CN110250162B - Biological sample cryoprotectant and application thereof - Google Patents
Biological sample cryoprotectant and application thereof Download PDFInfo
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- CN110250162B CN110250162B CN201910602073.0A CN201910602073A CN110250162B CN 110250162 B CN110250162 B CN 110250162B CN 201910602073 A CN201910602073 A CN 201910602073A CN 110250162 B CN110250162 B CN 110250162B
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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
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
The invention relates to the field of cell preservation solution, and particularly relates to a biological sample cryoprotectant and application thereof. The working solution of the biological sample cryoprotectant contains more than or equal to 5mM of active fragments; the active fragments are capable of undergoing phase separation; and comprising an RNA binding domain and a low complexity sequence domain. The biological sample cryoprotectant is nontoxic or low-toxic to a biological sample, can reduce the generation of ice crystals, balance the osmotic pressure inside and outside a cell membrane of the biological sample, and further maintain the function of the biological sample after cryopreservation.
Description
Technical Field
The invention relates to the field of cell preservation solution, and particularly relates to a biological sample cryoprotectant and application thereof.
Background
Storage of cells in a frozen state (cryopreservation) is a common technique used to maintain cell viability and genetic stability over long periods of time. Cryopreservation is typically a process in which a sample, such as a cell or tissue, is preserved by cooling to a temperature below zero, typically 77K (═ 196 ℃, the boiling point of liquid nitrogen). At such low temperatures, any biological activity, including biochemical reactions that cause cell death, is effectively prevented.
In conventional freezing techniques, a sample is collected, placed in a storage solution, and then cryopreserved. When the sample is used, it is thawed and then placed in cell culture medium. Cryopreservation protocols subject cells to various stresses and damage (e.g., damage caused by ice crystal formation) during whole cell harvesting, freezing, and thawing. These stresses and damages can cause irreversible damage to the cells.
Cell cryopreservation has conventionally been carried out using a cell membrane permeable cryopreservative such as DMSO (dimethyl sulfoxide), ethylene glycol, or glycerol, and a cell membrane non-permeable cryopreservative such as polyvinylpyrrolidone has also been generally used. However, these cryopreservative agents generally have a problem of toxicity to cells when the concentration is high. In addition, since the above-mentioned preservative inhibits the proliferation of cells, the cryopreservative must be removed from the cells in advance during the culture, which results in a complicated operation.
Therefore, reducing the use or concentration of cryoprotectants that are toxic to cells and reducing stress and damage during freezing while providing the necessary protective effects during freezing is a technical problem that needs to be addressed in the art.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The present inventors have conducted extensive experimentation and inventive work to obtain a freezing fluid containing phase change proteins or fragments thereof, and have surprisingly found that the freezing fluid can significantly improve the viability of biological samples after cryopreservation and reduce the amount of other toxic refrigerants used. The following invention is thus provided:
one aspect of the invention relates to a biological sample cryoprotectant, wherein a working solution of the cryoprotectant contains more than or equal to 5mM of active fragments;
the active fragment is a polypeptide and/or protein capable of Phase separation (Phase separation or Phase transition) and comprising an RNA binding domain (RNA binding domain) and a low-complexity sequence domain (LCD).
The biological sample cryoprotectant is an effective cryoprotectant for a biological sample, such as an organ, tissue, or cell, and the biological sample that has been cryopreserved remains functional.
Another aspect of the invention relates to the use of a biological sample cryoprotectant as described above for the cryopreservation of a biological sample.
In a further aspect, the present invention relates to a cryopreservation composition for a biological sample cryoprotectant as described above, said cryopreservation composition further comprising the biological sample.
In yet another aspect, the present invention relates to a method for the cryopreservation of a biological sample comprising the steps of: contacting a biological sample to be cryopreserved with a biological sample cryoprotectant as described above, thereby obtaining a cryopreserved composition, followed by reducing the temperature of the cryopreserved composition to a cryopreservation temperature.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the expression result of Rbm46 in human egg cells according to one embodiment of the present invention;
FIG. 2 is a biotin-isox induced precipitation of Rbm46 in one embodiment of the present invention; in the figure, input represents a lysate of a whole cell, supnatant represents a supernatant obtained after adding biotin-isox with a corresponding concentration, and pellet represents a precipitate obtained after adding biotin-isox with a corresponding concentration; reserve group indicates the results after 37 degree incubation;
FIG. 3 shows the intracellular distribution of Rbm46 at 4 ℃ in an embodiment of the invention;
FIG. 4 shows the intracellular distribution of Rbm46 at 37 ℃ in an embodiment of the invention;
FIG. 5 is a comparison of the separation of RbmC, N at different temperatures in one embodiment of the present invention;
a: rbm C-terminal, 37 degrees celsius; b: rbm C-terminal, 4 ℃; c: rbm N-terminal, 37 degrees celsius; d: rbm N-terminal, 4 ℃;
FIG. 6 shows the protective effect of Rbm46, Rbm15 and Rbm47 on HEK293T cells in freezing storage according to an embodiment of the present invention.
Detailed Description
The invention relates to a biological sample cryoprotectant, wherein the working solution contains active fragments with the concentration of more than or equal to 5 mM;
the active fragment is a polypeptide and/or protein capable of Phase separation (Phase separation or Phase transition) and comprising an RNA binding domain (RNA binding domain) and a low-complexity sequence domain (LCD).
The biological sample cryoprotectant may be a solid (e.g., lyophilized powder or spray dried powder) or a solution;
when it is a solution, the biological sample cryoprotectant may further comprise a solvent such as sterile water. In one aspect, the solution comprises from 10 w/w% to 90 w/w% of said biological sample cryoprotectant, such as from 40 w/w% to 65 w/w% or from 50 w/w% to 60 w/w% of said biological sample cryoprotectant.
The proteins that phase separate are mostly RNA binding proteins. RNA molecules can undergo phase changes as storage sites for species RNA assembled by protein higher order complexes and assemble into higher order structures upon stimulation by RNA. This process is often affected in human diseases. Many recent studies have shown that RNA promotes nucleolar aggregation, regulates the condensation of P particles (Pgranule), regulates viscosity and dynamics. The role of RNA for phase separation is currently unclear and remains to be studied.
The inventors have surprisingly found that, since cells also usually contain a large amount of phase change proteins, these proteins phase separate during freezing and thawing, further exacerbating the osmotic pressure imbalance across the cell membrane. Therefore, the phase change protein is added into the cells and the resuscitation solution, the formation of ice crystals can be reduced or inhibited at the temperature lower than 0 ℃, the osmotic pressure at two sides of cell membranes can be balanced, and the cell survival rate is improved.
It should be noted that, based on the above principle, the "biological sample cryoprotectant" is not limited to be regarded as a freezing solution, but also includes a balancing solution used in the freezing process, or a thawing solution.
As used herein, the term "functional after cryopreservation" in relation to a sample generally means that the sample, e.g., organ, cell or tissue, retains acceptable and/or desired functionality after cryopreservation. In one aspect, the sample retains all of its functionality after cryopreservation. In another aspect, the sample, e.g. a cell, retains at least 50% of the desired function, e.g. at least 60% of the desired function, e.g. at least 70% of the desired function, e.g. at least 80% of the desired function, e.g. at least 90% of the desired function, e.g. at least 95% of the desired function, e.g. 100% of the desired function, as an example for a cell, the important function to be preserved is the viability of the cell. As another example in relation to an organ, an important function to be preserved is a physiological function of the organ, such as a cardiac pump function. As another example with respect to tissue, in the case of transplantation, an important function to be preserved is the ability to maintain the integration of such tissue with surrounding tissue (e.g., skin). As another example of the cell, an egg cell includes a fertilization rate, a cleavage rate, a blastocyst rate, and the like.
In some embodiments, the working solution of the biological sample cryoprotectant comprises 10mM or more of the active fragment, e.g., 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 600mM, 700mM, 800mM, 900mM, or 1000mM, or a range of any two values therebetween.
In some embodiments, the active fragment is an RNA binding protein or a fragment thereof.
In some embodiments, the RNA binding protein is selected from one or more of Rbm15, Rbm47, and Rbm 46.
In some embodiments, a fragment of an RNA-binding protein should comprise a "RRM-LCS" domain.
For example, the fragment of Rbm46 is the C-terminal fragment (310 aa-485aa of Rbm 46), and the structure thereof should include the "RRM 1-RRM2-RRM 3-LCS" domain.
Taking the preferred phase separation protein of the present application, Rbm46, as an example, in one embodiment, the combination of alternative biological sample cryoprotectants may comprise:
balance liquid: buffer solution, 7-13% of human serum albumin, 3-7% of propylene glycol, 3-7% of ethylene glycol, 0.3-0.7M of sucrose, 5-7 mM of Rbm46 protein and/or fragments thereof;
refrigerating fluid: buffer solution, 25-35% of human serum albumin, 3-7% of propylene glycol, 3-7% of ethylene glycol, 0.3-0.7M of cane sugar, 17-23 mM of Rbm46 protein and/or fragments thereof;
thawing the liquid: buffer solution, 3 to 7 percent of propylene glycol, 3 to 7 percent of ethylene glycol, 0.7 to 1.3M of cane sugar, 5 to 7mM of Rbm46 protein and/or fragments thereof;
preferably:
balance liquid: buffer solution, 8-12% of human serum albumin, 4-6% of propylene glycol, 4-6% of ethylene glycol, 0.4-0.6M of sucrose, 5-6 mM of Rbm46 protein and/or fragments thereof;
refrigerating fluid: buffer solution, 27-33% of human serum albumin, 4-6% of propylene glycol, 4-6% of ethylene glycol, 0.4-0.6M of sucrose, 18-22 mM of Rbm46 protein and/or fragments thereof;
thawing the liquid: buffer solution, 4 to 6 percent of propylene glycol, 4 to 6 percent of ethylene glycol, 0.7 to 1.3M of cane sugar, 5 to 7mM of Rbm46 protein and/or fragments thereof;
more preferably:
balance liquid: buffer + 10% human serum albumin + 5% propylene glycol + 5% ethylene glycol +0.5M sucrose +5mM Rbm46 protein and/or fragments thereof;
refrigerating fluid: buffer + 30% human serum albumin + 5% propylene glycol + 5% ethylene glycol +0.5M sucrose +20mM Rbm46 protein and/or fragments thereof;
thawing the liquid: buffer + 5% propylene glycol + 5% ethylene glycol +1M sucrose +5mM Rbm46 protein and/or fragments thereof.
The combination of the above-specified biological sample cryoprotectants is preferably used for cryopreservation of egg cells or fertilized eggs.
In some embodiments, the biological sample cryoprotectant further comprises at least one additional cryoprotectant.
In some embodiments, the additional cryoprotectant is selected from the group consisting of acetamide, agar, alginate, isomalto-oligosaccharide, 1-aniline, albumin, amino acids, ammonium acetate, butylene glycol, chondroitin sulfate, chloroform, choline, diethylene glycol, dimethylacetamide, dimethylformamide, dimethyl sulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, alpha-glycerophosphate, glycerol monoacetate, hyaluronic acid, glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methylacetamide, methyl formamide, methylurea, hydrogels, dextrins, dextrans, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol, serine, sodium bromide, sodium chloride, any one of sodium iodide, pyridine N-oxide, ribose, sodium nitrate, sucrose, trehalose, sodium sulfate, sorbitol, triethylene glycol, trimethylamine acetate, urea, valine, and xylose, or a group thereof;
in some embodiments, the additional cryoprotectant may also be selected from derivatives of the above compounds.
In some embodiments, the biological sample cryoprotectant further comprises a pH buffering agent capable of maintaining substantial neutrality at low temperatures.
The term "low temperature" or "cryopreservation temperature" as used herein generally means the following temperatures: from sub-zero to-196 deg.C, such as from-50 deg.C to-196 deg.C, such as from-80 deg.C to-196 deg.C, for example less than-55 deg.C, such as less than-60 deg.C, such as less than-65 deg.C, such as less than-70 deg.C, such as less than-75 deg.C, such as less than-80 deg.C, such as less than-85 deg.C, such as less than-90 deg.C, such as less than-95 deg.C, such as less than-100 deg.C, such as less than-105 deg.C, such as less than-110 deg.C, such as less than-115 deg.C, such as less than-120 deg.C, such as less than-, e.g., a temperature of less than-165 deg.c, e.g., less than-170 deg.c, e.g., less than-175 deg.c, e.g., less than-180 deg.c, e.g., less than-185 deg.c, e.g., less than-190 deg.c.
The term "pH buffering agent" as used herein 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 buffer properties of such solutions. Thus, a solution or composition that exhibits buffering activity is referred to as a buffer or buffer solution. pH buffering agents generally do not have the unlimited ability to maintain the pH of a solution or composition. Rather, they are generally capable of maintaining a pH within a specified range. The buffer reagents employed herein should maintain a functional pH of the cryoprotected biological sample, such pH typically being "substantially neutral," e.g., about 6.8 to 7.6, or 7.0 to 7.4, or 7.1 to 7.3.
Generally, Buffers are capable of maintaining a pH at their pKa and within the next logarithm (see, e.g., Mohan, Buffers, A guide for the preparation and use of Buffers in biological systems, CALBIOCHEM, 1999). Buffers and buffer solutions are generally prepared from buffered salts or preferably non-ionic buffer components such as TRIS and HEPES. The buffer which can be used in the method of the invention is preferably selected from the group consisting of 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).
In some embodiments, the biological sample cryoprotectant may also include appropriate concentrations of certain ions, such as sodium, potassium, calcium, magnesium, chloride, as would be readily apparent to one of skill in the art, provided that the biological sample is functional after cryopreservation.
In some embodiments, the biological sample cryoprotectant is substantially free of DMSO, generally understood to be DMSO at less than 0.01 w/w%.
In some embodiments, the biological sample cryoprotectant may include other substances to enhance the viability of the sample. Examples of such substances may be constituted by IAPs (apoptosis inhibitors), inhibitors of the rho-associated protein kinase (ROCK) signaling pathway, growth factors such as EGF, FGF, PDGF, IGF, EPO, BDNF, TGF, TNF, VEGF. In another aspect, it may be mentioned that the biological sample cryoprotectant may comprise any serum fraction of human, bovine, equine, canine origin. The cryoprotective agent or composition may also contain a growth medium. In one aspect, growth media comprising a catenin/P300 antagonist and an activator protein/TGFP ligand, such as ID-8 bound to activator protein and TGFI3, may be used. Such media are particularly suitable for the culture of pluripotent stem cells, in particular embryonic stem cells, as described for example in WO 2013/054112. Another example is a standard knock out medium) It includes a knock-out Serum Replacement (KnockOut Serum Replacement) with GlutamaxTMDMEM/F12 supplemented with FGF, NEAA and BME. Another example is mTSERTMProvided is a system. Other examples of growth media according to the sample to be preserved are well known to the skilled person.
According to one aspect of the invention, the invention relates to the use of said cryoprotectant for biological samples for the cryopreservation of biological samples.
According to one aspect of the invention, the invention also relates to a cryopreservation composition comprising a biological sample cryoprotectant as described above, said cryopreservation composition further comprising the biological sample.
The cryopreservation composition is in a process of preparing for cryopreservation (e.g., equilibration process), is in a process of being cryopreserved, has been cryopreserved, or is in a process of thawing.
The amount of the biological sample cryoprotectant present in the cryopreservation composition is typically from 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 preferably from 6 to 10% w/w, or most preferably from 7 to 9% w/w.
According to one aspect of the invention, the invention also relates to a method for the cryopreservation of a biological sample, comprising the steps of: contacting a biological sample to be cryopreserved with a biological sample cryoprotectant as described above, thereby obtaining a cryopreserved composition, followed by reducing the temperature of the cryopreserved composition to a cryopreservation temperature.
According to any one of the embodiments of the present invention, the biological sample comprises an organ, a tissue or a cell.
The species source of the biological sample may be selected from animals including humans and all animal breeds (e.g., livestock and pets) and wild animals and birds including, without limitation, cows, horses, cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, banisters, and the like; the species of the biological sample may also be a plant or a microorganism having a cellular structure.
According to any embodiment of the invention, the organ, tissue is selected from the group consisting of: any of the spleen, heart, liver, brain and other neural tissue, 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, gall bladder, cornea, skin, valve, vein, artery, umbilical cord, placenta, organ-like structures from cell cultures and tumors, or a group thereof;
according to any one of the embodiments of the present invention, the cell is selected from any one of cells from vertebrate or invertebrate tissue, preferably epithelial cells, endothelial cells, fibroblasts, myofibroblasts, hepatocytes, hepatic astrocytes, cardiac myocytes, podocytes, keratinocytes, melanocytes, erythrocytes, neuronal cells (including neurons, astrocytes, microglia, and oligodendrocytes), leukocytes [ 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 type cells) ], tissue stem cells (including mesenchymal stem cells and progenitors of the above-mentioned cells), or a group thereof.
According to any embodiment of the invention, the cell is preferably a eukaryotic cell, more preferably the cell is capable of expressing a protein comprising the active fragment.
According to any embodiment of the present invention, the cells may be isolated single cells, homogeneous or heterogeneous cell aggregates, or cell-containing body fluids such as blood (e.g., umbilical cord blood, peripheral blood), amniotic fluid, semen, cerebrospinal fluid, bone marrow aspirate and menstrual fluid.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
This example illustrates the use of Rbm46 as a cryoprotectant component; the biological sample to be protected is an egg cell.
If not specifically described, Rbm46 used in the examples of the present application is derived from human (Homo sapiens) and has an NCBI Reference Sequence of NP-001264100.1.
Expression of Rbm46 in human egg cells
The distribution of Rbm46 in the egg cells at different stages of meiosis is observed by the inventor, and Rbm46 shows a punctate aggregation distribution in the cells (FIG. 1). There have been some previous articles reporting that RNA binding proteins are capable of intracellular aggregation and participate in the formation of germ cell granules (germ cell granules). We believe that this distribution of Rbm46 is most likely involved in the formation of germ cell grains.
Rbm46 is a phase change protein
It has been previously reported that proteins involved in RNA particle formation in vivo are specifically precipitated by biotin-isox. Biotin-isox spontaneously crystallizes at low temperatures, with a groove in the crystal of exactly the same size as the beta-sheet domain, which induces the protein capable of forming the beta-sheet structure to intercalate into it and catalyse its formation into the beta-sheet conformation. We added biotin-isox to the ovarian cell lysates and after incubation on ice found that Rbm46 was present in the pellet. And this precipitation was reversible, heating the precipitate to 37 degrees celsius, and the Rbm46 was found to re-dissolve again (fig. 2). This suggests that Rbm46 is involved in germ cell granule formation in germ cells as an RNA binding protein.
At higher temperatures, the Rbm46 protein is a single molecule protein in irregular motion; when the temperature is lowered, the two are bonded to each other to form a droplet-like structure (i.e., phase transition). The end of the purified recombinant protein Rbm46C can be used as an additive of an egg freezing solution to remarkably improve the thawing recovery survival rate of eggs.
3. At low temperature, Rbm46 shows punctate distribution in cells
In classical proteins involved in the formation of RNA particles, the domain of the non-fixed conformational region is responsible for the intracellular localization of the protein. After mutation of the non-fixed conformation domain, the protein can not be specifically positioned in the particle region, and the point distribution is changed into the dispersion distribution. We transfected the constructed expression vectors with different segment mutations into HK293T cells, and found that the single C-terminal (310 aa-485aa of Rbm 46) and normal proteins have the same localization pattern, but the protein lacking the C-terminal, i.e., the N-terminal (14 aa-309aa of Rbm 46), is distributed in a dispersed manner in the cells. This suggests that, like classical RNA binding proteins involved in RNA particle formation, the intracellular distribution of Rbm46 is also C-terminal related.
4. The end of the purified recombinant protein Rbm46C can form microphase separation
When the end of purified Rbm46N and the C-terminus were concentrated to about 5mg/ml using ultrafiltration tubes, respectively, significant turbidity was observed in the C-terminal protein. This turbidity was due to the formation of water-insoluble droplets in the protein solution, indicating a typical microphase-segregation of the solution, observed under light microscopy. Over time, these droplets undergo ostwald ripening. After centrifugal enrichment of the droplets, the droplets settled at the bottom of the tube aggregate to form a solid-like hydrogel (hydrogel). The phase separation phenomenon exhibits a temperature sensitive nature, when incubated to 37 degrees celsius, the droplets disappear and the protein turns into a completely dissolved state. This is almost the same as the phase separation of the RNA binding proteins FUS, Hnrnp A, etc. reported in the past. Meanwhile, this phenomenon does not occur at the N-terminus (FIG. 5). As described above, researchers now generally believe that the formation of ribonucleic acid particles that have no membrane structure in vivo, but are clearly distinct from the surrounding cytoplasmic environment, is caused by phase separation. The term "phase separation" means that when the polymer solution is blended, an incomplete thermodynamically stable system is formed, and thus the polymer solvent and the solution are separated from each other, but the polymer remains partially soluble and thus no macroscopic delamination occurs, and the polymer solution has a suspended droplet particle structure. As a physicochemical process, phase separation follows the laws of thermodynamics and occurs spontaneously when the gibbs free energy is less than zero. Gibbs free energy is determined by both enthalpy and entropy changes, so in general phase separation is affected by temperature and concentration. Protein is a kind of high molecular polymer, and the theory of the current research suggests that the component proteins involved in forming ribonucleic acid particles are recruited by certain signals and aggregated in cells, and high concentration is formed in local areas, so that the protein molecules are separated and converted from a completely soluble state into suspended droplets, and the droplets composed of the ribonucleic acid protein and RNA are ribonucleic acid particles (RNA granules). Ribonucleic acid particles are not surrounded by a membrane structure, and exchange substances with the outside at any moment, so that high dynamic property is kept in cells.
5. Oocyte freeze-thaw experiments.
The main reagents used included:
1. balance liquid: 80% phosphate buffer + 10% human serum albumin + 5% propylene glycol + 5% ethylene glycol +0.5M sucrose +5mM Rbm46 protein
2. Refrigerating fluid: 60% phosphate buffer + 30% human serum albumin + 5% propylene glycol + 5% ethylene glycol +0.5M sucrose +20mM Rbm46 protein
3. Thawing the liquid: 90% phosphate buffer + 5% propylene glycol + 5% ethylene glycol +1M sucrose +5mM Rbm46 protein
And (3) freezing process:
transferring the oocyte into a balance liquid, standing at room temperature for 10min, then transferring into a refrigerating fluid (VM) liquid, standing for 5min, transferring the oocyte into a cryopreservation tube, and quickly putting into liquid nitrogen for preservation at-196 ℃.
And (3) unfreezing:
taking out the carrier containing oocyte from liquid nitrogen tank, adding into resuscitation solution rapidly, standing at room temperature for 3min, transferring oocyte into gamete culture solution, and culturing at 37 deg.C under 6% CO2 and saturated humidity.
Subsequently, oocyte survival, fertilization, embryo development and blastocyst development were observed and recorded.
96 oocytes are collected in total, thawing recovery is carried out after freezing, 94 oocytes survive after recovery, and the survival rate is 98%; ICSI (intracytoplasmic injection of single sperm) is then carried out, 92 fertilized eggs are obtained, and the fertilization rate is 95.8%; cracking 90 pieces, with cracking rate 93.7%; 84 blastocysts develop, and the rate of blastocyst formation is 87.5 percent.
Meanwhile, oocyte freeze-thawing of the cryopreserved fluid without Rbm46 was performed in the same manner as described above: in the example, 63 oocytes are collected, 63 oocytes are unfrozen, 46 oocytes survive, the survival rate of the frozen and thawed ova is 73.0%, 41 oocytes are fertilized, the fertilization rate is about 89.1%, the cleavage rate is about 36 oocytes, the cleavage rate is about 87.8%, and the blastocyst rate is about 67.3%.
Example 2
Four sample cryopreserved solutions were prepared using HEK293T cells for cryopreservation testing.
The first sample cryopreservation solution contained Ethylene Glycol (EG) at a concentration of 22.5% (v/v) and phosphate buffer;
the second sample cryopreserving solution contained EG [ 22.5% (v/v) ], 5mM Rbm46 and buffer;
the third sample cryopreserving solution contains 5mM Rbm15 and buffer solution;
the fourth sample cryopreserved solution contained 5mM Rbm47 and buffer.
When HEK293 cells were grown to 80% confluence, the cells were treated with 0.25% trypsin to single cells, digestion was stopped with complete medium and the cells were resuspended, centrifuged at 200g and the supernatant removed. Adding appropriate amount of phosphate buffer solution to resuspend cells, counting cells with hemocytometer, and adjusting cell concentration to 1 × 106And/ml. The cells were split into 4 centrifuge tubes, 1ml per tube, centrifuged at 200g and the supernatant removed.
The cells were resuspended in each of the four sample lysates, 500. mu.L/tube. Then the cells are put into a refrigerator with the temperature of 80 ℃ below zero for quick freezing, the cells are taken out after one week, the cells are quickly re-warmed and thawed in a water bath with the temperature of 37 ℃, and a complete culture medium is added. The positive control sample was 293T cells that were maintained in culture at 37 ℃ without freezing. The negative control sample is added with cell culture medium only in the freezing and thawing process and is subjected to freezing and rewarming treatment.
After thawing, cells were stained with LIVE/DEAD cell viability assay kit (Thermo Fisher) and cell viability was measured using flow cytometry.
The experimental results are shown in fig. 6, and the addition of RNA-binding protein greatly improved the cell viability (statistical differences between # 2, # 3, and # 4 compared to # 1), which indicates that RNA-binding protein has strong ability to cryopreserve cells.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (12)
1. The working solution of the biological sample cryoprotectant contains more than or equal to 5mM of active fragments;
the active fragment is a protein capable of Phase separation (Phase separation or Phase transition) and comprising an RNA binding domain (RNA binding domain) and a low-complexity sequence domain (LCD), the protein being selected from one or more of RNA binding proteins Rbm15, Rbm47 and Rbm 46.
2. The biological sample cryoprotectant of claim 1, wherein the working solution comprises greater than or equal to 10mM of the active fragment.
3. The biological sample cryoprotectant of claim 1, further comprising at least one additional cryoprotectant.
4. The biological sample cryoprotectant of claim 3, said additional cryoprotectant being selected from the group consisting of acetamide, agar, alginate, isomalto-oligosaccharide, 1-aniline, albumin, amino acids, ammonium acetate, butylene glycol, chondroitin sulfate, chloroform, choline, diethylene glycol, dimethylacetamide, dimethylformamide, dimethyl sulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, alpha-glycerophosphate, glycerol monoacetate, hyaluronic acid, glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methylacetamide, methyl formamide, methyl urea, hydrogels, dextrins, dextrans, phenol, pluronic polyols, polyethylene glycols, polyvinylpyrrolidone, proline, any one of propylene glycol, serine, sodium bromide, sodium chloride, sodium iodide, pyridine N-oxide, ribose, sodium nitrate, sucrose, trehalose, sodium sulfate, sorbitol, triethylene glycol, trimethylamine acetate, urea, valine, and xylose, or a group thereof.
5. The biological sample cryoprotectant of claim 1, further comprising a pH buffering agent capable of maintaining approximately neutral at 0 ℃ to-196 ℃.
6. Use of the biological sample cryoprotectant of any one of claims 1 to 5 for the preparation of a cryoprotectant for cryopreservation of a biological sample.
7. The use of claim 6, wherein the biological sample is an organ, tissue or cell, or a combination thereof.
8. The use according to claim 7, wherein the organ, tissue is selected from the group consisting of: any of the spleen, heart, liver, brain and other neural tissue, 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, gall bladder, cornea, skin, valve, vein, artery, umbilical cord, placenta, organ-like structures from cell cultures and tumors, or a group thereof;
the cells are selected from cells from vertebrate or invertebrate tissue.
9. The use of claim 8, wherein the cell is any one of epithelial cells, endothelial cells, fibroblasts, myofibroblasts, hepatocytes, hepatic stellate cells, cardiomyocytes, podocytes, keratinocytes, melanocytes, erythrocytes, neuronal cells, leukocytes, tissue stem cells, or a group thereof.
10. The use according to claim 9, wherein the leukocyte is any one of a dendritic cell, neutrophil, sperm, egg cell, fertilized egg, macrophage or lymphocyte.
11. A cryopreservation composition comprising a biological sample cryoprotectant as claimed in any one of claims 1 to 5, said cryopreservation composition further comprising a biological sample; the biological sample is as defined in claim 7 or 8.
12. A method for the cryopreservation of a biological sample comprising the steps of: contacting a cryopreserved biological sample with the biological sample cryoprotectant of any one of claims 1 to 5, thereby obtaining a cryopreserved composition, followed by reducing the temperature of the cryopreserved composition to a cryopreservation temperature; the biological sample is as defined in claim 7 or 8.
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