WO2020259194A1 - 生物样品冷冻保护剂及其应用 - Google Patents

生物样品冷冻保护剂及其应用 Download PDF

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WO2020259194A1
WO2020259194A1 PCT/CN2020/092972 CN2020092972W WO2020259194A1 WO 2020259194 A1 WO2020259194 A1 WO 2020259194A1 CN 2020092972 W CN2020092972 W CN 2020092972W WO 2020259194 A1 WO2020259194 A1 WO 2020259194A1
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cells
biological sample
cryoprotectant
stem cells
cryopreservation
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PCT/CN2020/092972
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English (en)
French (fr)
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李立东
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北京太东生物科技有限公司
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Priority claimed from CN201910602073.0A external-priority patent/CN110250162B/zh
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Publication of WO2020259194A1 publication Critical patent/WO2020259194A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts

Definitions

  • the present disclosure relates to the field of cell preservation solutions, and in particular to a cryoprotectant for biological samples and applications thereof.
  • Cryopreservation is a universal technique used to maintain cell viability and genetic stability for a long time.
  • cryopreservation protocol allows cells to experience various stresses and damages (such as damage caused by ice crystal formation) during the entire cell collection, freezing, and thawing process. These stresses and injuries can cause irreversible damage to cells.
  • cryopreservatives such as DMSO (dimethyl sulfoxide), ethylene glycol, and glycerol have been used for cryopreservation of cells, and cell membrane-impermeable cryopreservatives such as polyvinylpyrrolidone can also be generally used.
  • concentration of these cryopreservatives is large, there is usually a problem of showing toxicity to cells.
  • the cryopreservative since the above-mentioned preservative hinders the proliferation of cells, the cryopreservative must be removed from the cells before culturing, which makes the operation complicated.
  • the inventors conducted a lot of experiments and creative work, and obtained a freezing liquid containing phase change protein or its fragments, and surprisingly found that the freezing liquid can significantly improve the vitality of biological samples after freezing, and reduce The amount of other toxic refrigerants used. This provides the following disclosure:
  • the present disclosure relates to a cryoprotectant for biological samples, the working solution of which contains active fragments ⁇ 5mM;
  • the active fragment is a polypeptide and/or protein capable of phase separation (Phase Separation or Phase Transition) and comprising RNA binding domain (RNA binding domain) and low-complexity sequence domain (LCD) .
  • the biological sample cryoprotectant is an effective cryoprotective reagent for biological samples such as organs, tissues or cells, and the biological samples that have been cryopreserved are still functional.
  • the present disclosure also relates to the application of the above-mentioned cryoprotectant for biological samples in cryopreservation of biological samples.
  • the present disclosure also relates to the cryopreservation composition of the cryoprotectant for biological samples as described above, and the cryopreservation composition further includes a biological sample.
  • the present disclosure also relates to a method for cryopreserving a biological sample, including the steps of contacting the biological sample to be cryopreserved with the biological sample cryoprotectant as described above, thereby obtaining a cryopreservation composition, and then reducing the cryopreservation
  • the temperature of the composition is to the freezing storage temperature.
  • Figure 1 shows the expression result of Rbm46 in human egg cells in an embodiment of the present disclosure
  • Figure 2 shows the Rbm46 precipitation induced by biotin-isox in an embodiment of the present disclosure
  • INPUT represents the lysate of whole cells
  • SUPNATANT represents the supernatant obtained after adding the corresponding concentration of biotin-isox
  • PELLET represents the addition of the corresponding concentration of biotin -Precipitate obtained after isox
  • RESERVE group represents the result after 37°C incubation
  • Figure 3 shows the intracellular distribution of Rbm46 at 4°C in an embodiment of the present disclosure
  • Figure 4 shows the intracellular distribution of Rbm46 at 37°C in an embodiment of the disclosure
  • Figure 5 is a comparison of the phase separation of RbmC and N at different temperatures in an embodiment of the disclosure
  • a Rbm C-terminal, 37 degrees Celsius
  • b Rbm C-terminal, 4 degrees Celsius
  • c Rbm N-terminal, 37 degrees Celsius
  • d Rbm N-terminal, 4 degrees Celsius
  • Figure 6 shows the protective effects of Rbm46, Rbm15, and Rbm47 on HEK293T cells during cryopreservation in an embodiment of the disclosure.
  • the present disclosure relates to a cryoprotectant for biological samples, the working solution of which contains active fragments ⁇ 5mM;
  • the active fragment is a polypeptide and/or protein capable of phase separation (Phase Separation or Phase Transition) and comprising RNA binding domain (RNA binding domain) and low-complexity sequence domain (LCD) .
  • low-complexity sequence domain is a protein fragment that is rich in or consists of only a few types of amino acids. The combination of these fragments usually follows simple patterns, such as tandem repeats, and is related to the rapid evolution rate of the molecule.
  • phase separation and “phase transition” can be used interchangeably and refer to the spontaneous separation (reverse mixing) of homogeneous solutions of molecules (such as biological macromolecules, such as proteins) into two.
  • Two co-existing phases for example, two liquid phases
  • one is a dense phase rich in the molecules
  • the other is a lean phase.
  • RNA and RNA-binding proteins participate in phase separation in cells, which are related to protein aggregation, the formation and assembly of membraneless organelles, and even some disease mechanisms.
  • the cryoprotectant for biological samples may be solid (for example, freeze-dried powder or spray-dried powder) or solution;
  • the biological sample cryoprotectant may further include a solvent such as sterile water.
  • the solution includes from 10w/w% to 90w/w% of said biological sample cryoprotectant, such as from 40w/w% to 65w/w% or from 50w/w% to 60w/w% of said cryoprotectant Cryoprotectant for biological samples.
  • RNA molecules can be used as storage sites for the RNA assembled by protein higher-order complexes to undergo phase changes and assemble into higher-order structures under the stimulation of RNA. This process is often affected in human diseases. Many recent studies have shown that RNA promotes the aggregation of nucleoli, regulates the condensation of P granules, and regulates viscosity and dynamics. The role of RNA in phase separation is still not very clear, and further research is needed.
  • phase change protein can reduce or inhibit the formation of ice crystals at temperatures below 0°C, and can balance the osmotic pressure on both sides of the cell membrane and improve cell survival.
  • cryoprotectant refers to the ability to minimize the formation of ice crystals in cells/tissues/organs when the tissue is cooled to sub-zero temperature compared with the freezing effect of no cryoprotectant, and basically does not affect the cells after heating. /Tissues/organs that cause damage.
  • biological sample cryoprotectant cannot be regarded as a cryopreservation liquid, but should also include the balance liquid or thawing liquid used in the freezing process.
  • the term "functional after cryopreservation" as used herein in connection with a sample generally means that the sample, such as an organ, cell, or tissue after cryopreservation, retains an acceptable and/or desired function after cryopreservation. In one aspect, the sample preserves all its functions after cryopreservation. On the other hand, a sample, such as a cell, retains at least 50% of the required functions, such as at least 60% of the required functions, such as at least 70% of the required functions, such as at least 80% of the required functions, For example, at least 90% of required functions, such as at least 95% of required functions, such as 100% of required functions, as an example of cells, the important function to be preserved is cell viability.
  • the important function to be preserved is the physiological function of the organ, such as the heart pump function.
  • tissue in the case of transplantation, an important function to be preserved is to maintain the ability of such tissue to integrate with surrounding tissues (such as skin).
  • a cell such as an egg cell, it includes its fertilization rate, cleavage rate, and blastocyst rate.
  • the working solution of the biological sample cryoprotectant contains ⁇ 10 mM active fragments, such as 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM , 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 600mM, 700mM, 800mM, 900mM or 1000mM, or any two values in between.
  • active fragments such as 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM , 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 600mM, 700
  • the active fragment is an RNA binding protein or a fragment thereof.
  • the RNA binding protein is selected from one or more of Rbm15, Rbm47, and Rbm46.
  • the fragment of the RNA binding protein should contain the "RRM-LCS" domain.
  • the fragment of Rbm46 is its C-terminal fragment (310aa-485aa of Rbm46), and its structure should contain the "RRM1-RRM2-RRM3-LCS" domain.
  • cryoprotectants for biological samples may include:
  • Freezing fluid buffer + 25% ⁇ 35% human serum albumin + 3% ⁇ 7% propylene glycol + 3% ⁇ 7% ethylene glycol + 0.3M ⁇ 0.7M sucrose + 17mM ⁇ 23mM Rbm46 protein and/or fragments thereof;
  • Freezing fluid buffer + 27% ⁇ 33% human serum albumin+4% ⁇ 6% propylene glycol+4% ⁇ 6% ethylene glycol+0.4M ⁇ 0.6M sucrose+18mM ⁇ 22mM Rbm46 protein and/or fragments thereof;
  • Balance solution buffer solution + 10% human serum albumin + 5% propylene glycol + 5% ethylene glycol + 0.5M sucrose + 5mM Rbm46 protein and/or fragments thereof;
  • Freezing fluid buffer + 30% human serum albumin + 5% propylene glycol + 5% ethylene glycol + 0.5M sucrose + 20mM Rbm46 protein and/or fragments thereof;
  • Thawing solution buffer + 5% propylene glycol + 5% ethylene glycol + 1M sucrose + 5mM Rbm46 protein and/or fragments thereof.
  • the above specific biological sample cryoprotectant combination is used for but not limited to cryopreservation protection of egg cells or fertilized eggs.
  • the biological sample cryoprotectant further includes at least one additional cryoprotectant.
  • the additional cryoprotectant is selected from acetamide, agar, alginate or ester, isomalt oligosaccharide, 1-aniline, albumin, amino acid, ammonium acetate, butanediol, chondroitin sulfate , Chloroform, choline, diethylene glycol, dimethylacetamide, dimethylformamide, dimethylsulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, ⁇ -Glycerol phosphate or ester, glycerol monoacetate, hyaluronic acid, glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methyl acetamide, methyl Amide, methylurea, hydrogel, dextrin, de
  • the additional cryoprotectant can also be a derivative of the above-mentioned compound.
  • the biological sample cryoprotectant further includes a pH buffering agent capable of maintaining a substantially neutral pH at a low temperature.
  • low temperature or “freezing storage temperature” as used herein generally means the following temperature: from below zero to -196°C, such as from -50°C to -196°C, such as from -80°C to -196°C, For example, lower than -55°C, such as lower than -60°C, such as lower than -65°C, such as lower than -70°C, such as lower than -75°C, such as lower than -80°C, such as lower than -85°C, for example Lower than -90°C, such as lower than -95°C, such as lower than -100°C, such as lower than -105°C, such as lower than -110°C, such as lower than -115°C, such as lower than -120°C, such as lower At -125°C, such as lower than -130°C, such as lower than -135°C, such as lower than -140°C, such as lower than -145°C, such as lower
  • pH buffering agent refers to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffering nature of such solutions. Therefore, solutions or compositions that exhibit buffering activity are called buffers or buffer solutions.
  • pH buffering reagents generally do not have unlimited ability to maintain the pH of a solution or composition. Instead, they are generally able to maintain a pH within a specific range.
  • the buffer reagent used in this application should maintain the functional pH of the cryoprotected biological sample. Such a pH is usually “substantially neutral”, such as 6.8 to 7.6, or 7.0 to 7.4, or about 7.1 to 7.3.
  • the buffer can maintain the pH within the upper and the next logarithm of its pKa (see, for example, Mohan, Buffers, A guide for the preparation and use of buffers in biological systems, CALBIOCHEM, 1999).
  • Buffers and buffer solutions are generally prepared from buffer salts or, for example, non-ionic buffer components such as TRIS and HEPES.
  • the buffer that can be used in the method of the present disclosure is, for example, selected from phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer, TRIS Buffered saline solution (TBS) and TRIS/EDTA (TE).
  • the cryoprotectant for biological samples may also include certain ions at appropriate concentrations, such as sodium ions, potassium ions, calcium ions, magnesium ions, and chloride ions, provided that the biological sample is functional after cryopreservation. This is easy for those skilled in the art.
  • the biological sample cryoprotectant does not substantially contain DMSO, and it is generally understood that the DMSO content is less than 0.01 w/w%.
  • the biological sample cryoprotectant may include other substances to improve the viability of the sample.
  • examples of such substances may consist of IAPs (inhibitors of apoptosis), inhibitors of the signaling pathway of rho-related protein kinase (ROCK), growth factors such as EGF, FGF, PDGF, IGF, EPO, BDNF, TGF, TNF, VEGF.
  • IAPs inhibitors of apoptosis
  • ROCK rho-related protein kinase
  • growth factors such as EGF, FGF, PDGF, IGF, EPO, BDNF, TGF, TNF, VEGF.
  • the biological sample cryoprotectant may include any serum components derived from human, bovine, equine, and dog.
  • the cryoprotective agent or composition may also contain growth medium.
  • a growth medium that includes the following, a catenin/P300 antagonist and an activated protein/TGFP ligand, such as ID-8 that binds to activated protein and TGFI3, can be used.
  • This type of medium is particularly suitable for the cultivation of pluripotent stem cells, especially embryonic stem cells, for example as described in WO 2013/054112.
  • Another example is a standard knock out medium, which includes KnockOut Serum Replacement, DMEM/F12 with Glutamax TM supplement, FGF, NEAA and BME.
  • Another example is the mTSER TM system.
  • Other examples of growth media according to the sample to be preserved are well known to the skilled person.
  • the present invention relates to the application of the cryoprotectant for biological samples in cryopreservation of biological samples.
  • the present disclosure also relates to a cryopreservation composition including the biological sample cryoprotectant as described above, the cryopreservation composition further including a biological sample.
  • the cryopreservation composition is in the process of preliminary cryopreservation (e.g., equilibration process), in the process of being cryopreserved, has been cryopreserved or in the process of thawing.
  • preliminary cryopreservation e.g., equilibration process
  • the amount of cryoprotectant for biological samples present in the cryopreservation composition is usually 1 to 80% w/w, such as from 2 to 70% w/w, such as from 4 to 45% w/w, or from 6 To 20% w/w, or from 6 to 12% w/w, or for example from 6 to 10% w/w, or for example from 7 to 9% w/w.
  • the present disclosure also relates to a method for cryopreserving a biological sample, including the steps of contacting the biological sample to be cryopreserved with the biological sample cryoprotectant as described above, thereby obtaining a cryopreservation composition, and then The temperature of the cryopreservation composition is lowered to the cryopreservation temperature.
  • the biological sample includes an organ, tissue, or cell, or a combination thereof.
  • the species source of the biological sample can be selected from animals, including humans and all livestock (such as domestic animals and pets) and wild animals and poultry, which include, without limitation, cattle, horses, dairy cows, pigs, sheep, goats, Rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, cockfighting, etc.; the species of the biological sample can also be plants or microorganisms with cell structures.
  • livestock such as domestic animals and pets
  • wild animals and poultry which include, without limitation, cattle, horses, dairy cows, pigs, sheep, goats, Rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, cockfighting, etc.
  • the species of the biological sample can also be plants or microorganisms with cell structures.
  • organ refers to any organ, such as liver, lung, kidney, intestine, heart, pancreas, testis, placenta, thymus, adrenal gland, artery, vein, lymph node, bone or skeletal muscle.
  • tissue includes any tissue type, including any kind of cell types (for example from one of the above-mentioned organs) and combinations thereof, including, for example, ovarian tissue, testicular tissue, umbilical cord tissue, placental tissue, connective tissue, heart tissue , From muscle, cartilage and bone tissue, endocrine tissue and nerve tissue.
  • tissue may also include adipose tissue or dental pulp tissue.
  • the tissue or organ is obtained from humans, such as human liver, human lung, human kidney, human intestine, human heart, human pancreas, human testis, human placenta, human thymus, human adrenal gland, human artery, human vein , Human lymph nodes, human bones or human skeletal muscles.
  • humans such as human liver, human lung, human kidney, human intestine, human heart, human pancreas, human testis, human placenta, human thymus, human adrenal gland, human artery, human vein , Human lymph nodes, human bones or human skeletal muscles.
  • the organ and tissue are selected from: spleen, heart, liver, brain and other nervous tissues, kidney, lung, pancreas, breast, umbilical cord, skin, placenta, ovary, fallopian tube, uterus, prostate , Tonsil, thymus, stomach, testis, trachea, cartilage, tendon, bone, skeletal muscle, smooth muscle, intestine, bladder, urethra, eye, gallbladder, cornea, skin, valve, vein, artery, umbilical cord, placenta, from cell culture Any of the organ-like structures of tumors and tumors, or their group.
  • the term "cell” includes any type of cell, such as somatic cells (including all cells in a tissue or organ), fibroblasts, keratinocytes, hepatocytes, cardiomyocytes, smooth muscle cells, stem cells, progenitor cells, Oocytes, sperm and germ cells. These cells may be in isolated form or in non-isolated form, for example in the form of body fluids, tissues or organs containing cells. In some embodiments, the cells are derived from mammalian tissues or organs, such as human tissues or organs.
  • the cell is selected from cells derived from vertebrate tissue or invertebrate tissue, such as epithelial cells, endothelial cells, fibroblasts, myofibroblasts, hepatocytes, hepatic stellate cells, Cardiomyocytes, podocytes, keratinocytes, melanocytes, red blood cells, neuronal cells (including neurons, astrocytes, microglia and oligodendrocytes), white blood cells (including dendritic cells, Neutrophils, sperm, egg cells, fertilized eggs, macrophages and lymphocytes (including T cells, B cells, NK cells, NKT cells and congenital lymphoid 1-3 cells)), tissue stem cells (including mesenchymal cells) Any one of stem cells and progenitor cells of the above-mentioned cells), or a group of them.
  • tissue stem cells including mesenchymal cells
  • the organ or tissue is an embryo.
  • the cell is an induced pluripotent stem cell, embryonic stem cell or adult stem cell.
  • the cell is selected from any one of bone marrow mesenchymal stem cells, hematopoietic stem cells, neural stem cells, peripheral blood stem cells, adipose stem cells, cardiac stem cells, placental subtotipotent stem cells, and amniotic stem cells, or The group of them.
  • the cell is an embryonic cell, such as an embryonic kidney cell.
  • the cell is a germ cell.
  • the cell is sperm, egg cell, fertilized egg or any combination thereof.
  • the cryopreserved biological sample is vitrified or non-vitrified.
  • the cell such as a eukaryotic cell, can express the protein containing the active fragment.
  • the cell may be an isolated single cell, a homogenous or heterogeneous cell aggregate, or a body fluid containing cells, such as blood (such as cord blood, peripheral blood). ), amniotic fluid, semen, cerebrospinal fluid, bone marrow extract and menstrual fluid.
  • blood such as cord blood, peripheral blood.
  • amniotic fluid such as amniotic fluid, semen, cerebrospinal fluid, bone marrow extract and menstrual fluid.
  • the Rbm46 used in the embodiments of the present application is of Homo sapiens origin, and its NCBI Reference Sequence is: NP_001264100.1.
  • Rbm46 is expressed in human egg cells
  • Rbm46 The inventors observed the distribution of Rbm46 in egg cells at different stages of meiosis and found that Rbm46 presents a point-like aggregated distribution in the cells (Figure 1). Some previous articles reported that RNA-binding proteins can accumulate in cells and participate in the formation of germ cell granules. We believe that this distribution of Rbm46 is most likely involved in the formation of germ cell granules.
  • Rbm46 is a phase change protein
  • Rbm46 protein When the temperature is high, Rbm46 protein is a single-molecule protein in irregular motion; when the temperature is lowered, they combine with each other to form a droplet-like structure (ie, phase transition).
  • the purified recombinant protein Rbm46 C-terminus can be used as an egg freezing solution additive to significantly improve the survival rate of thawed eggs.
  • Rbm46 presents a dot-like distribution in the cell at low temperature
  • the non-fixed conformational domain is responsible for the intracellular localization of the protein. After the non-fixed conformation domain is mutated, the protein cannot be specifically localized in the granular area, and changes from a punctate distribution to a diffuse distribution.
  • the C-terminal of the purified recombinant protein Rbm46 can form microphase separation
  • the purified Rbm46 N-terminus and C-terminus were concentrated to about 5mg/ml with an ultrafiltration tube, and the C-terminus protein was obviously turbid. Observed under a light microscope, this turbidity is due to the formation of water-insoluble droplets in the protein solution, indicating that the solution has a typical microphase-separate. Over time, these droplets will undergo ostwald ripening. After centrifugal enrichment of the droplets, the droplets deposited on the bottom of the tube aggregate to form a solid-like hydrogel (hydrogel). The phase separation phenomenon presents a temperature-sensitive characteristic. When it is incubated to 37 degrees Celsius, the droplets disappear and the protein turns into a completely dissolved state.
  • phase separation means that when the polymer solution is blended, it presents an incomplete thermodynamically stable system, so the polymer solvent and the solution will separate, but the polymer polymer remains partially soluble, so no macroscopic delamination occurs Phenomenon, but as a suspended droplet particle structure.
  • phase separation follows the laws of thermodynamics and proceeds spontaneously when the Gibbs free energy is less than zero. Gibbs free energy is determined by both enthalpy change and entropy change. Therefore, in general, phase separation is affected by temperature and concentration. Protein is a kind of high molecular polymer.
  • the current research theory believes that the component proteins involved in the formation of ribonucleic acid particles are recruited by certain signals and accumulate in the cell, forming high concentrations in local areas, resulting in the formation of phases in protein molecules. Separation, from a completely soluble state to a suspended droplet, this droplet composed of ribonucleoprotein and RNA is a ribonucleic acid granule. Ribonucleic acid particles are not surrounded by a membrane structure and exchange materials with the outside world at all times, maintaining a high degree of dynamics in the cell.
  • the main reagents used include:
  • the carrier containing the oocytes was taken out of the liquid nitrogen tank and quickly added to the resuscitation solution. After being placed at room temperature for 3 minutes, the oocytes were transferred into the gamete culture solution and cultured at 37° C., 6% CO2 and saturated humidity.
  • a total of 96 oocytes were collected. After freezing, they were thawed and resuscitated. After resuscitation, 94 survived, with a survival rate of 98%; then underwent ICSI (Intracytoplasmic Sperm Injection), 92 fertilized, with a fertilization rate of 95.8%; cleavage 90 The cleavage rate was 93.7%; 84 blastocysts developed and the blastocyst formation rate was 87.5%.
  • the freezing and thawing of oocytes without Rbm46 was carried out in the same way as above: in this example, 63 oocytes were collected, 63 were thawed, 46 survived, and the survival rate of eggs after freezing and thawing was 73.0%. 41 were fertilized, the fertilization rate was about 89.1%, 36 were cleavage, the cleavage rate was about 87.8%, and the blastocyst rate was about 67.3%.
  • the first sample cryopreservation solution contains ethylene glycol (EG) and phosphate buffer at a concentration of 22.5% (v/v);
  • the second sample cryopreservation solution contains EG[22.5%(v/v)], 5mM Rbm46 and buffer;
  • the third sample cryopreservation solution contains 5mM Rbm15 and buffer
  • the fourth sample cryopreservation solution contains 5mM Rbm47 and buffer.
  • HEK293 cells grow to 80% confluence, the cells are treated with 0.25% trypsin to form single cells, complete medium to stop the digestion and resuspend the cells, centrifuge at 200g, and remove the supernatant. Add an appropriate amount of phosphate buffer to resuspend the cells, use a hemocytometer to count the cells, and adjust the cell concentration to 1 ⁇ 10 6 /ml. Divide the cells into 4 centrifuge tubes, each with 1ml, centrifuge at 200g, and remove the supernatant.
  • the positive control sample is 293T cells that are continuously cultured without freezing at 37°C.
  • the negative control sample is only added with cell culture medium during the freezing and thawing process and subjected to freezing and rewarming treatment.
  • RNA binding protein greatly improves the cell survival rate (2#, 3#, 4# and 1# are statistically different), which shows that RNA binding protein has a strong The ability to freeze cells.
  • the biological sample cryoprotectant of the present disclosure can protect the biological sample by reducing or inhibiting the formation of ice crystals and balancing the osmotic pressure on both sides of the cell membrane during the cryopreservation of the biological sample, and improve the survival rate of the biological sample, especially the cell.

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Abstract

本公开涉及细胞保存液领域，具体而言，涉及一种生物样品冷冻保护剂及其应用。所述的生物样品冷冻保护剂工作液中含有≥5mM的活性片段；所述活性片段为能够进行相分离；且包含RNA结合结构域及低复杂度序列结构域的多肽和/或蛋白质。该生物样品冷冻保护剂对生物样品无毒或者是低毒的，且能减少冰晶产生，平衡生物样品细胞膜内外的渗透压，进而保持冻存后生物样品的功能。

Description

生物样品冷冻保护剂及其应用
相关申请的交叉引用
本申请要求于2019年6月27日提交中国专利局的申请号为201910569836.6、名称为“生物样品冷冻保护剂及其应用”的中国专利申请，以及于2019年7月5日提交中国专利局的申请号为201910602073.0、名称为“生物样品冷冻保护剂及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及细胞保存液领域，具体而言，涉及一种生物样品冷冻保护剂及其应用。
背景技术
冷冻状态(冷冻保存)细胞的贮藏是用在长期维持细胞活力和遗传稳定性的一种通用的技术。冷冻保存通常是一个过程，其中样品，例如细胞或组织，通过冷却到零度以下的温度，通常为77K(＝-196℃，液态氮的沸点)而被保存。在这样的低温下，任何生物活性——包括会引起细胞死亡的生化反应——都会被有效阻止。
在传统的冷冻技术中，样品被采集，放置在存储溶液，然后冷冻保存。当样品被使用时，其会被解冻然后被放置在细胞培养基中。冷冻保存方案使细胞在整个细胞采集，冻结，解冻过程中经历多种应力和损伤(例如冰晶形成所造成的损伤)。这些应力和损伤，可以对细胞造成不可逆的损伤。
一直以来，细胞冷冻保存采用DMSO(二甲基亚砜)、乙二醇、甘油等细胞膜通透型冷冻保存剂，并通常也可采用聚乙烯吡咯烷酮等细胞膜非通透型冷冻保存剂。但是，若这些冷冻保存剂浓度大，则通常存在对细胞显示毒性的问题。并且由于上述保存剂阻碍细胞的增殖，因而在进行培养时必须事先从细胞中除去冷冻保存剂，造成操作繁琐。
因此，在冷冻过程中，在提供必要的防护效果的前提下，减少对细胞有毒害的冷冻保护剂的使用或降低其浓度，并减小冷冻过程中的应力和损伤是本领域亟待解决的技术问题。
发明内容
本发明人进行了大量的实验和创造性的劳动，得到了一种含有相变蛋白或其片段的冷冻液，并且惊奇地发现，该冷冻液能够显著地提高生物样品冻存后的活力，并降低其他产生毒性的冷冻剂的使用量。由此提供了下述公开：
本公开涉及一种生物样品冷冻保护剂，其工作液中含有≥5mM的活性片段；
所述活性片段为能够进行相分离(Phase separation or phase transition)，且包含RNA结合结构域(RNA binding domain)及低复杂度序列结构域(low-complexity sequence domain,LCD)的多肽和/或蛋白质。
所述生物样品冷冻保护剂是有效的生物样品例如器官、组织或细胞的冷冻保护试剂，并且经过冷冻保存的所述生物样品仍然是有功能的。
本公开还涉及如上所述的生物样品冷冻保护剂在冷冻保存生物样品中的应用。
本公开还涉及如上所述的生物样品冷冻保护剂的冷冻保存组合物，所述冷冻保存组合物进一步包括生物样品。
本公开还涉及一种冷冻保存生物样品的方法，包括如下步骤：使将被冷冻保存的生物样品与如上所述的生物样品冷冻保护剂接触，从而获得冷冻保存组合物，随后降低所述冷冻保存组合物的温度至冷冻保存温度。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本公开一个实施例中Rbm46在人类卵细胞中表达结果；
图2为本公开一个实施例中生物素-isox诱导的Rbm46沉淀；图中INPUT表示全细胞的裂解产物，SUPNATANT表示加入对应浓度生物素-isox后得到的上清，PELLET表示加入对应浓度生物素-isox后得到的沉淀；RESERVE组表示37℃孵育后的结果；
图3为本公开一个实施例中Rbm46在4℃的胞内分布；
图4为本公开一个实施例中Rbm46在37℃的胞内分布；
图5为本公开一个实施例中RbmC、N在不同温度下相分离的对比；
a：Rbm C末端，37摄氏度；b：Rbm C末端，4摄氏度；c：Rbm N末端，37摄氏度；d：Rbm N末端，4摄氏度；
图6为本公开一个实施例中Rbm46、Rbm15、Rbm47对HEK293T细胞冻存时的保护效果。
具体实施方式
为使本公开实施方式的目的、技术方案和优点更加清楚，下面将对本公开实施方式中 的技术方案进行清楚、完整地描述。实施方式中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
除非本文另有定义，否则结合本公开使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。以下描述示例性方法和材料，但是与本文描述的那些类似或等同的方法和材料也可以用于本公开中。
本公开涉及一种生物样品冷冻保护剂，其工作液中含有≥5mM的活性片段；
所述活性片段为能够进行相分离(Phase separation or phase transition)，且包含RNA结合结构域(RNA binding domain)及低复杂度序列结构域(low-complexity sequence domain,LCD)的多肽和/或蛋白质。
如本文所用，一般来说，术语“低复杂度序列结构域(low-complexity sequence domain,LCD)”是一种富含或仅由少数类型氨基酸组成的蛋白质片段。这些片段的组合通常遵循简单模式，如串联重复，并且与分子的快速进化速率相关。
如本文所用，“相分离(phase separation)”和“相变(phasetransition)”可以互换使用，是指分子(例如生物大分子，例如蛋白质)的均相溶液自发地分离(反混合)成两种共同存在的相(例如两种液相)，一种是富含所述分子的密相，一种是贫相。一般认为，RNA以及RNA结合蛋白参与了细胞内的相分离，其与蛋白质聚集，无膜细胞器的形成和组装有关，甚至与一些疾病机理相关联。
所述生物样品冷冻保护剂可以是固体(例如冻干粉或喷雾干燥粉)或溶液；
当其为溶液时，所述生物样品冷冻保护剂可以进一步包括溶剂例如无菌水。在一方面，溶液包括从10w/w％到90w/w％的所述生物样品冷冻保护剂，如从40w/w％至65w/w％或从50w/w％到60w/w％的所述生物样品冷冻保护剂。
相分离的蛋白大多数是RNA结合蛋白。RNA分子可以作为蛋白高阶复合体组装的种RNA的存储位点进行相变并且在RNA的刺激下组装成为高阶结构。这个过程在人类疾病中经常受到影响。最近很多研究表明RNA促进核仁的聚集，调节P颗粒(P granule)的凝缩，调节粘度和动态性。RNA对于相分离起到的作用目前还是不很清楚，有待继续研究。
发明人惊奇地发现，由于细胞当中通常也含有大量的相变蛋白，这些蛋白在冷冻及复苏过程中发生相分离，进一步加剧细胞膜两侧的渗透压不平衡。因此，在细胞及复苏液中加入相变蛋白，能够在低于0℃的温度下减少或抑制冰晶形成，并且可以平衡细胞膜两侧渗透压，提高细胞存活率。
一般来说，“冷冻保护剂”是指与没有冷冻保护剂的冷冻效果相比，当组织冷却至零下温度时能够使细胞/组织/器官中的冰晶形成最小化并且在加热后基本上不对细胞/组织/器官 造成损伤的物质。但需要注意的是，基于上述原理，“生物样品冷冻保护剂”并不能够局限地被认为是冻存液，还应当包括冻存过程中所采用的平衡液，或者解冻液。
本文所使用的术语与样品相关的“冷冻保存后是有功能的”通常意味着，样品例如器官、细胞或组织在冷冻保存后保留在冷冻保存后可接受的和/或所需的功能。在一方面，样品在冷冻保存后保存其所有功能。在另一方面，样品，例如细胞，至少保留50％的所需的功能，例如至少60％的所需的功能，例如至少70％的所需的功能，例如至少80％的所需的功能，例如至少90％的所需的功能，例如至少95％的所需的功能，例如100％所需的功能，作为关于细胞的例子，将被保存的重要功能是细胞的生存力。作为关于器官的另一个实例，将被保存的重要功能是所述器官的生理功能，如心脏泵功能。作为关于组织的另一个实例，在移植的情况下，将被保存的重要功能是保持这样的组织与周围组织(如皮肤)整合的能力。作为关于细胞的另一个实例，如卵细胞，则包括其受精率、卵裂率和囊胚率等。
在一些实施方式中，所述生物样品冷冻保护剂的工作液中含有≥10mM的活性片段，例如10mM、15mM、20mM、25mM、30mM、40mM、50mM、60mM、70mM、80mM、90mM、100mM、150mM、200mM、250mM、300mM、350mM、400mM、450mM、500mM、600mM、700mM、800mM、900mM或1000mM，或它们之间任意两个数值组成的范围值。
在一些实施方式中，所述活性片段为RNA结合蛋白或其片段。
在一些实施方式中，所述RNA结合蛋白选自Rbm15、Rbm47以及Rbm46中的一种或多种。
在一些实施方式中，RNA结合蛋白的片段应当包含“RRM-LCS”结构域。
例如Rbm46的片段为其C末端片段(Rbm46的第310aa-485aa)，其结构中应当包含“RRM1-RRM2-RRM3-LCS”结构域。
以相分离蛋白——Rbm46——为例，在一个具体的实施方式中，可选用的生物样品冷冻保护剂的组合可以包括：
平衡液：缓冲液+7％～13％人血清白蛋白+3％～7％丙二醇+3％～7％乙二醇+0.3M～0.7M蔗糖+5mM～7mM Rbm46蛋白质和/或其片段；
冷冻液：缓冲液+25％～35％人血清白蛋白+3％～7％丙二醇+3％～7％乙二醇+0.3M～0.7M蔗糖+17mM～23mM Rbm46蛋白质和/或其片段；
解冻液：缓冲液+3％～7％丙二醇+3％～7％乙二醇+0.7M～1.3M蔗糖+5mM～7mM Rbm46蛋白质和/或其片段；
例如：
平衡液：缓冲液+8％～12％人血清白蛋白+4％～6％丙二醇+4％～6％乙二醇+0.4M～0.6M蔗糖+5mM～6mM Rbm46蛋白质和/或其片段；
冷冻液：缓冲液+27％～33％人血清白蛋白+4％～6％丙二醇+4％～6％乙二醇+0.4M～0.6M蔗糖+18mM～22mM Rbm46蛋白质和/或其片段；
解冻液：缓冲液+4％～6％丙二醇+4％～6％乙二醇+0.7M～1.3M蔗糖+5mM～7mM Rbm46蛋白质和/或其片段；
例如：
平衡液：缓冲液+10％人血清白蛋白+5％丙二醇+5％乙二醇+0.5M蔗糖+5mM Rbm46蛋白质和/或其片段；
冷冻液：缓冲液+30％人血清白蛋白+5％丙二醇+5％乙二醇+0.5M蔗糖+20mM Rbm46蛋白质和/或其片段；
解冻液：缓冲液+5％丙二醇+5％乙二醇+1M蔗糖+5mM Rbm46蛋白质和/或其片段。
上述特定的生物样品冷冻保护剂的组合用于但不限于卵细胞或者受精卵的冻存保护。
在一些实施方式中，所述生物样品冷冻保护剂还包括至少一种额外的冷冻保护剂。
在一些实施方式中，所述额外的冷冻保护剂选自乙酰胺，琼脂，海藻酸盐或酯，异麦芽糖寡糖，1-苯胺，白蛋白，氨基酸，醋酸铵，丁二醇，硫酸软骨素，氯仿，胆碱，二甘醇，二甲基乙酰胺，二甲基甲酰胺，二甲基亚砜(DMSO)，赤藓糖醇，乙醇，乙二醇，甲酰胺，葡萄糖，甘油，α-甘油磷酸盐或酯，单乙酸甘油，透明质酸、甘氨酸，羟乙基淀粉，肌醇，乳糖，氯化镁，硫酸镁，麦芽糖，甘露醇，甘露糖，甲醇，甲基乙酰胺，甲基甲酰胺，甲基脲，水凝胶、糊精、葡聚糖、苯酚，普流尼克多元醇，聚乙二醇，聚乙烯吡咯烷酮，脯氨酸，丙二醇，丝氨酸，溴化钠，氯化钠，碘化钠，吡啶N-氧化物，核糖，硝酸钠，蔗糖，海藻糖，硫酸钠，山梨糖醇，三甘醇，乙酸三甲基胺，脲，缬氨酸和木糖中的任一种，或它们所组成的组；
在一些实施方式中，所述额外的冷冻保护剂还可以选择上述化合物的衍生物。
在一些实施方式中，所述生物样品冷冻保护剂还包括能够在低的温度下维持大致中性的pH缓冲试剂。
本文所使用的术语“低的温度”或“冷冻保存温度”通常意指如下的温度：从零下到-196℃，如从-50℃至-196℃，如从-80℃至-196℃，例如低于-55℃，例如低于-60℃，例如低于-65℃，例如低于-70℃，例如低于-75℃，例如低于-80℃，例如低于-85℃，例如低于-90℃，例如低于-95℃，例如低于-100℃，例如低于-105℃，例如低于-110℃，例如低于-115℃，例如低于-120℃，例如低于-125℃，例如低于-130℃，例如低于-135℃，例如低于-140℃，例如低于-145℃，例如低于-150℃，例如低于-155℃，例如低于-160℃，例如低于-165℃，例如低于-170℃，例如低于-175℃，例如低于-180℃，例如低于-185℃，例如低于-190℃的温度。
如本文使用的术语“pH缓冲试剂”指水溶液或组合物，当酸或碱加入该溶液或组合物中时，所述水溶液或组合物抵抗pH中的变化。这种对pH变化的抗性是由于此类溶液的缓冲性质。因此，显示出缓冲活性的溶液或组合物被称为缓冲液或缓冲溶液。pH缓冲试剂一般不具有无限的维持溶液或组合物的pH的能力。相反，它们一般能够维持在特定范围内的pH。本申请所采用的缓冲试剂应当维持所冷冻保护的生物样品是有功能的pH，这样的pH通常为“大致中性”的，例如6.8～7.6，或者7.0～7.4，或者7.1～7.3左右。
一般地，缓冲液能够维持在其pKa上和下一个对数内的pH(参见例如，Mohan，Buffers，A guide for the preparation and use of buffers in biological systems，CALBIOCHEM，1999)。缓冲液和缓冲溶液一般由缓冲盐或例如非离子缓冲液组分如TRIS和HEPES制备。可以在本公开的方法中使用的缓冲液例如选自磷酸盐缓冲液、磷酸盐缓冲盐水缓冲液(PBS)、2-氨基-2羟甲基-1,3-丙二醇(TRIS)缓冲液、TRIS缓冲盐水溶液(TBS)和TRIS/EDTA(TE)。
在一些实施方式中，所述生物样品冷冻保护剂还可以在生物样品冷冻保存后是有功能的前提下包括适当浓度的某些离子，例如钠离子、钾离子、钙离子、镁离子、氯离子，这对于本领域技术人员是容易的。
在一些实施方式中，所述生物样品冷冻保护剂基本不含有DMSO，一般理解为DMSO含量少于0.01w/w％。
在一些实施方式中，所述生物样品冷冻保护剂可以包括其它物质以提高样品的可行性。这样物质的实施例可以由以下构成，IAPs(凋亡抑制剂)，对rho相关蛋白激酶(ROCK)信号通路(signaling pathway)抑制剂，生长因子例如EGF，FGF，PDGF，IGF，EPO，BDNF，TGF，TNF，VEGF。在另在一方面，可以提到的是，所述生物样品冷冻保护剂可以包括人，牛，马，犬来源的任何血清成分。冷冻保护试剂或组合物还可以含有生长培养基。在一方面，可以使用的是包括以下的生长培养基，连环蛋白/P300拮抗剂和活化蛋白/TGFP配体，例如与活化蛋白和TGFI3结合的ID-8。这类培养基特别适用于多能干细胞，特别是胚胎干细胞的培养，例如在WO 2013/054112中所描述的。另一个实施例是标准的剔除培养基(knock out medium)，其包括剔除血清取代物(KnockOut Serum Replacement)，具有Glutamax TM补充物的DMEM/F12,FGF，NEAA和BME。另一个实施例是mTSER TM系统。根据要保存的样品的生长培养基的其他的实施例是技术人员众所周知的。
根据本公开的一方面，本发涉及所述的生物样品冷冻保护剂在冷冻保存生物样品中的应用。
根据本公开的一方面，本公开还涉及包括如上所述的生物样品冷冻保护剂的冷冻保存组合物，所述冷冻保存组合物进一步包括生物样品。
所述冷冻保存组合物是在预备冷冻保存的过程中(例如平衡过程)、被冷冻保存的过程 中、已经被冷冻保存或在解冻过程过程中。
在所述冷冻保存组合物中存在的生物样品冷冻保护剂的量通常是1至80％w/w，如从2至70％w/w，如从4至45％w/w，或从6至20％w/w，或从6至12％w/w，或者例如从6至10％w/w，或者例如从7至9％w/w。
根据本公开的一方面，本公开还涉及冷冻保存生物样品的方法，包括如下步骤：使将被冷冻保存的生物样品与如上所述的生物样品冷冻保护剂接触，从而获得冷冻保存组合物，随后降低所述冷冻保存组合物的温度至冷冻保存温度。
根据本公开任一项实施方式，所述生物样品包括器官、组织或细胞，或它们的组合。
所述生物样品的种属来源可以选择为动物，包括人类及所有畜养(如家畜和宠物)和野生的动物及禽鸟，其非限制性地包括牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡等；所述生物样品的种属也可以为植物或具有细胞结构的微生物。
如本文所用，术语“器官”是指任何器官，例如肝，肺，肾，肠，心脏，胰腺，睾丸，胎盘，胸腺，肾上腺，动脉，静脉，淋巴结，骨或骨骼肌。
如本文所用，术语“组织”包括任何组织类型，包括任何种类的细胞类型(例如来自上述器官之一)及其组合，包括例如卵巢组织，睾丸组织，脐带组织，胎盘组织，结缔组织，心脏组织，来自肌肉，软骨和骨骼的组织，内分泌组织和神经组织。术语“组织”还可包括脂肪组织或牙髓组织。在一些实施方式中，组织或器官是从人获得的，例如人肝，人肺，人肾，人肠，人心，人胰腺，人睾丸，人胎盘，人胸腺，人肾上腺，人动脉，人体静脉，人淋巴结，人骨骼或人骨骼肌。
根据本公开任一项实施方式，所述器官、组织选自：脾、心脏、肝脏、脑和其它神经组织、肾、肺、胰腺、乳房、脐带、皮肤、胎盘、卵巢、输卵管、子宫、前列腺、扁桃体、胸腺、胃、睾丸、气管、软骨、腱、骨、骨骼肌、平滑肌、肠、膀胱、尿道、眼、胆囊、角膜、皮肤、瓣膜、静脉、动脉、脐带、胎盘、来自细胞培养物和肿瘤的器官样结构中的任一种，或它们所组成的组。
如本文所用，术语“细胞”包括任何类型的细胞，例如体细胞(包括组织或器官中的所有细胞)，成纤维细胞，角质形成细胞，肝细胞，心肌细胞，平滑肌细胞，干细胞，祖细胞，卵母细胞，精子和生殖细胞。这些细胞可以是分离的形式或非分离的形式，例如含有细胞的体液，组织或器官的形式。在一些实施方式中，细胞来自哺乳动物组织或器官，例如人组织或器官。
根据本公开任一项实施方式，所述细胞选自来自脊椎动物组织或无脊椎动物组织的细胞，例如上皮细胞、内皮细胞、成纤维细胞、肌成纤维细胞、肝细胞、肝星形细胞、心肌 细胞、足细胞、角质形成细胞、黑素细胞、红细胞、神经元细胞(包括神经元、星形胶质细胞、小胶质细胞和少突胶质细胞)、白细胞[包括树突细胞、嗜中性白细胞、精子、卵细胞、受精卵、巨噬细胞和淋巴细胞(包括T细胞、B细胞、NK细胞、NKT细胞和先天性淋巴样1-3型细胞)]、组织干细胞(包括间充质干细胞和以上提及的细胞的祖细胞)中的任一种，或它们所组成的组。
根据本公开任一项实施方式，所述器官、组织为胚胎。
根据本公开任一项实施方式，所述细胞为诱导性多能干细胞、胚胎干细胞或成体干细胞。
根据本公开任一项实施方式，所述细胞选自骨髓间充质干细胞、造血干细胞、神经干细胞、周边血干细胞、脂肪干细胞、心脏干细胞、胎盘亚全能干细胞和羊膜干细胞中的任一种，或它们所组成的组。
根据本公开任一项实施方式，所述细胞为胚胎细胞，例如胚胎肾细胞。
根据本公开任一项实施方式，所述细胞为生殖细胞。
根据本公开任一项实施方式，所述细胞为精子、卵细胞、受精卵或其任意组合。
根据本公开任一项实施方式，所述冷冻保存生物样品为玻璃化冷冻或非玻璃化冷冻。
根据本公开任一项实施方式，所述细胞例如真核细胞能够表达含有所述活性片段的蛋白质。
根据本公开任一项实施方式，其中所述细胞可以是被分离的单个细胞，也可以为同质或异质的细胞团聚物，或者是含有细胞的体液，例如血液(如脐带血，外周血)，羊水，精液，脑脊液，骨髓抽出物和月经液。
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
本实施例用于说明Rbm46作为冷冻保护剂组分的应用；被保护的生物样品为卵细胞。
若无特别描述，本申请实施例中所采用的Rbm46均为人(Homo sapiens)来源，其NCBI Reference Sequence为:NP_001264100.1。
1.Rbm46在人类卵细胞中表达
发明人对处于减数分裂不同时期卵细胞中Rbm46的分布进行观察发现，Rbm46在细胞中呈现点状的聚集式分布(图1)。之前有一些文章报道，RNA结合蛋白能够在细胞内聚集 并参与生殖细胞颗粒(germ cell granules)的形成。我们认为Rbm46的这种分布极有可能是参与形成了生殖细胞颗粒。
2.Rbm46是一种相变蛋白
此前有文章报道，在体内参与RNA颗粒形成的蛋白会被生物素-isox特异性沉淀。生物素-isox在低温状态下会自发形成结晶，其晶体上存在大小恰好和beta折叠结构域相同的沟，这种沟的存在会诱导能够形成beta折叠结构的蛋白嵌入其中并催化其形成beta折叠构象。我们在卵巢细胞裂解液中加入生物素-isox并在冰上孵育后发现，Rbm46存在于沉淀中。并且这种沉淀是可逆的，将沉淀加热至37摄氏度，发现Rbm46又会重新溶解(图2)。这表明Rbm46作为一种RNA结合蛋白，参与了生殖细胞中生殖细胞颗粒的形成。
在温度较高时，Rbm46蛋白是一种处在不规则运动中的单分子蛋白；温度降低时，发生相互结合，生成液滴状结构(即相变)。纯化的重组蛋白Rbm46C末端能够作为卵子冷冻液添加剂能够显著提高卵子解冻复苏存活率。
3.低温下Rbm46在细胞内呈现点状分布
在经典的参与RNA颗粒形成的蛋白中，无固定构象区域结构域负责蛋白在胞内的定位。将无固定构象结构域突变后，蛋白无法特异性定位于颗粒区域，而从点状分布变为弥散分布。我们将构建的不同区段突变的表达载体转染进入HK293T细胞中，发现单独的C末端(Rbm46的第310aa-485aa)和正常蛋白定位模式相同，但是缺少C末端的蛋白，即N末端(Rbm46的第14aa-309aa)却在细胞内弥散分布。这表明与经典的参与RNA颗粒形成的RNA结合蛋白一样，Rbm46在胞内的分布也是和C末端相关的。
4.纯化的重组蛋白Rbm46C末端能够形成微相分离
将纯化的Rbm46N末端以及C末端用超滤管分别浓缩至约5mg/ml，可以观察到C末端蛋白出现明显浑浊。在光镜下观察，这种浑浊是由于在蛋白溶液中形成了不溶于水的液滴，表明溶液发生了典型的微相分(microphase-separate)。随着时间的进行，这些液滴会经历ostwald熟化。当对液滴进行离心富集之后，沉淀在管底的液滴聚集形成了类固态的水凝胶(hydrogel)。相分离现象呈现出温度敏感的特质，当将其孵育到37摄氏度时，液滴消失，蛋白转变为完全溶解状态。这与以往报道的RNA结合蛋白FUS、Hnrnp A等形成的相分离几乎一致。同时，N末端并没有这种现象发生(图5)。如上文所述，目前研究人员普遍认为，体内无膜结构、但是和周围细胞质环境又有明显区别的核糖核酸颗粒的形成是由相分离(phase separation)所导致的。所谓相分离是指高分子溶液共混时，呈现出一种不完全的热力学稳定体系，因此高分子溶剂和溶液会发生分离，但是高分子高分子保持了部分可溶性，因此不出现宏观的分层现象，而是表现为悬浮的液滴微粒结构。作为一个物理化学过程，相分离遵循热力学定律，在吉布斯自由能小于零时自发进行。吉布斯自由能是 由焓变和熵变共同决定的，因此一般来说，相分离受温度和浓度的影响。蛋白质是高分子聚合物的一种，目前研究的理论认为，参与形成核糖核酸颗粒的组分蛋白受到某些信号的募集而在胞内聚集，在局部地区形成高浓度，导致蛋白质分子产生了相分离，由完全可溶状态转变为悬浮的液滴，这个由核糖核蛋白以及RNA组成的液滴就是核糖核酸颗粒(RNA granule)。核糖核酸颗粒没有膜结构包围，时刻与外界进行物质交换，在细胞中保持了高度的动态性。
5.卵母细胞冻融实验。
所用的主要试剂包括：
1、平衡液：80％磷酸盐缓冲液+10％人血清白蛋白+5％丙二醇+5％乙二醇+0.5M蔗糖+5mM Rbm46蛋白质
2、冷冻液：60％磷酸盐缓冲液+30％人血清白蛋白+5％丙二醇+5％乙二醇+0.5M蔗糖+20mM Rbm46蛋白质
3、解冻液：90％磷酸盐缓冲液+5％丙二醇+5％乙二醇+1M蔗糖+5mM Rbm46蛋白质
冷冻过程：
将卵母细胞转移进入平衡液，室温静置10min,然后转移至冷冻液(VM)液中，静置5min后将卵母细胞转移至冻存管内，迅速放入液氮内，-196℃保存。
解冻过程：
将含卵母细胞的载体从液氮罐中取出，迅速加入复苏液中，室温下放置3min后，将卵母细胞被移入配子培养液，在37℃、6％CO2及饱和湿度条件下培养。
随后，观察和记录卵母细胞存活状况、受精状况、胚胎发育状况及囊胚发育状况。
共收集卵母细胞96个，冷冻后进行解冻复苏，复苏后存活94枚，存活率98％；然后进行ICSI(单精子卵胞浆内注射)，受精92枚，受精率95.8％；卵裂90枚，卵裂率93.7％；发育囊胚84个，囊胚形成率87.5％。
同时，不含Rbm46的冻存液进行卵母细胞冻融实施采用与上述相同的方式：本例共收集卵母细胞63枚，解冻63枚，存活46枚，卵子冻融后存活率73.0％，受精41枚，受精率89.1％左右，卵裂36枚，卵裂率87.8％左右，囊胚率67.3％左右。
由以上数据可以看出，与不含Rbm46的冻存液相比，使用Rbm46的冻存液的情况下，卵细胞不仅在复苏后存活率方面显著提高(98％vs.73.0％)。而且使用Rbm46的冻存液的情况下，在受精率、卵裂率以及囊胚形成率方面都显著优于不含Rbm46的冻存液的情况。这说明Rbm46还更大程度地保护了复苏后的卵细胞的功能。
实施例2
利用HEK293T细胞进行冷冻保存测试，制备四种样品冻存液。
第一个样品冻存溶液含有浓度为22.5％(v/v)的乙二醇(EG)和磷酸盐缓冲液；
第二个样品冻存溶液含有EG[22.5％(v/v)]，5mM的Rbm46和缓冲液；
第三个样品冻存溶液含有5mM的Rbm15和缓冲液；
第四个样品冻存溶液含有5mM的Rbm47和缓冲液。
当HEK293细胞生长直至80％汇合度时，用0.25％的胰蛋白酶处理细胞成单细胞，完全培养基终止消化并重悬细胞，200g离心，除去上清。加入适量的磷酸盐缓冲液重悬细胞，使用血细胞计数器对细胞进行计数，调整细胞浓度为1×10 6/ml。将细胞分装4个离心管，每管1ml，200g离心，去除上清。
分别利用四种样品冻存液重悬细胞，500μL/管。然后将细胞放入-80℃冰箱中快速冷冻，一周后取出细胞，在37℃水浴中快速复温融化，加入完全培养基。阳性对照样品为在37℃下不经过冷冻，持续培养的293T细胞。阴性对照样品仅在冻融过程中加入细胞培养基，并对其进行冷冻复温处理。
细胞解冻后，用LIVE/DEAD细胞活性检测试剂盒(Thermo Fisher)染色，并使用流式细胞仪检测细胞活性。
实验结果如图6所示，RNA结合蛋白的加入极大地改善了细胞存活率(2#、3#、4#与1#相比均有统计学差异)，这说明RNA结合蛋白具有很强的冷冻保存细胞的能力。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开的生物样品冷冻保护剂可以通过在生物样品的冷冻保存过程中减少或抑制冰晶形成以及平衡细胞膜两侧渗透压来保护生物样品，提高生物样品，特别是细胞的存活率。

Claims (19)

  1. 生物样品冷冻保护剂，其工作液中含有≥5mM的活性片段；优选≥10mM；更优选为10nM和100mM之间；
    所述活性片段为能够进行相分离，且包含RNA结合结构域及低复杂度序列结构域的多肽和/或蛋白质。
  2. 根据权利要求1所述的生物样品冷冻保护剂，所述活性片段为RNA结合蛋白或其片段，所述RNA结合蛋白优选选自Rbm15、Rbm47以及Rbm46中的一种或多种。
  3. 根据权利要求1所述的生物样品冷冻保护剂，其还包括至少一种额外的冷冻保护剂。
  4. 根据权利要求3所述的生物样品冷冻保护剂，所述额外的冷冻保护剂选自乙酰胺，琼脂，海藻酸盐或酯，异麦芽糖寡糖，1-苯胺，白蛋白，氨基酸，醋酸铵，丁二醇，硫酸软骨素，氯仿，胆碱，二甘醇，二甲基乙酰胺，二甲基甲酰胺，二甲基亚砜(DMSO)，赤藓糖醇，乙醇，乙二醇，甲酰胺，葡萄糖，甘油，α-甘油磷酸盐或酯，单乙酸甘油，透明质酸、甘氨酸，羟乙基淀粉，肌醇，乳糖，氯化镁，硫酸镁，麦芽糖，甘露醇，甘露糖，甲醇，甲基乙酰胺，甲基甲酰胺，甲基脲，水凝胶、糊精、葡聚糖、苯酚，普流尼克多元醇，聚乙二醇，聚乙烯吡咯烷酮，脯氨酸，丙二醇，丝氨酸，溴化钠，氯化钠，碘化钠，吡啶N-氧化物，核糖，硝酸钠，蔗糖，海藻糖，硫酸钠，山梨糖醇，三甘醇，乙酸三甲基胺，脲，缬氨酸和木糖中的任一种，或它们的组合。
  5. 根据权利要求1所述的生物样品冷冻保护剂，其还包括能够在0℃～-196℃下维持大致中性的pH缓冲试剂。
  6. 权利要求1～5任一项所述的生物样品冷冻保护剂在冷冻保存生物样品中的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述生物样品包括器官、组织或细胞，或它们的组合物。
  8. 根据权利要求7所述的应用，其特征在于，所述器官、组织选自：脾、心脏、肝脏、脑和其它神经组织、肾、肺、胰腺、乳房、脐带、皮肤、胎盘、卵巢、输卵管、子宫、前列腺、扁桃体、胸腺、胃、睾丸、气管、软骨、腱、骨、骨骼肌、平滑肌、肠、膀胱、尿道、眼、胆囊、角膜、皮肤、瓣膜、静脉、动脉、脐带、胎盘、来自细胞培养物和肿瘤的器官样结构中的任一种，或它们的组合；
    所述细胞选自来自脊椎动物组织或无脊椎动物组织的细胞，更优选上皮细胞、内皮细胞、成纤维细胞、肌成纤维细胞、肝细胞、肝星形细胞、心肌细胞、足细胞、角 质形成细胞、黑素细胞、红细胞、神经元细胞(包括神经元、星形胶质细胞、小胶质细胞和少突胶质细胞)、白细胞[包括树突细胞、嗜中性白细胞、精子、卵细胞、受精卵、巨噬细胞和淋巴细胞(包括T细胞、B细胞、NK细胞、NKT细胞和先天性淋巴样1-3型细胞)]、组织干细胞(包括间充质干细胞和以上提及的细胞的祖细胞)中的任一种，或它们的组合。
  9. 根据权利要求7所述的应用，其中，所述器官、组织为胚胎。
  10. 根据权利要求7所述的应用，其中，所述细胞为诱导性多能干细胞、胚胎干细胞或成体干细胞。
  11. 根据权利要求7所述的应用，其中，所述细胞选自骨髓间充质干细胞、造血干细胞、神经干细胞、周边血干细胞、脂肪干细胞、心脏干细胞、胎盘亚全能干细胞和羊膜干细胞中的任一种，或它们的组合。
  12. 根据权利要求7所述的应用，其中，所述细胞为胚胎细胞，优选为胚胎肾细胞。
  13. 根据权利要求7所述的应用，所述细胞为能够表达含有所述活性片段的蛋白质的细胞。
  14. 根据权利要求7所述的应用，其中，所述细胞为生殖细胞。
  15. 根据权利要求7所述的应用，其中，所述细胞为精子、卵细胞、受精卵或其任意组合。
  16. 根据权利要求6～15中任一项所述的应用，其中，所述冷冻保存生物样品为玻璃化冷冻或非玻璃化冷冻。
  17. 包括权利要求1～5任一项所述的生物样品冷冻保护剂的冷冻保存组合物，所述冷冻保存组合物进一步包括生物样品；
    优选的，所述生物样品是权利要求7～15中任一项所定义的生物样品。
  18. 冷冻保存生物样品的方法，包括如下步骤：将被冷冻保存的生物样品与权利要求1～5任一项所述的生物样品冷冻保护剂接触，从而获得冷冻保存组合物，随后降低所述冷冻保存组合物的温度至冷冻保存温度；
    优选的，所述生物样品是权利要求7～15中任一项所定义的生物样品。
  19. 根据权利要求18所述的方法，其中所述降低所述冷冻保存组合物的温度至冷冻保存温度的步骤以玻璃化冷冻或非玻璃化冷冻的方法操作。
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