General preserving fluid for tissues and organs

Abstract

The invention belongs to the technical field of tissue and organ preservation, and particularly relates to tissue and organ general preservation liquid. The general preserving fluid for tissues and organs is prepared from the following components: hydroxyethyl starch, sodium chloride, lactobionic acid, potassium phosphate, magnesium sulfate, bombax pentahydrate, adenosine, allopurine, glutathione, glycerol, heparin, fructose-1, 6-diphosphate, mannitol, an antibacterial component, insulin, simethicone and the balance of distilled water. The general preserving fluid for tissues and organs can preserve the overall physiological activity of the tissues and organs for a long time in a non-frozen state or a frozen state.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue and organ preservation, and specifically relates to a universal tissue and organ preservation solution. The present invention also relates to a preparation method of the universal tissue and organ preservation solution and a tissue and organ preservation method. Background Art

[0002] In recent years, the field of tissue and organ preservation has developed rapidly. At present, tissue and organ preservation technology has covered various organs such as the heart, liver, pancreas, kidneys, intestines, etc. However, generally speaking, organs cannot be used directly and quickly after being removed, and need to be stored and transported for a long time. In this process, the preservation of tissues and organs is extremely important. Tissue and organ preservation fluid is mainly used to maintain the physiological activity of tissues and organs for a long time in an environment of ischemia and hypoxia.

[0003] University Wisconsin solution (UW solution) invented by the University of Wisconsin is a multi-organ preservation solution that is currently widely used in hospitals for organ preservation, but UW is expensive and has a short shelf life. HTK (Histidine Tryptophan Ketoglutarate) multi-organ preservation solution has a poor effect on kidney preservation and is not widely used in practice.

[0004] Therefore, there is still a demand for preservation fluids that are low-cost, highly versatile, and can preserve tissues and organs for longer periods of time. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a universal preservation solution for tissues and organs.

[0006] The universal tissue and organ preservation solution provided by the present invention comprises the following components and their contents per liter:

[0007]

[0008]

[0009] Preferably, the universal tissue and organ preservation solution comprises the following components and their contents per liter:

[0010]

[0011] The antibacterial components are: fennel and star anise extract and penicillin.

[0012] Preferably, the content of the fennel and star anise extract is 40-70 g, and the content of penicillin is 20-60 U per liter.

[0013] The antibacterial component can inhibit most bacteria and effectively prolong the shelf life of the preservation solution and the preservation time of tissues and organs.

[0014] Preferably, the universal tissue and organ preservation solution comprises the following components and their contents per liter:

[0015]

[0016] In a specific embodiment, the universal tissue and organ preservation solution comprises the following components and their contents per liter:

[0017]

[0018]

[0019] The universal tissue and organ preservation solution can preserve tissues and organs in a -20°C frozen state or in a 0-10°C non-frozen state.

[0020] Preferably, the universal tissue and organ preservation solution can preserve tissues and organs in a -20°C frozen state for at least 6 months.

[0021] Preferably, the universal tissue and organ preservation solution can preserve tissues and organs for at least 10 hours at 10°C in a non-frozen state; and can preserve tissues and organs for at least 72 hours at 4°C in a non-frozen state, more preferably at least 7 days.

[0022] The pH value of the universal tissue and organ preservation solution is 6-8, preferably 6.5-7.5, and more preferably 6.8-7.3.

[0023] Optionally, the pH value of the universal tissue and organ preservation solution can be adjusted by adding sodium hydroxide solution.

[0024] Those skilled in the art will appreciate that the above components can achieve the purpose of the present invention by using the same reagent from different manufacturers or using aliases of the same reagent.

[0025] The present invention also provides a method for preparing the universal tissue and organ preservation solution, which comprises the following steps: adding each component into water (such as distilled water) according to the prescription, mixing evenly, sterilizing, and preserving to obtain the solution.

[0026] Preferably, the mixing is performed at 10-40°C.

[0027] Preferably, the sterilization is performed using ultraviolet rays (for example, using a Huilangpu ultraviolet disinfection cabinet).

[0028] Preferably, the storage temperature is 0-10°C, preferably 4°C.

[0029] The present invention also provides a tissue and organ preservation method, which comprises the following steps: after obtaining the tissue and organ, completely replacing the blood in the tissue and organ with physiological saline containing heparin, and then perfusing the tissue and organ with the universal tissue and organ preservation solution according to the circulation pathway; after the perfusion is completed, completely immersing the tissue and organ in the universal tissue and organ preservation solution and storing it in a refrigerator.

[0030] Preferably, the storage may be in a -20°C frozen state or in a 0-10°C non-frozen state. The tissue organ may be one organ or multiple organs or a part of one organ or multiple parts of multiple organs. For example, it may include a whole organ, tissue or tissue group.

[0031] The tissue organ can be selected from all organs and tissues, for example, one or more selected from the heart, liver, brain, lung, kidney, pancreas, intestine (including large intestine and small intestine), etc.

[0032] Preferably, the tissue organ is selected from one or more of the liver, kidney and small intestine.

[0033] Beneficial effects of the present invention: The universal tissue and organ preservation solution of the present invention can preserve the overall physiological activity of tissues and organs for a long time in a non-frozen state of 0-10°C or a frozen state of -20°C. That is, after long-term preservation, the tissues and organs can still show the same physiological elasticity, color, and smell as normal tissues and organs; and in microscopic examination, the damage of tissue cells is controlled within a reasonable range. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1A This is a HE staining photograph of a liver treated with the universal tissue and organ preservation solution of the present invention and preserved for 72 hours.

[0035] Figure 1B These are HE staining photos of livers treated with UW solution and stored for 72 hours.

[0036] Figure 2A This is a HE staining photograph of a liver treated with the universal tissue and organ preservation solution of the present invention and preserved for 7 days.

[0037] Figure 2B These are HE staining photos of livers treated with UW solution and stored for 7 days.

[0038] Figure 3A The picture is HE staining of kidney treated with the universal tissue and organ preservation solution of the present invention and preserved for 72 hours.

[0039] Figure 3B These are HE staining photos of kidneys treated with UW solution and stored for 72 hours.

[0040] Figure 4AThe picture is HE staining of kidney treated with the universal tissue and organ preservation solution of the present invention and preserved for 7 days.

[0041] Figure 4B These are HE staining photos of kidneys treated with UW solution and stored for 7 days.

[0042] Figure 5A This is a HE staining photograph of the small intestine treated with the universal tissue and organ preservation solution of the present invention and preserved for 72 hours.

[0043] Figure 5B These are HE staining photos of the small intestine treated with UW solution and stored for 72 hours.

[0044] Fig. 6A This is a HE staining photograph of the small intestine treated with the universal tissue and organ preservation solution of the present invention and preserved for 7 days.

[0045] Figure 6B These are HE staining photos of the small intestine treated with UW solution and stored for 7 days.

[0046] Figure 7 The damage to liver cells was compared after being treated with the universal tissue and organ preservation solution of the present invention and UW solution and preserved for 72 hours and 7 days.

[0047] Figure 8 The damage to kidney cells was compared after being treated with the universal tissue and organ preservation solution of the present invention and UW solution and preserved for 72 hours and 7 days.

[0048] Fig. 9 The damage of small intestinal cells treated with the universal tissue and organ preservation solution of the present invention and UW solution and preserved for 72 hours and 7 days was compared.

[0049] Fig. 10A The photographs are of pig abdominal viscera that have been thawed after being cryopreserved for 6 months in the universal tissue and organ preservation solution of the present invention.

[0050] Fig. 10B The photograph is of pig chest organs thorax viscera thawed after being cryopreserved for 6 months in the universal tissue and organ preservation solution of the present invention. DETAILED DESCRIPTION

[0051] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the raw materials used in the present invention are all known products on the market and can be purchased online or offline. The following embodiments are only to make the technical advantages of the present invention clearer, but the application of the present invention is not limited to the following embodiments.

[0052] Those skilled in the art will appreciate that increasing or decreasing the content / concentration of various components of the universal tissue and organ preservation solution of the present invention in equal proportions, and adjusting the working concentration back to the proportion of the present invention by means of dilution, reduced pressure concentration, etc. during use, is essentially a conventional alternative form of the present invention in this field and still falls within the scope of the present invention.

[0053] The aliases of raffinose pentahydrate are: raffinose, raffinose, and raffinose. The aliases of adenosine are: adenosine glycoside, adenosine. The aliases of allopurine are: tongfengping, allopurinol, allopurinol, seroli, tongfengning, allopurine, allopurinol, viluolin, Milurit, Zyioric, Isopurionl, Zyloprim, Adenock, Anzief. The aliases of mannitol are: D-mannitol, D-mannitol, D-mannitol, D-xylemitol. The aliases of fructose-1,6-diphosphate are: 1,6-diphosphate fructose. The above raw materials are all commercially available.

[0054] Fennel and star anise extracts were purchased from Lvyuan Natural Spice Oil Refinery, Qingyuan District, Ji'an City, CAS: 68952-43-2.

[0055] The universal tissue and organ preservation solution of the present invention uses water (such as distilled water) as a solvent and other components as solutes.

[0056] Preparation Example

[0057] The components and contents / concentrations of the universal tissue and organ preservation solution are shown in Table 1.

[0058] Table 1 Components and contents / concentrations of tissue and organ universal preservation solution

[0059]

[0060]

[0061] According to the prescription in Table 1, add each component to an appropriate amount of distilled water, mix evenly at room temperature, then add distilled water to make up to 1L, sterilize with ultraviolet light using a Huilangpu ultraviolet ozone disinfection cabinet, and then store at 4°C. The pH value of the tissue and organ universal preservation solution is 6.8-7.3. Optionally, sodium hydroxide solution can be added to adjust the pH value.

[0062] Experimental Example 1

[0063] Preparation before the experiment: (1) Preparation of the universal preservation solution for tissues and organs of the present invention: The universal preservation solution for tissues and organs of the present invention prepared in the above preparation example is stored at 4°C for 1-2 hours. (2) Preparation of instruments: 1 bag of physiological saline (appropriate amount), 1 bag of UW treatment solution (appropriate amount), 1 small sterile operating table, 1 set of instrument consumables (dissecting forceps, scissors, hemostatic forceps, sutures), 1 sterile three-way pipe, and 2 sterile self-sealing bags.

[0064] Establish the perfusion model: Select 5-8 week old mice, weighing about 15-25g. Do not eat or drink for 12 hours before the operation. Use an animal anesthesia machine to anesthetize with isoflurane. After the mouse is anesthetized and stable, fix it on a small sterile operating table, prepare the skin on the mouse abdomen, and disinfect the abdomen again. After disinfection, separate the abdominal skin along the midline of the abdomen, and the incision is about 3cm. Take the aorta from the descending aorta below the iliac artery and remove a full set of abdominal organs (liver, gallbladder, stomach, spleen, intestines, kidneys, uterus, bladder, ureter, etc.) to ensure the integrity of the relevant organs and blood vessels.

[0065] Organ harvesting, perfusion and preservation. Details are as follows:

[0066] Treatment 1 of the present invention: 30 minutes before surgery, 1 mL of 100 U / mL heparin saline was injected into the abdomen of the mouse. After the animal was anesthetized with an anesthetic, the mouse was fixed on a small operating table, the skin of the mouse abdomen was prepared, and the abdomen was disinfected again. After disinfection, the abdominal skin was separated along the midline of the abdomen, and the incision was about 3 cm. The aorta below the descending aorta and above the iliac artery was taken, and a full set of abdominal organs (liver, gallbladder, stomach, spleen, kidney, uterus, bladder, ureter, etc.) were removed to ensure the integrity of the relevant organs and blood vessels. The descending aorta was closed, a tube was placed upward from the abdominal aorta, and the universal preservation solution of the tissues and organs of the present invention was connected. The flow rate was about 12 ml per minute, the pressure was about 7.8-9.8 kPa, about 80-100 cm of water column, until the organ turned pale and the liquid at the venous drainage outlet was clear, indicating that the lavage was sufficient. After the lavage was completed, it was stored in the universal preservation solution of the tissues and organs of the present invention at 4°C, and sealed in a sterile self-sealing bag for 72 hours.

[0067] Control treatment 1: except that UV liquid was used to replace the universal tissue and organ preservation liquid of the present invention, the same operation as treatment 1 of the present invention was used for treatment.

[0068] Treatment 2 of the present invention: 1 mL of 100 U / mL heparin saline was injected into the abdomen of the mouse 30 minutes before surgery. After anesthesia was stabilized by an animal anesthetic, the mouse was fixed on a small operating table, the skin of the mouse abdomen was prepared, and the abdomen was disinfected again. After disinfection, the abdominal skin was separated along the midline of the abdomen, and the incision was about 3 cm. The aorta below the descending aorta and above the iliac artery was taken, and a full set of abdominal organs (liver, gallbladder, stomach, spleen, kidney, uterus, bladder, ureter, etc.) were removed to ensure the integrity of the relevant organs and blood vessels. The descending aorta was closed, a tube was placed upward from the abdominal aorta, and the universal preservation solution of the tissues and organs of the present invention was connected. The flow rate was about 12 ml per minute, the pressure was about 7.8-9.8 kPa, about 80-100 cm of water column, until the organ turned pale and the liquid at the venous drainage outlet was clear, indicating that the lavage was sufficient. After the lavage was completed, it was stored in the universal preservation solution of the tissues and organs of the present invention at 4°C and sealed in a sterile self-sealing bag for 7 days.

[0069] Control treatment 2: except that UV liquid was used to replace the universal tissue and organ preservation liquid of the present invention, the same operation as treatment 2 of the present invention was used for treatment.

[0070] (1) After the mouse organs were preserved in the preservation solution of the present invention treatment 1 and the control treatment 1 for 72 hours, the tissue sections of the liver, kidney and small intestine of the two groups of mice were taken, fixed in 4% paraformaldehyde, and used for paraffin sectioning and pathological observation.

[0071] (2) After the mouse organs were stored in the preservation solution of the present invention treatment 2 and control treatment 2 for 7 days, the tissue sections of the liver, kidney and small intestine of the two groups of mice were taken, fixed in 4% paraformaldehyde, and used for paraffin sectioning and pathological observation.

[0072] HE staining of mouse organ tissue: (1) After the mouse organ tissue was fixed with formalin, it was dehydrated in a dehydrator and embedded in an embedding machine to prepare wax blocks; (2) Sectioning: The thickness of each section was 4 μm; (3) Baking: The cut wax slices were placed in 42°C warm water, and the slices were placed on a glass slide; and the slices were baked at 70°C for 1 hour; (4) Dewaxing: Dewaxing with xylene 1 and xylene 2 for 15 minutes each; (5) Hydration: Dewaxing with 100% ethanol 1 and 100% ethanol 2 for 5 minutes each, and then hydrating with 95%, 85%, and 75% ethanol for 5 minutes in sequence. n, and finally washed with distilled water for 5 minutes; (6) Hematoxylin staining: After staining for 8 minutes, rinse with pure water; (7) Differentiation: Differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with pure water for 20 minutes; (8) Eosin staining: Stain for 10 minutes; (9) Dehydration: First dehydrate with 80%, 90% and 95% ethanol for 5 minutes respectively, then dehydrate with 100% ethanol 1 and 100% ethanol 2 for 5 minutes respectively, and finally dehydrate with xylene 1 and xylene 2 for 5 minutes each; (10) Sealing: After sealing with neutral gum, dry it for 15 minutes and observe and take pictures under an optical microscope.

[0073] The pathologist randomly selected 5 fields of view at 400× magnification for single-blind analysis of the pathological results. The pathological manifestations under the light microscope were: cell vacuolation, cell necrosis, and shedding. Tissue inflammatory cell infiltration.

[0074] According to the tissue damage score: no damage = 0 points; damage <5% = 1 point; damage 5%-25% = 2 points; damage 25%-55% = 3 points; damage 50%-75% = 4 points; damage >75% = 5 points.

[0075] Statistical processing method: The scores of each visual field were accumulated and the average was calculated as the final score of the sample. Statistical processing method Experimental data were expressed as mean ± standard deviation, and the data were analyzed using GraphpadPrism 9 statistical software. The differences between the groups were analyzed using t-test, and P < 0.05 indicated that the differences were statistically significant.

[0076] result:

[0077] Pathological staining of tissue sections showed that, compared with the UW preservation solution, the degree of tissue damage of mouse organs was alleviated after being preserved in the universal tissue and organ preservation solution of the present invention for 72 hours.

[0078] Pathological staining of tissue sections showed that, compared with the UW preservation solution, the degree of tissue damage of mouse organs was alleviated after being preserved in the universal tissue and organ preservation solution of the present invention for 7 days.

[0079] Figure 1A This is a HE staining photograph of a liver treated with the universal tissue and organ preservation solution of the present invention and preserved for 72 hours. Figure 1BThese are HE staining photos of livers treated with UW solution and stored for 72 hours. Figure 2A This is a HE staining photograph of a liver treated with the universal tissue and organ preservation solution of the present invention and preserved for 7 days. Figure 2B These are HE staining photos of livers treated with UW solution and stored for 7 days. Figure 3A The picture is HE staining of kidney treated with the universal tissue and organ preservation solution of the present invention and preserved for 72 hours. Figure 3B These are HE staining photos of kidneys treated with UW solution and stored for 72 hours. Figure 4A The picture is HE staining of kidney treated with the universal tissue and organ preservation solution of the present invention and preserved for 7 days. Figure 4B These are HE staining photos of kidneys treated with UW solution and stored for 7 days. Figure 5A This is a HE staining photograph of the small intestine treated with the universal tissue and organ preservation solution of the present invention and preserved for 72 hours. Figure 5B These are HE staining photos of the small intestine treated with UW solution and stored for 72 hours. Fig. 6A This is a HE staining photograph of the small intestine treated with the universal tissue and organ preservation solution of the present invention and preserved for 7 days. Figure 6B These are HE staining photos of the small intestine treated with UW solution and stored for 7 days. Figure 7 The damage to liver cells was compared after being treated with the universal tissue and organ preservation solution of the present invention and UW solution and preserved for 72 hours and 7 days. Figure 8 The damage to kidney cells was compared after being treated with the universal tissue and organ preservation solution of the present invention and UW solution and preserved for 72 hours and 7 days. Fig. 9 The damage of small intestinal cells treated with the universal tissue and organ preservation solution of the present invention and UW solution and preserved for 72 hours and 7 days was compared.

[0080] The above experimental results show that the universal tissue and organ preservation solution of the present invention has a protective effect on the preservation of mouse organs in vitro, causes less damage to cells, and has a longer preservation time than UW solution and a lower cost than UW solution.

[0081] Experimental Example 2

[0082] This experimental example aims to explore the preservation effect of the tissue and organ universal preservation solution of the present invention in a long-term frozen state. The ideal effect is that after thawing, the tissue and organ show the same physiological elasticity, color, and smell as normal tissue and organ. Due to the long experimental time, in order to facilitate observation, the experiment uses pig abdominal organs.

[0083] 30 minutes before the operation, an appropriate amount of heparinized saline was injected into the pig's abdomen. After the animal anesthetic was used for anesthesia and the anesthesia was stable, the pig was placed on the operating table, the abdominal skin was prepared, and the abdomen was disinfected again. After disinfection, the abdominal skin was separated along the midline of the abdomen. A full set of organs (heart, lungs, liver, gallbladder, stomach, spleen, kidneys, uterus, bladder, ureters, etc.) were taken to ensure the integrity of the relevant organs and blood vessels. The descending aorta was closed, a catheter was placed upward from the abdominal aorta, and the universal preservation solution for tissues and organs of the present invention was connected. The universal preservation solution for tissues and organs of the present invention was perfused until the organ turned pale and the liquid at the venous drainage outlet was clear, indicating that the lavage was sufficient. After the lavage is completed, it was placed in the universal preservation solution for tissues and organs of the present invention at 4°C, and sealed in a sterile self-sealing bag and frozen at -20°C for 6 months. After thawing, the tissues and organs showed the same physiological elasticity, color, and odor as normal tissues and organs. See Fig. 10A , 10B . Fig. 10A The photographs are of pig abdominal viscera that have been thawed after being cryopreserved for 6 months in the universal tissue and organ preservation solution of the present invention. Fig. 10B The photograph is of pig chest organs thorax viscera thawed after being cryopreserved for 6 months in the universal tissue and organ preservation solution of the present invention.

[0084] In summary, the universal tissue and organ preservation solution of the present invention can preserve the overall physiological activity of tissues and organs for a long time in a -20°C frozen state and a 0-10°C non-frozen state. That is, frozen storage at -20°C for 6 months, during which the temperature is restored, and the physiological elasticity, color, and smell are consistent with those of normal tissues and organs. Non-frozen storage at 4°C for 7 days; during the storage period, the physiological elasticity, color, and smell are maintained consistent with those of normal tissues and organs.

[0085] Based on the above description of the invention, those skilled in the art can fully apply the present invention, and the same principles and similar modifications used should be considered to be within the scope of the present invention.

Claims

1. A universal preservation solution for tissues and organs, characterized in that: Each liter contains the following components and their contents:

2. The universal tissue and organ preservation solution according to claim 1, characterized in that: Each liter contains the following components and their contents:

3. The universal tissue and organ preservation solution according to claim 1 or 2, characterized in that: The antibacterial components are: fennel star anise extract and penicillin; Preferably, the content of the fennel and star anise extract is 40-70 g, and the content of penicillin is 20-60 U per liter.

4. The universal tissue and organ preservation solution according to claim 3, characterized in that: Each liter contains the following components and their contents:

5. The universal tissue and organ preservation solution according to claim 4, characterized in that: Each liter contains the following components and their contents:

6. The universal tissue and organ preservation solution according to any one of claims 1 to 5, characterized in that: The universal tissue and organ preservation solution preserves tissues and organs in a -20°C frozen state or a 0-10°C non-frozen state.

7. The universal tissue and organ preservation solution according to claim 6, characterized in that: The universal tissue and organ preservation solution preserves tissues and organs in a frozen state at -20°C for at least 6 months; or The universal tissue and organ preservation solution can preserve tissues and organs in a non-frozen state at 10° C. for at least 10 hours; and can preserve tissues and organs in a non-frozen state at 4° C. for at least 72 hours, preferably at least 7 days; or The pH value of the universal tissue and organ preservation solution is 6-8, preferably 6.5-7.5, and more preferably 6.8-7.

3.

8. A method for preserving tissues and organs, characterized in that: The method comprises the following steps: after obtaining tissue organs, completely replacing the blood in the tissue organs with physiological saline containing heparin, and then perfusing the tissue organs with the universal tissue organ preservation solution according to the circulation pathway; after the perfusion is completed, completely immersing the tissue organs in the universal tissue organ preservation solution and storing them in a refrigerator.

9. The tissue and organ preservation method according to claim 8, characterized in that: The storage is in a -20°C frozen state or a 0-10°C non-frozen state.

10. The tissue and organ preservation method according to claim 8, characterized in that: The tissue organ is selected from one or more of the heart, liver, brain, lung, kidney, pancreas, and intestine; preferably, the tissue organ is selected from one or more of the liver, kidney, and small intestine.

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