WO2020207151A1 - 一种无dmso的冷冻保存液及其制备方法与应用 - Google Patents
一种无dmso的冷冻保存液及其制备方法与应用 Download PDFInfo
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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
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06086—Dipeptides with the first amino acid being basic
- C07K5/06095—Arg-amino acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
- C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
Definitions
- the invention belongs to the technical field of biomedical materials, and specifically relates to a DMSO-free cryopreservation liquid and a preparation method thereof.
- cryopreservation technology Since the advent of cryopreservation technology, it has become one of the indispensable research methods in the field of natural science and has been widely adopted. With the improvement of living standards and the development of medical technology, the cryopreservation of human germ cells (sperm, oocyte) and gonadal tissue has become an important means of preserving fertility. In addition, with the aging of the world population, the demand for cryopreservation of donated human-derived cells, tissues or organs that can be used in regenerative medicine and organ transplantation is also rapidly increasing. Therefore, how to efficiently cryopreserve precious cells, tissues and organ resources has become an important topic in the field of life sciences.
- the most commonly used cryopreservation method is vitrification. Although the vitrification technology can make the liquid inside and outside the cell directly become vitreous during the rapid freezing process, it avoids the damage caused by the formation of ice crystals during the freezing process. However, during the rewarming process, the existing cryopreservation reagents cannot effectively control the growth of ice crystals, thereby damaging cells. Dimethyl sulfoxide (DMSO) is a commonly used flux and permeable cell cryopreservation agent for in vitro cell culture. However, DMSO has adverse side effects in clinical patient trials.
- DMSO Dimethyl sulfoxide
- DMSO also has strong cytotoxicity and different kinds of The sensitivity of cells to the concentration of DMSO is different, leading to the toxic side effects of cryopreservation reagents with DMSO as the main protective agent component on cells, and its application is limited.
- the high concentration ( ⁇ 15%) of DMSO commonly used in the current vitrification method seriously affects the survival rate, even (offspring) safety and functional expression of cryopreserved objects after resuscitation.
- the currently used cryopreservation reagents do not have the ability to effectively control the growth of ice crystals during the rewarming process, and at the same time have the problem of high toxicity of the reagents.
- the present invention provides a DMSO-free cryopreservation solution and a preparation method thereof.
- a DMSO-free cryopreservation solution per 100mL volume, containing 0.01-50.0g of bionic ice-controlling material, 5.0-45mL of polyol, 0.1-1mol L -1 of water-soluble sugar, 0-30mL of serum, and buffer
- the bionic ice control material is selected from polyvinyl alcohol PVA and/or amino acid bionic ice control material, and the cryopreservation liquid does not contain dimethyl sulfoxide (DMSO).
- the amino acid type bionic ice control material is selected from one of polyamino acids (polymerization degree ⁇ 2, preferably polymerization degree 8-40, for example, polymerization degree 8, 15, 20, etc.), amino acids, peptide compounds One or more than two.
- the peptide compound is a polypeptide (preferably a peptide composed of 2-8 different amino acids, such as 2 peptides, 3 peptides, 4 peptides), glycopeptide derivatives, compounds represented by formula (I),
- R is selected from substituted or unsubstituted alkyl, and the substituted group may be selected from -OH, -NH 2 , -COOH, -CONH 2 etc., for example, R is substituted or unsubstituted C 1-6 Alkyl, preferably R is -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 COOH; n is an integer greater than or equal to 1 and less than or equal to 1000, for example, an integer in the range of 1-100. In some embodiments of the present invention, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10.
- the polyol may be a C2-5 polyol, preferably a C2-C3 diol or triol, such as any one of ethylene glycol, propylene glycol, and glycerol.
- the water-soluble sugar may be at least one of non-reducing disaccharides, water-soluble polysaccharides, water-soluble cellulose, and sugar anhydrides, for example selected from sucrose, trehalose, polysucrose, and hydroxypropyl methyl Cellulose.
- the water-soluble sugar can protect the cell membrane and prevent cell sedimentation.
- the buffer may be at least one of DPBS, hepes-buffered HTF buffer, and other cell buffers.
- the serum can be selected from human serum albumin or its substitutes for human-derived cryopreserved objects, such as sodium dodecyl sulfonate SDS, and for non-human-derived cryopreserved objects, fetal bovine serum or bovine can be selected.
- Serum albumin for human-derived cryopreserved objects, such as sodium dodecyl sulfonate SDS, and for non-human-derived cryopreserved objects, fetal bovine serum or bovine can be selected.
- Serum albumin such as sodium dodecyl sulfonate SDS
- the bionic ice-controlling material may be PVA, and the PVA content may be 0.1-6.0g, for example 0.5-5.0g; specifically, it may be 1.0g, 2.0g, 3.0g, 4.0g .
- the bionic ice-controlling material may be a polyamino acid or an amino acid, and the content of the polyamino acid or amino acid is 0.01-50g, for example 1.5-50g; specifically, it may be 8.0g, 10g, 15g , 20g, 30g, 40g.
- the ice-controlling material may be a combination of PVA and polyamino acid, for example, composed of 0.1-5.0 g PVA and 1.0-9.0 g polyamino acid.
- the ice-controlling material may be a combination of PVA and amino acids, for example, composed of 0.1-5.0 g of PVA and 8.0-35 g of amino acids.
- the content of the polyol is 6.0-28 mL, such as 7.0-20 mL, 10-15 mL.
- the serum content is 0.1-30 mL per 100 mL, such as 5.0-20 mL, 10-15 mL.
- the cryopreservation solution is calculated per 100 mL, and preferably the serum content is 0;
- cryopreservation liquid according to the present invention cryopreservation solution per 100mL of said water-soluble sugar content is 0.1-1.0mol L -1, e.g. 0.1-0.8mol L -1, 0.2-0.6mol L -1; specifically, e.g. 0.25mol L -1 , 0.5mol L -1 , 1.0mol L -1 .
- the cryopreservation solution according to the present invention has a pH of 6.5-7.6, for example, 6.9-7.2.
- the cryopreservation solution is composed of the following components per 100 mL volume:
- the cryopreservation liquid is composed of the following components based on a volume of 100 mL:
- the cryopreservation solution is composed of the following components per 100 mL volume:
- it is composed of the following components per 100 mL volume:
- the cryopreservation solution is composed of the following components per 100 mL volume:
- the cryopreservation liquid is composed of the following components based on a volume of 100 mL:
- the cryopreservation solution is composed of the following components per 100 mL volume:
- the cryopreservation liquid is composed of the following components based on a volume of 100 mL:
- the present invention also provides a method for preparing the cryopreservation solution, which includes the following steps: dissolving the bionic ice control material in DPBS, adjusting the pH after cooling to room temperature, and dissolving other components except serum in the remaining DPBS, After cooling, mix, reconfirm or adjust the pH and make up the remaining buffer. Serum is added during use.
- the preparation method according to the present invention includes the following steps:
- the solution 1 optionally the solution 2, and the solution 3 are cooled to room temperature, they are mixed, the pH value is adjusted, and the volume of the remaining buffer is made up to a predetermined volume to obtain the cryopreservation solution.
- the preparation method according to the present invention includes the following steps:
- solution 1 Dissolve polyamino acid or amino acid in a part of buffer solution, adjust pH after cooling to room temperature, to form solution 1;
- the solution 1 optionally the solution 2, and the solution 3 are cooled to room temperature, they are mixed, the pH value is adjusted, and the volume of the remaining buffer is made up to a predetermined volume to obtain the cryopreservation solution.
- the serum is added when the cryopreservation solution is used.
- the step (1) PVA is dissolved in warm bath heating, such as oil bath or water bath heating; for example, the temperature of the water bath is 60-95°C, preferably 80°C.
- the dissolution includes a stirring step.
- the dissolution is ultrasonic assisted dissolution.
- a DMSO-free frozen balance solution per 100mL volume, containing 0-5.0g PVA, 5.0-45mL polyol, 0-30mL serum, and buffer balance.
- the PVA content is 0.1-5.0g, for example, 0.1g, 0.5g, 1.0g, 2.0g.
- the content of the polyol is 6.0-28 mL, for example 7.0-20 mL, 10-15 mL.
- the serum content is 0.1-30 mL, such as 5.0-20 mL, 10-15 mL.
- the serum content is zero.
- the freezing balance solution contains 7.5-15 mL of polyol, 10-20 mL of serum, and the remaining amount of DPBS per 100 mL volume.
- the freezing balance solution contains 1.0-5.0 g of PVA, 7.5-15 mL of polyol, and a buffer balance per 100 mL volume.
- the PVA, polyol and serum can be selected from the types of corresponding components of the freezing solution.
- the present invention also provides a preparation method of the above freezing balance solution, which includes dissolving each component in a buffer solution, storing the serum separately, and adding it during use.
- a DMSO-free reagent for cryopreservation includes the above freezing balance solution and the above freezing preservation solution, and the freezing balance solution and the freezing preservation solution exist independently.
- the serum content of the cryopreservation solution is 0, and the freezing balance solution contains 1.0-5.0 g of PVA, 7.5-15 mL of polyol, and a buffer balance per 100 mL volume.
- the freezing balance liquid includes the following components per 100 mL volume:
- the cryopreservation solution is based on a total volume of 100 mL and includes the following components:
- the PVA is selected from one or a combination of two or more of isotactic PVA, syndiotactic PVA and random PVA, for example, the syndiotacticity of the PVA is 15%-60%, preferably 45%- 60%, for example, 50%-55%.
- the PVA may be selected from PVA with a molecular weight of 10-500 kDa or higher, for example, a molecular weight of 10-30 kDa, 30-50 kDa, 80-90 kDa, 200-500 kDa.
- the PVA may be selected from PVA with a degree of hydrolysis greater than 80%, for example, the degree of hydrolysis is 80%-99%, 82-87%, 87%-89%, 89%-99%, 98%-99 %.
- the polyamino acid can be selected from homopolymers of at least one of lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, glycine, etc. (Polymerization degree ⁇ 2).
- the peptide compound is a polypeptide, which is composed of two or more amino acids, which can be selected from L-Thr-L-Ar G(TR), L-Thr-L-Pro(TP), L-Arg -L-Thr(RT), L-Pro-L-Thr(PT), L-Thr-L-Arg-L-Thr(TRT), L-Thr-L-Pro-L-Thr(TPT), L -One or more of Ala-L-Ala-L-Thr (AAT).
- Polypeptide synthesis methods known in the art can be used, such as solid phase synthesis to synthesize these polypeptides.
- the glycopeptide derivatives are synthesized from carbohydrates and amino acids, for example, molecules composed of glucose lactone (GDL) and ice-philic amino acids through chemical bonding, such as: GDL-L-Thr, GDL-L-Gln , GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Thr, etc.
- the glycopeptide compound can be prepared by a reaction method of sugars and amino acids known in the art, for example, solid-phase synthesis or reaction of sugars and amino acids in an organic solvent.
- the peptide compound has any structure shown in formula (1) to formula (8):
- the compound represented by formula (I) has any of the following structures:
- the compound represented by formula (9) is prepared by the following synthetic route:
- the dosage of each component is based on the total volume of 100 mL of the solution, and the remainder is buffer solution.
- the present invention also provides the application of the above cryopreservation solution in cryopreservation of various cells, organs and tissues, including cryopreservation of oocytes, embryos, various types of stem cells, organs and tissues.
- Organs and tissues include but are not limited to ovarian organs and ovarian tissues.
- the present invention further provides a method for freezing and thawing cells or embryos, including:
- the cells or embryos are put in a balance solution for balance before being placed in the cryopreservation solution.
- the present invention further provides a method for cryopreservation of stem cells using a droplet method.
- the method for cryopreservation of stem cells includes the following steps: adding the cryopreservation solution to the stem cells, pipetting and dispersing to prepare a stem cell suspension, and removing the stem cell suspension Placed on a frozen slide, stored in liquid nitrogen (-196°C).
- the thawing of cryopreserved stem cells includes placing the frozen slide with the stem cells in a-MEM medium and thawing at 37°C.
- the stem cells are various stem cells known in the art that have differentiation functions, such as totipotent stem cells, pluripotent stem cells or multipotent stem cells, including but not limited to embryonic stem cells and various types of mesenchymal stem cells ( For example, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, etc.), hematopoietic stem cells, etc.
- differentiation functions such as totipotent stem cells, pluripotent stem cells or multipotent stem cells, including but not limited to embryonic stem cells and various types of mesenchymal stem cells ( For example, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, etc.), hematopoietic stem cells, etc.
- the present invention also provides a cryopreservation method for organs and/or tissues, which includes: balancing the organs and/or tissues in a cryopreservation solution, then putting the organs and/or tissues in the cryopreservation solution, and then putting the organs and/or tissues Place it on a frozen slide and store in liquid nitrogen.
- the organ and/or tissue is an ovarian tissue or an ovarian organ, which may be an ovarian tissue section or a complete ovarian tissue.
- cryopreservation and “cryopreservation” have the same meaning and can be used interchangeably. It refers to the preservation of certain substances or cells, tissues, and organs at low temperatures to maintain their original physical and chemical and/or biological activities. Physiological and biochemical functions.
- the type of "stem cells” is not particularly limited.
- the cryopreservation solution of the present invention can be used to cryopreserve various types of stem cells known in the art, including but not limited to umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and fat Mesenchymal stem cells, hematopoietic stem cells, etc.
- biological tissues may be derived from animals, including warm-blooded mammals, such as humans and primates; birds; domesticated domestic or farm animals, such as cats, dogs, sheep, goats, cows, horses, and pigs. ; Laboratory animals, such as mice, rats and guinea pigs; fish; reptiles; zoo animals and wild animals.
- the cryopreservation solution and freezing balance solution provided by the present invention do not contain DMSO. When used for cryopreservation of mouse oocytes and embryos, they can reach the same level as or even better than commercial cryopreservation solutions (containing 15% DMSO by volume). High cell and tissue survival rate and functional expression stability, with high preservation efficiency. Among them, the cryopreservation solution without DMSO or serum further solves the problems of poor stability of the commercial cryopreservation solution commonly used in clinical practice due to the presence of serum and easy introduction of parasitic biological contaminants.
- the cryopreservation solution of the present invention has simple composition, convenient source of raw materials, low cost, and can be widely used in cryopreservation of oocytes, cell-like cells (such as embryos, etc.), stem cells, tissues and organs.
- Figure 1 is a stained picture of fresh (unfrozen) ovarian organ slices of a 3-day-old mouse
- Fig. 2 is a picture of section staining after thawing of frozen whole ovarian organs in Comparative Example 7;
- Figure 3 is a picture of section staining after thawing of frozen whole ovarian organs in Application Example 14;
- Figure 4 is a picture of section staining after thawing of frozen whole ovarian organs in Application Example 15;
- Figure 5 is a sectioned stained picture of the frozen ovarian organs of Application Example 16 after thawing;
- Figure 6 is a stained picture of a fresh (unfrozen) ovarian tissue section of a sexually mature mouse
- Fig. 7 is a section staining picture of a frozen ovarian tissue section of Comparative Example 8 after thawing;
- Figure 8 is a section staining picture of the frozen ovarian tissue section of Application Example 17 after thawing;
- Figure 9 is a section staining picture of the frozen ovarian tissue section of Application Example 18 after thawing;
- Fig. 10 is a section staining picture of the frozen ovarian tissue section of Application Example 19 after thawing.
- the PVA used in the embodiment of the present invention has a syndiotacticity of 50%-55%, a molecular weight of 13-23 kDa, and a degree of hydrolysis of 98%.
- poly-L-proline has a degree of polymerization of 8 or 15, and a molecular weight of 795 and 1475;
- poly-L-arginine has a degree of polymerization of 8, and a molecular weight of 1267.
- the degree of polymerization of poly-L-proline in the thawing solution is 8, and the molecular weight is 795.
- the survival rate in the embodiment of the present invention is the average survival rate of 3-12 repeated experiments.
- Cryopreservation Solution A Each 100ml contains the following components:
- Cryopreservation Solution B Each 100ml contains the following components:
- Freeze preservation solution preparation steps Heat 2.0g of PVA in a water bath at 80°C and dissolve it in 20mL of DPBS with magnetic stirring, adjust the pH to 7.1, which is solution 1; mix 8.0g of L-Arg and 4.0g of L- Thr was dissolved in 20mL of DPBS, adjusted to pH 7.1, and was solution 2; 17g (0.05mol) of sucrose (the final concentration of sucrose in the cryopreservation solution was 0.5mol L -1 ) was dissolved in 20mL of DPBS by ultrasound, and the sucrose After all is dissolved, add 10 mL of ethylene glycol to make solution 3; after solution 1, solution 2 and solution 3 return to room temperature, mix the three solutions evenly, adjust the pH to 7.1 and use DPBS to make up the balance to the total 80% of the volume, add 20 mL of serum during use.
- Each 100ml contains the following components:
- Each 100ml contains the following components:
- Frozen balance solution a Heat 2.0 g of PVA in a water bath at 80°C and dissolve it in 50 mL of DPBS with magnetic stirring. After all the PVA is dissolved, adjust the pH to 7.0, add 7.5 mL of ethylene glycol, mix well, and determine with DPBS. Make up to 100mL, set aside.
- Frozen balance solution b The total volume is 100 mL. Dissolve 7.5 mL of ethylene glycol in 72.5 mL of DPBS, mix well, and add 20 mL of serum during use.
- Frozen Balance Solution 1# Each 1mL contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, and the balance is DPBS;
- Cryopreservation solution 1# each 1mL contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, the balance For DPBS.
- Frozen balance solution b each 1mL contains 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, and the balance is DPBS;
- Cryopreservation Solution 2# Each 1 mL contains 10% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, and the balance is DPBS.
- Example 1 There are three types of thawing solution formulas used in Example 1 and Comparative Example 1 of the present invention as follows:
- Thaw solution 1# Thaw solution I (containing 1.0mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw solution II (containing 0.5mol L -1 sucrose, 20% serum, the balance is DPBS) ; Thaw Solution III (containing 0.25mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw Solution IV (20% serum, the balance is DPBS).
- Thaw solution 2# Thaw solution I (contains 1.0mol L -1 sucrose, 20mg mL -1 PVA, the balance is DPBS); Thaw solution II (contains 0.5mol L -1 sucrose, 20mg mL -1 PVA, The remainder is DPBS); Thaw Solution III (contains 0.25mol L -1 sucrose, 20 mg mL -1 PVA, the balance is DPBS); Thaw Solution IV (20 mg mL -1 PVA, the remainder is DPBS).
- thawing solution I containing 1.0mol L -1 of sucrose, 20mg mL PVA -1 and polyproline 10mg mL -1 of balance DPBS
- thawed solution II containing 0.5mol L - 1 sucrose, 20mg mL PVA -1 and polyproline 5.0mg mL -1, with the balance of DPBS
- thawed solution III containing 0.25mol L -1 sucrose, 20mg mL PVA -1's, 2.5mg mL - 1 polyproline, the balance is DPBS
- thawing solution IV (20mg mL -1 PVA, the balance is DPBS).
- the oocytes and embryos were cryopreserved according to the schemes in Table 1 and Table 2, respectively.
- the mouse oocytes are first placed in the cryopreservation solution to equilibrate for 5 minutes; then they are placed in the prepared cryopreservation solution for 1 minute, and the oocytes that have been equilibrated in the cryopreservation solution are placed on the freezing rod, and then Quickly put it into liquid nitrogen (-196°C), and close the carrier rod and continue to save; when thawing, put the frozen oocytes in 37°C thawing solution I for 5 minutes, and then sequentially in the thawing solution II- Equilibrate each for 3 minutes in IV; culture the thawed oocytes for 2 hours and observe the number of viable cells, and calculate the survival rate (see Table 1).
- the mouse embryos are first placed in the cryopreservation solution to equilibrate for 5 minutes, and then placed in the prepared cryopreservation solution for 50 seconds.
- the embryos that have been equilibrated in the cryopreservation solution are placed on the freezing rod, and then quickly put into liquid nitrogen (- 196°C), and seal the loading rod and continue to save; when thawing, put the frozen embryos in 37°C Thawing Solution I for 3 minutes, and then equilibrate in Thawing Solution II-IV for 3 minutes; the thawing is complete
- the embryos were cultured for 2 hours, the number of surviving embryos was observed, and the survival rate was calculated (see Table 2).
- the DMSO-free cryopreservation solution and cryopreservation solution of the present invention through the synergistic effect of each component, can be used in oocytes even without adding DMSO.
- the cryopreservation of cells and embryos still has a good preservation effect, which solves the defect that the existing cryopreservation solution is toxic to cells or embryos due to the addition of a larger concentration of DMSO.
- Example 2 Cryopreservation of human umbilical cord mesenchymal stem cells
- Cryopreservation Solution E Total volume 100mL, containing 10mL of ethylene glycol, 20mL of serum, 17g of sucrose (0.5mol L -1 ), 4.0g of poly-L-arginine (polymerization degree 8), 1.0g of PVA, and the remaining amount of DPBS .
- Cryopreservation solution F total volume 100 mL, containing 20 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L -1 ), 16 g of L-Arg, 8.0 g of L-Thr, and the remainder of DPBS.
- Cryopreservation solution G total volume 100 mL, containing 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L -1 ), 2.0 g of PVA, and the remainder of DPBS.
- Cryopreservation solution H total volume 100 mL, containing 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L -1 ), 28 g of TR, and the remainder of DPBS.
- Cryopreservation Solution I The total volume is 100mL, containing 10mL of ethylene glycol, 17g of sucrose (0.5mol L-1), 2.0g of PVA, and the balance of DPBS.
- the preparation method of the cryopreservation solution is the same as in Example 1.
- the preparation method of TR is as follows:
- Cryopreservation solution 3# each 1 mL contains 10% (v/v) DMSO, 15% (v/v) fetal bovine serum, and the balance is a-MEM medium (USA, Invitrogen, C12571500BT).
- the above cryopreservation solution was used to perform cryopreservation of human umbilical cord-filled mesenchymal stem cells according to the protocol in Table 3.
- the cryopreservation method of human umbilical cord stem cells is specifically the microdrop method: the human umbilical cord mesenchymal stem cells on the culture dish are digested with 25% trypsin for 2 minutes, and then placed in an equal volume of culture medium (10% FBS + a-MEM medium ), gently pipette until all the stem cells fall off, add a 1.5ml centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, separate the cells from the culture medium, add 10uL of freezing solution to the bottom of the centrifuge tube, gently pipette to disperse the stem cells, this 10uL
- the freezing solution with stem cells is placed on a frozen slide and stored in liquid nitrogen (-196 degrees Celsius).
- the survival rate of stem cells can reach 92.4% even without DMSO (Application Example 9), and even when DMSO and serum are not added at all, the survival rate can reach 77.1% (Application example 13) shows that the freezing reagent can not only achieve the effectiveness of conventional freezing liquid freeze-dried cells, but also much higher than the cryopreservation recovery rate of the commonly used cryopreservation solution containing 10% DMSO (Comparative Example 5) , The cryopreservation effect based on PVA is significantly better than Comparative Example 6 without PVA.
- Example 3 Cryopreservation of intact ovarian organs and ovarian tissue sections
- Cryopreservation solution J a total volume of 100 mL, containing 10 mL of ethylene glycol, 17 g of sucrose (0.5 mol L -1 ), 2.0 g of PVA, and the remainder of DPBS.
- Cryopreservation solution K total volume 100 mL, containing 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L -1 ), 1.0 g of PVA, and the remainder of DPBS.
- Cryopreservation solution L contains 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L -1 ), 4.0 g of poly-L-arginine (polymerization degree 8), 1.0 g of PVA, and the balance of DPBS.
- each 1mL cryopreservation solution contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) serum, 0.5M sucrose, and the balance is DPBS.
- Frozen balance solution a Heat 2.0 g of PVA in a water bath at 80°C and dissolve it in 50 mL of DPBS with magnetic stirring. After all the PVA is dissolved, adjust the pH to 7.0, add 7.5 mL of ethylene glycol, mix well, adjust the pH and Make up the remaining volume to 100mL and set aside.
- Frozen balance solution b 7.5 mL of ethylene glycol is added to 72.5 mL of DPBS, mixed well, and 20 mL of serum is added during use;
- Frozen Balance Solution 1# Each 1mL contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, and the balance is DPBS;
- Cryopreservation solution 1# each 1mL contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5mol L -1 sucrose , The balance is DPBS.
- Thaw solution 1# Thaw solution I (containing 1.0mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw solution II (containing 0.5mol L -1 sucrose, 20% serum, the balance is DPBS) ; Thaw Solution III (containing 0.25mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw Solution IV (20% serum, the balance is DPBS).
- Thaw solution 2# Thaw solution I (contains 1.0mol L -1 sucrose, 20mg mL -1 PVA, the balance is DPBS); Thaw solution II (contains 0.5mol L -1 sucrose, 20mg mL -1 PVA, The remainder is DPBS); Thaw Solution III (contains 0.25mol L -1 sucrose, 20 mg mL -1 PVA, the balance is DPBS); Thaw Solution IV (20 mg mL -1 PVA, the remainder is DPBS).
- cryopreservation solution and the freezing balance solution and cryopreservation solution of the comparative example were used to cryopreserve intact ovarian organs of mice and ovarian tissue sections of sexually mature mice within 3 days of newborn according to the schemes in Table 4 and Table 5.
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Abstract
Description
物质 | 用量 |
聚-L-脯氨酸(g) | 1.5 |
PVA(g) | 2.0 |
乙二醇(mL) | 10 |
蔗糖(mol L -1) | 0.5 |
DPBS(mL) | 余量 |
物质 | 含量 |
L-Arg(g) | 8.0 |
L-Thr(g) | 4.0 |
PVA(g) | 2.0 |
乙二醇(mL) | 10 |
蔗糖(mol L -1) | 0.5 |
胎牛血清(mL) | 20 |
DPBS(mL) | 余量 |
物质 | 用量 |
PVA(g) | 2.0 |
乙二醇(mL) | 10 |
蔗糖(mol L -1) | 0.5 |
胎牛血清(mL) | 20 |
DPBS(mL) | 余量 |
物质 | 用量 |
PVA(g) | 2.0 |
乙二醇(mL) | 10 |
蔗糖(mol L -1) | 0.5 |
DPBS(mL) | 余量 |
编号 | 平衡液 | 冷冻保存液 | 解冻液 | 冻卵总数 | 2小时后存活率 |
应用实例1 | a | A | 解冻液1# | 39 | 89.7% |
应用实例2 | a | A | 解冻液3# | 60 | 98.6% |
应用实例3 | b | B | 解冻液1# | 109 | 94.8% |
应用实例4 | b | C | 解冻液1# | 90 | 97.7% |
应用实例5 | a | D | 解冻液1# | 50 | 93.4% |
应用实例6 | a | D | 解冻液2# | 53 | 96.5% |
对比实例1 | 平衡液1# | 冷冻液1# | 解冻液1# | 146 | 95 |
对比实例2 | b | 冷冻液2# | 解冻液1# | 96 | 81.9% |
编号 | 平衡液 | 冷冻保存液 | 解冻液 | 冻胚胎总数 | 2小时后存活率 |
应用实例7 | a | A | 解冻液1# | 42 | 95.2% |
应用实例8 | a | D | 解冻液1# | 41 | 95.8% |
对比实例3 | 平衡液1# | 冷冻液1# | 解冻液1# | 38 | 94.3% |
对比实例4 | b | 冷冻液2# | 解冻液1# | 39 | 82.4% |
编号 | 冷冻保存液 | 冷冻保存方法 | 存活率 |
应用实例9 | 冷冻保存液E | 微滴法 | 92.4% |
应用实例10 | 冷冻保存液F | 微滴法 | 71.0% |
应用实例11 | 冷冻保存液G | 微滴法 | 72.2% |
应用实例12 | 冷冻保存液H | 微滴法 | 75.1% |
应用实例13 | 冷冻保存液I | 微滴法 | 77.1% |
对比实例5 | 冷冻保存液3# | 微滴法 | 76.6% |
对比实例6 | 冷冻保存液1# | 微滴法 | 63.9% |
编号 | 平衡液 | 冷冻保存液 | 解冻液 | 形态 |
应用实例14 | a | J | 解冻液2# | 图3 |
应用实例15 | b | K | 解冻液1# | 图4 |
应用实例16 | b | L | 解冻液1# | 图5 |
对比实例7 | 平衡液1# | 冷冻液1# | 解冻液1# | 图2 |
编号 | 平衡液 | 冷冻保存液 | 解冻液 | 形态 |
应用实例17 | a | J | 解冻液2# | 图8 |
应用实例18 | b | K | 解冻液1# | 图9 |
应用实例19 | b | L | 解冻液1# | 图10 |
对比实例8 | 平衡液1# | 冷冻液1# | 解冻液1# | 图7 |
Claims (12)
- 一种无DMSO的冷冻保存液,其特征在于,以每100mL体积计,含有仿生控冰材料0.01-50g,多元醇5.0-45mL,水溶性糖0.1-1.0mol L-1,血清0-30mL,余量为缓冲液,所述仿生控冰材料选自PVA和/或氨基酸类仿生控冰材料。
- 根据权利要求1或2所述的冷冻保存液,所述控冰材料为PVA,所述PVA含量为0.1-6.0g;优选,所述多元醇可以为C2-5的多元醇,例如乙二醇,丙二醇,丙三醇中的任一种;优选,所述水溶性糖可以为非还原性双糖、水溶性多糖、糖酐中的至少一种,例如选自蔗糖、水溶性纤维素(例如羟丙基甲基纤维素)、海藻糖、聚蔗糖;优选,所述缓冲液可以为DPBS、hepes-buffered HTF缓冲液、其他细胞缓冲液中的至少一种;根据本发明,所述血清针对人源性冷冻保存对象可选人血清白蛋白或其替代物,例如十二烷基磺酸钠,针对非人源性冷冻保存对象可选胎牛血清或牛血清白蛋白。
- 根据权利要求1-3任一项所述的冷冻保存液,其特征在于,所述仿生控冰材料为聚氨基酸或氨基酸,所述控冰材料的含量为0.01-50g;优选地,所述控冰材料为PVA与氨基酸和/或聚氨基酸的组合,例如由0.1-5.0g的PVA和8.0-35g的氨基酸和/或1.0-9.0g聚氨基酸组成。
- 根据权利要求1-4任一项所述的冷冻保存液,其特征在于,所述PVA选自等规PVA、间规PVA和无规PVA的一种或两种以上的组合,例如所述PVA的间同规整度为15%-60%,优选50%-60%;优选,PVA选自分子量为10-500kDa或者更高分子量的PVA;优选,所述PVA选自水解度为大于80%的PVA;优选,所述聚氨基酸选自赖氨酸、精氨酸、脯氨酸、苏氨酸、组氨酸、谷酰胺酸、天冬氨酸、甘氨酸等中至少一种的均聚物(聚合度≥2);优选,所述多肽选自2-8个不同的氨基酸组成的肽,例如2肽、3肽、4肽,例如L-Thr-L-Arg(TR),L-Thr-L-Pro(TP),L-Arg-L-Thr(RT),L-Pro-L-Thr(PT),L-Thr-L-Arg-L-Thr(TRT),L-Thr-L-Pro-L-Thr(TPT),L-Ala-L-Ala-L-Thr(AAT)中的一种或两种以上;优选,所述糖肽衍生物为糖类与氨基酸合成,例如为葡萄糖内酯(GDL)与亲冰氨基酸通过化学键合而组成的分子,例如:GDL-L-Thr,GDL-L-Gln,GDL-L-Asn,GDL-L-Phe,GDL-L-Tyr,GDL-L-Val,GDL-L-Ser中的至少一种。
- 根据权利要求1-5任一项所述的冷冻保存液,其特征在于,所述多元醇含量为6.0-28mL;优选,所述血清含量为0。优选,所述水溶性糖含量为0.1-1.0mol L -1;优选,所述冷冻保存液pH为6.5-7.6。
- 如权利要求1-6任一项所述的冷冻保存液的制备方法,其特征在于,包括如下步骤:将控冰材料溶解于缓冲液中,冷却到室温后调节pH,将其他组分溶解于剩下的缓冲液中,冷却后混合;优选,包括如下步骤:(1)将PVA溶解于一部分缓冲液中,冷却到室温后调节pH,得到溶液1;(2)任选地,将聚氨基酸或氨基酸溶解于一部分缓冲液,冷却到室温后调节pH,形成溶液2;(3)将水溶性糖溶解于一部分缓冲液中,待水溶性糖全部溶解后加入除血清的其他组分,制得溶液3;(4)待溶液1、任选地溶液2、和溶液3冷却至室温后混合,调节pH并采用缓冲液定容至预定体积,得到所述冷冻保存液;任选地,当所述冷冻保存液含有血清时,所述血清在所述冷冻保存液使用时添加。
- 一种无DMSO的冷冻平衡液,其特征在于,以每100mL计,含有PVA 0-5.0g,多元醇5.0-45mL,血清0-30mL,缓冲液余量;优选地,所述PVA含量为0.1-4.0g。
- 根据权利要求8所述的冷冻平衡液,其特征在于,所述冷冻平衡液以每100mL计,含有多元醇7.5-15mL,血清10-20mL,缓冲液余量;优选地,所述冷冻平衡液以每100mL计,含有PVA 0.5-3.5g,多元醇7.5-15mL,缓冲液余量。
- 一种无DMSO的冷冻保存试剂,其特征在于,包括上权利要求1-6任一项所述冷冻保存液和权利要求8-9任一项所述的冷冻平衡液,所述冷冻保存液和冷冻平衡液独立存在。优选地,血清含量为0,所述冷冻平衡液以每100mL计,含有PVA 0.5-2.5g,多元醇7.5-15mL,缓冲液余量。
- 如权利要求1-6任一项所述的冷冻保存液和/或权利要求8-9任一项所述的冷冻平衡液在生物组织冷冻保存中的应用。
- 根据权利要求11所述的应用,所述生物组织选自卵母细胞、胚胎、干细胞、器官或组织中的至少一种。
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AU2020256502A AU2020256502B2 (en) | 2019-04-09 | 2020-03-02 | Cryopreservation solution without DMSO, preparation method therefor and application thereof |
SG11202110871UA SG11202110871UA (en) | 2019-04-09 | 2020-03-02 | Dmso-free cryopreservation solution and preparation method and use thereof |
EP20787356.3A EP3939427A4 (en) | 2019-04-09 | 2020-03-02 | CRYOPRESERVATION SOLUTION WITHOUT DMSO, PROCESS FOR ITS PREPARATION AND ITS USE |
JP2021560653A JP2022528772A (ja) | 2019-04-09 | 2020-03-02 | Dmsoフリーの凍結保存液及びその調製方法と応用 |
KR1020217033905A KR20210143833A (ko) | 2019-04-09 | 2020-03-02 | Dmso가 없는 동결 보존액 및 그 제조방법과 응용 |
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