Cryopreservation liquid of umbilical cord tissue, cryopreservation method and method for extracting umbilical cord mesenchymal stem cells

Abstract

The invention provides an umbilical cord tissue cryopreservation solution, a cryopreservation method and a method for extracting umbilical cord mesenchymal stem cells. The freezing medium for the umbilical cord tissue comprises a basic permeable protective agent with the concentration of 5% v / v to 20% v / v, a non-permeable protective agent with the concentration of 5% v / v to 30% v / v, an antioxidant with the concentration of 0.1% v / v to 2% v / v, an ice control molecular material with the concentration of 0.001% v / v to 5% v / v, a buffer system with the concentration of 10 mM to 100 mM, an energy substance or antioxidant with the concentration of 0.1 mM to 10 mM, an ion carrier with the concentration of 0.1 mM to 5 mM and a membrane stabilizer. The cryopreservation solution for the umbilical cord tissue can ensure that cells in the umbilical cord tissue keep a survival state at low temperature, the preservation time of the cryopreservation solution is prolonged to several months and even one year, the preservation time of the umbilical cord is effectively prolonged, and the cryopreservation solution is far superior to an existing cryopreservation scheme.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to cryopreservation solutions, cryopreservation methods, and methods for extracting umbilical cord mesenchymal stem cells. Background Technology

[0002] The umbilical cord is the connecting structure between the fetus and the placenta. It is rope-shaped, smooth and transparent, containing connective tissue, one umbilical vein, and two umbilical arteries. Between the epidermis and blood vessels lies a gelatinous substance called Wharton's jelly, which is rich in mesenchymal stem cells, collagen fibers, hyaluronidase, and other components. Umbilical cord mesenchymal stem cells (MSCs) are pluripotent stem cells found in the umbilical cord tissue of newborns. They possess high differentiation potential and can differentiate in multiple directions. They have broad clinical application prospects in tissue engineering for bone, cartilage, muscle, nerve, and myocardium.

[0003] The general method for preserving umbilical cords is to store them at 4°C, but this method has a short preservation time. The longest preservation time currently is 48 hours. Extending the preservation time will affect the survival of umbilical cord tissue cells, making it impossible to extract umbilical cord mesenchymal stem cells or resulting in poor growth of the extracted umbilical cord mesenchymal stem cells. Summary of the Invention

[0004] The main objective of this invention is to provide a cryopreservation solution, cryopreservation method, and method for extracting umbilical cord mesenchymal stem cells to solve the technical problem of short preservation time in existing preservation methods.

[0005] To achieve the above objectives, the present invention provides a cryopreservation solution for umbilical cord tissue, comprising a 5% v / v to 20% v / v basic permeability protectant, a 5% v / v to 30% v / v non-permeability protectant, a 0.1% v / v to 2% v / v antioxidant, a 0.001% v / v to 5% v / v ice-controlling molecular material, a 10mM to 100mM buffer system, a 0.1mM to 10mM energy substance or antioxidant, and a 0.1mM to 5mM ion carrier and membrane stabilizer.

[0006] The basic permeability protectant is dimethyl sulfoxide, glycerol, ethylene glycol, or propylene glycol.

[0007] The non-permeable protective agent is sucrose, trehalose, glucose, or mucopolysaccharide.

[0008] The antioxidant is glutathione, ascorbic acid and its salts, lipoic acid, histidine, N-acetylcysteine ​​or a metal chelating agent.

[0009] The ice-controlling molecule is polyvinyl alcohol, polyvinylpyrrolidone, amino acids, polyamino acids, or nonionic surfactants.

[0010] According to the embodiments of this application, at least one of the following conditions is met: (1) The basic permeability protectant is 5% v / v~10% v / v dimethyl sulfoxide, 6% v / v~19% v / v glycerol, 5% v / v~15% v / v ethylene glycol or 9% v / v~14% v / v propylene glycol.

[0011] (2) The non-permeable protective agent is 6.8% v / v~28% v / v sucrose, 9% v / v~30% v / v trehalose, 5% v / v~26% v / v glucose or 8% v / v~29% v / v mucopolysaccharide.

[0012] (3) The antioxidant is 0.1% w / v~2% w / v glutathione, 0.3% w / v~1.8% w / v ascorbic acid and its salts, 0.2% w / v~2% w / v lipoic acid, 0.1% w / v~1.3% w / v histidine, 0.1% w / v~2% w / v N-acetylcysteine ​​or 0.5% w / v~2.3% w / v metal chelating agent.

[0013] (4) The ice-controlling molecule is 0.001% w / v~1% w / v polyvinyl alcohol, 0.01% w / v~2% w / v polyvinylpyrrolidone, 0.001% w / v~4% w / v amino acid, 0.001% w / v~3.9% w / v polyamino acid or 0.001% w / v~5% w / v nonionic surfactant.

[0014] This application also provides a method for cryopreserving umbilical cord tissue, including the following steps: After pretreatment, the umbilical cord is cut into tissue blocks.

[0015] The tissue block and the above-mentioned cryopreservation solution were mixed evenly and then frozen at -80°C.

[0016] According to an embodiment of this application, the step of freezing the tissue mixture obtained by mixing the tissue block and the cryopreservation solution at -80°C includes: The tissue mixture was directly frozen at -80°C.

[0017] According to an embodiment of this application, the tissue block and the cryopreservation solution are mixed at a volume ratio of 1:4 to 1:10.

[0018] According to an embodiment of this application, the pretreatment step of the umbilical cord includes: The umbilical cord was soaked in 75% ethanol for 30-60 seconds and then removed.

[0019] The middle portion of the umbilical cord is cleaned in antibacterial saline to remove blood and impurities, and the umbilical vein and artery are removed. The antibacterial saline is a saline solution containing 1%-6% penicillin-streptomycin and 0.1%-0.2% gentamicin.

[0020] According to an embodiment of this application, the side length of each side of the tissue block is ≤0.5cm.

[0021] The step of mixing the tissue block and the cryopreservation solution evenly includes: The tissue block was centrifuged in antibacterial saline, the supernatant was removed, and a portion of cryopreservation solution was added to resuspend the tissue block.

[0022] Add the remaining cryopreservation solution to the resuspended tissue block and mix well.

[0023] This application also provides a method for extracting umbilical cord mesenchymal stem cells, comprising the following steps: The umbilical cord was cryopreserved using the methods described above.

[0024] The frozen tissue blocks were then revived.

[0025] After reviving, remove the cryopreservation solution from the tissue blocks and add them to a pre-prepared complete culture medium containing 1%-3% penicillin-streptomycin and 0.1%-0.2% gentamicin for culture.

[0026] According to an embodiment of this application, the step of thawing the frozen tissue block includes: The frozen tissue blocks were thawed at 37°C.

[0027] According to an embodiment of this application, the step of removing the cryopreservation solution from the thawed tissue block and culturing it includes: After the tissue blocks were revived, the cryopreservation solution was removed, and the tissues were washed with physiological saline and dried.

[0028] After adding culture medium and culturing continuously for 72 hours, replace the culture medium every two days.

[0029] The aforementioned cryopreservation solution for umbilical cord tissue can maintain the survival of cells in the umbilical cord tissue at low temperatures, extending its preservation time to more than two weeks, several months, or even a year. This effectively improves the preservation time of umbilical cord tissue and is far superior to existing cryopreservation methods. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0031] Figure 1 This is a growth diagram of P2 generation umbilical cord cells from the fresh group not frozen in this application.

[0032] Figure 2 This application describes the growth of P2 generation cells after umbilical cord resuscitation using the tissue cryopreservation solution from Example 1.

[0033] Figure 3 This is a growth diagram of P0 generation cells after umbilical cord resuscitation in the cryopreservation group using the cryopreservation solution of Comparative Example 2.

[0034] Figure 4 This is a P0 generation cell growth diagram of the umbilical cord after thawing from the tissue cryopreservation group using the cryopreservation solution of Example 1 in this application.

[0035] Figure 5 These are the cell growth curves of umbilical cord tissue from the fresh group and the frozen group in this application.

[0036] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] It should be noted that all directional indicators (such as up, down, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indicator will also change accordingly.

[0039] Furthermore, in this invention, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature.

[0040] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0041] This invention provides a cryopreservation solution for umbilical cord tissue, comprising a 5% v / v to 20% v / v basic permeability protectant, a 5% v / v to 30% v / v non-permeability protectant, a 0.1% v / v to 2% v / v antioxidant, a 0.001% v / v to 5% v / v ice-controlling molecular material, a 10mM to 100mM buffer system, a 0.1mM to 10mM energy substance or antioxidant, and a 0.1mM to 5mM ion carrier and membrane stabilizer. The basic permeability protectant is dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, or propylene glycol. The non-permeability protectant is sucrose, trehalose, glucose, or a mucopolysaccharide. The antioxidant is glutathione, ascorbic acid and its salts, lipoic acid, histidine, N-acetylcysteine, or a metal chelating agent. The ice-controlling molecules are polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), amino acids, polyamino acids, or nonionic surfactants.

[0042] Basic permeability protectants are mostly small molecules that can freely penetrate cell membranes. During the cooling process, they can increase the concentration of intracellular solutes, lower the cell freezing point, and inhibit the formation of intracellular ice crystals, thereby reducing mechanical damage to cells caused by intracellular ice crystal formation. At the same time, they can balance osmotic pressure and prevent cells from excessively dehydrating and shrinking.

[0043] Non-permeable cryoprotectants are polysaccharides or macromolecules that cannot penetrate the cell membrane and remain extracellularly. They create a hypertonic environment outside the cell, promoting the outward permeation of intracellular water molecules and causing moderate, controlled dehydration of the cell before freezing. The reduced intracellular free water content decreases the probability of ice crystal formation. High concentrations of sugars increase solution viscosity, promoting the formation of amorphous "vitrified" solids rather than regular ice crystals at deep cryogenic temperatures, thus reducing physical damage. They also maintain membrane stability; for example, trehalose is believed to stabilize the phospholipid bilayer on the cell membrane surface through hydrogen bonds, preventing membrane fusion and rupture.

[0044] Antioxidants can mitigate severe oxidative damage to proteins, lipids, and DNA caused by reactive oxygen species bursts due to cellular metabolic disorders and hypothermia during cell cryopreservation and thawing. At the same time, these oxidants construct a comprehensive oxidative defense system by directly neutralizing free radicals, decomposing peroxides, or chelating metal ions involved in oxidation reactions, thus protecting the cellular structure and co-energy integrity.

[0045] Antioxidants include glutathione, ascorbic acid and its salts, lipoic acid, histidine, N-acetylcysteine, or metal chelators. For example, metal chelators include ethylenediaminetetraacetic acid, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or ethylene glycol diethyl ether diaminetetraacetic acid.

[0046] Ice-controlling molecules adsorb onto the nucleus or surface of ice crystals through interactions such as hydrogen bonds, physically preventing further growth of ice crystals. They can also inhibit the highly destructive recrystallization process of ice crystals during rewarming and interact with cell membranes to stabilize cell membranes at the ice-water interface, preventing mechanical tearing of the membrane by ice crystals.

[0047] The ice-controlling molecules are polyvinyl alcohol, polyvinylpyrrolidone, amino acids, polyamino acids, or nonionic surfactants. For example, a nonionic surfactant is Pluronic acid. TM F-68, Pluronic TM F-127, polyether F-68.

[0048] Buffer systems provide cells with a stable physiological pH environment, preventing cell poisoning or metabolic disorders caused by drastic pH fluctuations. For example, a buffer system is a buffer solution that maintains a pH of 7.2–7.4. Exemplary buffer systems include organic buffer solutions, bicarbonate / CO2 buffer solutions, or phosphate buffer solutions.

[0049] Energy substances can provide cells with readily available energy substrates in the early stages after cell recovery, supporting the rapid restoration of cellular metabolic activity, membrane repair, and ion pump operation. Examples of energy substances include carbohydrates and organic acid salts.

[0050] Ion carriers and membrane stabilizers can help stabilize cell membranes and cytoskeleton structures, maintain ion balance, and prevent cell membrane rupture or permeation under changes in osmotic pressure and low temperatures. Examples of ion carriers and membrane stabilizers include melitoxin, maltodextrin, trehalose, polyethylene glycol, and betaine.

[0051] The above components work synergistically within the specified range to reduce ice crystal damage, stabilize cell membranes, and maintain pH environment, thereby significantly improving the survival rate and functional integrity of tissues after cryopreservation.

[0052] The aforementioned cryopreservation solution for umbilical cord tissue can maintain the survival of cells in the umbilical cord tissue at low temperatures, extending its preservation time to more than two weeks, up to several months or even a year, effectively improving the preservation time of umbilical cord tissue and far surpassing current cryopreservation methods.

[0053] According to embodiments of this application, the cryopreservation solution for the umbilical cord tissue satisfies at least one of the following conditions: (1) The basic permeability protectant is 5% v / v~10% v / v dimethyl sulfoxide, 6% v / v~19% v / v glycerol, 5% v / v~15% v / v ethylene glycol or 9% v / v~14% v / v propylene glycol.

[0054] (2) The non-permeable protective agent is 6.8% v / v~28% v / v sucrose, 9% v / v~30% v / v trehalose, 5% v / v~26% v / v glucose or 8% v / v~29% v / v mucopolysaccharide.

[0055] (3) The antioxidant is 0.1% w / v~2% w / v glutathione, 0.3% w / v~1.8% w / v ascorbic acid and its salts, 0.2% w / v~2% w / v lipoic acid, 0.1% w / v~1.3% w / v histidine, 0.1% w / v~2% w / v N-acetylcysteine ​​or 0.5% w / v~2.3% w / v metal chelating agent.

[0056] (4) The ice-controlling molecule is 0.001% w / v~1% w / v polyvinyl alcohol, 0.01% w / v~2% w / v polyvinylpyrrolidone, 0.001% w / v~4% w / v amino acid, 0.001% w / v~3.9% w / v polyamino acid or 0.001% w / v~5% w / v nonionic surfactant.

[0057] The cryopreservation solution for umbilical cord tissue described above can ensure that the cells in the umbilical cord tissue remain viable at low temperatures, extending the preservation time to several months or even a year, effectively improving the preservation duration of umbilical cord tissue, which is far superior to the existing cryopreservation methods.

[0058] This application also provides a method for cryopreserving umbilical cord tissue, including the following steps: S100: After pre-processing the umbilical cord, cut it into tissue blocks.

[0059] The core of pretreatment is "cleaning and impurity removal," which removes contaminants from the surface and interior of the umbilical cord. Umbilical cord pretreatment typically includes aseptic cleaning and the removal of non-target tissues.

[0060] In aseptic cleaning procedures, the umbilical cord surface is repeatedly rinsed with sterile saline (or saline containing antibiotics such as penicillin and streptomycin to enhance the antibacterial effect) to remove visible impurities such as attached blood, amniotic fluid, and dirt.

[0061] Procedures involving the removal of non-target tissues include, for example, using sterile surgical scissors and forceps to peel off and remove the amnion (the outermost transparent membrane) and any attached residual placental tissue from the surface of the umbilical cord. Another example is cutting the cleaned umbilical cord into 1-3 cm segments and removing the umbilical vein and artery. Yet another example is removing tissue from both ends of the umbilical cord (this part may have experienced local ischemia and necrosis due to ligation).

[0062] The specific procedures for umbilical cord pretreatment are not limited; one or more procedures can be selected as needed. After pretreatment, the umbilical cord is cut into tissue blocks, such as blocks with sides ≤0.5cm. For ease of operation, the cleaned umbilical cord can be cut into 1-3cm segments first, and then into tissue blocks of the required size.

[0063] S200: Mix the tissue block and the above-mentioned cryopreservation solution evenly, and freeze at -80°C.

[0064] In this step, the volume of the cryopreservation solution is larger than the volume of the tissue block. The tissue block and cryopreservation solution are placed into a cryopreservation container, such as a cryovial, and mixed thoroughly by inverting or other methods. This ensures that the tissue block is submerged in the cryopreservation solution and evenly dispersed. Then, it is frozen at -80°C, such as in a -80°C freezing apparatus.

[0065] The method of cryopreservation is not specifically limited. In some embodiments, the step of freezing the tissue mixture obtained by mixing the tissue block and the cryopreservation solution at -80°C includes: The tissue mixture was directly frozen at -80°C.

[0066] This method is a direct freezing cryopreservation method, which involves directly placing the tissue mixture in an environment of -80°C for cryopreservation. It has the advantages of being easy to operate and having a short procedure.

[0067] In other embodiments, the tissue mixture is cryopreserved using a programmed freezing method. In this embodiment, the tissue mixture is cryopreserved in multiple stages, with the freezing temperature gradually decreasing in each stage, and the temperature of the last cryopreservation stage being 80°C.

[0068] In some embodiments, the tissue block and the cryopreservation solution are mixed at a volume ratio of 1:4 to 1:10.

[0069] In some embodiments, the pretreatment step of the umbilical cord includes: The umbilical cord was soaked in 75% ethanol for 30 seconds and then removed.

[0070] The middle portion of the umbilical cord is cleaned in antibacterial saline to remove blood and impurities, and the umbilical vein and artery are removed. The antibacterial saline is a saline solution containing 6% penicillin-streptomycin and 0.2% gentamicin.

[0071] For example, in a sterile biosafety cabinet, the umbilical cord is completely immersed in 75% ethanol for 30 seconds. The cord is then removed from the ethanol and both ends are cut off. The remaining cord (the middle portion) is transferred to a pre-prepared saline solution containing 6% penicillin-streptomycin and 0.2% gentamicin and rinsed 5-10 times to completely remove blood. The cleaned cord is then cut into 1-3 cm segments, and the umbilical vein and artery are removed.

[0072] The umbilical cord is then cut into tissue blocks, in some embodiments, wherein the side length of each tissue block is ≤0.5 cm. In some embodiments, the step of resuspending the tissue blocks and mixing them thoroughly with the cryopreservation solution includes: The tissue block was centrifuged in antibacterial saline, the supernatant was removed, and a portion of cryopreservation solution was added to resuspend the tissue block.

[0073] Add the remaining cryopreservation solution to the resuspended tissue block and mix well.

[0074] For example, the cut tissue blocks are immediately centrifuged in pre-prepared physiological saline containing 6% penicillin-streptomycin and 0.2% gentamicin, the supernatant is removed, and 1 mL of cryopreservation solution is added. The resuspended tissue blocks are transferred to cryovials, and 4 mL of cryopreservation solution is added for every 1 mL of tissue block. The tubes are inverted and mixed thoroughly to ensure complete mixing of the tissue blocks and cryopreservation solution. The tubes are then placed in a freezer at -80°C for freezing. For long-term freezing, the tissues should be transferred to liquid nitrogen for preservation.

[0075] This application also provides a method for extracting umbilical cord mesenchymal stem cells, comprising the following steps: S10: The umbilical cord is cryopreserved using the cryopreservation method described above.

[0076] This step involves freezing the food using the same method as the previous method, and will not be repeated here.

[0077] S20: Resuscitate the frozen tissue blocks.

[0078] In some embodiments, the step of thawing the frozen tissue block includes: The frozen tissue blocks were thawed at 37°C.

[0079] For example, the frozen tissue is quickly placed into a preheated 37°C water bath and shaken rapidly to ensure that the cryopreservation solution thaws as quickly as possible.

[0080] S30: Remove the cryopreservation solution from the revived tissue block and culture it.

[0081] In some embodiments, the step of removing the cryopreservation solution from the revived tissue block and culturing it includes: S31: After removing the cryopreservation solution from the revived tissue block, wash it with physiological saline and dry it.

[0082] For example, after the umbilical cord tissue is revived, the cryopreservation solution is removed, the tissue is cleaned with physiological saline, and then the tissue is transferred to a culture flask. The culture flask is then inverted and placed in an incubator to dry for 30 minutes to 2 hours. Culture medium is then slowly added, and the flask is gently placed in the incubator for culture.

[0083] S32: After adding culture medium and culturing continuously for 72 hours, replace the culture medium every two days.

[0084] For example, change the medium on day 3 of culture, and then change the medium every two days until most of the cells have crawled out of the vicinity of the tissue. Gently tap the tissue to detach it, and replace it with fresh culture medium to culture the cells until the confluence rate reaches 80-90%. Cells were digested with trypsin; under a microscope, the cells were observed to become rounded and detach. Fetal bovine serum was immediately added for further digestion. The cells were then transferred to centrifuge tubes, centrifuged, and the supernatant was discarded. The cell pellet was mixed with culture medium and counted at 8000 cells / cm³. 2 Inoculate back into the culture flask and culture continuously for 72 hours. Repeat this step 6 times to complete the proliferation test, and plot the proliferation curve based on the 6 counts.

[0085] In some specific embodiments, the step of removing the cryopreservation solution from the revived tissue block and culturing it includes: After removing the cryopreservation solution from the revived tissue blocks, wash them with physiological saline, and dry them in a 37°C incubator containing 5% carbon dioxide. After 30-120 minutes, remove them from the incubator and add pre-prepared complete culture medium containing 1%-3% penicillin-streptomycin and 0.1%-0.2% gentamicin. Let them stand in the incubator for 120 hours, then replace the culture medium with fresh medium. Change the medium every three days thereafter until primary mesenchymal stem cells grow, at which point proliferation testing can be performed.

[0086] The technical solution of this application will be described below with reference to specific embodiments and comparative examples.

[0087] 1. Materials Serum-free culture medium, CAS number RP02010, was purchased from Shouning Biotechnology Co., Ltd. PBS CAS number C10010500BT, purchased from Gibco Penicillin-streptomycin CAS number 15140-122, purchased from Gibco 0.9% sodium chloride injection was purchased from Shijiazhuang No. 4 Pharmaceutical Co., Ltd. Trypsin-EDTA, CAS number 25200056, purchased from Gibco Fetal Bovine Serum, CAS number FSD500, purchased from Excell Bio. Gentamicin was purchased from Chenxin Pharmaceutical. Methods for preparing umbilical cord mesenchymal stem cells include the tissue block method and the digestion method.

[0088] The following embodiments and comparative examples include: Preparation before experiment 1. Remove the culture medium from the 2-8℃ refrigerator and allow it to warm to room temperature for at least 60 minutes.

[0089] 2. After cleaning your hands with 75% ethanol spray, wipe the outer surfaces of culture flasks, pipettes, sodium chloride injection solution, and other material packaging with a lint-free cloth containing 75% ethanol. Transfer the wiped materials into the biosafety cabinet. Turn on the UV lamp in the biosafety cabinet and sterilize for at least 30 minutes.

[0090] 3. After cleaning your hands with 75% ethanol spray, wipe the outer surface of the packaging materials such as culture media with a lint-free cloth containing 75% ethanol. Open the biosafety cabinet and transfer the wiped materials into the biosafety cabinet for later use.

[0091] Clean up the area after the experiment 1. After the experiment is completed, pour out the waste liquid, take out and organize the reagents and consumables related to this experiment, clean the biosafety cabinet surface, wipe the biosafety cabinet surface and pipette with a lint-free cloth containing 75% ethanol, and then sterilize the biosafety cabinet with ultraviolet light for no less than 30 minutes.

[0092] Example 1 The cryopreservation solution for umbilical cord tissue consisted of: 9.5% v / v DMSO, 16.3% v / v sucrose, 1.5% v / v glutathione, 0.9% v / v polyvinyl alcohol, a 60 mM buffer system, 7 mM energy source, 4.8 mM ionopeptide and membrane stabilizer, with the balance made up with physiological saline. The energy source was glucose. The ionopeptide and membrane stabilizer was polyethylene glycol.

[0093] The cryopreservation method for umbilical cord tissue in this embodiment includes the following steps: I. Organizational Measures 1. Prepare cryopreservation solution for umbilical cord tissue and prepare physiological saline solution containing 6% penicillin-streptomycin + 0.2% gentamicin; 2. Wash the received umbilical cord three times with physiological saline containing 6% penicillin-streptomycin + 0.2% gentamicin to remove blood and dirt, and cut off both ends of the umbilical cord; 3. Measure and record the umbilical cord length; 4. Cut the umbilical cord into 1-3cm segments and wash it three times again with physiological saline containing 6% penicillin-streptomycin + 0.2% gentamicin; 5. Remove the two veins and one artery from the umbilical cord; II. Tissue cryopreservation 1. Cut the umbilical cord into 0.1-0.5cm pieces. 3 Cut the tissue into small pieces (complete the cutting process within 2 hours), and immediately place the cut tissue pieces into centrifuge tubes containing 6% penicillin-streptomycin + 0.2% gentamicin saline to prevent the tissue pieces from drying out. Centrifuge at 2000 rpm for 5 minutes. 2. Discard the supernatant, add physiological saline containing 6% penicillin-streptomycin + 0.2% gentamicin, and centrifuge at 2000 rpm for 5 min; 3. Discard the supernatant and resuspend the tissue block in a small amount of cryopreservation solution; 4. Add 1 mL of tissue block resuspended in cryopreservation solution to a 5 mL cryopreservation tube, then add 4 mL of cryopreservation solution, and invert to mix thoroughly to ensure the tissue block and cryopreservation solution are fully mixed. 5. Store in a -80℃ freezer for two weeks.

[0094] Example 2 The only difference from Example 1 is that in the tissue cryopreservation step, the tissue is frozen at -80°C for one month. The remaining steps are the same as in Example 1.

[0095] Example 3 The only difference from Example 1 is that in the tissue cryopreservation step, the tissues are directly frozen in a -80°C freezer for one week and then transferred to liquid nitrogen for storage, with a total duration of 3 months. The remaining steps are the same as in Example 1.

[0096] Example 4 The only difference from Example 1 is that in the tissue cryopreservation step, the tissues were directly frozen in a -80°C freezer for one week and then transferred to liquid nitrogen for storage, for a total duration of 6 months. The remaining steps are the same as in Example 1.

[0097] Example 5 The only difference from Example 1 is the cryopreservation solution for the umbilical cord tissue. The remaining steps are the same as in Example 1. The specific composition of the cryopreservation solution for the umbilical cord tissue in this example is as follows: The solution consisted of 9.5% v / v DMSO, 20% v / v sucrose, 1.5% v / v glutathione, 5% v / v polyvinyl alcohol, a 60 mM buffer system, 7 mM energy source or antioxidant, and 4.8 mM ion carrier and membrane stabilizer, with the balance made up with physiological saline. The energy source was glucose. The ion carrier and membrane stabilizer was polyethylene glycol.

[0098] Example 6 The only difference from Example 1 is the cryopreservation solution for the umbilical cord tissue. The remaining steps are the same as in Example 1. The specific composition of the cryopreservation solution for the umbilical cord tissue in this example is as follows: The solution consisted of 18% v / v DMSO, 16.3% v / v sucrose, 0.5% v / v glutathione, 4% v / v polyvinyl alcohol, a 60 mM buffer system, 7 mM energy source or antioxidant, and 4.8 mM ion carrier and membrane stabilizer, with the balance made up with physiological saline. The energy source was glucose. The ion carrier and membrane stabilizer was polyethylene glycol.

[0099] Example 7 The only difference from Example 1 is the cryopreservation solution for the umbilical cord tissue. The remaining steps are the same as in Example 1. The specific composition of the cryopreservation solution for the umbilical cord tissue in this example is as follows: The solution consists of 9.5% v / v DMSO, 6.8% v / v sucrose, 0.1% v / v glutathione, 1% v / v polyvinyl alcohol, a 60 mM buffer system, 7 mM energy source or antioxidant, and 4.8 mM ionopeptide and membrane stabilizer, with the balance made up with physiological saline. The energy source is glucose. The ionopeptide and membrane stabilizer is polyethylene glycol.

[0100] Comparative Example 1 Fresh seed bottle culture Prepare a serum-free culture medium containing 2% penicillin-streptomycin + 0.2% gentamicin; After centrifuging the umbilical cord tissue block twice, place it in a culture flask with Wharton's jelly facing down, invert it in an incubator to dry for 30 min to 2 h, then slowly add serum-free culture medium containing 2% penicillin-streptomycin + 0.2% gentamicin, and let it stand.

[0101] Comparative Example 2 The only difference from Example 1 is that in the tissue cryopreservation step, a conventional cryopreservation solution (90% v / v FBS + 10% v / v DMSO) was used, instead of the tissue cryopreservation solution described in this patent. The remaining steps are the same as in Example 1.

[0102] Example 8 In this embodiment, the effects of fresh group, frozen for 2 weeks, frozen for 1 month, frozen for 3 months and frozen for 6 months on the proliferation of MSCs isolated from umbilical cord to P6 were tested respectively.

[0103] (1) Umbilical cord resuscitation and seed bottle ① Quickly place the umbilical cord tissues frozen in Examples 1-4 into a 37°C water bath and shake them rapidly to ensure the cryopreservation solution thaws as soon as possible; ② Pour the umbilical cord tissue block containing cryopreservation solution into a centrifuge tube, discard the cryopreservation solution, add physiological saline containing 6% penicillin-streptomycin + 0.2% gentamicin, and centrifuge at 2000 rpm for 5 min; ③ Discard the supernatant, add physiological saline containing 6% penicillin-streptomycin + 0.2% gentamicin again, and centrifuge at 2000 rpm for 5 min; ④ Discard the supernatant, place the umbilical cord tissue block in a culture bottle, invert it in an incubator to dry for 30 min to 2 h, and then slowly add serum-free culture medium containing 2% penicillin-streptomycin + 0.2% gentamicin.

[0104] (2) Proliferation culture of umbilical cord mesenchymal stem cells ① After the umbilical cord tissue block is seeded into the culture bottle, it needs to be placed in the incubator for 3 days. Do not move the culture bottle during this period to prevent the tissue block from falling off due to external force. After 3 days, change the medium, aspirate the culture medium in the culture bottle, add fresh culture medium, and then change the medium every two days. During this period, observe whether the tissue block has crawled out of the cells. ② After cells have grown near most of the tissue blocks, count the number of tissue blocks that have grown out of the cells, gently tap the tissue blocks to remove them, discard the culture medium containing the tissue blocks, replace it with fresh culture medium, and culture the cells until the confluence rate is 80-90%; ③ Discard the culture medium and add an appropriate amount of PBS to wash; ④ Add 0.05% trypsin for digestion. Observe under a microscope that the cells gradually become rounder and brighter. When you gently tap the cells, some cells will slip off. Immediately add FBS to stop the digestion. ⑤ Transfer the cell suspension into a centrifuge tube, centrifuge at 1300 rpm for 5 min, discard the supernatant, add culture medium, and count using AOPI; ⑥ Based on the counting results, calculate at 8000 cells / cm 2 After being seeded into culture flasks and multiplied for 72 hours, the proliferation data were detected, and the results were continuously passaged until P6.

[0105] ⑦ Using the aforementioned method, umbilical cord tissue was cryopreserved, and the differences between the fresh group (not cryopreserved) and the cryopreserved group were detected. The following conclusions were drawn: The growth status of P2 generation umbilical cord cells from the unfrozen fresh group is as follows: Figure 1 As shown, the growth status of P2 generation cells in the umbilical cord cryopreservation group after thawing using the tissue cryopreservation solution described in Example 1 is as follows: Figure 2 As shown, the growth status of P0 generation cells in the umbilical cord cryopreservation group after thawing using the cryopreservation solution of Comparative Example 2 is as follows: Figure 3 As shown, the growth status of P0 generation cells in the umbilical cord cryopreservation group after thawing using the tissue cryopreservation solution described in Example 1 is as follows: Figure 4 As shown in the figure, experimental results indicate that umbilical cord tissue cryopreserved using the cryopreservation solution described in this patent achieved a 99% survival rate after thawing and cultured to P2 generation in culture flasks, with excellent cell growth and morphology. In contrast, umbilical cord tissue cryopreserved using commercially available cryopreservation solutions showed sporadic cell migration around the periphery after thawing, and the cells differentiated but could not continue growing. The growth curves of the umbilical cord tissue before and after cryopreservation are shown in the figure. Figure 5 As shown, the growth state of umbilical cord tissue before and after cryopreservation is basically similar.

[0106] Therefore, this invention solves the problem of short preservation time of umbilical cord tissue, which affects the survival of cells in umbilical cord tissue, and provides a cryopreservation solution, cryopreservation method and method for extracting umbilical cord mesenchymal stem cells.

[0107] The above technical solutions of the present invention are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made under the technical concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A cryopreservation solution for umbilical cord tissue, characterized in that, Includes 5% v / v~20% v / v basic permeability protectant, 5% v / v~30% v / v non-permeability protectant, 0.1% v / v~2% v / v antioxidant, 0.001% v / v~5% v / v ice-controlling molecular material, 10mM~100mM buffer system, 0.1mM~10mM energy substance or antioxidant, and 0.1mM~5mM ion carrier and membrane stabilizer; The basic permeability protectant is dimethyl sulfoxide, glycerol, ethylene glycol or propylene glycol; The non-permeable protective agent is sucrose, trehalose, glucose, or mucopolysaccharide; The antioxidant is glutathione, ascorbic acid and its salts, lipoic acid, histidine, N-acetylcysteine ​​or a metal chelating agent; The ice-controlling molecule is polyvinyl alcohol, polyvinylpyrrolidone, amino acids, polyamino acids, or nonionic surfactants.

2. The cryopreservation solution for umbilical cord tissue according to claim 1, characterized in that, At least one of the following conditions must be met: (1) The basic permeability protectant is 5% v / v~10% v / v dimethyl sulfoxide, 6% v / v~19% v / v glycerol, 5% v / v~15% v / v ethylene glycol or 9% v / v~14% v / v propylene glycol; (2) The non-permeable protective agent is 6.8% v / v~28% v / v sucrose, 9% v / v~30% v / v trehalose, 5% v / v~26% v / v glucose or 8% v / v~29% v / v mucopolysaccharide; (3) The antioxidant is 0.1% w / v~2% w / v glutathione, 0.3% w / v~1.8% w / v ascorbic acid and its salts, 0.2% w / v~2% w / v lipoic acid, 0.1% w / v~1.3% w / v histidine, 0.1% w / v~2% w / v N-acetylcysteine ​​or 0.5% w / v~2.3% w / v metal chelating agent; (4) The ice-controlling molecule is 0.001% w / v~1% w / v polyvinyl alcohol, 0.01% w / v~2% w / v polyvinylpyrrolidone, 0.001% w / v~4% w / v amino acid, 0.001% w / v~3.9% w / v polyamino acid or 0.001% w / v~5% w / v nonionic surfactant.

3. A method for cryopreserving umbilical cord tissue, characterized in that, Includes the following steps: After pretreatment, the umbilical cord is cut into tissue blocks; The tissue block is mixed evenly with the cryopreservation solution according to claim 1 or 2, and then cryopreserved at -80°C.

4. The method for cryopreserving umbilical cord tissue according to claim 3, characterized in that, The step of freezing the tissue mixture obtained by mixing the tissue block and the cryopreservation solution at -80°C includes: The tissue mixture was directly frozen at -80°C.

5. The method for cryopreserving umbilical cord tissue according to claim 3, characterized in that, The tissue block and the cryopreservation solution are mixed at a volume ratio of 1:4 to 1:

10.

6. The method for cryopreserving umbilical cord tissue according to claim 3, characterized in that, The steps for pre-treating the umbilical cord include: The umbilical cord was soaked in 75% ethanol for 30-60 seconds and then removed. The middle part of the umbilical cord is cleaned in antibacterial saline to remove blood and dirt, and the umbilical vein and umbilical artery of the umbilical cord are removed; wherein, the antibacterial saline is a saline containing 1%-6% penicillin-streptomycin and 0.1%-0.2% gentamicin.

7. The method for cryopreserving umbilical cord tissue according to claim 6, characterized in that, The side length of each side of the tissue block is ≤0.5cm; The step of mixing the tissue block and the cryopreservation solution evenly includes: The tissue block was centrifuged in antibacterial saline, the supernatant was removed, and a portion of cryopreservation solution was added to resuspend the tissue block. Add the remaining cryopreservation solution to the resuspended tissue block and mix well.

8. A method for extracting umbilical cord mesenchymal stem cells, characterized in that, Includes the following steps: The umbilical cord is cryopreserved using the cryopreservation method described in any one of claims 1 to 7; The frozen tissue blocks were revived; After reviving, remove the cryopreservation solution from the tissue blocks and add them to a pre-prepared complete culture medium containing 1%-3% penicillin-streptomycin and 0.1%-0.2% gentamicin for culture.

9. The method for extracting umbilical cord mesenchymal stem cells according to claim 8, characterized in that, The step of reviving the frozen tissue block includes: The frozen tissue blocks were thawed at 37°C.

10. The method for extracting umbilical cord mesenchymal stem cells according to claim 8 or 9, characterized in that, The step of removing the cryopreservation solution from the revived tissue block and culturing it includes: After the tissue blocks were revived, the cryopreservation solution was removed, and the tissues were washed with physiological saline and dried. After adding culture medium and culturing continuously for 72 hours, replace the culture medium every two days.

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