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 PDF

Info

Publication number
WO2020207151A1
WO2020207151A1 PCT/CN2020/077473 CN2020077473W WO2020207151A1 WO 2020207151 A1 WO2020207151 A1 WO 2020207151A1 CN 2020077473 W CN2020077473 W CN 2020077473W WO 2020207151 A1 WO2020207151 A1 WO 2020207151A1
Authority
WO
WIPO (PCT)
Prior art keywords
solution
cryopreservation
pva
serum
balance
Prior art date
Application number
PCT/CN2020/077473
Other languages
English (en)
French (fr)
Inventor
严杰
乔杰
闫丽盈
李蓉
王健君
金晟琳
吕健勇
Original Assignee
北京大学第三医院（北京大学第三临床医学院）
中国科学院化学研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京大学第三医院（北京大学第三临床医学院）, 中国科学院化学研究所 filed Critical 北京大学第三医院（北京大学第三临床医学院）
Priority to AU2020256502A priority Critical patent/AU2020256502B2/en
Priority to KR1020217033905A priority patent/KR20210143833A/ko
Priority to EP20787356.3A priority patent/EP3939427A4/en
Priority to US17/594,202 priority patent/US20220167610A1/en
Priority to JP2021560653A priority patent/JP2022528772A/ja
Priority to SG11202110871UA priority patent/SG11202110871UA/en
Publication of WO2020207151A1 publication Critical patent/WO2020207151A1/zh

Links

Images

Classifications

    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

本发明公开了一种无DMSO的冷冻保存液。所述冷冻保存液以每100mL体积计，含有仿生控冰材料0.01-50.0g，多元醇5.0-30mL，水溶性糖1-30g，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。本发明的冷冻保存液和冷冻平衡液不含有DMSO，在用于冷冻保存小鼠卵母细胞、胚胎时，能达到与商业化冷冻保存液（含有15％DMSO）同等甚至更高的细胞、组织存活率和功能表达稳定性，具有较高的保存效率。其中，既无DMSO也无血清的冷冻保存液，进一步解决了目前临床普遍使用的商业化冷冻保存液因含有血清而造成的稳定性差，可带入寄生性生物污染物质等问题。本发明的冷冻保存液组成简单，且原料来源方便，成本低廉，可广泛应用于各类细胞冷冻保存。

Description

一种无DMSO的冷冻保存液及其制备方法与应用
本申请要求2019年4月9日向中国国家知识产权局提交的专利申请号为201910281978.2，发明名称为“一种无DMSO的冷冻保存液及其制备方法”的在先申请，以及专利申请号为201910281986.7，发明名称为“一种肽类化合物和含有该化合物的冷冻保存液”的在先申请的优先权。上述两件在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于生物医用材料技术领域，具体涉及一种无DMSO的冷冻保存液及其制备方法。
背景技术
冷冻保存技术自问世以来，成为自然科学领域不可缺少的研究方法之一，已被广泛采用。随着生活水平的提高和医学技术的发展，人类生殖细胞(精子、卵母细胞)、性腺组织等的冷冻保存成为保存生育力的重要手段。另外，随着世界人口老龄化加剧，对捐赠的可用于再生医学和器官移植的人源性细胞、组织或器官的冷冻保存的需求也极速增加。因此，如何高效的冷冻保存珍贵的细胞、组织以及器官资源已成为生命科学领域的重要课题。
目前最常用的冷冻保存方法为玻璃化冷冻。玻璃化冷冻技术虽然在快速冷冻过程中可使细胞内外的液体直接成为玻璃态而避免了冷冻过程中因冰晶形成而导致的损伤。但是，在复温过程中，现有的冷冻保存试剂不能有效的控制冰晶的生长，从而损害细胞。二甲基亚砜(DMSO)是体外细胞培养常用的助熔剂和渗透类细胞冻存保护剂，然而，DMSO在临床患者试验时产生不良副作用；同时DMSO还具有强的细胞毒性，且不同种类的细胞对于DMSO浓度的敏感性不同，导致DMSO为主要保护剂成分的冻存试剂对细胞的毒副作用，其应用受到限制。目前玻璃化冷冻方法通常使用的高浓度(≥15％)的DMSO，严重影响冷冻保存对象复苏后的存活率甚至(子代)安全性以及功能表达。综上，目前采用的冷冻保存试剂存在不具备复温过程中有效控制冰晶生长的能力，同时存在试剂毒性大的问题。
发明内容
为改善现有技术的上述缺陷，本发明提供一种无DMSO的冷冻保存液及其制备方法。
本发明提供如下技术方案:
一种无DMSO的冷冻保存液，以每100mL体积计，含有仿生控冰材料0.01-50.0g，多元醇5.0-45mL，水溶性糖0.1-1mol L -1，血清0-30mL，余量为缓冲液，所述仿生控冰材料选自聚乙烯醇PVA和/或氨基酸类仿生控冰材料，所述冷冻保存液中不含有二甲基亚砜(DMSO)。
根据本发明，所述氨基酸类仿生控冰材料选自聚氨基酸(聚合度≥2，优选聚合度为8～40，例如聚合度为8、15、20等)、氨基酸、肽类化合物中的一种或两种以上。
根据本发明，所述肽类化合物为多肽(优选2-8个不同的氨基酸组成的肽，例如2肽、3肽、4肽)、糖肽衍生物、式(I)所示化合物，
Figure PCTCN2020077473-appb-000001
其中，R选自取代或未取代的烷基，所述取代的基团可以选自-OH、-NH 2、-COOH、-CONH 2等，例如，R为取代或未取代的C 1-6烷基，优选R为-CH 3、-CH 2CH 3、-CH 2CH 2COOH；n为大于等于1而小于等于1000的整数，例如可以为1～100范围内的整数。在本发明的一些实施方式中，n为2、3、4、5、6、7、8、9、10的整数。
根据本发明，所述多元醇可以为C2-5的多元醇，优选C2-C3的二元醇、三元醇，例如乙二醇，丙二醇，丙三醇中的任一种。
根据本发明，所述水溶性糖可以为非还原性双糖、水溶性多糖、水溶性纤维素、糖酐中的至少一种，例如选自蔗糖、海藻糖、聚蔗糖、羟丙基甲基纤维素。所述水溶性糖可以起到保护细胞膜和避免细胞沉降的作用。
根据本发明，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SDS，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。
根据本发明的冷冻保存液，所述仿生控冰材料可以为PVA，所述PVA含量为0.1-6.0g，例如0.5-5.0g；具体地，可以为1.0g、2.0g、3.0g、4.0g。
根据本发明的冷冻保存液，所述仿生控冰材料可以为聚氨基酸或氨基酸，所述聚氨基酸或氨基酸的含量为0.01-50g，例如1.5-50g；具体地，可以为8.0g、10g、15g、20g、30g、40g。
根据本发明的冷冻保存液，所述控冰材料可以为PVA与聚氨基酸的组合，例如由0.1-5.0g的PVA和1.0-9.0g的聚氨基酸组成。
根据本发明的冷冻保存液，所述控冰材料可以为PVA与氨基酸的组合，例如由0.1-5.0g的PVA和8.0-35g的氨基酸组成。
根据本发明的冷冻保存液，以每100mL计，所述多元醇含量为6.0-28mL，例如7.0-20mL，10-15mL。
根据本发明的冷冻保存液，以每100mL计，所述血清含量为0.1-30mL，例如5.0-20mL，10-15mL。
根据本发明的冷冻保存液，所述冷冻保存液以每100mL计，优选血清含量为0；
根据本发明的冷冻保存液，每100mL冷冻保存液中所述水溶性糖含量为0.1-1.0mol L -1，例如0.1-0.8mol L -1，0.2-0.6mol L -1；具体地，例如0.25mol L -1，0.5mol L -1，1.0mol L -1.
根据本发明的冷冻保存液，其pH为6.5-7.6，例如为6.9-7.2。
作为本发明的一个实施方案，所述冷冻保存液以每100mL体积计，由如下组分组成：
Figure PCTCN2020077473-appb-000002
优选，所述冷冻保存液以每100mL体积计，由如下组分组成：
Figure PCTCN2020077473-appb-000003
作为本发明的一个实施方案，所述冷冻保存液以每100mL体积计，由如下组分组成：
Figure PCTCN2020077473-appb-000004
Figure PCTCN2020077473-appb-000005
优选，以每100mL体积计，由如下组分组成：
Figure PCTCN2020077473-appb-000006
作为本发明的一个实施方案，所述冷冻保存液以每100mL体积计，由如下组分组成：
Figure PCTCN2020077473-appb-000007
优选，所述冷冻保存液以每100mL体积计，由如下组分组成：
Figure PCTCN2020077473-appb-000008
作为本发明的一个实施方案，所述冷冻保存液以每100mL体积计，由如下组分组成：
Figure PCTCN2020077473-appb-000009
优选，所述冷冻保存液以每100mL体积计，由如下组分组成：
Figure PCTCN2020077473-appb-000010
Figure PCTCN2020077473-appb-000011
本发明还提供所述冷冻保存液的制备方法，包括如下步骤：将仿生控冰材料溶解于DPBS中，冷却到室温后调节pH，将除血清外的其他组分溶解于剩下的DPBS中，冷却后混合，再次确认或调节pH并补齐余量缓冲液，血清于使用时加入。
根据本发明的制备方法，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)将水溶性糖溶解于另一部分缓冲液中，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值并定容补足余量缓冲液至预定体积，得到所述冷冻保存液。
根据本发明的制备方法，包括如下步骤：
(1)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1；
(2)任选地，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2；
(3)将水溶性糖溶解于另一部分缓冲液中，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值并定容补足余量缓冲液至预定体积，得到所述冷冻保存液。
根据本发明的制备方法，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。
根据本发明的制备方法，所述步骤(1)中，PVA在温浴加热中溶解，例如油浴或水浴加热；例如水浴温度为60-95℃，优选80℃。所述步骤(1)中，所述溶解包括搅拌步骤。
根据本发明的制备方法，所述步骤(2)中，所述溶解为超声辅助溶解。
一种无DMSO的冷冻平衡液，以每100mL体积计，含有PVA 0-5.0g，多元醇5.0-45mL，血清0-30mL，缓冲液余量。
根据本发明的冷冻平衡液，所述PVA含量为0.1-5.0g，例如0.1g、0.5g、1.0g、2.0g。
根据本发明的冷冻平衡液，所述多元醇含量为6.0-28mL，例如7.0-20mL，10-15mL。
根据本发明的冷冻平衡液，所述血清含量为0.1-30mL，例如5.0-20mL，10-15mL。作为本发明的一个实施方案，所述血清的含量为0。
作为本发明的一个实施方案，所述冷冻平衡液以每100mL体积计，含有多元醇7.5-15mL，血清10-20mL，DPBS余量。
作为本发明的一个实施方案，所述冷冻平衡液以每100mL体积计，含有PVA 1.0-5.0g，多元醇7.5-15mL，缓冲液余量。
本发明的冷冻平衡液中，所述PVA、多元醇和血清可从冷冻保存液相应组分的种类中选择。
本发明还提供上述冷冻平衡液的制备方法，包括将各组分溶解于缓冲溶液中，血清单独存放，在使用时添加。
一种无DMSO的冷冻保存用试剂，包括上述冷冻平衡液和上述冷冻保存液，所述冷冻平衡液和冷冻保存液分别独立存在。
根据本发明的冷冻保存用试剂，所述冷冻保存液的血清含量为0，所述冷冻平衡液以每100mL体积计，含有PVA 1.0-5.0g，多元醇7.5-15mL，缓冲液余量。
根据本发明的冷冻保存用试剂，所述冷冻平衡液以每100mL体积计，包括如下组分：
Figure PCTCN2020077473-appb-000012
所述冷冻保存液以总体积100mL计，包括如下组分:
Figure PCTCN2020077473-appb-000013
根据本发明，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45％-60％，例如为50％-55％。
根据本发明，所述PVA可选自分子量为10-500kDa或者更高分子量的PVA，例如分子量为10-30kDa、30-50kDa、80-90kDa、200-500kDa。
根据本发明，所述PVA可选自水解度为大于80％的PVA，例如水解度为80％-99％、82-87％、87％-89％、89％-99％、98％-99％。
根据本发明，所述聚氨基酸可选自赖氨酸、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)。
根据本发明，所述肽类化合物为多肽，是由两种以上的氨基酸组成，可选自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-Ala-L-Ala-L-Thr(AAT)中的一种或两种以上。可采用本领域已知的多肽合成方法，例如采用固相合成法合成这些多肽。
根据本发明，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-Thr等。可采用本领域已知的糖类与氨基酸的反应方法制备所述糖肽化合物，例如采用固相合成法或者使糖类和氨基酸在有机溶剂中反应。
根据本发明，所述肽类化合物具有式(1)-式(8)所示任一结构：
Figure PCTCN2020077473-appb-000014
Figure PCTCN2020077473-appb-000015
根据本发明，所述式(I)所示化合物具有如下任一所示的结构：
Figure PCTCN2020077473-appb-000016
根据本发明，所述式(9)所示的化合物采用如下合成路线制备：
Figure PCTCN2020077473-appb-000017
本发明冷冻保存液和冷冻平衡液中，各组分用量均以100mL溶液总体积为基准，余量为缓冲液。
本发明还提供上述冷冻保存液在冷冻保存各类细胞、器官以及组织的应用，包括冷冻保存卵母细胞、胚胎、各类干细胞、器官和组织。其中器官和组织包括但不限于卵巢器官、卵巢组织。
本发明进一步提供一种细胞或胚胎的冷冻和复苏方法，包括：
(1)将细胞或胚胎置于本发明的冷冻保存液中，制成细胞悬浮液，进行冷冻；
(2)将冷冻的细胞或胚胎放入解冻液中进行解冻复苏。
本发明所述的冷冻和复苏方法，所述细胞或胚胎在置于冷冻保存液中之前，先放入平衡液中进行平衡。
本发明进一步提供一种干细胞冷冻保存的方法，采用微滴法，例如所述干细胞冷冻保存的方法包括如下步骤:将冷冻保存液加入干细胞中，吹打分散，制成干细胞悬液，将干细胞悬液置于冷冻载片上，液氮(-196℃)冷冻保存。
根据本发明的实施方案，冷冻保存的干细胞的解冻包括将置有干细胞的冷冻载片置于 a-MEM培养基中，37℃解冻。
根据本发明的实施方案，所述干细胞为本领域已知的具有分化功能的各种干细胞，例如全能干细胞、多能干细胞或者专能干细胞，包括但不限于胚胎干细胞、各类间充质干细胞(例如脐带间充质干细胞、脂肪间充质干细胞、骨髓间充质干细胞等)、造血干细胞等。
本发明还供器官和/或组织的冷冻保存方法，包括：将器官和/或组织在冷冻平衡液中平衡，然后将器官和/或组织放入冷冻保存液中，再将器官和/或组织置于冷冻载片上，液氮冷冻保存。
在一个实施方案中，所述器官和/或组织为卵巢组织或者卵巢器官，可以为卵巢组织切片或者完整的卵巢组织。
本发明中“冻存”和“冷冻保存”具有相同含义，可互换使用，指通过低温对某种物质或者细胞、组织、器官进行保存，使其保持其本来的理化和/或生物活性、生理生化功能。
本发明中，“干细胞”的种类没有特别的限定，本发明的冷冻保存液可用于冷冻保存本领域已知的各类干细胞，包括但不限于脐带间充质干细胞、骨髓间充质干细胞、脂肪间充质干细胞、造血干细胞等。
本发明中，生物组织可以是来源于动物，包括温血哺乳动物，例如人和灵长类动物；鸟类；驯养的家养或农场动物，例如猫、狗、绵羊、山羊、牛、马和猪；实验室动物，例如小鼠、大鼠和豚鼠；鱼；爬行动物；动物园动物和野生动物等。
有益效果
本发明提供的冷冻保存液和冷冻平衡液，不含有DMSO，在用于冷冻保存小鼠卵母细胞、胚胎时，能达到与商业化冷冻保存液(含有体积浓度为15％DMSO)同等甚至更高的细胞、组织存活率和功能表达稳定性，具有较高的保存效率。其中，既无DMSO也无血清的冷冻保存液，进一步解决了目前临床普遍使用的商业化冷冻保存液因含有血清而造成的稳定性差，易带入寄生性生物污染物质等问题。本发明的冷冻保存液组成简单，且原料来源方便，成本低廉，可广泛应用于卵母细胞、类细胞(例如胚胎等)、干细胞、组织以及器官冷冻保存。
附图说明
图1为新生3天的小鼠新鲜(未冷冻)卵巢器官切片染色图片；
图2为对比实例7冻存的完整卵巢器官解冻后切片染色图片；
图3为应用实例14冻存的完整卵巢器官解冻后切片染色图片；
图4为应用实例15冻存的完整卵巢器官解冻后切片染色图片；
图5为应用实例16冻存的卵巢器官解冻后切片染色图片；
图6为性成熟的小鼠新鲜(未冷冻)卵巢组织切片染色图片；
图7为对比实例8冻存的卵巢组织切片解冻后切片染色图片；
图8为应用实例17冻存的卵巢组织切片解冻后切片染色图片；
图9为应用实例18冻存的卵巢组织切片解冻后切片染色图片；
图10为应用实例19冻存的卵巢组织切片解冻后切片染色图片。
具体实施方式
下文将结合具体实施例对本发明的制备方法做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、材料等，如无特殊说明，均可从商业途径得到。
本发明实施例中采用的PVA为间同规整度为50％-55％，分子量为13-23kDa，水解度98％。
本发明实施例中冷冻液中采用聚-L-脯氨酸聚合度为8或者15，分子量为795和1475；聚L-精氨酸聚合度为8，分子量为1267。解冻液中聚-L-脯氨酸聚合度为8，分子量为795。
本发明实施例中存活率为3-12次重复实验的存活率平均值。
实施例1.小鼠卵母细胞和胚胎冷冻保存
1.制备冷冻保存液：按以下配方配制冷冻保存液
冷冻保存液A:每100ml中含有如下组分：
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
将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于25mL的DPBS中，调节pH为7.0，为溶液1；将1.5g的聚-L-脯氨酸超声溶于另外20mL的DPBS中，调节pH为7.0，为溶液 2；将17g(0.05mol)的蔗糖(蔗糖在冷冻保存液中终浓度为0.5mol L -1)超声溶解于25mL的DPBS中，待蔗糖全部溶解后依次加入10mL的乙二醇，为溶液3，待溶液1、溶液2及溶液3恢复至室温，再将三种溶液混匀，调节pH为7.0，并用DPBS定容补齐余量至总体积100mL，备用。
冷冻保存液B:每100ml中含有如下组分：
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
冷冻保存液配液步骤：将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于20mL的DPBS中，调节pH为7.1，为溶液1；将8.0g的L-Arg和4.0g的L-Thr溶于20mL的DPBS中，调节pH为7.1，为溶液2；17g(0.05mol)的蔗糖(蔗糖在冷冻保存液中终浓度为0.5mol L -1)超声溶解于20mL的DPBS中，待蔗糖全部溶解后加入10mL的乙二醇，为溶液3；待溶液1、溶液2及溶液3恢复至室温，再将三种溶液混均，调节pH值为7.1并用DPBS定容补齐余量至总体积的80％，使用时加入20mL的血清。
冷冻保存液C:
每100ml中含有如下组分：
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
将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于25mL的DPBS中，调节pH为6.9， 为溶液1；17g(0.05mol)的蔗糖(蔗糖在冷冻保存液中终浓度为0.5mol L -1)超声溶解于25mL的DPBS中，待蔗糖全部溶解后加入10mL的乙二醇，为溶液2，待溶液1及溶液2恢复至室温，再将两种溶液混均，调节pH值并定容补齐余量至总体积的80％，20mL血清单独存放待保存液使用时添加。
冷冻保存液D：
每100ml中含有以下组分：
物质 用量
PVA(g) 2.0
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
DPBS(mL) 余量
将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于30mL的DPBS中，调节pH为7.0，为溶液1；17g(0.05mol)的蔗糖(蔗糖在冷冻保存液中终浓度为0.5mol L -1)超声溶解于25mL的DPBS中，待蔗糖全部溶解后加入10mL的乙二醇，为溶液2，待溶液1及溶液2恢复至室温，再将两种溶液混均，调节pH值并定容补齐余量至总体积100mL，备用。
2.制备冷冻平衡液：按以下配方配制冷冻平衡液
冷冻平衡液a：将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于50mL的DPBS中，待PVA全部溶解，调节pH为7.0，加入7.5mL的乙二醇，混匀，用DPBS定容至100mL，备用。
冷冻平衡液b：总体积100mL，将7.5mL的乙二醇溶于72.5mL的DPBS中，混匀，使用时加20mL的血清。
对比例1：
冷冻平衡液1#：每1mL中含有7.5％(v/v)的DMSO,7.5％(v/v)的乙二醇，20％(v/v)的胎牛血清，余量为DPBS；
冷冻保存液1#：每1mL中含有15％(v/v)的DMSO,15％(v/v)的乙二醇，20％(v/v)的胎牛血清，0.5M蔗糖，余量为DPBS。
冷冻平衡液b：每1mL中含有7.5％(v/v)的乙二醇，20％(v/v)的胎牛血清，余量为 DPBS；
冷冻保存液2#：每1mL中含有10％(v/v)的乙二醇，20％(v/v)的胎牛血清，0.5M蔗糖，余量为DPBS。
本发明实施例1和对比例1采用的解冻液配方有如下三种：
解冻液1#：解冻液I(含有1.0mol L -1蔗糖，20％的血清，余量为DPBS)；解冻液II(含有0.5mol L -1蔗糖，20％的血清，余量为DPBS)；解冻液III(含有0.25mol L -1蔗糖，20％的血清，余量为DPBS)；解冻液IV(20％的血清，余量为DPBS)。
解冻液2#：解冻液Ⅰ(含有1.0mol L -1的蔗糖，20mg mL -1的PVA，余量为DPBS)；解冻液Ⅱ(含有0.5mol L -1蔗糖，20mg mL -1的PVA，余量为DPBS)；解冻液Ⅲ(含有0.25mol L -1蔗糖，20mg mL -1的PVA，余量为DPBS)；解冻液Ⅳ(20mg mL -1的PVA，余量为DPBS)。
解冻液3#：解冻液I(含有1.0mol L -1的蔗糖，20mg mL -1的PVA，10mg mL -1的聚脯氨酸，余量为DPBS)；解冻液II(含有0.5mol L -1蔗糖，20mg mL -1的PVA，5.0mg mL -1的聚脯氨酸，余量为DPBS)；解冻液III(含有0.25mol L -1蔗糖，20mg mL -1的PVA，2.5mg mL -1的聚脯氨酸，余量为DPBS)；解冻液IV(20mg mL -1的PVA，余量为DPBS)。
应用例1：
采用上述实施例及对比例的冷冻平衡液以及冷冻保存液按表1和表2中的方案分别进行卵母细胞和胚胎冷冻保存。
1.卵母细胞冷冻保存
小鼠卵母细胞先置于冷冻平衡液中平衡5分钟；然后置于所制备的冷冻保存液中1分钟，将已在冷冻保存液中平衡后的卵母细胞放置于冷冻载杆上，然后快速投入液氮中(-196℃)中，并封闭载杆后继续保存；解冻时，将冻存的卵母细胞置于37℃的解冻液Ⅰ中平衡5分钟，再依次在解冻液Ⅱ-Ⅳ中各平衡3分钟；将解冻完毕的卵母细胞培养2小时后观察存活细胞数量，计算存活率(参见表1)。
2.胚胎冷冻保存
小鼠胚胎先置于冷冻平衡液平衡5分钟，然后置于所制备的冷冻保存液中50秒，将已在冷冻保存液中平衡的胚胎放置于冷冻载杆上，然后快速投入液氮(-196℃)中，并封闭载杆后继续保存；解冻时，将冷冻的胚胎置于37℃的解冻液Ⅰ中平衡3分钟，再依次在解冻液Ⅱ-Ⅳ中各平衡3分钟；将解冻完毕胚胎培养2小时，观察存活胚胎数量，计算存活率(参见表2)。
表1小鼠卵母细胞冷冻保存存活率
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％
表2小鼠胚胎冷冻保存存活率
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％
根据实施例1和对比例1中各实例的数据可以看出，本发明无DMSO的冷冻保存液和冷冻平衡液，通过各个组分协同作用，即便在不添加DMSO的情况下，用于卵母细胞和胚胎的冷冻保存均仍有良好的保存效果，解决了现有冻存液中因加入较大浓度的DMSO对于细胞或胚胎产生毒性的缺陷。而且由应用实例2，6-8可以看出，即便在平衡液，冷冻液以及解冻液同时不加血清以及DMSO的情况下，在本发明中的仿生控冰材料和渗透性保护剂乙二醇等共同作用下，仍能达到优于现有商业化冻存液的冷冻保存卵母细胞和胚胎的的存活率，进一步解决了目前临床普遍使用的商业化冷冻保存液因含有血清而造成的保质期短、易带入寄生性生物污染物质等问题。
实施例2：人脐带间充质干细胞的冷冻保存
1.制备冷冻保存液：按以下配方配制冷冻保存液
冷冻保存液E：总体积100mL，含有乙二醇10mL、血清20mL、蔗糖17g(0.5mol L -1)、聚L-精氨酸(聚合度为8)4.0g、PVA 1.0g、DPBS余量。
冷冻保存液F：总体积100mL，含有乙二醇20mL、血清20mL、蔗糖17g(0.5mol L -1)、L-Arg 16g、L-Thr 8.0g、DPBS余量。
冷冻保存液G：总体积100mL，含有乙二醇10mL、血清20mL、蔗糖17g(0.5mol L -1)、PVA 2.0g、DPBS余量。
冷冻保存液H：总体积100mL，含有乙二醇10mL，血清20mL、蔗糖17g(0.5mol L -1)、TR 28g、DPBS余量。
冷冻保存液I：总体积100mL，含有乙二醇10mL、蔗糖17g(0.5mol L-1)、PVA 2.0g、DPBS余量。
冷冻保存液配制方法同实施例1。
其中，TR的制备方法如下：
(1)将2-氯三苯甲基氯树脂放入反应管中，加DCM(20mL g -1)，振荡30分钟。砂芯抽滤除去溶剂，加入三倍摩尔过量Fmoc-L-Thr(tBu)-OH，再加入8倍摩尔过量的DIEA,最后加入DMF溶解，振荡30分钟。甲醇封头30分钟。
(2)除去溶剂DMF，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液。取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(3)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后，向反应管中加入用尽量少的DMF溶解的Fmoc-Arg(Pbf)-OH；两倍过量，HBTU两倍过量。之后，立刻加入8倍过量的DIEA，反应30分钟。
(4)抽掉溶液后，取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，无色为阴性反应，即反应完全。
(5)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后除去溶剂，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液，取少量树脂，用乙醇清洗后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(6)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DCM(15mL g -1，两次)清洗后抽干树脂。
(7)使用切割液(15mL g -1，TFA：水：EDT：Tis＝95:1:2:2,V/V)将产物切割90 分钟。并将切割液用氮气吹干，之后冻干，得到多肽粗品。
(8)用HPLC纯化多肽并转盐或脱盐，HPLC：tR＝4.8mins(纯化柱子型号：Kromasil100-5C18,4.6mm*250mm；梯度洗脱液：0.1％TFA乙腈溶液和0.1％TFA水溶液,0mins-1:99，20mins-1:4)。将纯化后的溶液冻干，既得到成品L-Thr-L-Arg(TR)。产率约为80％。质谱鉴定276.2为[M+H] +。
对比例2：
冷冻保存液3#：每1mL中含有10％(v/v)的DMSO，15％(v/v)的胎牛血清，余量为a-MEM培养基(USA，Invitrogen，C12571500BT)。
采用上述冷冻保存液按表3中的方案分别进行人脐带充间质干细胞的冷冻保存。所用人脐带干细胞冷冻保存方法具体为微滴法：将培养皿上的人脐带间充质干细胞用25％胰酶消化2分钟后，放入等体积培养液(10％FBS+a-MEM培养基)，轻柔吹打至干细胞全部脱落，加入1.5ml离心管，1000rmp离心5分钟，弃上清，将细胞与培养基分离，将10uL冷冻液加入离心管底部，轻柔吹打使干细胞团分散，将此10uL带有干细胞的冷冻液置于冷冻载片上，置于液氮(-196摄氏度)冻存。解冻时，将带有细胞及冷冻液的冷冻载杆直接放入37℃的a-MEM培养基中进行解冻。解冻后，台盼蓝染色察看其存活率，并使用仪器JIMBIO-FIL计数细胞数量，存活率＝活细胞数/细胞总数(参见表3)。
表3人脐带充间质干细胞冷冻保存存活率
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％
本发明的冷冻保存液进行人脐带充间质干细胞冷冻保存时，即使不使用DMSO干细胞存活率可达92.4％(应用实例9)，甚至在完全不添加DMSO和血清时，存活率可达到77.1％(应用实例13)，表明该冷冻用试剂不仅能达到常规冷冻液冷冻干细胞的有效性，甚至远高于目前普遍使用的含有10％的DMSO的冷冻保存液(对比实例5)的冷冻保存复苏率，基于PVA的冷冻保存效果显著优于不添加PVA的对比实例6。
实施例3：完整卵巢器官和卵巢组织切片的冷冻保存
冷冻保存液J：总体积100mL，含有乙二醇10mL、蔗糖17g(0.5mol L -1)、PVA 2.0g、DPBS余量。
冷冻保存液K:总体积100mL，含有乙二醇10mL、血清20mL、蔗糖17g(0.5mol L -1)、PVA 1.0g、DPBS余量。
冷冻保存液L：含有乙二醇10mL、血清20mL、蔗糖17g(0.5mol L -1)、聚L-精氨酸(聚合度为8)4.0g、PVA 1.0g、DPBS余量。
对比例3：冷冻保存液每1mL中含有15％(v/v)的DMSO,15％(v/v)的乙二醇，20％(v/v)的血清，0.5M蔗糖，余量为DPBS。
冷冻平衡液a：将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于50mL的DPBS中，待PVA全部溶解，调节pH为7.0，加入7.5mL乙二醇，混匀，调节pH值并定容补齐余量至100mL，备用。
冷冻平衡液b：7.5mL的乙二醇加入72.5mL的DPBS中，混匀，使用时加入20m L的血清；
对比例3：
冷冻平衡液1#：每1mL中含有7.5％(v/v)的DMSO,7.5％(v/v)的乙二醇，20％(v/v)的胎牛血清，余量为DPBS；
冷冻保存液1#：每1mL中含有15％(v/v)的DMSO,15％(v/v)的乙二醇，20％(v/v)的胎牛血清，0.5mol L -1蔗糖，余量为DPBS。
解冻液1#：解冻液Ⅰ(含有1.0mol L -1蔗糖，20％的血清，余量为DPBS)；解冻液Ⅱ(含有0.5mol L -1蔗糖，20％的血清，余量为DPBS)；解冻液Ⅲ(含有0.25mol L -1蔗糖，20％的血清，余量为DPBS)；解冻液Ⅳ(20％的血清，余量为DPBS)。
解冻液2#：解冻液Ⅰ(含有1.0mol L -1的蔗糖，20mg mL -1的PVA，余量为DPBS)；解冻液Ⅱ(含有0.5mol L -1蔗糖，20mg mL -1的PVA，余量为DPBS)；解冻液Ⅲ(含有0.25mol L -1蔗糖，20mg mL -1的PVA，余量为DPBS)；解冻液Ⅳ(20mg mL -1的PVA，余量为DPBS)。
采用上述冷冻保存液及对比例的冷冻平衡液以及冷冻保存液按表4、表5中的方案分别对新生3天内的小鼠完整卵巢器官和性成熟小鼠的卵巢组织切片进行冷冻保存。
整个卵巢器官或者卵巢组织切片先置于平衡液室温平衡25分钟，然后置于所制备的冷冻保存液中15分钟，之后将完整卵巢器官或卵巢组织切片放置于冷冻载杆上，投入液氮中保存。解冻后，完整卵巢器官或卵巢组织切片放入培养液(10％FBS+a-MEM)后置于37℃、5％CO2培养箱中复苏2小时后使用4％多聚甲醛固定、石蜡包埋、HE染色观察形态，结果如图1-10所示，图1为新鲜未冷冻的卵巢器官切片照片，图6为新鲜未冷冻的卵巢组织的切片照片。
表4卵巢器官冷冻保存方案
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
表5卵巢组织冷冻保存方案
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
根据图1-5可知，与使用不添加氨基酸类仿生控冰材料的对比实例7以及新鲜未冷冻的卵巢器官相比，实施例14-16中原始卵泡结构相对完整，间质结构相对完整，细胞胞浆均质、淡染相对较多，胞核皱缩、深染相对较少；血管管壁结构完整，管腔塌陷较少，内皮细胞胞浆均质、淡染相对较多，胞核皱缩、深染相对较少。可见，实施例14-16组对于卵巢器官的冻存效果更好。
根据图6-10可知，实施例17-19的方案和对比实例8以及新鲜未冷冻的卵巢组织相比，窦卵泡结构相对完整，间质结构相对完整，细胞胞浆均质、淡染相对较多，胞核皱缩、深染相对较少，可见本发明的冻存液用于冻存卵巢组织也比现有技术具有更好的效果。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (12)

  1. 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。
  2. 根据权利要求1所述的冷冻保存液，其特征在于，所述氨基酸类仿生控冰材料选自聚合度≥2的聚氨基酸、氨基酸、肽类化合物中的一种或两种以上；
    优选地，所述肽类化合物为多肽、糖肽衍生物、或式(I)所示化合物，
    Figure PCTCN2020077473-appb-100001
    其中，R选自取代或未取代的烷基，所述取代基可以选自-OH、-NH 2、-COOH、-CONH 2等，例如，R为取代或未取代的C1-6烷基，优选R为-CH3、-CH 2CH 3、-CH 2CH 2COOH；n为大于等于1而小于等于1000的整数。
  3. 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。
  4. 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。
  5. 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。
  6. 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。
  7. 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。
  8. 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。
  9. 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。
  10. 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。
  11. 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。
  12. 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。
PCT/CN2020/077473 2019-04-09 2020-03-02 一种无dmso的冷冻保存液及其制备方法与应用 WO2020207151A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
AU2020256502A AU2020256502B2 (en) 2019-04-09 2020-03-02 Cryopreservation solution without DMSO, preparation method therefor and application thereof
KR1020217033905A KR20210143833A (ko) 2019-04-09 2020-03-02 Dmso가 없는 동결 보존액 및 그 제조방법과 응용
EP20787356.3A EP3939427A4 (en) 2019-04-09 2020-03-02 CRYOPRESERVATION SOLUTION WITHOUT DMSO, PROCESS FOR ITS PREPARATION AND ITS USE
US17/594,202 US20220167610A1 (en) 2019-04-09 2020-03-02 Dmso-free cryopreservation solution and preparation method and use thereof
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
SG11202110871UA SG11202110871UA (en) 2019-04-09 2020-03-02 Dmso-free cryopreservation solution and preparation method and use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201910281978 2019-04-09
CN201910281978.2 2019-04-09
CN201910281986 2019-04-09
CN201910281986.7 2019-04-09

Publications (1)

Publication Number Publication Date
WO2020207151A1 true WO2020207151A1 (zh) 2020-10-15

Family

ID=72750834

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/077473 WO2020207151A1 (zh) 2019-04-09 2020-03-02 一种无dmso的冷冻保存液及其制备方法与应用

Country Status (8)

Country Link
US (1) US20220167610A1 (zh)
EP (1) EP3939427A4 (zh)
JP (1) JP2022528772A (zh)
KR (1) KR20210143833A (zh)
CN (3) CN111793107B (zh)
AU (1) AU2020256502B2 (zh)
SG (1) SG11202110871UA (zh)
WO (1) WO2020207151A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112514892B (zh) * 2020-12-25 2022-06-14 广州赛莱拉干细胞科技股份有限公司 一种外泌体冻存保护液及其制备方法
CN112715533B (zh) * 2021-02-26 2022-04-29 康妍葆(北京)干细胞科技有限公司 一种间充质干细胞的冻存液及冻存方法
CN113229274A (zh) * 2021-05-20 2021-08-10 郑州优倍得生物科技有限公司 一种诱导多能干细胞冻存液及冻存方法
CN115399311A (zh) * 2021-05-26 2022-11-29 北京大学深圳医院 玻璃化冷冻液套装及其制备方法和应用
CN114391535B (zh) * 2022-01-19 2024-02-02 宋伟 用于培养类器官的样本保存液

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991010361A1 (en) * 1990-01-17 1991-07-25 The Regents Of The University Of California Composition to improve survival of biological materials
CN101250499A (zh) * 2008-04-11 2008-08-27 中山大学 一种干细胞无毒冻存液
CN108207930A (zh) * 2016-12-15 2018-06-29 中国科学院理化技术研究所 一种鸡尾酒式冷冻保护剂及其应用
WO2018191411A1 (en) * 2017-04-12 2018-10-18 X-Therma, Inc. Novel peptoid polymers and methods of use
CN109221082A (zh) * 2018-09-14 2019-01-18 上海慧存医疗科技有限公司 细胞冻存液、冻存复苏方法及其应用

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001275392A1 (en) * 2000-06-09 2001-12-24 Organ Recovery Systems Cryopreservation method using cryoprotective composition of propanediol and a vehicle solution
JP2005261413A (ja) * 2004-03-19 2005-09-29 Yamaguchi Technology Licensing Organization Ltd 動物胚凍結保存液とそれを用いた動物胚凍結保存法
US20050250088A1 (en) * 2004-03-25 2005-11-10 Boldt Jeffrey P Cryopreservation media
JP4588598B2 (ja) * 2004-09-24 2010-12-01 セーレン株式会社 細胞凍結保存用組成物
US20070042337A1 (en) * 2005-07-20 2007-02-22 The Regents Of The University Of California Isochoric method and device for reducing the probability of ice nucleation during preservation of biological matter at subzero centigrade temperatures
CN103254453B (zh) * 2013-04-24 2015-09-09 华中科技大学 一种聚合物分散有机凝胶光致变色薄膜的制备方法
US9458424B2 (en) * 2013-12-19 2016-10-04 FertiPro N.V. Methods for cryopreservation of stem cells via slow-freezing
SG11201802777XA (en) * 2015-10-14 2018-05-30 X Therma Inc Compositions and methods for reducing ice crystal formation
US11701389B2 (en) * 2016-03-09 2023-07-18 Emory University Methods of preserving mesenchymal stem cells
CN105961374A (zh) * 2016-07-04 2016-09-28 深圳市合康生物科技股份有限公司 细胞冻存液
TWI664412B (zh) * 2016-10-04 2019-07-01 全崴生技股份有限公司 維持細胞活性之組成物和方法
US20220125042A1 (en) * 2017-05-17 2022-04-28 Universidade Nova De Lisboa Cryoprotectant and/or cryopreservant composition, methods and uses thereof
CN107183008A (zh) * 2017-05-27 2017-09-22 魏方萌 一种胎盘间充质干细胞冻存液及其冻存方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991010361A1 (en) * 1990-01-17 1991-07-25 The Regents Of The University Of California Composition to improve survival of biological materials
CN101250499A (zh) * 2008-04-11 2008-08-27 中山大学 一种干细胞无毒冻存液
CN108207930A (zh) * 2016-12-15 2018-06-29 中国科学院理化技术研究所 一种鸡尾酒式冷冻保护剂及其应用
WO2018191411A1 (en) * 2017-04-12 2018-10-18 X-Therma, Inc. Novel peptoid polymers and methods of use
CN109221082A (zh) * 2018-09-14 2019-01-18 上海慧存医疗科技有限公司 细胞冻存液、冻存复苏方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
黄凤玲 等 (HUANG, FENGLING ET AL.): "快速冷冻牛体外受精胚胎 (Non-official translation: Quick Freezing of Bovine IVF Embryos)", 广西农业大学学报 (JOURNAL OF GUANGXI AGRICULTURAL UNIVERSITY), vol. 13, no. 1, 30 March 1994 (1994-03-30), XP55742126, ISSN: 1005-2011, DOI: 20200410172547X *

Also Published As

Publication number Publication date
CN111793110A (zh) 2020-10-20
EP3939427A1 (en) 2022-01-19
AU2020256502A1 (en) 2021-11-11
US20220167610A1 (en) 2022-06-02
CN111793112A (zh) 2020-10-20
AU2020256502B2 (en) 2023-08-17
CN111793110B (zh) 2022-11-11
KR20210143833A (ko) 2021-11-29
JP2022528772A (ja) 2022-06-15
CN111793107A (zh) 2020-10-20
CN111793107B (zh) 2022-04-12
SG11202110871UA (en) 2021-10-28
EP3939427A4 (en) 2022-07-13

Similar Documents

Publication Publication Date Title
WO2020207151A1 (zh) 一种无dmso的冷冻保存液及其制备方法与应用
US10815456B2 (en) Composition, kit and method for cryopreserving cells
CN111789105B (zh) 一种氨基酸类冷冻保存液在干细胞冻存中的应用
CN111789104B (zh) 一种冷冻保存液在干细胞冻存中的应用
CN111793108B (zh) 一种含肽类化合物的冷冻保存液在器官和组织冷冻保存中的应用
WO2020207152A1 (zh) 一种无血清冷冻保存液及其制备方法与应用
CN111789100B (zh) 一种无dmso的冷冻保存液在卵母细胞或胚胎冷冻保存中的应用
RU2785227C1 (ru) Криоконсервирующий раствор, не содержащий дмсо, и способ его получения и применение
JP7459130B2 (ja) 融解液及びその調製方法並びに応用
CN111789102B (zh) 一种解冻液在冷冻保存的卵母细胞或胚胎解冻中的应用
CN111789103B (zh) 一种冷冻保存用解冻液及解冻方法
RU2791227C1 (ru) Размораживающая жидкость, способ ее приготовления и ее применение

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20787356

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021560653

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20217033905

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020256502

Country of ref document: AU

Date of ref document: 20200302

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020787356

Country of ref document: EP

Effective date: 20211011