A serum-free and DMSO-free cryopreservation solution for mesenchymal stem cells, its preparation method and application

Abstract

The present invention belongs to the field of cell culture and preservation, and specifically discloses a serum-free and DMSO-free mesenchymal stem cell cryopreservation solution, its preparation method and application. The cryopreservation solution contains the following components: a basic diluent 40-80 v / v%, a permeable cryoprotectant 5-20 v / v%, a non-permeable cryoprotectant 1-10 v / v%, a cell membrane protectant 5-20 v / v%, a cell activity additive 5-20 v / v%, and a plant-derived natural antioxidant composition; the basic diluent contains the following components: sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose, HEPES, and sodium pyruvate; the plant-derived natural antioxidant composition is cyanidin-3-O-glucoside C3G and epigallocatechin gallate EGCG; this cryopreservation solution does not contain serum or DMSO, and creatively combines the two plant-derived natural antioxidants C3G and EGCG. The sources and dosages of all components are clear, it does not contain any cytotoxic components, is easy to use, has a high cell recovery viability after cryopreservation, and has a wide range of applications.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture and preservation, and specifically discloses a serum-free and DMSO-free mesenchymal stem cell cryopreservation solution, a preparation method thereof, and an application thereof. Background Art

[0002] Stem cells are a type of cells with self-renewal ability and multi-lineage differentiation potential. Under certain conditions, they can differentiate into various functional cells. According to the developmental stage of stem cells, they are divided into embryonic stem cells and adult stem cells. According to the functions of adult stem cells, they can be divided into neural stem cells, hematopoietic stem cells, mesenchymal stem cells, etc. The sources of stem cells are usually embryos, bone marrow, peripheral blood, umbilical cord blood, etc. Stem cells can undergo mitosis and differentiation under certain conditions, divide into a variety of specialized cells, have the ability to repair the structure and function of various tissues and organs, and can use self-renewal to provide more stem cells. Therefore, stem cells are called "universal cells" in the medical field and have very important significance in the medical field.

[0003] Mesenchymal stem cells (MSCs) are an important member of the stem cell family. They are a type of adult stem cells with self-renewal and multi-lineage differentiation potential, originating from the mesoderm and ectoderm in the early stage of development, and widely existing in bone marrow, adipose tissue, and the stroma of various tissues and organs. Based on their important biological characteristics in multi-lineage differentiation, immune regulation, tissue repair, cell implantation, etc., mesenchymal stem cells have been widely used in the treatment research of various acute and chronic diseases. For example, mesenchymal stem cells can differentiate into a variety of tissue cells such as adipose, bone, cartilage, muscle, nerve, and liver under specific induction conditions in vivo or in vitro. They still have multi-lineage differentiation potential after continuous passage culture and cryopreservation, and have no immune rejection reaction when implanted in the body. They can be used for the repair of tissue and organ damage caused by aging and diseases, and are ideal seed cells for tissue engineering and the core of regenerative medicine technology.

[0004] Mesenchymal stem cells are a very precious biological resource with extremely broad application prospects in many fields such as developmental biology and medicine. After being amplified and cultured in vitro, mesenchymal stem cells can be used in clinical and various scientific research experiments. Inevitably, the prepared cells need to be cryopreserved. Existing stem cell cryopreservation is usually carried out with common ordinary cell cryopreservation solutions, and its composition is: dimethyl sulfoxide (DMSO), serum and cell culture medium. DMSO is the most commonly used permeating cryoprotectant, but it has cytotoxicity and can denature intracellular proteins. Moreover, DMSO will also damage the potency and differentiation potential of stem cells. The components of serum are very complex. It not only has a great possibility of bringing infectious substances such as viruses and other allergens, but also has an impact on the differentiation of stem cells. The presence of other unknown components in serum will also have unpredictable effects on the growth and differentiation of stem cells. Summary of the Invention

[0005] In order to solve the above problems, the present invention discloses a serum-free and DMSO-free mesenchymal stem cell cryopreservation solution, its preparation method and application, so that the recovery survival rate of mesenchymal stem cells from various tissue sources after cryopreservation remains above 90%, and various biological characteristics of the mesenchymal stem cells after recovery can be maintained.

[0006] A serum-free and DMSO-free mesenchymal stem cell cryopreservation solution contains the following components: basic diluent 40-80 v / v%, permeating cryoprotectant 5-20 v / v%, non-permeating cryoprotectant 1-10 v / v%, cell membrane protectant 5-20 v / v%, cell activity additive 5-20 v / v% and plant-derived natural antioxidant composition.

[0007] The basic diluent contains the following components: sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose, HEPES and sodium pyruvate.

[0008] The composition of the plant-derived natural antioxidant composition is: cyanidin-3-O-glucoside C3G L and epigallocatechin gallate EGCG.

[0009] During the freezing process, cells will undergo oxidative stress and generate excessive reactive oxygen species (ROS). These ROS have strong oxidizing properties and will snatch electrons from biological macromolecules such as nucleic acids and proteins in the cells, causing oxidative damage and molecular distortion, resulting in cell damage and aging. Therefore, during the cell cryopreservation process, it is very necessary to appropriately supplement exogenous antioxidants to assist cells in resisting free radical damage. Currently, most of the antioxidants that can scavenge intracellular ROS are chemically synthesized and have certain toxic side effects. Researchers have increasingly turned their attention to more efficient and safe natural products. During the screening of natural antioxidants, it was found that the combined application of multiple antioxidants is beneficial for the antioxidants to play their roles in different aspects, thereby greatly enhancing the antioxidant effect as a whole, producing a synergistic effect, and avoiding the damage caused by overloading cells with only a single antioxidant. The present invention creatively combines two plant-derived natural antioxidants, cyanidin-3-O-glucoside (C3G) and epigallocatechin gallate (EGCG), which can efficiently maintain the activity and various biological functions of mesenchymal stem cells after cryopreservation and recovery, and is applicable to non-programmed cryopreservation, and can be conveniently and quickly widely applied to the cryopreservation of various mesenchymal stem cells.

[0010] Furthermore, for the above-mentioned serum-free and DMSO-free mesenchymal stem cell cryopreservation solution, the basic diluent contains the following components: sodium chloride 5 - 10 g / L, potassium chloride 0.1 - 0.8 g / L, potassium dihydrogen phosphate 0.01 - 0.1 g / L, disodium hydrogen phosphate 0.02 - 0.1 g / L, sodium bicarbonate 0.1 - 0.5 g / L, D-glucose 0.2 - 2 g / L, HEPES 1.0 - 5.0 g / L, sodium pyruvate 0.05 - 0.2 g / L. The basic diluent in the cell cryopreservation solution contains various salt ions, which can effectively maintain the balance of key ions and osmotic pressure inside and outside the cells and maintain the stability of the pH environment.

[0011] Furthermore, for the above-mentioned serum-free and DMSO-free mesenchymal stem cell cryopreservation solution, the basic diluent contains the following components: sodium chloride 8 g / L, potassium chloride 0.4 g / L, potassium dihydrogen phosphate 0.06 g / L, disodium hydrogen phosphate 0.048 g / L, sodium bicarbonate 0.35 g / L, D-glucose 1 g / L, HEPES 3.57 g / L, sodium pyruvate 0.11 g / L.

[0012] Furthermore, for the above-mentioned serum-free and DMSO-free mesenchymal stem cell cryopreservation solution, the permeating cryoprotectant is 1,3-propanediol; the non-permeating cryoprotectant is hydroxyethyl starch; the cell membrane protectant is trehalose; the cell activity additive is GlutaMAX.

[0013] The cryopreservation solution of the present invention contains a variety of cell cryoprotectants, which can increase the ability of cells to resist cryogenic damage and ensure the good state of cells before and after cryopreservation. Using 1,3-propanediol as an osmotic intracellular cryoprotectant can quickly penetrate the cell membrane into the cells, lower the freezing point, delay the cryopreservation process, at the same time increase the ion concentration inside the cells, reduce the formation of intracellular ice crystals, thereby reducing the degree of cell damage and ensuring the cell recovery survival rate. Using trehalose and hydroxyethyl starch as non-osmotic extracellular cryoprotectants; trehalose is a typical stress metabolite, which can form a unique protective film on the cell surface under harsh environments such as low temperature and high osmotic pressure, effectively protecting the biomolecular structure of the cell membrane from being damaged; hydroxyethyl starch is a macromolecular substance that cannot penetrate cells. It can reduce solute damage by diluting the extracellular electrolyte concentration, preferentially bind to water molecules in the solution before ice crystal formation, reduce the content of extracellular free water, and reduce the formation of ice crystals. L-glutamine is an important substance and energy source during cell culture and thus becomes an important component of the culture medium. L-glutamine can play a role in stabilizing the cell membrane and protein structure by maintaining and increasing the intracellular glutathione (GSH) reserve, protecting the normal functions of cells. L-alanyl-L-glutamine dipeptide (GlutaMAX) is a substitute for L-glutamine, which has better stability, does not degrade spontaneously, can significantly reduce the accumulation of toxic ammonia, and improve cell viability and growth.

[0014] Furthermore, the above-mentioned serum-free and DMSO-free mesenchymal stem cell cryopreservation solution contains the following components: basic diluent 60 v / v%, 1,3-propanediol 10 v / v%, hydroxyethyl starch 5 v / v%, trehalose 10 v / v%, GlutaMAX 10 v / v% and a plant-derived natural antioxidant composition;

[0015] The content of each component of the plant-derived natural antioxidant composition is: cyanidin-3-O-glucoside C3G 10 μg / mL, epigallocatechin gallate EGCG 50 μg / mL.

[0016] Under normal physiological conditions, the intracellular oxidation and antioxidant systems maintain a dynamic balance. However, during cell freezing, oxidative stress is induced, leading to the accumulation of excessive reactive oxygen species (ROS). The production and clearance of ROS in cells become unbalanced, and the accumulation of ROS in cells causes various oxidative damages. Mammalian cells have a powerful endogenous ROS defense system, which consists of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione transferase (GSH), and can promptly and rapidly scavenge the excess ROS in the body. SOD catalyzes the disproportionation reaction of ROS and is an important antioxidant enzyme in cells and the main scavenger of ROS. GSH is one of the main members of the intracellular antioxidant defense system and is an important enzyme for scavenging H2O2 and many organic hydroperoxides. Together, SOD and GSH can reduce and block the primary initiation effect of ROS peroxidation and the secondary initiation effect by reducing peroxides through scavenging ROS. Therefore, the levels of SOD and GSH activities in the body directly affect the clearance of ROS. Cyanidin-3-O-glucoside (C3G) and epigallocatechin gallate (EGCG) are natural antioxidants derived from plants, which are safe and non-toxic and have various effects such as significant antioxidant, anti-inflammatory, and anti-tumor effects. C3G and EGCG can significantly inhibit the decrease in cell viability induced by oxidative stress, significantly reduce the accumulation of intracellular reactive oxygen species (ROS), the level of malondialdehyde (MDA), and the release of nitric oxide (NO), and significantly increase the activities of superoxide dismutase (SOD) and glutathione transferase (GSH). It was found in the examples of the present invention that the combined application of the two natural antioxidants C3G and EGCG can greatly enhance the antioxidant effect as a whole, produce a synergistic effect, and avoid the damage caused by overloading cells with only a single antioxidant, thereby better achieving the purpose of protecting cell membranes and biological macromolecules such as nucleic acids and proteins in cells during cell freezing.

[0017] Further, the preparation method of the above-mentioned serum-free and DMSO-free mesenchymal stem cell cryopreservation solution includes the following steps:

[0018] 1) Prepare a basic diluent, and sequentially add sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose, and sodium pyruvate, and adjust the pH to 7.2 with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer.

[0019] 2) Prepare 30% hydroxyethyl starch stock solution, 1M trehalose stock solution, 1mg / mL C3G stock solution, 5mg / mL EGCG stock solution, and 200mM GlutaMAX product.

[0020] 3) Mix the basic diluent, 1,3-propanediol, hydroxyethyl starch, trehalose, GlutaMAX, C3G, and EGCG evenly in proportion.

[0021] 4) Filter and sterilize the solution obtained in step 3) with a filter having a pore size of 0.22 μm to obtain a serum-free and DMSO-free mesenchymal stem cell non-programmed cryopreservation solution.

[0022] Furthermore, the application of the above-mentioned serum-free and DMSO-free mesenchymal stem cell cryopreservation solution includes the following steps:

[0023] Step 1: Culture the cells to be cryopreserved. When the cells grow to the required number, select the cells with good growth status, take pictures of the cells for record, collect the cells and count them.

[0024] Step 2: According to the number of cell tubes to be cryopreserved, resuspend the cell clumps with the cryopreservation solution at 1.6×10^6 cells per tube.

[0025] Step 3: After gently pipetting the cell cryopreservation solution evenly, insert the cell cryopreservation tubes into the hundred-well box and directly place them in a -80°C refrigerator for cryopreservation or transfer the cells into a liquid nitrogen tank.

[0026] Step 4: Before cell resuscitation, turn on the water bath in advance and heat it to 37°C. Incubate the required culture medium to 37°C. After taking out the cells from the -80°C refrigerator or liquid nitrogen tank, quickly place them in the water bath to melt, and then resuspend the cells with the culture medium and centrifuge.

[0027] Step 5: Resuspend the cell clumps with an appropriate amount of fresh culture medium, and then use a cell counter or trypan blue staining to count the cells, and statistically analyze the number and viability of the cells after resuscitation.

[0028] Step 6: Inoculate the cells into a culture flask at an appropriate density. Take pictures for record 24 hours after resuscitation, observe the cell morphology and adhesion situation, and perform various cell function tests.

[0029] Furthermore, according to the above application, the cells include but are not limited to human-derived MSC cells, such as other mammalian MSC cells and various cancer cell lines.

[0030] Furthermore, according to the above application, the human-derived MSC cells are derived from tissues including but not limited to bone marrow, dental pulp, adipose tissue, or umbilical cord, etc.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a serum-free and DMSO-free cryopreservation solution for mesenchymal stem cells, its preparation method and application. This cryopreservation solution does not contain serum or DMSO, and creatively combines two plant-derived natural antioxidants, C3G and EGCG. The sources and dosages of all components are clear, without any cytotoxic components, and it is convenient to use. The recovery viability of cryopreserved cells is high, and the application range is wide. It can be used for the cryopreservation of various mesenchymal stem cells, as well as some other types of stem cells and tumor cell lines.

[0033] (1) Using the cell cryopreservation solution of the present invention, there are no any exogenous serum components, avoiding the introduction of animal-derived components and allergens, avoiding the unpredictable effects of unknown serum components on cell growth and differentiation, and avoiding the possibility of virus contamination.

[0034] (2) The cell cryopreservation solution of the present invention does not contain DMSO, and uses 1,3-propanediol as a permeable intracellular cryoprotectant. It can quickly penetrate the cell membrane into the cells, lower the freezing point, delay the cryopreservation process, reduce the formation of intracellular ice crystals, and has no any cytotoxic effect, which is safe and efficient.

[0035] (3) The cell cryopreservation solution of the present invention creatively combines two plant-derived natural antioxidants, C3G and EGCG, to scavenge excessive reactive oxygen free radicals in cells, significantly reducing the cell viability damage caused by oxidative stress during cell cryopreservation.

[0036] (4) Using the cell cryopreservation solution of the present invention, it is specially developed for mesenchymal stem cells from various tissue sources, and is not only applicable to the cryopreservation of various MSCs (such as bone marrow / dental pulp / umbilical cord, etc.), but also can be used for the cryopreservation of other types of stem cells and tumor cell lines.

[0037] (5) Using the cell cryopreservation solution of the present invention, the sources and dosages of all components are very clear. The dosages and concentrations of various reagents have been repeatedly proportioned and optimized, with stable performance and convenient and fast preparation and production.

[0038] (6) Using the cell cryopreservation solution of the present invention, the cryopreserved cell suspension can be directly placed in a refrigerator at -80°C and below for cryopreservation, without the need for a programmed cooling device and without cumbersome cryopreservation steps, saving a lot of time and effort.

[0039] (7) Using the cell cryopreservation solution of the present invention, it does not contain serum and cell culture medium, greatly reducing the cost, and the cryopreservation solution has a long shelf life. When using the cryopreservation solution prepared 12 months later to cryopreserve cells, the cell recovery viability still remains above 90%. Description of the Drawings

[0040] Figure 1Photographs were taken of HBMSC cells before cryopreservation and after resuscitation (Example 5). a and b are before cryopreservation (40X, 100X), and c and d are after resuscitation (40X, 100X).

[0041] Figure 2 Photographs were taken of HBMSC cells after resuscitation and induced differentiation (Example 5). a and b are the alizarin red staining effects of osteogenic induced differentiation (100X, 200X), c and d are the oil red O staining effects of adipogenic induced differentiation (100X, 200X), and e and f are the alcian blue staining effects of chondrogenic induced differentiation (40X, 100X).

[0042] Figure 3 Detection of cell markers of HBMSC cells after resuscitation (Example 5).

[0043] Figure 4 Photographs were taken of HADSC cells before cryopreservation and after resuscitation (Example 5). a and b are before cryopreservation (40X, 100X), and c and d are after resuscitation (40X, 100X).

[0044] Figure 5 Photographs were taken of HADSC cells after resuscitation and induced differentiation (Example 5). a and b are the alizarin red staining effects of osteogenic induced differentiation (100X, 200X), c and d are the oil red O staining effects of adipogenic induced differentiation (100X, 200X), and e and f are the alcian blue staining effects of chondrogenic induced differentiation (40X, 100X).

[0045] Figure 6 Detection of cell markers of HADSC cells after resuscitation (Example 5).

[0046] Figure 7 Photographs were taken of HUMSC cells before cryopreservation and after resuscitation (Example 5). a and b are before cryopreservation (40X, 100X), and c and d are after resuscitation (40X, 100X).

[0047] Figure 8 Photographs were taken of HUMSC cells after resuscitation and induced differentiation (Example 5). a and b are the alizarin red staining effects of osteogenic induced differentiation (100X, 200X), c and d are the oil red O staining effects of adipogenic induced differentiation (100X, 200X), and e and f are the alcian blue staining effects of chondrogenic induced differentiation (50X, 100X).

[0048] Figure 9 Detection of cell markers of HUMSC cells after resuscitation (Example 5).

[0049] Figure 10 Proliferation detection of three types of mesenchymal stem cells after resuscitation in Test Examples 1-3. Detailed implementation methods

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0051] For the reagents or instruments used in the embodiments of the present invention, if the manufacturer is not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0052] The preparation method of the serum-free and DMSO-free mesenchymal stem cell non-programmed cryopreservation solution is carried out by feeding materials according to the raw material formula of each embodiment. The method includes the following steps:

[0053] 1) Prepare a basic diluent, and sequentially add sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose, and sodium pyruvate, and adjust the pH to 7.2 with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution;

[0054] 2) Prepare a 30% hydroxyethyl starch mother liquor, a 1M trehalose mother liquor, a 1mg / mL C3G mother liquor, a 5mg / mL EGCG mother liquor, and a 200mM GlutaMAX product;

[0055] 3) Mix the basic diluent, 1,3-propanediol, hydroxyethyl starch, trehalose, GlutaMAX, C3G, and EGCG evenly in proportion;

[0056] 4) Filter and sterilize the solution obtained in step 3) with a filter with a pore size of 0.22μm to obtain the serum-free and DMSO-free mesenchymal stem cell non-programmed cryopreservation solution.

[0057] The application of the serum-free and DMSO-free mesenchymal stem cell non-programmed cryopreservation solution includes the following steps:

[0058] Step 1: Culture the cells to be cryopreserved. When the cells grow to the required number, select the cells with good growth status, take pictures of the cells for record, collect the cells and count them;

[0059] Step 2: According to the number of cell tubes to be cryopreserved, resuspend the cell clumps with the cryopreservation solution of the present invention at 1.6×10^6 cells per tube. The viscosity of the cryopreservation solution of the present invention is relatively high, and the adhesion loss (about 10%) of the cell number and the volume of the cryopreservation solution should be considered when using;

[0060] Step 3: After gently pipetting the cell cryopreservation solution evenly, insert the cell cryopreservation tube into the hundred-well box and directly store it in a -80°C refrigerator. If long-term storage is required, the cells can be transferred to a liquid nitrogen tank;

[0061] Step 4: Before cell resuscitation, turn on the water bath in advance and heat it to 37°C. Incubate the required culture medium to 37°C as well. After taking out the cells from the -80°C refrigerator or liquid nitrogen tank, place them in the water bath to melt as soon as possible, then resuspend the cells with the culture medium and centrifuge them.

[0062] Step 5: Resuspend the cell pellet with an appropriate amount of fresh culture medium, then use a cell counter or trypan blue staining to count the cells, and statistically analyze the number and viability of the resuscitated cells.

[0063] Step 6: Inoculate the cells into a culture flask at an appropriate density. After 24 hours of resuscitation, take a photo for record, observe the cell morphology and adhesion situation, and perform various cell function tests (such as cell proliferation, cell induced differentiation, etc.).

[0064] Example 1

[0065] The cell cryopreservation solution used in the present invention is composed of: basic diluent 60 v / v%, 1,3-propanediol 10 v / v%, hydroxyethyl starch 5 v / v%, trehalose 10 v / v%, GlutaMAX 10%, C3G 10 μg / mL.

[0066] Example 2

[0067] The cell cryopreservation solution used in the present invention is composed of: basic diluent 60 v / v%, 1,3-propanediol 10 v / v%, hydroxyethyl starch 5 v / v%, trehalose 10 v / v%, GlutaMAX 10%, EGCG 50 μg / mL.

[0068] Example 3

[0069] The cell cryopreservation solution used in the present invention is composed of: basic diluent 60 v / v%, 1,3-propanediol 10 v / v%, hydroxyethyl starch 5 v / v%, trehalose 10 v / v%, GlutaMAX 10%, C3G 1 μg / mL, EGCG 100 μg / mL.

[0070] Example 4

[0071] The cell cryopreservation solution used in the present invention is composed of: basic diluent 60 v / v%, 1,3-propanediol 10 v / v%, hydroxyethyl starch 5 v / v%, trehalose 10 v / v%, GlutaMAX 10%, C3G 20 μg / mL, EGCG 10 μg / mL.

[0072] Example 5

[0073] The cell cryopreservation solution used in the present invention has the following composition: 60 v / v% of basic diluent, 10 v / v% of 1,3-propanediol, 5 v / v% of hydroxyethyl starch, 10 v / v% of trehalose, 10% of GlutaMAX, 10 μg / mL of C3G, and 50 μg / mL of EGCG.

[0074] Comparative Example 1

[0075] CRD0 serum-free and DMSO-free cell cryopreservation solution product (product number: CF0301) purchased from Cellregen.

[0076] Comparative Example 2

[0077] Common cell cryopreservation solution containing serum and DMSO commonly used in the laboratory.

[0078] Test Example 1

[0079] Human bone marrow mesenchymal stem cells (HBMSC)

[0080] 1. Count the cells before cryopreservation and after resuscitation, as shown in Table 1;

[0081] 2. Take photos of the cells before cryopreservation and after resuscitation (Example 5), see appendix Figure 1 : The cells all grow in long spindle shapes, with good polarity and good three-dimensional sense;

[0082] 3. Detect the proliferation of the cells after resuscitation (Example 5), as shown in Table 2;

[0083] 4. Induce the differentiation of the cells after resuscitation (Example 5), see appendix Figure 2 : When the cell confluence reaches about 70%, add the osteogenic induction solution for mesenchymal stem cells. After 23 days, perform alizarin red staining. Alizarin red combines with osteoid to form concentric dark red nodules (a, b); when the cell confluence reaches about 90%, add the adipogenic induction solution for mesenchymal stem cells. After 23 days, perform oil red O staining, and visible relatively standard lipid droplets stained red can be seen (c, d); the cells are cultured in a mass, and induced and cultured with the chondrogenic induction solution for mesenchymal stem cells. The cells gradually change from flat cell masses attached to the bottom of the centrifuge tube into cell spheres. After 21 days of induction, the cell masses become larger, rounder, and the surface becomes smoother (e, f);

[0084] 5. Detect the cell markers after resuscitation (Example 5), see appendix Figure 3 : The flow cytometry detection results of cell surface marker molecules are CD29, 92.86%; CD44, 98.66%; CD34, 0.70%; CD45, 0.80%; the detection results of cell surface marker molecules are qualified;

[0085] Table 1. Cell count of HBMSC after resuscitation (cryopreserved with 1.5×10^6 cells before cryopreservation)

[0086] HBMSC Cell count Cell viability Example 1 1.27E+6 84.9％ Example 2 1.29E+6 86.2％ Example 3 1.34E+6 89.5％ Example 4 1.37E+6 91.3％ Example 5 1.41E+5 93.8％ Comparative Example 1 1.35E+5 90.3％ Comparative Example 2 1.42E+5 94.7％

[0087] Table 2. Detection of cell proliferation after resuscitation (Example 5, inoculated with 5.0×10^4 cells)

[0088] Unit: 10^4 1d 2d 3d 4d 5d 6d HBMSC 5.13 8.62 18.3 29.8 50.8 56.4

[0089] From the above results, it can be seen that the serum-free and DMSO-free mesenchymal stem cell non-programmed cryopreservation solution provided by the present invention enables the resuscitation survival rate of human bone marrow mesenchymal stem cells (HBMSC) to remain above 90% after cryopreservation (the effect of Example 5 is the best), and can maintain the normal proliferation ability, induction differentiation ability and cell-specific markers of the cells after resuscitation.

[0090] Test Example 2

[0091] Human adipose-derived mesenchymal stem cells (HADSC)

[0092] 1. Counting of cells before and after cryopreservation, see Table 3;

[0093] 2. Photographing of cells before and after cryopreservation, see Appendix Figure 4 : Most cells grow in a long spindle shape, some cells have irregular shapes, good polarity and good three-dimensional sense;

[0094] 3. Detection of cell proliferation after resuscitation (Example 5), see Table 4;

[0095] 4. Induction differentiation of cells after resuscitation (Example 5), see Appendix Figure 5 : When the cell confluence reaches about 70%, add the osteogenic induction solution for mesenchymal stem cells. Calcium nodules can be seen after 21 days, and the positive osteogenic rate reaches 90% after alizarin red staining (a, b); when the cell confluence reaches about 90%, add the adipogenic induction solution for mesenchymal stem cells. After 21 days, a large number of lipid droplets appear. After oil red O staining, relatively standard lipid droplets stained red can be seen (c, d); after the cells are cultured in a mass and induced with the chondrogenic induction solution for mesenchymal stem cells, the cells gradually change from flat cell masses attached to the bottom of the centrifuge tube into cell spheres. After 21 days of induction, the cell masses become larger, rounder, and the surface becomes smooth, forming smooth chondrospheres (e, f);

[0096] 5. Detection of cell markers after resuscitation (Example 5), see Appendix Figure 6 : Flow cytometry detection results of cell surface marker molecules: CD29, 99.87%; CD44, 99.85%; CD34, 1.94%; CD45, 1.23%; the detection results of cell surface marker molecules are qualified;

[0097] Table 3. Cell counting after resuscitation (cryopreserved with 1.5×10^6 cells before cryopreservation)

[0098] HADSC Cell count Cell viability Example 1 1.23E+6 82.3％ Example 2 1.25E+6 83.5％ Example 3 1.30E+6 86.7％ Example 4 1.33E+6 88.9％ Example 5 1.36E+5 90.7％ Comparative Example 1 1.32E+5 88.1％ Comparative Example 2 1.38E+5 92.3％

[0099] Table 4, Detection of cell proliferation after resuscitation (Example 5, inoculated with 5.0*10^4 cells)

[0100] Unit: 10^4 1d 2d 3d 4d 5d 6d HADSC 5.66 6.78 11.3 19.8 35.8 40.4

[0101] From the above results, it can be seen that the serum-free and DMSO-free mesenchymal stem cell non-programmed cryopreservation solution provided by the present invention enables the resuscitation survival rate of human adipose mesenchymal stem cells (HADSC) after cryopreservation to remain above 90% (the effect of Example 5 is the best), and can maintain the normal proliferation ability, induction and differentiation ability, and cell-specific markers of the cells after resuscitation.

[0102] Test Example 3

[0103] Human umbilical cord mesenchymal stem cells (HUMSC)

[0104] 1. Counting of cells before and after cryopreservation, see Table 5;

[0105] 2. Photographing of cells before and after cryopreservation, see Appendix Figure 7 : The cells all grew in a long spindle shape, with good polarity and three-dimensional sense;

[0106] 3. Detection of cell proliferation after resuscitation (Example 5), see Table 6;

[0107] 4. Induction and differentiation of cells after resuscitation (Example 5), see Appendix Figure 8 : When the cell confluence reached about 70%, the mesenchymal stem cell osteogenic induction solution was added, and calcium nodules could be seen after 41 days. After alizarin red staining, the osteogenic positive rate reached 80% (a, b); when the cell confluence reached about 90%, the mesenchymal stem cell adipogenic induction solution was added. After 41 days, oil red O staining was performed, and relatively standard lipid droplets stained red could be seen (c, d); after the cells were cultured in a mass, and induced and cultured with the mesenchymal stem cell chondrogenic induction solution, the cells gradually changed from flat cell masses attached to the bottom of the centrifuge tube into cell spheres. After 21 days of induction, the cell masses became larger, rounder, and smoother, forming smooth cartilage spheres (e, f);

[0108] 5. Detection of cell markers after resuscitation (Example 5), see Appendix Figure 9 : Flow cytometry detection results of cell surface marker molecules: CD29, 99.91%; CD44, 99.97%; CD34, 0.44%; CD45, 0.31%; the detection results of cell surface marker molecules were qualified;

[0109] Table 5, Cell counting after resuscitation (cryopreserved with 1.5*10^6 cells before cryopreservation)

[0110] HUMSC Cell count Cell viability Example 1 1.25E+6 83.5％ Example 2 1.26E+6 84.1％ Example 3 1.31E+6 87.5％ Example 4 1.36E+6 90.7％ Example 5 1.38E+5 92.2％ Comparative Example 1 1.34E+5 89.6％ Comparative Example 2 1.40E+5 93.3％

[0111] Table 6. Detection of cell proliferation after resuscitation (Example 5, inoculated with 2.0×10^4 cells)

[0112] Unit: 10^4 1d 2d 3d 4d 5d 6d HUMSC 5.02 10.4 20.6 32.9 60.1 65.8

[0113] From the above results, it can be seen that the serum-free and DMSO-free mesenchymal stem cell non-programmed cryopreservation solution provided by the present invention enables the resuscitation survival rate of human umbilical cord mesenchymal stem cells (HUMSC) after cryopreservation to remain above 90% (the effect of Example 5 is the best), and can maintain the normal proliferation ability, induction and differentiation ability, and cell-specific markers of the cells after resuscitation.

[0114] Figure 10 For the detection of the proliferation of three types of mesenchymal stem cells after resuscitation in Test Examples 1-3.

[0115] From the above Test Examples 1-3, it can be seen that the present invention provides a new type of serum-free and DMSO-free non-programmed cell cryopreservation solution for cryopreserving mesenchymal stem cells, which excludes the adverse effects of exogenous serum and DMSO, and creatively combines two plant-derived natural antioxidants, cyanidin-3-O-glucoside (C3G) and epigallocatechin gallate (EGCG), can efficiently maintain the activity and various biological functions of mesenchymal stem cells after cryopreservation and resuscitation, and is suitable for non-programmed cryopreservation, and can be conveniently and quickly widely applied to the cryopreservation of various mesenchymal stem cells.

[0116] The above are only several limited preferred embodiments of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A serum-free and DMSO-free cryopreservation solution for mesenchymal stem cells, characterized in that, it contains the following components: basic diluent 40 - 80 v / v%, permeating cryoprotectant 5 - 20 v / v%, non-permeating cryoprotectant 1 - 10 v / v%, cell membrane protectant 5 - 20 v / v%, cell activity additive 5 - 20 v / v%, and plant-derived natural antioxidant composition; the basic diluent contains the following components: sodium chloride 5 - 10 g / L, potassium chloride 0.1 - 0.8 g / L, potassium dihydrogen phosphate 0.01 - 0.1 g / L, disodium hydrogen phosphate 0.02 - 0.1 g / L, sodium bicarbonate 0.1 - 0.5 g / L, D-glucose 0.2 - 2 g / L, HEPES 1.0 - 5.0 g / L, sodium pyruvate 0.05 - 0.2 g / L; the permeating cryoprotectant is 1,3-propanediol; the non-permeating cryoprotectant is hydroxyethyl starch; the cell membrane protectant is trehalose; the cell activity additive is GlutaMAX; the composition of the plant-derived natural antioxidant composition is: cyanidin-3-O-glucoside C3G 1 - 20 μg / mL and epigallocatechin gallate EGCG 10 - 100 μg / mL.

2. The serum-free and DMSO-free cryopreservation solution for mesenchymal stem cells according to claim 1, characterized in that, the basic diluent contains the following components: sodium chloride 8 g / L, potassium chloride 0.4 g / L, potassium dihydrogen phosphate 0.06 g / L, disodium hydrogen phosphate 0.048 g / L, sodium bicarbonate 0.35 g / L, D-glucose 1 g / L, HEPES 3.57 g / L, sodium pyruvate 0.11 g / L.

3. The serum-free and DMSO-free cryopreservation solution for mesenchymal stem cells according to claim 1, characterized in that, it contains the following components: basic diluent 60 v / v%, 1,3-propanediol 10 v / v%, hydroxyethyl starch 5 v / v%, trehalose 10 v / v%, GlutaMAX 10 v / v%, and plant-derived natural antioxidant composition; the content of each component of the plant-derived natural antioxidant composition is: cyanidin-3-O-glucoside C3G 10 μg / mL, epigallocatechin gallate EGCG 50 μg / mL.

4. The preparation method of the serum-free and DMSO-free cryopreservation solution for mesenchymal stem cells as claimed in claim 1, characterized in that, it includes the following steps: 1) Prepare the basic diluent, and successively add sodium chloride, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, D-glucose, sodium pyruvate, and adjust the pH to 7.2 with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HEPES buffer; 2) Prepare 30% hydroxyethyl starch stock solution, 1 M trehalose stock solution, 1 mg / mL C3G stock solution, 5 mg / mL EGCG stock solution, and 200 mM GlutaMAX commercial product; 3) Mix the basic diluent, 1,3-propanediol, hydroxyethyl starch, trehalose, GlutaMAX, C3G, and EGCG evenly according to the ratio; 4) Filter and sterilize the solution obtained in step 3) with a filter having a pore size of 0.22 μm to obtain a serum-free and DMSO-free mesenchymal stem cell non-programmed cryopreservation solution.

5. The application of the serum-free and DMSO-free mesenchymal stem cell cryopreservation solution according to any one of claims 1-3, characterized in that, it includes but is not limited to human MSC cells, other mammalian MSC cells or various cancer cell lines.

6. According to the application described in claim 5, characterized in that, the sources of the human MSC cells include but are not limited to bone marrow, dental pulp, adipose or umbilical cord tissue.

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