Application of harpagoside in preparation of low-temperature preserving fluid for biological materials

Abstract

The invention discloses an application of harpagoside in preparation of a low-temperature preservation solution of a biological material. On the first aspect, the invention provides application of harpagoside, a stereoisomer thereof or pharmaceutically acceptable ester thereof in preparation of a low-temperature preserving fluid for biological materials. The harpagoside component can effectively improve the preservation effect of the biological material at low temperature and prolong the preservation time.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and in particular to the application of harpagoside in the preparation of cryopreservation solutions for biological materials. Background Art

[0002] Among the many current treatment methods for end-stage organ failure, cancer, etc., organ transplantation is one of the most effective methods. However, the organ preservation time limit is an important issue restricting the development of organ transplantation. During the process of organ transplantation, the preservation of donor organs is an important factor affecting the success or failure of organ transplantation and the postoperative survival and quality of life of patients. Therefore, how to improve organ preservation technology has always been a research hotspot in this field.

[0003] Traditional organ preservation techniques are mainly static cryopreservation, that is, replacing the blood in the donor organ with an organ preservation solution and then quickly storing it in a low-temperature state, generally 4°C. Currently, the most widely used and effective organ preservation solution is UW solution. At the same time, in response to problems such as possible cell damage caused by it, HTK solution, IGL-1 solution, etc. have also emerged one after another. However, when these preservation solutions are applied to organ preservation, the effective cold preservation time limit is short, and the reperfusion injury after transplantation is also relatively serious. Therefore, it is necessary to improve these organ preservation solutions to better protect the activity of the graft, extend the preservation time limit, thereby improving the effect of organ transplantation and reducing complications. Summary of the Invention

[0004] This application aims to at least solve one of the technical problems existing in the prior art. For this reason, this application proposes an application of harpagoside in the preparation of cryopreservation solutions for biological materials.

[0005] In the first aspect of this application, there is provided an application of harpagoside, its stereoisomers or its pharmaceutically acceptable esters in the preparation of cryopreservation solutions for biological materials.

[0006] The application according to the embodiments of this application has at least the following beneficial effects: Harpagoside components can effectively improve the preservation effect of biological materials at low temperature and extend the preservation time.

[0007] Among them, the structural formula of harpagoside is as follows:

[0008] The CAS number is 19210-12-9.

[0009] Among them, stereoisomers refer to isomers with the same molecular formula and structure, but different arrangements of atoms in space.

[0010] In some embodiments of the present application, stereoisomers include configurational isomers and conformational isomers, and configurational isomers can be further divided into cis-trans isomers, enantiomers and diastereomers.

[0011] Among them, pharmaceutically acceptable esters can be, for example, esters that produce harpagoside or its stereoisomers by hydrolysis, and can be, for example, esters formed by at least one hydroxyl group on a six-membered heterocycle or a five-membered ring.

[0012] In some embodiments of the present application, pharmaceutically acceptable esters include at least one of inorganic esters and organic esters. Inorganic esters can be, for example, phosphate esters, and organic esters are, for example, saturated or unsaturated fatty acid esters, etc.

[0013] In some embodiments of the present application, the biological material is any one of cells, tissues, organs, and organoids.

[0014] Among them, an organoid refers to a cell aggregate developed in vitro from stem cells (such as adult stem cells, induced pluripotent stem cells) or organ progenitor cells (such as organ progenitor cells of adult small intestine progenitor cells, lung progenitor cells, etc.), which has a spatial and tissue structure similar to that in vivo. Organoids can be single organoids containing only one type of organ cell, or composite organoids with multiple types of organ cells or multiple organs.

[0015] In some embodiments of the present application, the biological material is at least one of cells, tissues, organs, and organoids of the liver, kidney, pancreas, small intestine, heart, lung, brain, limbs, etc. For example, it can be at least one cell derived from the liver, kidney, pancreas, small intestine, heart, lung, brain, limbs, at least one tissue derived from the liver, kidney, pancreas, small intestine, heart, lung, brain, limbs, the liver, kidney, pancreas, small intestine, heart, lung, brain, and at least one organoid of the liver, kidney, pancreas, small intestine, heart, lung, brain, etc.

[0016] In some embodiments of the present application, the biological material is any one of small intestine cells, small intestine tissues, small intestine, and small intestine organoids.

[0017] In some embodiments of the present application, the biological material is any one of kidney cells, kidney tissues, kidney, and kidney organoids. In some embodiments of the present application, the biological material is any one of pancreatic cells, islet cells, pancreatic tissues, islet tissues, pancreas, and pancreatic organoids.

[0018] In some embodiments of the present application, the cryopreservation solution includes an ionic buffer and an energy-providing substance.

[0019] In some embodiments of the present application, the ionic buffer in the cryopreservation solution includes at least one of sodium hydroxide, potassium hydroxide, sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, potassium bicarbonate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, etc. When the cryopreservation solution is mixed with biological materials by adding the ionic buffer, the osmotic pressure and environmental pH of the cells therein are maintained stable.

[0020] In some embodiments of the present application, the energy-providing substances in the cryopreservation solution include at least one of sugars, nucleosides, amino acids, ketoglutaric acid, etc. By adding the energy-providing substances to supply energy, the basic physiological metabolic conditions of cells under low-temperature conditions are maintained.

[0021] In some embodiments of the present application, the sugars include at least one of monosaccharides, disaccharides, trisaccharides, polysaccharides, etc., such as arabinose, xylose, ribose, lyxose, glucose, fructose, galactose, mannose, sucrose, raffinose, trehalose, lactose, maltose, dextran, hydroxyethyl starch, water-soluble starch, etc.

[0022] In some embodiments of the present application, the nucleosides include adenosine.

[0023] In some embodiments of the present application, the amino acids include at least one of glutamine, glycine, serine, tyrosine, asparagine, leucine, etc.

[0024] It can be understood that the energy-providing substances can also be derivatives of the above-mentioned sugars, nucleosides, amino acids, etc. that have an energy-providing effect. For example, for derivatives of lactose, they include lactitol, lactobionic acid, lactulose, lactitol, galactooligosaccharides, etc.

[0025] In some embodiments of the present application, the cryopreservation solution further includes at least one of an antioxidant, an osmotic stabilizer, an anticoagulant, and an antibiotic.

[0026] In some embodiments of the present application, the antioxidant includes at least one of reduced glutathione, vitamin B9, vitamin C, vitamin D, water-soluble vitamin E (Trolox), allopurinol, N-acetylcysteine, etc. By adding the antioxidant, free radicals generated during the metabolic process are scavenged, the redox environment is maintained, and apoptosis of cells and ischemia-reperfusion injury during preservation are prevented.

[0027] In some embodiments of the present application, the osmotic stabilizer includes at least one of dextran, hydroxyethyl starch, water-soluble starch, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone. By adding the osmotic stabilizer, problems such as ice crystal damage and cell membrane rupture under cryopreservation conditions are avoided.

[0028] In some embodiments of the present application, the anticoagulant includes at least one of heparin, hirudin, EDTA-2Na, sodium citrate, etc. The addition of the anticoagulant avoids the occurrence of blood clotting, thrombosis and other situations during the preservation process.

[0029] In some embodiments of the present application, the antibiotic includes at least one of penicillin, streptomycin, gentamicin, etc. The addition of the antibiotic avoids the reproduction of bacteria.

[0030] In some embodiments of the present application, the cryopreservation solution includes any one of the intracellular fluid type cryopreservation solution and the extracellular fluid type cryopreservation solution.

[0031] In some embodiments of the present application, the cryopreservation solution includes any one of UW solution, IGL-1 solution, IGL-2 solution, Gala solution, HTK solution, Custodiol-N solution, ST solution, KPS solution, Celsior solution, CRMB solution, Polysol solution, VSL solution, Marshall solution, HCA solution, HCA-II solution, Leeds solution, Somah solution, Perfadex solution, ET-Kyoto solution. For example, the composition of UW solution includes potassium hydroxide, potassium dihydrogen phosphate, magnesium sulfate, lactic acid, raffinose, adenosine, allopurinol, reduced glutathione, hydroxyethyl starch, etc.; the composition of Gala solution includes D-glucose, CaCl2, KCl, KH2PO4, MgCl2, MgSO4, NaCl, NaHCO3, Na2HPO4, vitamin C, reduced glutathione, L-arginine, heparin, etc.; the composition of HTK solution includes NaCl, KCl, CaCl2, MgCl2, mannitol, tryptophan, histidine, pentanedioic acid, etc.; the composition of Custodiol-N solution includes NaCl, KCl, CaCl2, MgCl2, sucrose, tryptophan, histidine, N-acetylhistidine, aspartic acid, L-arginine, glycine, L-alanine, pentanedioic acid, deferoxamine, LK614, etc.; the composition of KPS solution includes potassium hydroxide, potassium dihydrogen phosphate, magnesium sulfate, glucose, mannitol, ribose, adenosine, allopurinol, reduced glutathione, hydroxyethyl starch, etc.; the composition of Celsior solution includes calcium chloride, potassium chloride, magnesium chloride, sodium hydroxide, mannitol, lactic acid, glutamic acid, histidine, reduced glutathione, etc.; the composition of CRMB solution includes potassium dihydrogen phosphate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium hydroxide, glutamine, adenosine, L-arginine, mannitol, D-raffinose, lactitol, allopurinol, reduced glutathione, etc.; the composition of Somah solution includes potassium dihydrogen phosphate, potassium chloride, sodium chloride, sodium bicarbonate, calcium chloride, disodium hydrogen phosphate, magnesium chloride, magnesium sulfate, D-glucose, reduced glutathione, vitamin C, L-arginine, L-citrulline malate, adenosine, creatine orotate, creatine, L-carnosine, L-carnitine, dichloroacetate, insulin, etc.; the composition of Perfadex solution includes sodium chloride, potassium chloride, magnesium sulfate, disodium hydrogen phosphate, potassium phosphate, glucose, dextran 40, etc. It can be understood that the above different cryopreservation solutions have not completely consistent preservation effects on different organ tissues. For example, the general-purpose UW solution and HTK solution, which have good preservation effects in organs or related tissues and cells such as liver, kidney, pancreas, small intestine, heart, etc., the IGL-1 solution applicable to organs or related tissues and cells such as liver, kidney, small intestine, etc., the Celsior solution applicable to organs or related tissues and cells such as liver, pancreas, heart, etc.

[0032] Among them, low temperature generally refers to a storage temperature below 20 °C, such as a low temperature condition of 0 to 12 °C. It should be noted that according to different biological materials and different storage purposes, it can also be a temperature condition below 0 °C, such as an ultra-low temperature condition of -6 to -4 °C, or a lower temperature condition such as -80 °C or -196 °C. It can be understood that at different storage temperatures, the storage time corresponding to different biological materials to basically maintain their physiological functions or to be able to basically maintain their physiological functions after resuscitation also changes accordingly.

[0033] In some embodiments of the present application, the cryopreservation solution is used to maintain the activity, tight junctions, and cell stemness of cells at low temperature.

[0034] It should be noted that some biological materials need to be pre-washed with a perfusion solution to replace extracellular fluids such as blood before storage in order to be stored, or the physiological activity of the biological material is restored by perfusion with the perfusion solution after storage. And in some cases, the cryopreservation solution and the perfusion solution used before and after storage can have the same or different component ratios. Therefore, the cryopreservation solution in the embodiments of the present application also includes the use as a perfusion solution.

[0035] In a second aspect of the present application, a cryopreservation solution for biological materials is provided, and the cryopreservation solution further contains harpagoside, its stereoisomers, or pharmaceutically acceptable esters thereof.

[0036] In some embodiments of the present application, the cryopreservation solution includes an ionic buffer, an energy-providing substance, and harpagoside, its stereoisomers, or pharmaceutically acceptable esters thereof.

[0037] In some embodiments of the present application, stereoisomers include configurational isomers and conformational isomers, and configurational isomers can be further divided into cis-trans isomers, enantiomers, and diastereomers.

[0038] In some embodiments of the present application, pharmaceutically acceptable esters include at least one of inorganic esters and organic esters. Inorganic esters can be, for example, phosphate esters, and organic esters are, for example, saturated or unsaturated fatty acid esters, etc.

[0039] In some embodiments of the present application, the ionic buffer in the cryopreservation solution includes at least one of sodium hydroxide, potassium hydroxide, sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, potassium bicarbonate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, etc. By adding the ionic buffer, when the cryopreservation solution is mixed with biological materials, the osmotic pressure and environmental pH of the cells therein are maintained stable.

[0040] In some embodiments of the present application, the energy-providing substances in the cryopreservation solution include at least one of sugars, nucleosides, amino acids, ketoglutaric acid, etc. By adding the energy-providing substances to supply energy and maintain the basic physiological metabolic conditions of cells under low-temperature conditions.

[0041] In some embodiments of the present application, the sugars include at least one of monosaccharides, disaccharides, trisaccharides, polysaccharides, etc., such as arabinose, xylose, ribose, lyxose, glucose, fructose, galactose, mannose, sucrose, raffinose, trehalose, lactose, maltose, dextran, hydroxyethyl starch, water-soluble starch, etc.

[0042] In some embodiments of the present application, the nucleosides include adenosine.

[0043] In some embodiments of the present application, the amino acids include at least one of glutamine, glycine, serine, tyrosine, asparagine, leucine, etc.

[0044] It can be understood that the energy-providing substances can also be derivatives of the above-mentioned sugars, nucleosides, amino acids, etc. that have an energy-providing effect. For example, derivatives of lactose include lactitol, lactobionic acid, lactulose, lactosucrose, galactooligosaccharides, etc.

[0045] In some embodiments of the present application, the cryopreservation solution further includes at least one of an antioxidant, an osmotic stabilizer, an anticoagulant, and an antibiotic.

[0046] In some embodiments of the present application, the antioxidant includes at least one of reduced glutathione, vitamin B9, vitamin C, vitamin D, water-soluble vitamin E (Trolox), allopurinol, N-acetylcysteine, etc. By adding the antioxidant, free radicals generated during the metabolic process are scavenged, the redox environment is maintained, and apoptosis and ischemia-reperfusion injury of cells during preservation are prevented.

[0047] In some embodiments of the present application, the osmotic stabilizer includes at least one of dextran, hydroxyethyl starch, water-soluble starch, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone. By adding the osmotic stabilizer, problems such as ice crystal damage and cell membrane rupture under cryopreservation conditions are avoided.

[0048] In some embodiments of the present application, the anticoagulant includes at least one of heparin, hirudin, EDTA-2Na, sodium citrate, etc. By adding the anticoagulant, situations such as blood coagulation and thrombosis during preservation are avoided.

[0049] In some embodiments of the present application, the antibiotic includes at least one of penicillin, streptomycin, gentamicin, etc. By adding the antibiotic, bacterial reproduction is avoided.

[0050] In some embodiments of the present application, the cryopreservation solution includes any one of intracellular fluid-type cryopreservation solutions and extracellular fluid-type cryopreservation solutions.

[0051] In some embodiments of the present application, the cryopreservation solution includes any one of UW solution, IGL-1 solution, IGL-2 solution, Gala solution, HTK solution, Custodiol-N solution, ST solution, KPS solution, Celsior solution, CRMB solution, Polysol solution, VSL solution, Marshall solution, HCA solution, HCA-II solution, Leeds solution, Somah solution, Perfadex solution, ET-Kyoto solution. For example, the composition of UW solution includes potassium hydroxide, potassium dihydrogen phosphate, magnesium sulfate, lactic acid, raffinose, adenosine, allopurinol, reduced glutathione, hydroxyethyl starch, etc.; the composition of Gala solution includes D-glucose, CaCl2, KCl, KH2PO4, MgCl2, MgSO4, NaCl, NaHCO3, Na2HPO4, vitamin C, reduced glutathione, L-arginine, heparin, etc.; the composition of HTK solution includes NaCl, KCl, CaCl2, MgCl2, mannitol, tryptophan, histidine, pentanedioic acid, etc.; the composition of Custodiol-N solution includes NaCl, KCl, CaCl2, MgCl2, sucrose, tryptophan, histidine, N-acetylhistidine, aspartic acid, L-arginine, glycine, L-alanine, pentanedioic acid, deferoxamine, LK614, etc.; the composition of KPS solution includes potassium hydroxide, potassium dihydrogen phosphate, magnesium sulfate, glucose, mannitol, ribose, adenosine, allopurinol, reduced glutathione, hydroxyethyl starch, etc.; the composition of Celsior solution includes calcium chloride, potassium chloride, magnesium chloride, sodium hydroxide, mannitol, lactic acid, glutamic acid, histidine, reduced glutathione, etc.; the composition of CRMB solution includes potassium dihydrogen phosphate, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium hydroxide, glutamine, adenosine, L-arginine, mannitol, D-raffinose, lactitol, allopurinol, reduced glutathione, etc.; the composition of Somah solution includes potassium dihydrogen phosphate, potassium chloride, sodium chloride, sodium hydrogen carbonate, calcium chloride, disodium hydrogen phosphate, magnesium chloride, magnesium sulfate, D-glucose, reduced glutathione, vitamin C, L-arginine, L-citrulline malate, adenosine, orotic acid creatine, creatine, L-carnosine, L-carnitine, dichloroacetate, insulin, etc.; the composition of Perfadex solution includes sodium chloride, potassium chloride, magnesium sulfate, disodium hydrogen phosphate, potassium phosphate, glucose, dextran 40, etc.

[0052] In some embodiments of the present application, the concentration of harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof in the cryopreservation solution is 1 μM to 1 mM, and for example, it can be 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM.

[0053] In a third aspect of the present application, a method for treating a biological material is provided, and the treatment method includes mixing harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof, or a cryopreservation solution containing harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof with the biological material.

[0054] In some embodiments of the present application, stereoisomers include configurational isomers and conformational isomers, and configurational isomers can be further divided into cis-trans isomers, enantiomers and diastereoisomers.

[0055] In some embodiments of the present application, pharmaceutically acceptable esters include at least one of inorganic esters and organic esters. Inorganic esters can be, for example, phosphate esters, and organic esters are, for example, saturated or unsaturated fatty acid esters, etc.

[0056] In some embodiments of the present application, the biological material is any one of cells, tissues, organs, and organoids.

[0057] In some embodiments of the present application, the biological material is at least one of cells, tissues, organs, and organoids of the liver, kidney, pancreas, small intestine, heart, lung, brain, limbs, etc. For example, it can be at least one cell derived from the liver, kidney, pancreas, small intestine, heart, lung, brain, limbs, at least one tissue derived from the liver, kidney, pancreas, small intestine, heart, lung, brain, limbs, the liver, kidney, pancreas, small intestine, heart, lung, brain, and at least one organoid of the liver, kidney, pancreas, small intestine, heart, lung, brain, etc.

[0058] In some embodiments of the present application, the biological material is any one of small intestine cells, small intestine tissues, small intestine, and small intestine organoids.

[0059] In some embodiments of the present application, the biological material is any one of kidney cells, kidney tissues, kidney, and kidney organoids.

[0060] In some embodiments of the present application, the biological material is any one of pancreatic cells, islet cells, pancreatic tissues, islet tissues, pancreas, and pancreatic organoids.

[0061] In some embodiments of the present application, the treatment method includes at least one of washing, preservation, and resuscitation.

[0062] In some embodiments of the present application, washing includes washing the biological material with a solution containing harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof.

[0063] In some embodiments of the present application, the washing solution is a cryopreservation solution.

[0064] In some embodiments of the present application, the washing method is lavage.

[0065] In some embodiments of the present application, preservation includes mixing harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof, or a cryopreservation solution containing harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof with the biological material, and then cryopreserving.

[0066] In some embodiments of the present application, the cryopreservation solution is mixed with the biological material by perfusion.

[0067] In some embodiments of the present application, cryopreservation is performed at a temperature below 20 °C.

[0068] In some embodiments of the present application, cryopreservation is performed at 0 - 12 °C.

[0069] In some embodiments of the present application, cryopreservation is performed at -6 - -4 °C.

[0070] In some embodiments of the present application, the cryopreservation time is more than 4 hours, such as 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 hours.

[0071] In some embodiments of the present application, recovery includes mixing the cryopreserved biological material with harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof, or a cryopreservation solution containing harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof.

[0072] In some embodiments of the present application, recovery includes perfusing the cryopreserved biological material with a cryopreservation solution.

[0073] In some embodiments of the present application, the mixing temperature for recovery is 3 °C or higher, such as 3, 6, 9, 12, 15, 18, 21 °C.

[0074] It can be understood that in some of these embodiments, the above methods for treating biological materials do not include methods for treating diseases corresponding to the biological materials, such as methods for treating diseases related to the liver, kidney, pancreas, small intestine, heart, lungs, brain, and limbs.

[0075] In the embodiments of the present application, harpagoside components are added to the cryopreservation solution, so as to prolong the cryopreservation time limit of biological materials such as small intestine grafts, relieve the urgency of organ transplantation, reduce organ waste, and enable more patients to obtain transplantation opportunities. The added harpagoside protects the cellular mucosal barrier including small intestine cells by maintaining tight junctions between cells, prevents bacterial translocation, and is beneficial to reducing postoperative infections, inflammations, and rejection reactions. Moreover, the protective effect of harpagoside on the cellular mucosal barrier also protects the stem cells therein, is beneficial to preserving the activity of the graft, promoting post-transplant regeneration, and improving the postoperative survival rate and long-term survival rate of the graft.

[0076] In addition, at low temperatures, cells are in a state of low metabolism and energy deficiency, which affects the absorption and metabolism of substances in the preservation solution by biological materials. However, harpagoside has the characteristic of acting at low temperatures, making it particularly suitable for the cryopreservation of biological materials, especially organs. At the same time, harpagoside plays a protective role on many cells such as small intestine cells at low temperatures, and at the physiological temperature of 37 °C, it does not affect the activity and proliferation of small intestine cells, and has high safety.

[0077] Additional aspects and advantages of the present application will be given in part in the following description, will become apparent in part from the following description, or will be understood through the practice of the present application. Description of the Drawings

[0078] Figure 1 Shows the body temperature changes of Syrian hamsters before pre-torpor and after entering hibernation.

[0079] Figure 2 Is a schematic diagram of small intestine sampling of hamsters in the active state and the hibernation state.

[0080] Figure 3 Shows the H&E staining results of small intestine tissues of mice (Mus), hamsters in the active state (Ham), and hamsters in the hibernation state (Torpor Ham) after perfusion with UW solution and cryopreservation at 4 °C for 0 - 48 h. The scale bar is 200 μm.

[0081] Figure 4 Is the single-cell nuclear transcriptome atlas of the small intestine of hamsters in the active state and the hibernation state. Among them, A is the integrated atlas analyzed by UMAP, B is the independent atlases of 2 hamsters in the active state (Active-1, 2) and 2 hamsters in the hibernation state (Torpid-1, 2); C is the Dotplot diagram of the specific expression of marker genes of each cell population.

[0082] Figure 5Results of Calcian-AM / PI cell viability staining of small intestinal organoids of hamsters and mice after storage at 4°C for 0 - 48 h. Among them, green represents live cells, red represents dead cells, and the scale bar is 200 μm.

[0083] Figure 6 Results of storage of small intestinal organoids of hamsters and mice at 4°C for 24 h, resuscitation at 37°C for 72 h, and passage. The scale bar is 200 μm.

[0084] Figure 7 In [Figure 0], A is a schematic diagram of the process of hibernation simulation of small intestinal organoids, and B is a photo of cell states at different time points. The scale bar is 200 μm.

[0085] Figure 8 Results of screening genes related to hibernation protection of hamster small intestinal cells. Among them, A shows the expression of different genes in each cell population of hamster small intestine in the active state and hibernation state. B shows the expression of different genes in small intestinal organoids of mice and hamsters during the low temperature - rewarming process. Ace2 marked with an asterisk is a candidate functional target related to hibernation protection of hamster small intestinal cells.

[0086] Figure 9 Results of Calcian-AM / PI cell viability staining to detect the protective effects of 4 Ace2 agonists: harpagoside (Har), methazolamide (Met), imatinib (Ima), and diminazene (DIZE) on mouse small intestinal organoids treated at low temperature for 12 h and 24 h. DMSO treatment at 37°C was used as a positive control, and DMSO treatment at 4°C was used as a negative control. Among them, green represents live cells, red represents dead cells, and the scale bar is 200 μm.

[0087] Figure 10 Results of Calcian-AM / PI cell viability staining to detect the protective effects of 20 μM and 100 μM harpagoside (Har-20, Har-100) on mouse small intestinal organoids treated at low temperature for 24 h. DMSO treatment at 37°C was used as a positive control, and DMSO treatment at 4°C and blank treatment were used as negative controls. Among them, green represents live cells, red represents dead cells, and the scale bar is 200 μm.

[0088] Figure 11 Results of detection of the resuscitation and growth ability of small intestinal cells after treating mouse small intestinal organoids with DMSO, 20 μM and 100 μM harpagoside (Har-20, Har-100), subjecting them to low temperature treatment at 4°C for 56 h (4°C - 56 h), and then rewarming and culturing for 4 days and 7 days (37°C - 4 days / 7 days). The scale bar is 200 μm.

[0089] Figure 12H&E staining results of the small intestine tissues of mice in the HBSS, UW, and UW+Har groups after perfusion and cryopreservation. The scale bar is 100 μm.

[0090] Figure 13 Ace2 immunofluorescence staining results of the small intestine tissues of mice in the UW and UW+Har groups after perfusion and cryopreservation.

[0091] Figure 14 GO analysis results of the transcriptome data of mouse intestinal organoids after 24-hour hypothermic treatment with and without harpagoside.

[0092] Figure 15 In A, the expression of genes related to cell tight junctions in mouse intestinal organoids during the hibernation simulation process at 37°C normal culture state (M5_37), 4°C treatment for 6 hours (M5_4_6h), 4°C treatment for 12 hours (M5_4_12h), 4°C treatment for 24 hours (M5_4_24h), and 4°C treatment for 48 hours (M5_4_48h) is shown. In B, the expression of genes related to cell tight junctions in the blank group (M5_IO_37) of mouse intestinal organoids and after 24-hour hypothermic treatment with (M5_Har_100) and without (M5_DMSO) harpagoside is shown.

[0093] Figure 16 In A, the expression of genes related to cell stemness in mouse intestinal organoids during the hibernation simulation process at 37°C normal culture state (M5_37), 4°C treatment for 6 hours (M5_4_6h), 4°C treatment for 12 hours (M5_4_12h), 4°C treatment for 24 hours (M5_4_24h), and 4°C treatment for 48 hours (M5_4_48h) is shown. In B, the expression of genes related to cell stemness in the blank group (M5_IO_37) of mouse intestinal organoids and after 24-hour hypothermic treatment with (M5_Har_100) and without (M5_DMSO) harpagoside is shown.

[0094] Figure 17 H&E staining results of the pancreatic tissues of mice in the UW and UW+Har groups after perfusion and hypothermia-rewarming preservation. The scale bar is 200 μm, and the arrow indicates the damaged structure.

[0095] Figure 18 Ace2 immunofluorescence staining results of the pancreatic tissues of mice in the UW and UW+Har groups after perfusion and hypothermia-rewarming preservation.

[0096] Figure 19 H&E staining results of the kidney tissues of mice in the UW and UW+Har groups after perfusion and hypothermia-rewarming preservation. The scale bar is 100 μm.

[0097] Figure 20These are the results of immunofluorescence staining of β-Actin and Ace2 in the kidney tissues of mice in the UW and UW+Har groups after perfusion and hypothermic-rewarming preservation. Detailed implementation manners

[0098] The following will clearly and completely describe the concept and technical effects generated by this application in combination with the embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only part of the embodiments of this application, rather than all embodiments. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative efforts all fall within the scope of protection of this application.

[0099] The embodiments of this application are described in detail below. The described embodiments are exemplary and are only used to explain this application and should not be construed as a limitation of this application.

[0100] In the description of this application, the meaning of several is more than one, and the meaning of multiple is more than two. Understandings such as greater than, less than, exceeding, etc. do not include the present number, and understandings such as above, below, within, etc. include the present number. If there is a description of first and second, it is only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features or implicitly indicating the sequence relationship of the indicated technical features.

[0101] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art belonging to the technical field of this application. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0102] In the description of this application, descriptions with reference to terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples" or "some examples" mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0103] Example 1: Verification of the low-temperature tolerance of the small intestine of hibernating hamsters and mice

[0104] Subcutaneously implant an iButton temperature recorder into an active Syrian hamster, raise it in a climate chamber, and use a low temperature of 5°C and a dark environment to induce hibernation in the Syrian hamster. Refer to Figure 1 When its body temperature drops to ~4°C, it enters a stable hibernation cycle to obtain hibernating hamsters.

[0105] Reference Figure 2 Small intestine tissues of mice, active hamsters, and hibernating hamsters were taken respectively to compare their low-temperature tolerance. The small intestine tissues were perfused with UW solution and cryopreserved at 4 °C. After different preservation times, the tissue morphology and degree of damage were analyzed based on H&E staining of paraffin sections of the small intestine tissues.

[0106] The results are as Figure 3 shown. After 48 hours of cryopreservation, the small intestine of mice was the most severely damaged, with the villi completely falling off. The small intestine of active hamsters also had obvious damage, while the small intestine structure of hibernating hamsters was intact without obvious damage. The above results indicate that the small intestine of hibernating hamsters has stronger low-temperature adaptation and tolerance.

[0107] Example 2: Single-nucleus transcriptome atlas of the small intestine of hibernating hamsters

[0108] Small intestine tissues were taken from two active hamsters and two hibernating hamsters respectively for single-nucleus transcriptome sequencing to obtain the single-nucleus transcriptome atlas of the small intestine of active and hibernating hamsters. The results are as Figure 4 shown. Through clustering analysis and marker gene expression, 12 major small intestine cell populations were identified, including endothelial cells, smooth muscle cells, fibroblasts, immune cells, enteroendocrine cells, tuft cells, goblet cells, Paneth cells, intestinal epithelial cells, secretory progenitor cells, absorptive progenitor cells, and stem progenitor cells, providing a data basis for exploring the mechanism of small intestine protection during hibernation.

[0109] Example 3: Low-temperature and rewarming tolerance of small intestine organoids of hamsters and mice

[0110] Small intestine organoids of hamsters and mice were constructed respectively, and the specific steps are as follows:

[0111] (1) Small intestine tissues of hamsters and mice were taken respectively and placed in a petri dish containing pre-cooled PBS. The small intestine was cut along the intestine and the digestive contents inside were washed. The mucus and villi on the intestinal wall were gently scraped off with a sterilized coverslip, the tissue was cut into small pieces of 1 - 2 mm, and washed 4 - 6 times with cold PBS until the supernatant was clear.

[0112] (2) The small intestine tissue of hamsters was added with 10 mL of pre-cooled 10 mM EDTA (prepared with PBS), placed on a shaker together with an ice box, and digested at 80 rpm for 40 min. The difference in the digestion conditions for the small intestine tissue of mice was that it was digested with 2.5 mM EDTA for 20 min.

[0113] (3) The digestion solution was discarded, cold DMEM / F12 was added, and the crypts were detached by repeatedly pipetting with a 5 mL pipette. It was passed through a 100 μm cell sieve, centrifuged at 450 g for 5 min, and the supernatant was discarded.

[0114] (4) Resuspend the cells in Matrigel on ice and seed them into a 4-well plate at 50 μL per well. Invert the plate and incubate at 37 °C for 15 min until the Matrigel solidifies. Then add pre-warmed hamster and mouse intestinal organoid media and 10 μM Y27632 respectively, and culture in an incubator at 37 °C and 5% CO2. Change the medium every 3 - 4 days. Once the cells are stably passaged, they can be used for experiments.

[0115] The cultured hamster and mouse intestinal organoids were stored at 4 °C for 0 - 48 hours respectively, and then rewarmed at 37 °C for 72 h, and the cell viability was compared. The results are as Figure 5 and Figure 6 . The intestinal cells of hamsters can tolerate low temperature for a longer time. There is no obvious cell death after 24 hours of low temperature, and more dead cells appear after 48 hours of low temperature. Moreover, after rewarming culture after 24 hours of low temperature treatment, the cells can quickly recover growth and passage. While mouse intestinal cells are more sensitive to low temperature. Large-scale cell death occurs after 12 hours of low temperature, and all cells die after 24 hours of low temperature, and they cannot recover growth after rewarming. The above results prove that hamster intestinal organoids have stronger low temperature-rewarming tolerance than mouse intestinal organoids.

[0116] Example 4: Screening genes related to intestinal low temperature protection

[0117] 1. Simulation of low temperature-rewarming hibernation of intestinal organoids

[0118] Refer to Figure 7 , and let the intestinal organoids experience low temperature (4 °C)-rewarming (37 °C) to simulate the hibernation process. First, culture hamster and mouse intestinal organoids normally at 37 °C, then transfer the organoids to 4 °C for low temperature treatment for 48 hours, and then move the organoids back to 37 °C for rewarming culture for 48 hours. Seven time points were selected during the low temperature-rewarming process for Bulk RNAseq to analyze the transcriptome dynamic changes of hibernation simulation, including: normal culture state at 37 °C, 4 °C treatment for 6 hours, 12 hours, 24 hours and 48 hours, and 37 °C rewarming for 6 hours and 48 hours.

[0119] 2. Screening of candidate targets related to hamster intestinal low temperature protection

[0120] Using the small intestine scRNA-seq results of hamsters in the active and hibernating states in Example 2 as in vivo hibernation data, and the Bulk RNA-seq results of the hibernation simulation process of small intestine organoids of hamsters and mice as in vitro hibernation data, the two types of data were integrated. Through gene differential expression analysis, genes that met the following expression patterns were screened out as candidate research targets related to small intestine cryoprotection: 1. In the small intestine of hamsters, compared with the active state, the expression was upregulated during hibernation; 2. In small intestine organoids, compared with mice, hamsters had high expression at most time points; 3. In small intestine organoids, compared with 37 °C, hamsters maintained or upregulated expression during the low-temperature process, while mice maintained low expression or downregulated expression during the low-temperature process.

[0121] The results were as Figure 8 shown. Ace2 was one of the targets with the most conforming expression patterns and was selected as the functional target for subsequent research.

[0122] Example 5: Cryoprotective effects of different Ace2 agonists

[0123] Four reported agonists that activate Ace2 were selected: Harpagoside, Methazolamide, Imatinib, and Diminazene aceturate.

[0124] The mouse small intestine organoids cultured in Example 3 were respectively set with a positive control group (37 °C DMSO), a negative control group (4 °C DMSO), and experimental groups (4 °C 100 μM Har / Met / Ima / DIZE, with DMSO as the solvent) and treated for 12 hours and 24 hours. Calcian-AM / PI staining was used to detect cell viability.

[0125] The results were as Figure 9 shown. Compared with the negative control group, the low temperature for 12 hours had no significant effect on the cell viability of each experimental group. However, after 24 hours of low temperature, only the small intestine organoids treated with Harpagoside maintained cell viability comparable to that during normal culture at 37 °C, and the other three agonists had no obvious protective effect.

[0126] To further verify that Harpagoside has a cryoprotective effect, the low-temperature cell viability detection was repeated using the cultured mouse small intestine organoids. Treatment with 37 °C DMSO was used as the positive control, treatment with 4 °C DMSO and blank were used as the negative controls, and treatment with 4 °C DMSO + 20 / 100 μM Harpagoside was used as the experimental group. The three repeated experiments were 100 μM, 100 μM, and 20 μM respectively. The results were as Figure 10As shown, harpagoside at 20 μM can achieve a good cryoprotective effect and enable the intestinal organoids to maintain their three-dimensional structure even after 24 hours of low temperature, while the intestinal organoids in the negative control group became loose in structure and showed cell shedding after low temperature.

[0127] To detect the ability of small intestinal cells to resume growth after rewarming, mouse intestinal organoids were treated with DMSO, 20 μM, and 100 μM harpagoside, followed by low temperature at 4°C for 56 hours and then rewarming and culturing for 4 days and 7 days. The results are as Figure 11 shown. It can be seen from the figure that after rewarming 56 hours after low temperature, the small intestinal cells treated with 20 μM and 100 μM harpagoside can rapidly resume growth and grow in large numbers. In the control group treated with DMSO, there was no obvious cell growth after 4 days of rewarming, and only a small amount of cell growth appeared until 7 days later.

[0128] All of the above results prove that the agonist of Ace2, harpagoside, has a cryoprotective effect on intestinal organoids.

[0129] Example 6: Verification of the cryoprotective effect of harpagoside in intestinal tissue

[0130] Harpagoside with a final concentration of 100 μM was added to UW solution (SPS-1, Organ Recovery systems), and then intestinal vascular perfusion and cryopreservation at 4°C were performed on mouse intestinal tissue, with UW solution without harpagoside and cell balanced salt solution HBSS as controls. After cryopreservation at 4°C for 0 - 72 hours, the degree of injury was compared by H&E staining, and the expression level of Ace2 was identified by immunofluorescence staining.

[0131] The results are shown in Figure 12 and Figure 13 . The results of H&E staining showed that HBSS had the worst protective effect, with severe edema in the intestinal tissue occurring after 12 hours of low temperature and complete necrosis after 24 hours of low temperature; the intestinal tissue in UW solution was also severely damaged after 48 hours of low temperature, with complete shedding of villi; while UW solution supplemented with harpagoside could reduce the injury of intestinal tissue at low temperature and maintain the villus structure for up to 72 hours. The results of immunofluorescence staining showed that Ace2 was concentrated in the brush border of the intestinal villi. In UW solution, the expression was upregulated after 24 hours of low temperature, but significantly downregulated when the low temperature time was extended to 48 hours, accompanied by the shedding of villi. After preservation with harpagoside added, the expression of Ace2 was upregulated with the extension of low temperature time and protected the villus structure for 72 hours.

[0132] The above results prove that harpagoside can improve the cryopreservation effect of intestinal tissue, extend the preservation time, and its protective effect may be related to the function of Ace2.

[0133] Example 7: Verification of the cryoprotective mechanism of harpagoside in small intestinal organoids

[0134] Refer to the previous examples to treat mouse small intestinal organoids with harpagoside (using DMSO as a solvent), and perform transcriptome sequencing after 24 hours of low temperature. Normal culture at 37°C and treatment with DMSO at low temperature for 24 hours were used as controls. In addition, transcriptome data of continuous low temperature treatment (normal culture at 37°C, low temperature at 4°C for 6, 12, 24, and 48 hours) of mouse small intestinal organoids were selected from the hibernation simulation in Example 4, and these two groups of data were analyzed and compared.

[0135] Results reference Figures 14 to 16 , GO analysis showed that at low temperature for 24 hours, the addition of harpagoside significantly upregulated the Wnt signaling pathway in small intestinal cells. Gene differential expression analysis showed that genes related to cell tight junctions were downregulated with the prolongation of low temperature time, while harpagoside could maintain the expression of related genes in small intestinal cells after 24 hours of low temperature, which was equivalent to the expression level during normal culture at 37°C. This also explained the phenomenon of loose structure and cell shedding in mouse small intestinal organoids after low temperature in Example 5, while harpagoside could protect the three-dimensional structure of organoids by protecting cell-cell junctions. In addition, genes related to small intestinal cell stemness also showed a similar expression pattern, that is, they were downregulated with the prolongation of low temperature time, especially the small intestinal stem cell marker genes Lgr5 and Olfm4, which were significantly downregulated after 6 hours of low temperature. And harpagoside could maintain the expression of stemness genes in small intestinal cells after 24 hours of low temperature, which was equivalent to the expression level during normal culture at 37°C.

[0136] The above results prove that harpagoside protects the structure of small intestinal organoids and promotes cell survival by maintaining cell tight junctions and cell stemness at low temperature.

[0137] Example 8: Verification of the cryoprotective effect of harpagoside in the pancreas and kidney

[0138] Refer to Example 6, add 100 μM of harpagoside to UW solution, and then perform vascular perfusion and cryopreservation on the pancreas and kidneys of mice, using UW solution without harpagoside as a control. After cryopreservation at 4°C for 24 hours, transfer to 37°C for rewarming treatment for 1 hour, compare the degree of injury by H&E staining, and identify the expression of Ace2 and β-Actin by immunofluorescence staining.

[0139] Pancreatic results reference Figure 17 and Figure 18, the H&E staining results showed that after 24 hours of hypothermia and 1 hour of rewarming, the pancreatic tissues in UW solution were severely damaged, with acinar cell shrinkage, loose arrangement of duct cells, and islet rupture. However, UW solution supplemented with harpagoside could keep the pancreatic tissues structurally intact and cell connections tight. The immunofluorescence staining results showed that Ace2 was mainly expressed in islets, ducts, and capillaries, and its expression in islets was upregulated after preservation with harpagoside.

[0140] Kidney results reference Figure 19 and Figure 20 , the H&E staining results showed that the treatment of 24 hours of hypothermia and 1 hour of rewarming had no significant effect on the kidney structure. The immunofluorescence staining results showed that compared with 0 hour, the distribution of β-Actin was abnormal after preservation in UW solution, transferring to the edges of the renal tubule and glomerular structures, indicating the destruction of the cytoskeleton. However, after preservation with harpagoside, β-Actin was evenly distributed in the cells, comparable to that at 0 hour, indicating that harpagoside could maintain the cytoskeleton. The expression of Ace2 was concentrated on the surface of renal tubular cells, and there was no significant change in its expression after preservation.

[0141] Example 9:

[0142] Referring to Examples 6 and 8, the cryoprotective effects of HTK solution, IGL-1 solution, and Celsior solution supplemented with harpagoside were verified in small intestine tissues, pancreatic tissues, and kidneys. The results of small intestine tissues showed that the small intestine tissues in these cryopreservation solutions were severely damaged after hypothermia treatment, with complete villus shedding; while after adding harpagoside, the damage was reduced at low temperature and the retention time of the villus structure was prolonged. The results of pancreatic tissues showed that the pancreatic tissues in these cryopreservation solutions were severely damaged after hypothermia and rewarming treatments, with acinar cell shrinkage, loose arrangement of duct cells, and islet rupture; while after adding harpagoside, the pancreatic tissues could still maintain structural integrity and cell connections tight after hypothermia and rewarming treatments. The results of kidneys showed that the cytoskeletons of kidneys in these cryopreservation solutions were damaged after hypothermia and rewarming treatments; while after adding harpagoside, the kidneys could still maintain the cytoskeleton after hypothermia and rewarming treatments.

[0143] The above results prove that harpagoside has cryoprotective effects on various different organs, tissues, or related cells including the small intestine, pancreas, and kidneys.

[0144] By exploring the protective mechanism of the small intestine against cold ischemia during the hibernation of Syrian hamsters, this application identified the functional gene Ace2, and the activation of its function is related to the cryoprotection of small intestinal cells. Moreover, harpagoside, an effective agonist of Ace2, can play a role in protecting the mucosal barrier and cell stemness of the small intestine when added to the preservation solution as a safe drug, thereby improving the activity of small intestinal grafts and extending the preservation time. There are no reports on the cryoprotective mechanism of Ace2 and the cryoprotective application of harpagoside to cells and organs. Therefore, this application uses the effective drug harpagoside in the cold preservation of various organs, organoids, tissues, and cells.

[0145] The above has described this application in detail in conjunction with the embodiments. However, this application is not limited to the above embodiments, and various changes can be made without departing from the gist of this application within the scope of knowledge possessed by those of ordinary skill in the art. In addition, the embodiments of this application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof in preparing cryopreservation solutions for biological materials.

2. The application according to claim 1, wherein The biological material is any one of cells, tissues, organs, and organoids.

3. The application according to claim 2, characterized in that The biological material is at least one of cells, tissues, organs, and organoids of the liver, kidney, pancreas, small intestine, heart, lung, brain, and extremities; Optionally, the biological material is any one of small intestine cells, small intestine tissues, small intestine, and small intestine organoids; Optionally, the biological material is any one of kidney cells, kidney tissues, kidney, and kidney organoids; Optionally, the biological material is any one of pancreatic cells, islet cells, pancreatic tissues, islet tissues, pancreas, and pancreatic organoids.

4. The application according to claim 1, wherein The cryopreservation solution includes an ionic buffer and an energy source; Optionally, the cryopreservation solution further includes at least one of an antioxidant, an osmotic stabilizer, an anticoagulant, and an antibiotic.

5. The application according to claim 1, wherein The cryopreservation solution includes an intracellular fluid-type cryopreservation solution or an extracellular fluid-type cryopreservation solution; Optionally, the cryopreservation solution includes any one of UW solution, IGL-1 solution, IGL-2 solution, HBSS solution, Gala solution, HTK solution, Custodiol-N solution, ST solution, KPS solution, Celsior solution, CRMB solution, Polysol solution, VSL solution, Marshall solution, HCA solution, HCA-II solution, Leeds solution, Somah solution, Perfadex solution, ET-Kyoto solution.

6. The application according to claim 1, wherein the cryopreservation solution is used to maintain the activity, tight junctions, and cell stemness of cells at low temperatures.

7. Cryopreservation solution for biological materials, characterized in that, The cryopreservation solution contains harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof.

8. The cryopreservation solution according to claim 7, wherein, The concentration of harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof in the cryopreservation solution is 1 μM to 1 mM.

9. A method for treating a biological material, characterized in that, It includes mixing harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof, or a cryopreservation solution containing harpagoside, its stereoisomers or pharmaceutically acceptable esters thereof with the biological material.

10. The processing method according to claim 9, characterized in that, The treatment method is at least one of washing, preservation, and resuscitation.

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