A cryopreservation solution for stem cell cryopreservation and a method for preparing the same

Abstract

The application discloses a cryopreservation solution for stem cell cryopreservation and a preparation method thereof, and contains the following components in each 100 mL solution: 0.1-50 g of biomimetic ice-controlling material, 0.1-15 mL of DMSO, 0.1-30 mL of serum, and the balance of buffer solution. The cryopreservation solution of the application takes amino acid or polyvinyl alcohol as the main component, has low DMSO content and low toxicity, and has clear components, and can achieve the same or even higher cell survival rate as the existing cryopreservation solution. The cryopreservation solution of the application has simple composition, and the raw materials are convenient and low in cost, and can be widely applied to the cryopreservation of various cells, tissues or organs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a cryopreservation solution for stem cell cryopreservation and its preparation method. Background Technology

[0002] Cryopreservation refers to the preservation of biological materials at ultra-low temperatures, slowing down or stopping cell metabolism and division. Once normal physiological temperatures are restored, cell development can resume. Since its inception, this technology has become an indispensable research method in the natural sciences and has been widely adopted. Stem cells, due to their ability to repair damaged tissue cells, replace damaged cells, and stimulate the regeneration of the body's own cells, are widely used in clinical treatment and regenerative medicine. For example, human umbilical cord mesenchymal stem cells from newborn fetuses have broad clinical application prospects due to their low immunogenicity, high cell content, and strong proliferative capacity; cryopreservation of human umbilical cord mesenchymal stem cells makes their widespread clinical application possible. In addition, with the increasing aging of the world's population, the demand for cryopreservation of donated human cells, tissues, or organs that can be used for regenerative medicine and organ transplantation is also increasing rapidly. Therefore, how to efficiently cryopreserve precious cell, tissue, and organ resources for unforeseen needs has become an urgent scientific and technological problem to be solved.

[0003] Currently, vitrification is the most commonly used cryopreservation method. While vitrification allows the intracellular and extracellular fluids to become glassy during rapid freezing, preventing damage caused by ice crystal formation, existing cryopreservation reagents cannot effectively control ice crystal growth during thawing, thus damaging cells. Stem cells, such as mesenchymal stem cells, possess self-renewal and multi-lineage differentiation potential, and can be widely used in stem cell transplantation, autoimmune diseases, or gene therapy vectors. Current stem cell cryopreservation reagents typically consist of cryoprotectants and stem cell growth media to support cells and reduce ice crystal formation. However, growth media can easily lead to indiscriminate differentiation of stem cells, affecting their directed differentiation potential after thawing. Summary of the Invention

[0004] This invention provides a cryopreservation solution containing, per 100 mL: 0.1-50 g of biomimetic ice-controlling material, 0.1-15 mL of DMSO, 0.1-30 mL of serum, and the remainder of buffer solution.

[0005] According to the present invention, the cryopreservation solution does not contain polyols or sugars.

[0006] According to the present invention, the biomimetic ice-controlling material is selected from at least one of polyvinyl alcohol or amino acid-based biomimetic ice-controlling materials.

[0007] According to the present invention, the amount of polyvinyl alcohol contained in each 100 mL of the cryopreservation solution is preferably 0.1-6 g.

[0008] According to the present invention, the PVA is selected from one or more combinations of isotactic PVA, anaregular PVA, and random PVA, for example, the anaregularity of the PVA is 15%-65%, specifically, for example, 40%-60% or 53%-55%. Random PVA is preferred, for example, PVA with an anaregularity of 45%-65%.

[0009] According to the present invention, the PVA may be selected from PVA with a molecular weight of 10-500kDa or higher, such as 10-30kDa, 30-50kDa, 80-90kDa, or 200-500kDa.

[0010] According to the present invention, the PVA may be selected from PVA with a degree of hydrolysis greater than 80%, for example, with a degree of hydrolysis of 80%-99%, 82-87%, 87%-89%, 89%-99%, or 98%-99%.

[0011] According to the present invention, the amino acid-based biomimetic ice-controlling material is selected from one or more of amino acids, polyamino acids, or peptide compounds.

[0012] As one embodiment of the present invention, the amino acid-based biomimetic ice-controlling material can be an amino acid containing both glaucogenic and hydrophilic groups, or a polyamino acid or peptide compound composed of an amino acid containing glaucogenic groups and an amino acid containing hydrophilic groups.

[0013] The hydrophilic group is a functional group that can form non-covalent interactions with water molecules, such as hydrogen bonds, van der Waals interactions, electrostatic interactions, hydrophobic interactions, or π-π interactions with water; it can be selected from at least one of hydroxyl (-OH), amino (-NH2), carboxylic acid (-COOH), or amide (-CONH2);

[0014] The lyophilic group is a functional group that can form non-covalent interactions with ice, such as hydrogen bonds, van der Waals interactions, electrostatic interactions, hydrophobic interactions, or π-π interactions with ice; by way of example, the lyophilic group can be selected from at least one of hydroxyl (-OH), amino (-NH2), phenyl (-C6H5), or pyrrolyl (-C4H8N).

[0015] For example, the amino acid-based biomimetic ice-controlling material is selected from one or two of arginine, threonine, proline, lysine, histidine, glutamine, aspartic acid, glycine, etc., or a polyamino acid composed of the above amino acids; for example, the amino acid-based biomimetic ice-controlling material is a combination of arginine and threonine.

[0016] As one embodiment of the present invention, the amino acid-based biomimetic ice-controlling material is a polyamino acid (degree of polymerization ≥ 2), preferably with a degree of polymerization of 2 to 40 (such as a degree of polymerization of 6, 8, 15, 20, etc.), and the polyamino acid is an amino acid homopolymer, such as one or a combination of two or more of poly-L-proline, poly-L-arginine, etc.

[0017] In an embodiment of the present invention, the peptide compound is obtained by reacting glaucogenic amino acids, such as threonine (L-Thr), glutamine (L-Gln), aspartic acid (L-Asn), etc., with other hydrophilic amino acids or gluconolactone (GDL) or other sugars. The other hydrophilic amino acids may be selected from arginine, proline, alanine, etc.

[0018] The peptide compound is a peptide formed by two or more amino acid units, such as 2-8 amino acid units, specifically 2, 3, 4, or 5 amino acid units; each amino acid unit is different. The arrangement of the glaucophytic amino acid and other hydrophilic amino acids in the peptide compound is not particularly limited, and they can be linked by amino acid linking groups or chemical bonds known in the art. For example, glaucophytic amino acids and hydrophilic amino acids can be arranged individually or in sequence, or multiple glaucophytic amino acids or multiple hydrophilic amino acids can be linked to form glaucophytic amino acid fragments or hydrophilic amino acid fragments, which are then linked to hydrophilic amino acids (or fragments) or glaucophytic amino acids (or fragments), respectively.

[0019] In embodiments of the present invention, the peptide compound is a polypeptide, such as at least one selected from 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), and L-Thr-L-Cys-L-Thr(TCT). The polypeptide can be prepared using polypeptide synthesis methods known in the art, such as solid-phase synthesis.

[0020] In another embodiment, the peptide compound is a glycopeptide derivative, such as a glycopeptide derivative synthesized from gluconolactone or other sugars and amino acids, such as GDL-L-Thr, GDL-L-Ser, or GDL-L-Val. The glycopeptide derivative can be prepared according to methods known in the art for reacting sugars and amino acids, such as solid-phase synthesis or reacting sugars and amino acids in an organic solvent.

[0021] According to the present invention, the content of the amino acid-based biomimetic ice-controlling material in each 100 mL cryopreservation solution is 0.5-50 g, preferably 1.0-35 g. For example, when the amino acid-based biomimetic ice-controlling material is an amino acid, its content can be 5.0-35 g, preferably 15-25 g; when the amino acid-based biomimetic ice-controlling material is a polyamino acid, its content can be 0.5-9.0 g, preferably 1.0-5.0 g.

[0022] According to the present invention, the buffer may be selected from at least one of DPBS or hepes-buffered HTF buffer or other cell culture buffers known in the art.

[0023] According to the present invention, the serum may be human serum albumin or its alternative, such as sodium dodecyl sulfate, for human cryopreservation subjects, and fetal bovine serum or bovine serum albumin for non-human cryopreservation subjects.

[0024] According to the present invention, the DMSO content in each 100 mL cryopreservation solution is 0.1-15 mL, preferably 1.0-10 mL, more preferably 1-7.5 mL, for example 1.5-5 mL.

[0025] According to the present invention, the serum content in each 100 mL cryopreservation solution is 0.1-30 mL, for example 5.0-20 mL, 10-15 mL.

[0026] According to the present invention, the pH of the cryopreservation solution is 6.5-7.6, for example 6.9-7.2.

[0027] As one embodiment of the present invention, the cryopreservation solution contains the following components per 100 mL:

[0028] Amino acids 0.5-50g

[0029] DMSO 0.1-10mL

[0030] Serum 0.1-30 mL

[0031] Remaining buffer solution.

[0032] Preferably, the cryopreservation solution contains the following components per 100 mL:

[0033] L-Arg 2.0-20g

[0034] L-Thr 1.0-10g

[0035] DMSO 0.1-10mL

[0036] 5.0-20 mL of serum

[0037] Remaining DPBS.

[0038] As one embodiment of the present invention, the cryopreservation solution contains the following components per 100 mL volume:

[0039] Polyamino acids 0.5-9.0g

[0040] DMSO 0.1-10mL

[0041] 5.0-20 mL of serum

[0042] Remaining buffer solution.

[0043] Preferably, the cryopreservation solution contains the following components per 100 mL volume:

[0044] Poly-L-proline or poly-L-arginine 1.0-8.0g

[0045] DMSO 0.1-10mL

[0046] 5.0-20 mL of serum

[0047] Remaining DPBS.

[0048] As one embodiment of the present invention, the cryopreservation solution comprises the following components per 100 mL volume:

[0049] PVA 1.0-5.0g

[0050] DMSO 0.1-10mL

[0051] Serum 0.1-20 mL

[0052] Remaining buffer solution.

[0053] Preferably, the cryopreservation solution comprises the following components per 100 mL volume:

[0054] PVA 1.0-4.0g

[0055] DMSO 4-10mL

[0056] 10-20 mL of serum

[0057] Remaining DPBS.

[0058] The present invention also provides a method for preparing the above-mentioned cryopreservation solution, comprising the following steps:

[0059] (1) Dissolve the biomimetic ice-controlling material in a portion of a buffer solution, adjust the pH, and form solution 1;

[0060] (2) Dissolve DMSO in a portion of the buffer solution to prepare solution 2;

[0061] (3) After solution 1 and solution 2 are cooled to room temperature, they are mixed, the pH is adjusted and the volume is adjusted to a predetermined volume with buffer solution to obtain the cryopreservation solution.

[0062] According to the preparation method of the present invention, the serum is added when the cryopreservation solution is used.

[0063] According to the preparation method of the present invention, when the biomimetic ice-controlling material is polyvinyl alcohol, water bath heating and stirring are used; for example, the water bath temperature is 65-85℃ or 70-80℃; the stirring is mechanical stirring, such as magnetic stirring.

[0064] This invention also provides the application of the cryopreservation solution in the cryopreservation of stem cells. The cryopreservation of stem cells can be performed using any stem cell freezing method known in the art. In one embodiment, it includes adding the cryopreservation solution containing stem cells into a cryovial, placing it in an isopropanol cryopreservation box, and freezing. In another embodiment, the above-described cryopreservation solution is used to perform stem cell cryopreservation using the microdroplet method.

[0065] Beneficial effects

[0066] The cryopreservation solution provided by this invention uses polyvinyl alcohol or amino acid-based biomimetic ice-controlling materials as the main ice-controlling components. These materials are widely available, have good biocompatibility, and the prepared cryopreservation reagent can be prepared without cell culture medium. Its composition is well-defined and simple, allowing for higher cell viability. The cryopreservation solution of this invention uses readily available and inexpensive raw materials, making it widely applicable to the cryopreservation of various stem cells. Detailed Implementation

[0067] The preparation method of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0069] In this embodiment of the invention, poly-L-proline has a degree of polymerization of 8 or 15 and a molecular weight of 795 or 1475; poly-L-arginine has a degree of polymerization of 8 or 15 and a molecular weight of 1267 or 2361.

[0070] In this embodiment of the invention, the survival rate is the average survival rate of 2-3 repeated experiments.

[0071] Example

[0072] 1. Preparation of cryopreservation solution: Prepare the cryopreservation solution according to the following formula.

[0073] Cryopreservation solution A: Total volume 100 mL. Dissolve 2.0 g of poly-L-arginine in 25 mL of DPBS by sonication and adjust the pH to 7.0 to obtain solution 1. Dissolve 10 mL of DMSO in 20 mL of DPBS to obtain solution 2. After solutions 1 and 2 have returned to room temperature, mix the two solutions thoroughly, adjust the pH to 7.0, and bring the volume up to 85% of the total volume with DPBS. Store 15 mL of serum separately and add it to the cryopreservation solution before use.

[0074] Cryopreservation Solution B: Total volume 100mL. Dissolve 2.0g of PVA in 25mL of DPBS by heating and magnetically stirring in an 80℃ water bath. After all PVA has dissolved and cooled to room temperature, adjust the pH to 7.0. Dissolve 10mL of DMSO in 25mL of DPBS to form Solution 2. After Solution 1 and Solution 2 have returned to room temperature, mix the two solutions thoroughly, adjust the pH, and bring the volume to 85% of the total volume. Store 15mL of serum separately and add it when the cryopreservation solution is used.

[0075] Comparative example:

[0076] Cryopreservation solution #1: Each 1 mL contains 10% (v / v) DMSO, 15% (v / v) fetal bovine serum, and the remainder is α-MEM medium.

[0077] Application example:

[0078] Human umbilical cord mesenchymal stem cells were cryopreserved according to the cryopreservation solutions of the above embodiments and comparative examples, as shown in Table 1.

[0079] Human umbilical cord mesenchymal stem cells are cryopreserved in 300μL cryovials.

[0080] The method for cryopreservation of human umbilical cord stem cells used in this invention is as follows: human umbilical cord mesenchymal stem cells on a culture dish are digested with 25% trypsin for 2-3 minutes, then placed in an equal volume of culture medium (10% FBS + α-MEM medium), and gently pipetted until all stem cells are detached. After centrifugation at 1000 rpm for 5 minutes, the supernatant is discarded, 300 μL of cryopreservation solution is added to a centrifuge tube, and the stem cell cluster is gently dispersed by pipetting. The cryopreservation solution containing stem cells is added to a 2 ml cryopreservation tube, placed in an isopropanol cryopreservation box, and placed in a -80°C freezer. After thawing for 24 hours, the viability is observed by trypan blue staining (see Table 1).

[0081] Table 1. Survival rate of cryopreserved human umbilical cord mesenchymal stem cells.

[0082] serial number cryopreservation solution Survival rate Application Example 1 A 76.5％ Application Example 2 B 78.0％ Comparison Example 1 Coolant #1 76.4％

[0083] As can be seen from the data in Table 1, the cryopreservation solution of the present invention does not require the addition of stem cell culture medium, thus avoiding the introduction of complex culture medium. Only biomimetic ice-controlling material and a small amount of DMSO are added. When human umbilical cord mesenchymal stem cells are cryopreserved, the survival rate of stem cells can reach about 80%, indicating that it has the same effectiveness as existing cryopreservation solutions with added culture medium, and the composition is simpler and more controllable, which has broad application prospects.

[0084] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cryopreservation solution for stem cell cryopreservation, comprising per 100 mL: 1.0-8.0 g poly-L-arginine, 1.0-10 mL DMSO, 5.0-20 mL serum or human serum albumin or bovine serum albumin, and the remainder being buffer solution; The degree of polymerization of the poly-L-arginine is 8 to 15.

2. The cryopreservation solution according to claim 1, wherein the buffer solution is selected from at least one of DPBS or hepes-buffered HTF buffer.

3. The cryopreservation solution according to any one of claims 1 to 2, wherein the pH of the cryopreservation solution is 6.5-7.

6.

4. A method for preparing the cryopreservation solution according to any one of claims 1-3, comprising the following steps: (1) Dissolve the poly-L-arginine in a portion of the buffer solution, adjust the pH to form solution 1; (2) Dissolve DMSO in a portion of the buffer solution to prepare solution 2; (3) After solution 1 and solution 2 are cooled to room temperature, they are mixed, the pH is adjusted and the volume is adjusted to the predetermined volume with buffer solution. Before use, the serum or human serum albumin or bovine serum albumin is added to obtain the cryopreservation solution.

5. The use of the cryopreservation solution according to any one of claims 1-3 in the cryopreservation of stem cells.

6. The application according to claim 5 includes adding the cryosol containing stem cells into a cryovial, placing it in an isopropanol cryovial box, and freezing it.