <Manufacturing of biological sample preservatives>

[0055] Pure water was placed as a solvent in a heat-resistant bottle of a predetermined capacity (2L glass medium bottle (manufactured by Thermo Fisher Scientific)), and stirred at 60 rpm using a temperature-controlled stirrer (product name: Hot Stirrer REXIM RSH-4DN (manufactured by AS ONE)) set to 37°C. Subsequently, buffer solutions as described in Tables 1-3 below were added, and the mixture was stirred further until the buffer solutions were completely dissolved. Next, ammonium sulfate and other components as described in Tables 1-3 below were added at the concentrations described in Table 1 below, and the mixture was stirred until each component was completely dissolved, thereby producing each biological sample preservative solution (Examples 1-9).

[0056] [Table 1]

[0057] [Table 2]

[0058] [Table 3]

[0059] <Measurement of the preservation effect of biological samples> The preservation effect of each biological sample preservative manufactured as described above in Examples 1 to 9 was measured as follows.

[0060] [Experiment 1] For each of the biological sample preservatives from Example 1, 1 / 2 dilution, and 1 / 4 dilution were dispensed in 1.5 mL microcentrifuge tubes, with 1 mL each being the undiluted solution and 1.5 mL dilution. Then, 1.3 mg of mouse liver was taken as a biological sample and placed in the microcentrifuge tube containing each biological sample preservative, and immersed in the biological sample preservative. At this time, 1 mL of phosphate-buffered saline was dispensed into a 1.5 mL microcentrifuge tube as a negative control, and similarly, 1.3 mg of mouse liver was immersed in this phosphate-buffered saline. The biological samples in each microcentrifuge tube were then stored at 37°C under normal pressure for 24 hours.

[0061] After 24 hours, the liver samples were removed from each microcentrifuge tube, and RNA extraction was performed. RNA extraction was carried out using a commercially available RNA extraction kit (product name: RNeasy Plus Mini Kit (QIAGEN)) according to the method specified by the kit. The storage stability of the biological samples was then evaluated by measuring the concentration of RNA obtained from each biological sample in each microcentrifuge tube. RNA concentration was measured using an electrophoresis system (product name: Bioanalyzer 2100 (Agilent)) according to the method specified by the system.

[0062] Figure 1 shows images of the microtubes containing mouse livers stored at 37°C for 24 hours in the undiluted, 1 / 2 diluted, and 1 / 4 diluted solutions of the biological sample preservative from Example 1, as well as in phosphate-buffered saline prepared as a negative control. Table 4 shows the RNA concentrations obtained from mouse livers stored in the undiluted, 1 / 2 diluted, and 1 / 4 diluted solutions of the biological sample preservative from Example 1, as well as in phosphate-buffered saline.

[0063] [Table 4]

[0064] As shown in Figure 1, it was confirmed that as the concentration of components in each biological sample preservative decreased from the undiluted solution to the 1 / 2 diluted solution and then to the 1 / 4 diluted solution, the preservation state of the preserved biological sample deteriorated, and the biological sample preservative solution also became turbid. In particular, compared to preservation with phosphate-buffered saline, preservation with the 1 / 4 diluted biological sample preservative of Example 1 resulted in a similar level of turbidity as phosphate-buffered saline. On the other hand, the undiluted and 1 / 2 diluted biological sample preservatives of Example 1 showed significantly less turbidity compared to phosphate-buffered saline, confirming that the biological sample itself was in excellent condition. In particular, the undiluted biological sample preservative of Example 1 showed almost no turbidity, and the biological sample maintained almost its original state, confirming that it can preserve biological samples very well even at room temperature.

[0065] Furthermore, as shown in Table 4, it was confirmed that the concentrations of each RNA component extracted from the biological sample after preservation also increased in proportion to the concentration of each component of the biological sample preservative. Specifically, when the biological sample was preserved with a 1 / 4 dilution of the biological sample preservative of Example 1, the concentration of RNA extracted from the biological sample was equivalent to that of RNA extracted from a biological sample preserved in phosphate-buffered saline. However, when the biological sample was preserved with a 1 / 2 dilution of the biological sample preservative of Example 1, the concentration of RNA extracted from the biological sample was clearly higher than that of RNA extracted from a biological sample preserved in phosphate-buffered saline. Moreover, when the biological sample was preserved with the undiluted solution of the biological sample preservative of Example 1, the concentration of RNA extracted from the biological sample was extremely high (more than twice) compared to that of RNA extracted from a biological sample preserved in phosphate-buffered saline. In other words, these results indicate that the preservation effect of biological samples depends on the concentration of the biological sample preservative in Example 1, and that the undiluted and 1 / 2 diluted solutions of the biological sample preservative in Example 1 provide extremely high preservation effects for biological samples.

[0066] [Experiment 2] One mL of each biological sample preservative from Examples 1-9 was dispensed from a heat-resistant bottle and placed into a 1.5 mL microcentrifuge tube. Then, 40 mg of mouse liver was taken as the biological sample and placed in the microcentrifuge tube containing the biological sample preservative from Examples 1-9, and immersed in the biological sample preservative. At the same time, 1 mL of phosphate-buffered saline was dispensed into a 1.5 mL microcentrifuge tube as a control, and similarly, 40 mg of mouse liver was immersed in this phosphate-buffered saline. The biological samples in each microcentrifuge tube were then left to stand at 37°C under normal pressure for 24 hours for preservation.

[0067] After 24 hours, the liver, which was the biological sample, was removed from each microtube, and RNA extraction was performed. RNA extraction was carried out using the same method as in Experiment 1.

[0068] Figure 2 shows images of the microtubes containing mouse liver after 24 hours of storage at 37°C in each of the biological sample preservative solutions for Examples 1-9 and in phosphate-buffered saline prepared as a negative control. For the positive control, 40 mg of mouse liver was collected, frozen in a microtube at -80°C, and stored; images of this sample were also taken in the same manner. Table 5 shows the RNA concentrations obtained from the mouse livers stored in each of the biological sample preservatives and phosphate-buffered saline for Examples 1-9.

[0069] [Table 5]

[0070] As shown in Figure 2, in all cases, the biological samples stored at room temperature in each of the biological sample preservative solutions from Examples 1 to 9 maintained a condition equivalent to that of the biological sample stored at -80°C, which was prepared as a positive control, and no turbidity was observed in any of the biological sample preservative solutions. On the other hand, the biological sample stored in phosphate-buffered saline, which was prepared as a negative control, showed significantly more turbidity compared to the biological samples from Examples 1 to 9. In other words, it was confirmed that all of the biological sample preservation solutions from Examples 1 to 9 have an extremely good effect on preserving biological samples even at room temperature.

[0071] Furthermore, as shown in Table 5, the concentrations of RNA extracted from biological samples stored at room temperature using each of the biological sample preservative solutions in Examples 1 to 9 were all measured to be at least 2.9 times higher than the concentrations of RNA extracted from biological samples stored in phosphate-buffered saline, which was prepared as a negative control. In particular, the concentration of RNA extracted from biological samples stored at room temperature using the biological sample preservative solution of Example 4 was equivalent to the concentration of RNA extracted from biological samples frozen and stored at -80°C, which was prepared as a positive control, and the concentration of RNA extracted from biological samples stored at room temperature using the biological sample preservative solution of Example 9 was measured to be more than 1.3 times higher. In other words, these results confirm that all of the biological sample preservatives in Examples 1 to 9 have an extremely high preservation effect on biological samples.

Claims

1. A first addition step involves adding 1.0 to 10.0 mol / L of ammonium sulfate to a predetermined solvent, A second addition step involves adding an excipient to the solution obtained in the first addition step, The process includes a freeze-drying step in which the solution obtained by the second addition step is freeze-dried, The preservative obtained by dissolving the solid substance obtained by the freeze-drying process in a solvent is, The present invention enables the preservation of biological tissue or RNA within biological tissue at 37°C for 24 hours, and the preservation effect increases as the concentration of ammonium sulfate increases within the range of 1.0 to 10.0 mol / L. When biological tissue or RNA within biological tissue is stored at 37°C for 24 hours, less turbidity is observed compared to when it is stored in phosphate-buffered saline. A method for producing a biological tissue or an RNA preservative in a biological tissue, wherein the excipient is added in the second addition step to suppress the explosion of the ammonium sulfate in the freeze-drying step.

2. In the second addition step, 0.25 to 30% by weight of the excipient is added to the solution obtained in the first addition step. A method for producing a biological tissue or RNA preservative in a biological tissue, according to claim 1, wherein the excipient is added in the second addition step to suppress the explosion of ammonium sulfate in the freeze-drying apparatus due to rapidly reaching the freezing point in the freeze-drying step.

3. The method for producing a biological tissue or RNA preservative in a biological tissue according to claim 1, wherein the freeze-drying step involves freeze-drying the solution obtained in the second addition step at -10°C to -100°C.

4. In the first addition step, 1.7 to 5.0 mol / L of the ammonium sulfate is added to the predetermined solvent. A method for producing a preservative for biological tissue or RNA in biological tissue according to claim 1, wherein the preservative allows biological tissue or RNA in biological tissue to be preserved at 37°C for 24 hours, and the preservative effect increases as the concentration of ammonium sulfate increases within the range of 1.7 to 5.0 mol / L.

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