Serum-free cryopreservation solution and preparation method and application thereof

Abstract

A serum-free cryopreservation solution contains a biomimetic ice control material, a polyol, a water-soluble sugar, and a buffer solution margin. The bionic ice control material can be polyvinyl alcohol or an amino acid compound. The cryopreservation liquid of the present invention uses the bionic ice control material as the main component, does not contain serum, has low toxicity, and can achieve a cell survival rate that is the same as, or higher than, existing cryopreservation solutions.

Claims

1. A serum-free cryopreservation solution comprising, per 100 mL in volume, 0.01-50.0 g of a biomimetic ice growth inhibition material, 5.0-45 mL of a polyalcohol, a water-soluble saccharide at 0.1-1.0 mol L.sup.1, 0-15 mL of DMSO and the balance of a buffer, wherein the biomimetic ice growth inhibition material is a combination of an atactic polyvinyl alcohol (PVA) having a syndiotacticity of 15%-60% and a degree of hydrolysis of greater than 80% with an amino acid biomimetic ice growth inhibition material selected from GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Val, or GDL-L-Ser, and wherein the polyalcohol differs from the atactic PVA.

2. The cryopreservation solution according to claim 1, wherein: the polyalcohol is a polyalcohol with 2-5 carbon atoms; the water-soluble saccharide is selected from a non-reducing disaccharide, a water-soluble polysaccharide, a glycoside, and mixtures thereof; and the buffer is selected from DPBS, hepes-buffered HTF buffer, cell buffers, and mixtures thereof.

3. The cryopreservation solution according to claim 1, wherein: the content of DMSO is 1.0-10 mL per 100 mL of the cryopreservation solution; the content of the water-soluble saccharide is 0.1-0.8 mol L.sup.1 per 100 mL of the cryopreservation solution; and the content of the polyalcohol is 5.0-45 mL per 100 mL of the cryopreservation solution.

4. The cryopreservation solution according to claim 1, comprising, per 100 mL in volume, 0.1-50 g of the amino acid biomimetic ice growth inhibition material, 5.0-45 mL of the polyalcohol, 0-15 mL of DMSO, the water-soluble saccharide at 0.1-1 mol L.sup.1, and the balance of the buffer.

5. The cryopreservation solution according to claim 1, comprising, per 100 mL in volume, 0.01-6.0 g of the atactic PVA, 5.0-45 mL of the polyalcohol, the water-soluble saccharide at 0.1-1.0 mol L.sup.1, 0-15 mL of DMSO, and the balance of the buffer.

6. A preparation method of the cryopreservation solution according to claim 1, comprising the following steps: (1) dissolving the amino acid biomimetic ice growth inhibition material in a portion of the buffer, and adjusting the pH to form a solution 1; optionally, dissolving the atactic PVA in another portion of the buffer, and adjusting the pH to give a solution 2; (2) dissolving the sucrose in a third portion of the buffer, and adding other components after the sucrose is completely dissolved to prepare a solution 3; and (3) mixing the solution 1, optionally the solution 2 and the solution 3 after they are cooled to room temperature, and adjusting the pH and making up to a predetermined volume with the buffer to give the cryopreservation solution.

7. An amino acid cryopreservation reagent, comprising the cryopreservation solution according to claim 1 and a freezing equilibrium solution, wherein the freezing equilibrium solution comprises, per 100 mL in volume, 5.0-45 mL of the polyalcohol, the balance of the buffer, and optionally, 0-15 mL of DMSO, and 0.1-5.0 g of the atactic PVA having a syndiotacticity of 15%-60% and a degree of hydrolysis of greater than 80%, wherein the polyalcohol differs from the atactic PVA, wherein the freezing equilibrium solution and the preservation solution are present independently of each other.

8. A process for cryopreservation of a biological tissue, comprising placing the biological tissue in the cryopreservation solution according to claim 1 to prepare a cell suspension, and freezing the cell suspension.

9. The process according to claim 8, wherein the biological tissue is selected from an oocyte, an embryo, a stem cell, an organ, tissue, and mixtures thereof.

10. The cryopreservation solution according to claim 2, wherein: the polyalcohol is selected from ethylene glycol, propylene glycol, and glycerol; or the water-soluble saccharide is selected from sucrose and trehalose.

11. The cryopreservation solution according to claim 1, wherein: the polyalcohol is selected from ethylene glycol, propylene glycol, and glycerol; or the water-soluble saccharide is selected from sucrose, trehalose, water-soluble cellulose, polysucrose, and mixtures thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a picture of a stained slice of a fresh (unfrozen) ovarian organ;

(2) FIG. 2 is a picture of a stained slice of a cryopreserved intact ovarian organ of Comparative Embodiment 7 after thawing;

(3) FIG. 3 is a picture of a stained slice of a cryopreserved intact ovarian organ of Application Embodiment 9 after thawing;

(4) FIG. 4 is a picture of a stained slice of fresh (unfrozen) ovarian tissue;

(5) FIG. 5 is a picture of a stained slice of cryopreserved ovarian tissue of Comparative Embodiment 8 after thawing; and

(6) FIG. 6 is a picture of a stained slice of cryopreserved ovarian tissue of Application Embodiment 10 after thawing.

DETAILED DESCRIPTION

(7) The preparation method of the present invention will be further illustrated in detail with reference to the following specific examples. It should be understood that the following examples are merely exemplary illustration and explanation of the present invention, and should not be construed as limiting the protection scope of the present invention. All techniques implemented based on the aforementioned contents of the present invention are encompassed within the protection scope of the present invention.

(8) Unless otherwise stated, the experimental methods used in the following examples are conventional methods. Unless otherwise stated, the reagents, materials, and the like used in the following examples are commercially available.

(9) In the examples of the present invention, poly-L-proline used in the freezing solution has a degree of polymerization of 15 or 8 and a molecular weight of 1475 or 795, and poly-L-arginine used has a degree of polymerization of 8 and a molecular weight of 1267. Poly-L-proline in a thawing solution has a degree of polymerization of 8 and a molecular weight of 795.

(10) The survival rate in the examples of the present invention is the average survival rate of 3-12 repeated experiments.

Example 1. Cryopreservation of Oocytes and Embryos

(11) 1. Preparation of Cryopreservation Solutions: Cryopreservation Solutions were Prepared According to the Following Formulations.

(12) Cryopreservation solution A: 2.0 g of a PVA was dissolved in 30 mL of DPBS in a water bath at 80 C. by heating and magnetic stirring, and the pH was adjusted to 7.0 to give a solution 1; 17 g (0.05 mol) of sucrose (the final concentration of the sucrose in the cryopreservation solution was 0.5 mol L.sup.1) was ultrasonically dissolved in 25 mL of DPBS, and after the sucrose was completely dissolved, 10 mL of ethylene glycol was added to give a solution 2; after returning to room temperature, the solution 1 and the solution 2 were mixed homogeneously, the pH was adjusted, and the volume was made up to a total volume of 100 mL to give the cryopreservation solution A for later use.

(13) Cryopreservation solution B (total volume: 100 mL): 1.5 g of poly-L-proline (with a degree of polymerization of 15) was ultrasonically dissolved in 20 mL of DPBS, and the pH was adjusted to 7.0 to give a solution 1; 2.0 g of a PVA was dissolved in 25 mL of DPBS in a water bath at 80 C. by heating and magnetic stirring, and the pH was adjusted to 7.0 to give a solution 2; 17 g (0.05 mol) of sucrose (the final concentration of the sucrose in the cryopreservation solution was 0.5 mol L.sup.1) was ultrasonically dissolved in 25 mL of DPBS, and after the sucrose was completely dissolved, 10 mL of ethylene glycol was added to give a solution 3; after returning to room temperature, the solution 1, the solution 2 and the solution 3 were mixed homogeneously, the pH was adjusted, and the volume was made up to a total volume of 100 mL to give the cryopreservation solution B for later use.

(14) Cryopreservation solution C (total volume: 100 mL): 1.5 g of poly-L-arginine (with a degree of polymerization of 8) was ultrasonically dissolved in 20 mL of DPBS, and the pH was adjusted to 7.0 to give a solution 1; 2.0 g of a PVA was dissolved in 25 mL of DPBS in a water bath at 80 C. by heating and magnetic stirring, and the pH was adjusted to 7.0 to give a solution 2; 17 g (0.05 mol) of sucrose (the final concentration of the sucrose in the cryopreservation solution was 0.5 mol L.sup.1) was ultrasonically dissolved in 25 mL of DPBS, and after the sucrose was completely dissolved, 10 mL of ethylene glycol was added to give a solution 3; after returning to room temperature, the solution 1, the solution 2 and the solution 3 were mixed homogeneously, the pH was adjusted, and the volume was made up to a total volume of 100 mL to give the cryopreservation solution C for later use.

(15) 2. Preparation of Freezing Equilibration Solutions: The Freezing Equilibration Solutions were Prepared According to the Following Formulations.

(16) Freezing equilibration solution a: 7.5 mL of ethylene glycol and 7.5 mL of DMSO were added to 65 mL of DPBS, and mixed well, and 20 mL of serum was added when the freezing equilibration solution was used.

(17) Freezing equilibration solution b: 2.0 g of a PVA was dissolved in 50 mL of DPBS in a water bath at 80 C. by heating and magnetic stirring, the pH was adjusted to 7.0 after the PVA was completely dissolved, 7.5 mL of ethylene glycol was added, all the components were mixed homogeneously, the pH was adjusted, and the volume was made up to 100 mL to give the freezing equilibration solution b for later use.

(18) Comparative Example 1:

(19) A freezing equilibration solution a comprises, per 1 mL, 7.5% (v/v) of DMSO, 7.5% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum and the balance of DPBS; A cryopreservation solution 1# comprises, per 1 mL: 15% (v/v) of DMSO, 15% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum, sucrose at 0.5 mol L.sup.1, and the balance of DPBS. A freezing equilibration solution 2# comprises, per 1 mL, 7.5% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum and the balance of DPBS;

(20) A cryopreservation solution 2# comprises, per 1 mL, 10% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum, sucrose at 0.5 mol L.sup.1, and the balance of DPBS.

(21) The three formulations of the thawing solutions used in the Example 1 and Comparative Example 1 were as follows: A thawing solution 1# comprises a thawing solution I (comprising sucrose at 1.0 mol.Math.L.sup.1, 20% of serum and the balance of DPBS), a thawing solution II (comprising sucrose at 0.5 mol.Math.L.sup.1, 20% of serum and the balance of DPBS), a thawing solution III (comprising sucrose at 0.25 mol.Math.L.sup.1, 20% of serum and the balance of DPBS), and a thawing solution IV (comprising 20% of serum and the balance of DPBS). A thawing solution 2# comprises a thawing solution I (comprising sucrose at 1.0 mol L.sup.1, a PVA at 20 mg mL.sup.1, polyproline at 10 mg mL.sup.1, and the balance of DPBS), a thawing solution II (comprising sucrose at 0.5 mol L.sup.1, a PVA at 20 mg mL.sup.1, polyproline at 5.0 mg mL.sup.1, and the balance of DPBS), a thawing solution III (comprising sucrose at 0.25 mol L.sup.1, a PVA at 20 mg mL.sup.1, polyproline at 2.5 mg mL.sup.1, and the balance of DPBS), and a thawing solution IV (a PVA at 20 mg mL.sup.1 and the balance of DPBS). A thawing solution 3# comprises a thawing solution I (comprising sucrose at 1.0 mol.Math.L.sup.1, a PVA at 20 mg.Math.mL.sup.1 and the balance of DPBS), a thawing solution II (comprising sucrose at 0.5 mol.Math.L.sup.1, a PVA at 20 mg.Math.mL.sup.1 and the balance of DPBS), a thawing solution III (comprising sucrose at 0.25 mol.Math.L.sup.1, a PVA at 20 mg mL.sup.1 and the balance of DPBS), and a thawing solution IV (comprising a PVA at 20 mg.Math.mL.sup.1 and the balance of DPBS).

Application Example 1

(22) The freezing equilibration solutions and the cryopreservation solutions of the example and comparative example described above were used to cryopreserve oocytes and embryos according to the schemes in Table 1 and Table 2, respectively.

(23) 1. Cryopreservation of Oocytes

(24) Mouse oocytes were firstly equilibrated in a freezing equilibration solution for 5 min, and then equilibrated in the prepared cryopreservation solution for 1 m. The oocytes equilibrated in the cryopreservation solution were loaded onto straws, and the straws were quickly put into liquid nitrogen (96 C.) and then closed for cryopreservation. At the time of thawing, the frozen oocytes were equilibrated in the thawing solution I at 37 #C for 5 mi, and then equilibrated in the thawing solutions II-IV in sequence for 3 m each. After the thawed oocytes were incubated for 2 h, the number of the survived cells was observed, and the survival rates were calculated (see Table 1).

(25) 2. Cryopreservation of Embryos

(26) Mouse embryos were firstly equilibrated in a freezing equilibration solution for 5 m, and then equilibrated in the cryopreservation solution prepared according to the above formulation for 50 s. The embryos equilibrated in the cryopreservation solution were loaded onto straws, and the straws were quickly put into liquid nitrogen (196 C.) and closed for cryopreservation. At the time of thawing, the frozen embryos were equilibrated in the thawing solution I at 37 C. for 3 m, and then equilibrated in the thawing solutions II-IV in sequence for 3 m each. After the thawed embryos were incubated for 2 h, the number of survived embryos was observed, and the survival rates were calculated (see Table 2).

(27) TABLE-US-00001 TABLE 1 Survival rates of cryopreserved mouse oocytes Equilibration Cryopreservation Thawing Total number of Survival rates No. solution solution solution frozen oocytes after 2 h Application b A Thawing 50 93.4% Embodiment 1 solution 1# Application b A Thawing 53 96.5% Embodiment 2 solution 3# Application b B Thawing 39 89.7% Embodiment 3 solution 1# Application b B Thawing 60 98.6% Embodiment 4 solution 2# Comparative a Freezing Thawing 146 95.0% Embodiment 1 solution 1# solution 1# Comparative Equilibration Freezing Thawing 96 81.9% Embodiment 2 solution 2# solution 2# solution 1#

(28) TABLE-US-00002 TABLE 2 Survival rates of cryopreserved mouse embryos Equilibration Cryopreservation Thawing Total number of Survival rates No. solution solution solution frozen embryos after 2 h Application b B Thawing 42 95.2% Embodiment 5 solution 1# Application b C Thawing 30 96.67% Embodiment 6 solution 3# Application b A Thawing 41 95.8% Embodiment 7 solution 1# Comparative a Freezing Thawing 38 94.30% Embodiment 3 solution 1# solution 1# Comparative Equilibration Freezing Thawing 39 82.40% Embodiment 4 solution 2# solution 2# solution 1#

(29) As can be seen from the data in Table 1 and Table 2, when the cryopreservation solution disclosed herein is used for cryopreservation of oocytes and embryos, the survival rates of the embryos and the oocytes can reach more than 95%, and the survival rate of the oocytes can reach 96.5% and 98.6%, which are far higher than the post-thaw survival rate of the oocytes cryopreserved by a serum-containing commercial cryopreservation solution (Comparative Embodiments 1-4) generally used in clinic practice at present. When the equilibration solution, the freezing solution and the thawing solution all are free of serum and DMSO, the survival rates of cryopreserved oocytes and embryos can be superior to those of an existing commercial cryopreservation solution under the combined action of the biomimetic ice growth inhibition material disclosed herein, a permeable protectant, namely ethylene glycol, and the like. The problems are further solved that the commercial cryopreservation solutions commonly used in clinical practice at present are short in shelf life and prone to introduce parasitic biological contaminants due to the presence of serum.

Example 2. Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells

(30) 1. Preparation of Cryopreservation Solutions:

(31) Cryopreservation solution A: 2.0 g of a PVA was dissolved in 30 mL of DPBS in a water bath at 80 C. by heating and magnetic stirring, and the pH was adjusted to 7.0 to give a solution 1; 17 g (0.05 mol) of sucrose (the final concentration of the sucrose in the cryopreservation solution was 0.5 mol L.sup.1) was ultrasonically dissolved in 25 mL of DPBS, and after the sucrose was completely dissolved, 10 mL of ethylene glycol was added to give a solution 2; after returning to room temperature, the solution 1 and the solution 2 were mixed homogeneously, the pH was adjusted, and the volume was made up to a total volume of 100 mL to give the cryopreservation solution A for later use.

(32) 2. Comparative Example 2:

(33) A cryopreservation solution 1# comprises, per 1 mL: 15% (v/v) of DMSO, 15% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum, sucrose at 0.5 mol L.sup.1, and the balance of DPBS.

(34) A cryopreservation solution 3# comprises, per 1 mL, 10% (v/v) of DMSO, 15% (v/v) of fetal bovine serum, and the balance of a-MEM medium (C12571500BT, Invitrogen, USA).

Application Example 2

(35) Human umbilical cord mesenchymal stem cells were cryopreserved using the cryopreservation solutions of Example 2 and Comparative Example 2.

(36) 1. Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells by Microdroplet Method

(37) The cryopreservation method of the human umbilical cord stem cells used herein is specifically as follows: human umbilical cord mesenchymal stem cells on a culture dish were digested using 25% pancreatin for 3 min (the digestion time was controlled to be within 2-3 min), put into a culture solution (10% FBS+a-MEM culture medium) of the same volume, and gently pipetted until the stem cells completely fell off; the cells were added into a 1.5 mL centrifuge tube for centrifuging for 5 min at 1000 rpm, and the supernatant was discarded (to separate the cells from the supernatant); 10 L of freezing solution was added to the bottom of the centrifuge tube, the stem cells were gently pipetted to disperse stem cell clusters, and 10 L of freezing solution with the stem cells was placed on a freezing slide and then cryopreserved in liquid nitrogen (196 C.). At the time of thawing, the straw with the cells and the freezing solution was placed directly in a culture medium at 37 C. for thawing. After thawing, cells were stained with trypan blue to observe the survival rates, and the number of cells was counted using an instrument JIMBIO-FIL, survival rate=number of live cells/total number of cells (see Table 3).

(38) TABLE-US-00003 TABLE 3 Survival rates of cryopreserved human umbilical cord mesenchymal stem cells Cryopreservation Cryopreservation No. solution method Survival rates Application A Microdroplet 77.1% Embodiment 8 method Comparative Freezing Microdroplet 63.9% Embodiment 5 solution 1# method Comparative Freezing Microdroplet 76.6% Embodiment 6 solution 3# method

(39) When the cryopreservation solution disclosed herein is used for cryopreservation of the human umbilical cord mesenchymal stem cells, the survival rate of the stem cells can reach 77% or more although no serum is used. This means that the cryopreservation reagent can achieve the same effectiveness as a conventional freezing solution in freezing stem cells, and has a post-thaw survival rate even far higher than that of a cryopreservation solution (Comparative Embodiment 6) comprising 10% of DMSO and 20% of serum commonly used at present.

Example 3: Cryopreservation of Ovarian Organs and Ovarian Tissues

(40) 1. Preparation of Cryopreservation Solutions:

(41) Preparation of the cryopreservation solution: the cryopreservation solution and the freezing equilibration solution were prepared in the same manner as in Example 1 according to the formulations of the cryopreservation solution A and the freezing equilibration solution b of Example 1.

(42) 2. Comparative Example 3:

(43) A freezing equilibration solution a comprises, per 1 mL, 7.5% (v/v) of DMSO, 7.5% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum and the balance of DPBS; A cryopreservation solution 1# comprises, per 1 mL: 15% (v/v) of DMSO, 15% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum, sucrose at 0.5 mol L.sup.1, and the balance of DPBS.
3. Thawing Solution:

(44) The thawing solution was prepared according to the formulations of the thawing solution 1# and the thawing solution 3# of Example 1: A thawing solution 1# comprises a thawing solution I (comprising sucrose at 1.0 mol.Math.L.sup.1, 20% of serum and the balance of DPBS), a thawing solution II (comprising sucrose at 0.5 mol.Math.L.sup.1, 20% of serum and the balance of DPBS), a thawing solution III (comprising sucrose at 0.25 mol.Math.L.sup.1, 20% of serum and the balance of DPBS), and a thawing solution IV (comprising 20% of serum and the balance of DPBS). A thawing solution 3# comprises a thawing solution I (comprising sucrose at 1.0 mol.Math.L.sup.1, a PVA at 20 mg.Math.mL.sup.1 and the balance of DPBS), a thawing solution II (comprising sucrose at 0.5 mol.Math.L.sup.1, a PVA at 20 mg.Math.mL.sup.1 and the balance of DPBS), a thawing solution Ill (comprising sucrose at 0.25 mol.Math.L.sup.1, a PVA at 20 mg.Math.mL.sup.1 and the balance of DPBS), and a thawing solution IV (comprising a PVA at 20 mg.Math.mL.sup.1 and the balance of DPBS).
Application Example 3:

(45) The ovarian organs of mice newly born within 3 days and the ovarian tissue slices of sexually mature mice were cryopreserved using the freezing equilibration solutions and cryopreservation solutions of the above examples and comparative examples according to the schemes in Table 4 and Table 5.

(46) The whole ovarian organs or ovarian tissue slices were firstly equilibrated in an equilibration solution at room temperature for 25 min, then equilibrated in the prepared cryopreservation solution for 15 min, and then loaded onto straws, the straws were put into liquid nitrogen for preservation. After thawing, the ovarian organs or ovarian tissue slices were incubated in a culture solution (10% FBS+a-MEM) in an incubator at 37 C./5% CO.sub.2 for 2 h for further thawing, and then fixed with 4% paraformaldehyde, embedded in paraffin and stained with HE for morphological observation with microscope. The results are shown in FIGS. 1-6. FIG. 1 is a picture of a stained slice of a fresh unfrozen ovarian organ, and FIG. 4 is a picture of a stained slice of a fresh unfrozen ovarian tissue.

(47) TABLE-US-00004 TABLE 4 Ovarian organ cryopreservation scheme Equilibration Cryopreservation Thawing No. solution solution solution Morphology Application b A Thawing FIG. 3 Embodiment 9 solution 3# Comparative a Freezing Thawing FIG. 2 Embodiment 7 solution 1# solution 1#

(48) TABLE-US-00005 TABLE 5 Ovarian tissue cryopreservation scheme Equilibration Cryopreservation Thawing No. solution solution solution Morphology Application b A Thawing FIG. 6 Embodiment 10 solution 3# Comparative a Freezing Thawing FIG. 5 Embodiment 8 solution 1# solution 1#

(49) As can be seen from FIGS. 1-6, when the cryopreservation solution disclosed herein is used to preserve ovarian organs and ovarian tissues, compared with the cryopreservation solution commonly used in the prior art and fresh unfrozen ovarian organs, the ovarian organs and ovarian tissues treated with the cryopreservation solution described herein are characterized in that: the original follicle structure is relatively intact, the interstitial structure is relatively intact, the cytoplasm of cells is homogeneous and lightly stained in a relatively large amount, and nucleus shrinkage and deep staining are relatively mild, the structure of the vascular wall of the ovarian organ is intact, lumen collapse is mild, the cytoplasm of endothelial cells is homogeneous and lightly stained in a relatively large amount, and nucleus shrinkage and deep staining are relatively mild.

(50) It can be seen that cryopreservation solutions disclosed herein prepared with various biomimetic ice growth inhibition materials as main components have good inhibiting effect on the growth of ice crystals, can avoid the use of serum and maintain good biocompatibility, and can be applied to cryopreservation of oocytes, embryos, stem cells, reproductive organs and tissues, where high cell survival rates and good biological activity can be achieved.

(51) The examples of the present invention have been described above. However, the present invention is not limited to the above examples. Any modifications, equivalents, improvement and the like made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.