Serum-component-free cell freezing medium and preparation method thereof

Abstract

The invention relates to the technical field of cell cryopreservation solutions, in particular to a serum-component-free cell cryopreservation solution and a preparation method thereof.The serum-component-free cell cryopreservation solution is prepared from, by mass, 70%-80% of a basic culture medium, 5%-10% of dimethyl sulfoxide (DMSO) and 10%-20% of skim milk powder; according to the cell cryopreservation liquid, the milk powder is used for replacing serum, potential biological risks are reduced, in the biological sterilization link, compared with the serum, the technology is more mature, the sterilization effect is more thorough, the biological safety is higher, the production cost is further reduced, the milk source can be obtained through automatic equipment, operation of professional blood sampling personnel is not needed, and the production cost is reduced. The method has the advantages that the milk source can be immediately treated, the risk of pollution is reduced, the collected bovine blood needs to be subjected to standing and centrifugation, the difference between milk powder batches is smaller than that of the serum, and the quality is easier to guarantee.

Description

Technical Field

[0001] The present invention relates to the technical field of cell freezing solutions, and in particular to a serum-free cell freezing solution and a preparation method thereof. Background Art

[0002] Cryopreservation is the storage of living cells and tissues at extremely low temperatures to maintain their structural integrity for long-term preservation. This is achieved by cooling them to ultra-low temperatures (below -130°C) until extracellular ice forms. This process reduces the kinetic energy of the cells and molecular motion, thereby slowing down the degradation of biomolecules. However, inducing low-temperature conditions without intervention can lead to cell dehydration and gradual cell damage. Accumulated extracellular ice crystals can also cause changes in cell permeability, ultimately leading to cell damage and death. Therefore, the use of cryoprotectants is key to reducing cell damage and improving survival rates.

[0003] Cryoprotectants are divided into two categories: cell-permeable and non-cell-permeable. Cell-permeable agents, such as dimethyl sulfoxide (DMSO), glycerol, and 1,2-propylene glycol, can enter cells and reduce electrolyte concentrations, protecting them from ice crystals. Non-cell-permeable agents, such as 2-methyl-2,4-pentanediol and polymers such as polyvinylpyrrolidone, hydroxyethyl starch, and certain sugars, help prevent extracellular ice formation. DMSO is widely used due to its low cost and low cytotoxicity, making it suitable for a variety of cell types. DMSO can be used as a standard cryopreservation reagent for different cell types in biobanks, including pluripotent stem cells and progenitor stem cells. Human conjunctival cells cryopreserved in 10% DMSO showed no difference in proliferation capacity or expression of progenitor cell markers compared to cells that were not cryopreserved.

[0004] Serum is another important component of cryopreservation fluid, providing a variety of nutrients and growth factors required for cell growth. Traditional cell cryopreservation fluid is primarily composed of culture medium, serum, and dimethyl sulfoxide (DMSO) mixed in a specific ratio. However, serum is a complex mixture with incompletely defined components and large batch-to-batch variability, resulting in unstable composition of the prepared cell preservation fluid. The source of serum is generally subject to certain geographical and seasonal restrictions, and it is relatively expensive. More importantly, the greatest risk of using serum is the potential risk of contamination by blood-borne pathogens, which can affect the safety of subsequent experiments. Commonly used bovine serum is also toxic and inhibitory to cells. Its complex composition, while providing nutrients to cells, can negatively affect cell attachment to microcarrier surfaces, significantly reducing cell attachment rates. Therefore, a safe, inexpensive, and readily available alternative to serum is urgently needed to meet the urgent demand for safe and efficient culture protocols in the biomedicine, food, and other fields.

[0005] Disadvantages of using serum are as follows:

[0006] 1) There are huge potential biological risks

[0007] Serum is a light yellow transparent liquid separated after blood coagulation and the removal of fibrinogen and certain coagulation factors in the plasma. Its main functions are to provide basic nutrients, hormones and various growth factors, binding proteins, contact-promoting and growth factors to prevent cell adhesion from mechanical damage, and to play a certain protective role on cells in culture. As a biological product, serum inevitably faces biosafety-related issues. In recent years, biosafety issues have received widespread attention. my country's "Biosafety Law of the People's Republic of China" has been implemented since April 15, 2021. The promulgation of this decree has important guiding significance for laboratory biosafety. Many viruses are of animal origin, such as mad cow disease, bovine viral diarrhea virus, bovine parvovirus, rabies virus, etc. Fetal bovine serum, as an animal-derived biological product, flows into the market and must be biologically active and unquarantined. Once serum products that fail inspection and quarantine enter the market, the infectious substances they carry will pose a major threat to both humans and the environment. Take bovine herpesvirus type 1 as an example. It is the pathogen of infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV) and infectious pustular balanoposthitis (IPB). After cattle are infected with bovine herpesvirus type 1, the symptoms may be atypical. Seropositive cattle remain in a latent infection state for life. Stress can induce viral reactivation and intermittent viral excretion, leading to the risk of continued virus transmission to susceptible cattle herds. Direct contact between different cows is the main risk factor for reinfection. In addition, mad cow disease is a chronic degenerative disease that affects the central nervous system. Humans can be infected with mad cow disease by eating contaminated beef or coming into contact with sick cow serum. People of all ages are susceptible to the disease and it is difficult to diagnose until it is almost completely cured.

[0008] To save costs, some companies usually purchase newborn calves for blood collection from various dairy farms or small-scale breeders. The health status of the animals is not very clear. They may also carry various antibodies and inhibitory factors due to vaccination or recovery from illness and pass them on to the newborn calves. Therefore, finding a substitute for serum is a key factor in controlling the risk of potential infectious diseases.

[0009] 2) Expensive

[0010] To obtain high-quality calf serum, the cattle herd should first be properly monitored, including immune status, disease status, feed nutrition, cattle age, etc. Calves that show physical weakness, deformity, or suspected disease after birth should be discarded. The above strict screening process will also increase the cost of animal breeding.

[0011] Newborn calf serum refers to blood collected from newborn calves that have not eaten within 14 hours of birth, and the serum is separated and synthesized after sterilization and filtration. Therefore, the blood collection time must be controlled within 14 hours. Prolonging the time will change the protein content of the serum and reduce the quality of the serum. The quality control of the blood collection process must start with the preparation of equipment and personnel. The glassware and instruments used must be soaked in acid and alkali solutions and inactivation solutions in accordance with the requirements of medical and surgical procedures, rinsed with distilled water and deionized water that meet the standards for injection water, dry baked or high-pressure steamed. After pretreatment, store in a low-temperature, dry and clean place, and put in a sterile place in advance on the day of use. The blood collection room is sterile and irradiated with ultraviolet sterilization lamp for 30 minutes. The blood collection room must be clean and disinfected before surgery. After surgery, the wound must be cleaned and the blood vessels must be cleaned. When the blood collector operates, the blood collection tube must be inserted cleanly and accurately, inserted deeply and securely, and the operating personnel must strictly change clothes and wear masks to minimize the amount of floating bacteria and mycoplasma in the operating room. The blood collection and separation process must be strictly carried out in processed vessels, one cow and one container, and closed operation to minimize the degree of contamination. Control of production details is crucial to obtaining high-quality serum. The above-mentioned strict operating environment and personnel quality requirements further increase the production cost of bovine serum.

[0012] The chain reaction of cattle inventory and cow destocking will have a significant impact on beef prices. For example, since the end of 2024, China's domestic beef cattle inventory has begun to decline, with a year-on-year decrease of 4.39%. This has directly led to an 8% year-on-year decrease in the number of newborn calves during the same period. Based on the two-year calf rearing cycle, it is estimated that the number of beef cattle available for market will be significantly reduced by the second half of 2025. This supply gap will further drive up prices. Since 2024, corn prices have surged by 20% year-on-year, significantly increasing breeding costs. Rising feed costs have directly squeezed breeding profits. If corn prices continue to rise, breeding profits may be further squeezed, driving up domestic beef prices. At the same time, the imported beef market is also facing new changes. Rising import unit prices and strengthened policy regulation are further driving the scarcity premium of domestic beef. Modern farm workers generally earn more than 6,000 yuan per month, a 35% increase in 2024 compared to 2019. The serious aging population has led to an exacerbated labor shortage. These factors have led to rising bovine serum prices.

[0013] 3) The difference between serum batches affects the freezing effect

[0014] As an important culture medium component, the quality and stability of serum have a significant impact on experimental results. Simply judging by the naked eye, even serum of the same brand may have different colors. This color difference is mainly due to a variety of factors, including hemoglobin content, bilirubin level, health status of the donor animal, collection and processing methods, and storage conditions. The color of serum is mainly determined by hemoglobin (bright red) and bilirubin (yellow). When the hemoglobin content is low, the serum is light yellow or golden yellow; when the hemoglobin content is high, it is light red. This color change reflects the degree of solubility of hemoglobin in the serum, and hemolysis (the release of hemoglobin due to the rupture of red blood cells) is one of the main reasons for the color change. The physiological state, age, gender and nutritional conditions of different donor animals will affect the composition and color of the serum. For example, young fetal cattle have more fragile red blood cells and are more likely to undergo hemolysis during the blood collection process, resulting in a reddish serum color. In addition, pasture management and serum collection techniques in different regions will also affect the quality of the serum, and thus affect its color. The collection and processing methods of serum are also important factors affecting the color. The centrifugation speed, temperature and time after blood collection will affect the degree of red blood cell rupture, thereby affecting The hemoglobin content in serum. In addition, the storage conditions of serum, such as temperature, light and oxygen exposure, will also affect the oxidation and degradation of hemoglobin, further affecting the color of serum. Although color itself does not directly determine the quality of serum, it can be used as a rough indicator of the degree of hemolysis. Darker serum may indicate a higher degree of hemolysis, which may affect the accuracy and reproducibility of the experimental results. Chinese researchers have used MDCK cells to compare the culture effects of fetal bovine serum and newborn calf serum. The results showed that in terms of the number of colonies formed in cultured MDCK cells, newborn calf serum was lower than fetal bovine serum. Another study compared fetal bovine serum and newborn calf serum in the culture of MDCK, VERO, CHO-K1, and Hela cells. The four cell types, adherent subculture, suitable proliferation concentration and doubling time, and colony formation rate, showed that both sera met the requirements when culturing VERO and CHO-K1 cells, while fetal bovine serum performed better when culturing MDCK and Hela cells. Therefore, sera from different sources will have an important impact on the freezing effect of cryopreservatives.

[0015] Therefore, in view of the problems that the above-mentioned traditional cell freezing solution is a mixture of culture medium, serum and dimethyl sulfoxide, and has complex serum components and large differences between batches, its source is restricted by region and season, it is expensive, and there is a risk of contamination by blood-borne pathogens due to serum, it has toxic and inhibitory effects on cells, and affects the cell adhesion effect, a cell freezing solution without serum components and its preparation method can be designed. Summary of the Invention

[0016] In order to overcome the problems of complex serum components and large batch differences in traditional cell freezing solutions, geographical and seasonal restrictions on sources, high prices, and the risk of contamination by blood-borne pathogens introduced by serum, toxic and inhibitory effects on cells, and affected cell attachment effects.

[0017] The technical solution of the present invention is: a serum-free cell freezing solution, which comprises the following components in mass percentage: 70% to 80% of basal culture medium, 5% to 10% of dimethyl sulfoxide (DMSO), and 10% to 20% of skim milk powder.

[0018] Preferably, the basal culture medium comprises amino acids, inorganic salts, sugars and vitamins.

[0019] Preferably, the amino acids include L-arginine 250-320 mg / L, L-asparagine 30-70 mg / L, L-aspartic acid 10-30 mg / L, L-cystine dihydrochloride 50-70 mg / L, L-glutamic acid 10-30 mg / L, glycine 5-15 mg / L, L-histidine 10-40 mg / L, L-hydroxyproline 10-30 mg / L, L-isoleucine 30-70 mg / L, L-leucine 30-70mg / L, L-lysine hydrochloride 30-50mg / L, L-methionine 10-20mg / L, L-phenylalanine 10-20mg / L, L-proline 10-30mg / L, L-serine 20-40mg / L, L-threonine 10-30mg / L, L-tryptophan 3-4mg / L, L-tyrosine 22-24mg / L, L-valine 10-30mg / L.

[0020] Preferably, the inorganic salts include 50-120 mg / L of calcium nitrate, 44-50 mg / L of anhydrous magnesium sulfate, 500-700 mg / L of anhydrous sodium dihydrogen phosphate, 300-500 mg / L of potassium chloride, and 5000-7000 mg / L of sodium chloride.

[0021] Preferably, the sugars include 1500-3000 mg / L of glucose.

[0022] Preferably, the vitamins include reduced glutathione 0.5-1.5 mg / L, biotin 0.1-0.3 mg / L, D-calcium pantothenate 0.2-0.3 mg / L, folic acid 0.5-1.5 mg / L, inositol 30-40 mg / L, nicotinamide 0.5-1.5 mg / L, pyridoxine hydrochloride 0.5-1.5 mg / L, choline chloride 2-4 mg / L, thiamine hydrochloride 0.5-1.5 mg / L, vitamin B 0.1-0.2 mg / L, and para-aminobenzoic acid 0.5-1.5 mg / L.

[0023] Preferably, the skimmed milk powder is 50-70 g / L.

[0024] A method for preparing a serum-free cell freezing solution comprises the serum-free cell freezing solution described above, and the steps are as follows:

[0025] S1: Raw material preparation

[0026] S11: Prepare a basal culture medium according to a given formula, and uniformly dissolve and mix various amino acids, inorganic salts, sugars, and vitamins according to corresponding contents in a magnetic stirrer at a stirring speed of 300-500 rpm and a heating temperature of ≤40°C;

[0027] S12: Prepare 60 g / L skim milk powder solution, dissolve the skim milk powder in an appropriate amount of water, and stir thoroughly in a magnetic stirrer at a stirring speed of 400-600 rpm and a heating temperature of 50-60°C;

[0028] S2: Mixing preparation

[0029] According to the ratio of 70% to 80% basal culture medium, 10% to 20% skim milk powder, and 5% to 10% DMSO, measure the corresponding volumes of basal culture medium, skim milk powder solution, and DMSO, pour them into a magnetic stirrer, and mix at room temperature at a stirring speed of 200-300 rpm for 5 minutes. Stir thoroughly to prepare cell freezing solution.

[0030] The beneficial effects of the present invention are as follows: the cell freezing solution of the present invention uses milk powder instead of serum, thereby reducing potential biological risks. Milk powder is a powder made by removing moisture from animal milk and is suitable for storage. Milk powder is made from fresh cow's milk or goat's milk and is frozen or heated to remove almost all the moisture in the milk. Milk powder is easy to prepare, convenient to carry, and rich in nutrition. Instant milk powder has larger and looser particles than ordinary milk powder, good wettability, and high dispersion. When prepared, it can be quickly dissolved even with warm water. It is convenient to store, does not require special equipment, and is easy to carry. The production process is as follows: raw milk procurement → impurity filtration → degreasing → heat sterilization → ingredient adjustment → concentration → spray drying → packaging; therefore, compared with serum, the technology in the biological sterilization link is more mature, the sterilization effect is more thorough, and the biological safety is higher.

[0031] Reduced production costs: The production cost of milk powder includes many aspects, such as raw material cost, production cost, equipment cost, labor cost, etc.; among them, the raw material cost mainly includes milk, and the price of these raw materials varies due to factors such as market supply and demand and production cost; production costs include processing, mixing, packaging and other costs; equipment costs include production equipment purchase, maintenance and operation costs; labor costs include labor costs of production staff, management personnel and R&D personnel; in 2024, the production cost of one kilogram of milk powder is between 10 yuan and 20 yuan, among which the raw material cost accounts for the majority of the production cost, which is related to the acquisition of blood Compared with serum, animal milk is easy to obtain. Cows can collect milk every day. The collection of serum requires a certain period of time, otherwise the cows are likely to die, and the source cost is significantly reduced. The milk source can be obtained by fully automated equipment, which does not require more professional blood collection personnel to operate. However, the personnel who collect cattle serum need to undergo long-term scientific and professional training, which significantly increases the labor cost. The milk source can be processed immediately after it is obtained, reducing the risk of contamination. The collected cattle blood needs to be left to stand and centrifuged. Excessive operating procedures can easily increase the risk of serum contamination. In order to avoid such risks, the cost investment will inevitably increase.

[0032] The difference between milk powder batches is smaller: Generally speaking, the nutritional composition of milk powder is very complex. Sn-2 palmitic acid is one of the saturated fatty acids that exists in large quantities in milk powder, accounting for about 25% of the total fat content. Protein provides nutrition to the body, and fat provides nutrition and energy to the body to provide the rich flavor of milk. Milk contains lactose, which is very important for cell development. Minerals, also known as inorganic salts, are indispensable substances for cell composition, including calcium, iron, phosphorus, zinc, copper, manganese, molybdenum, etc. These substances are commonly known as divalent minerals. Divalent minerals are indispensable nutrients for cells, and this nutrient element cannot be directly produced in cells, but can only be obtained through external channels. Milk powder contains all known types of vitamins. For example, vitamin A promotes normal cell growth and reproduction, and vitamin B is involved in sugar and energy metabolism in the body. Vitamin E has antioxidant properties, and soy lecithin is a fundamental substance of life. It is not only an important component of human biological membranes but also a source of choline and fatty acids. It plays a key role in maintaining the physiological activity of biological membranes and normal metabolism. Soy lecithin is a component of cell membranes, enhancing cellular information transmission, improving cell vitality, and enhancing the self-repair ability of cell membranes, thereby protecting against external damage. This nutrient is uniquely added to Inno formula milk. In February 2024, the National Health Commission and the State Administration for Market Regulation jointly issued GB 19644-2024, "National Food Safety Standard for Milk Powder and Modified Milk Powder" (hereinafter referred to as the "New Standard"). The new standard will replace GB 19644-2010 (hereinafter referred to as the "Old Standard") and will officially take effect on February 8, 2025. Compared with the old standard, the new standard has revised and improved terminology definitions, sensory requirements, technical requirements, and product labeling. It can more scientifically and rigorously guarantee the product quality and safety of milk powder. Therefore, the variability between milk powder batches is smaller than that of serum, and the quality is more easily guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown is an observation image of 4T1 cells frozen for 3 months and then revived and cultured for 24 hours in the experiment of the present invention;

[0034] Figure 2 Shown is an observation diagram of CT26 cells frozen for 3 months and then revived and cultured for 24 hours in the experiment of the present invention. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the embodiments.

[0036] The present invention provides an embodiment: a serum-free cell freezing solution, which comprises the following components, calculated by mass percentage: 70% to 80% basal culture medium, 5% to 10% dimethyl sulfoxide (DMSO), and 10% to 20% skim milk powder.

[0037] The basal culture medium includes amino acids, inorganic salts, sugars and vitamins.

[0038] Amino acids include L-arginine 250-320mg / L, L-asparagine 30-70mg / L, L-aspartic acid 10-30mg / L, L-cystine dihydrochloride 50-70mg / L, L-glutamic acid 10-30mg / L, glycine 5-15mg / L, L-histidine 10-40mg / L, L-hydroxyproline 10-30mg / L, L-isoleucine 30-70mg / L, L-leucine L-amino acid 30-70mg / L, L-lysine hydrochloride 30-50mg / L, L-methionine 10-20mg / L, L-phenylalanine 10-20mg / L, L-proline 10-30mg / L, L-serine 20-40mg / L, L-threonine 10-30mg / L, L-tryptophan 3-4mg / L, L-tyrosine 22-24mg / L, L-valine 10-30mg / L.

[0039] Inorganic salts include calcium nitrate 50-120 mg / L, anhydrous magnesium sulfate 44-50 mg / L, anhydrous sodium dihydrogen phosphate 500-700 mg / L, potassium chloride 300-500 mg / L, and sodium chloride 5000-7000 mg / L.

[0040] Sugars include glucose 1500-3000 mg / L.

[0041] Vitamins include reduced glutathione 0.5-1.5 mg / L, biotin 0.1-0.3 mg / L, D-calcium pantothenate 0.2-0.3 mg / L, folic acid 0.5-1.5 mg / L, inositol 30-40 mg / L, niacinamide 0.5-1.5 mg / L, pyridoxine hydrochloride 0.5-1.5 mg / L, choline chloride 2-4 mg / L, thiamine hydrochloride 0.5-1.5 mg / L, vitamin B 0.1-0.2 mg / L, and para-aminobenzoic acid 0.5-1.5 mg / L.

[0042] Skimmed milk powder is 50-70g / L.

[0043] A method for preparing a serum-free cell freezing solution comprises the serum-free cell freezing solution described above, and the steps are as follows:

[0044] S1: Raw material preparation

[0045] S11: Prepare a basal culture medium according to a given formula, and uniformly dissolve and mix various amino acids, inorganic salts, sugars, and vitamins according to corresponding contents in a magnetic stirrer at a stirring speed of 300-500 rpm and a heating temperature of ≤40°C;

[0046] S12: Prepare 60 g / L skim milk powder solution, dissolve the skim milk powder in an appropriate amount of water, and stir thoroughly in a magnetic stirrer at a stirring speed of 400-600 rpm and a heating temperature of 50-60°C;

[0047] S2: Mixing preparation

[0048] According to the ratio of 70% to 80% basal culture medium, 10% to 20% skim milk powder, and 5% to 10% DMSO, measure the corresponding volumes of basal culture medium, skim milk powder solution, and DMSO, pour them into a magnetic stirrer, and mix at room temperature at a stirring speed of 200-300 rpm for 5 minutes. Stir thoroughly to prepare cell freezing solution.

[0049] Example 1

[0050] The present invention provides an embodiment: a serum-free cell freezing solution, which comprises the following components, calculated by mass percentage: 70% basal culture medium, 10% dimethyl sulfoxide (DMSO), and 20% skim milk powder.

[0051] The basal culture medium includes amino acids, inorganic salts, sugars and vitamins.

[0052] Amino acids include L-arginine 290 mg / L, L-asparagine 50 mg / L, L-aspartic acid 20 mg / L, L-cystine dihydrochloride 65 mg / L, L-glutamic acid 20 mg / L, glycine 10 mg / L, L-histidine 15 mg / L, L-hydroxyproline 20 mg / L, L-isoleucine 50 mg / L, L-leucine 50 mg / L, L-lysine hydrochloride 40 mg / L, L-methionine 15 mg / L, L-phenylalanine 15 mg / L, L-proline 20 mg / L, L-serine 30 mg / L, L-threonine 20 mg / L, L-tryptophan 4 mg / L, L-tyrosine 23 mg / L, and L-valine 20 mg / L.

[0053] Inorganic salts include calcium nitrate 100 mg / L, anhydrous magnesium sulfate 48 mg / L, anhydrous sodium dihydrogen phosphate 676 mg / L, potassium chloride 400 mg / L, and sodium chloride 6000 mg / L.

[0054] Sugars include glucose 2000 mg / L.

[0055] Vitamins include reduced glutathione 1mg / L, biotin 0.2mg / L, D-calcium pantothenate 0.25mg / L, folic acid 1mg / L, inositol 35mg / L, niacinamide 1mg / L, pyridoxine hydrochloride 1mg / L, choline chloride 3mg / L, thiamine hydrochloride 1mg / L, vitamin B 0.1mg / L, and para-aminobenzoic acid 1mg / L.

[0056] Skimmed milk powder is 60g / L.

[0057] A method for preparing a serum-free cell freezing solution comprises the serum-free cell freezing solution described above, and the steps are as follows:

[0058] S1: Raw material preparation

[0059] S11: Prepare a basal culture medium according to a given formula, and uniformly dissolve and mix various amino acids, inorganic salts, sugars, and vitamins according to corresponding contents in a magnetic stirrer at a stirring speed of 300-500 rpm and a heating temperature of ≤40°C;

[0060] S12: Prepare 60 g / L skim milk powder solution, dissolve the skim milk powder in an appropriate amount of water, and stir thoroughly in a magnetic stirrer at a stirring speed of 400-600 rpm and a heating temperature of 50-60°C;

[0061] S2: Mixing preparation

[0062] According to the ratio of 70% basal culture medium, 20% skim milk powder, and 10% DMSO, measure the corresponding volumes of basal culture medium, skim milk powder solution, and DMSO, pour them into a magnetic stirrer, and mix at room temperature at a stirring speed of 200-300 rpm for 5 minutes. Stir thoroughly to prepare cell freezing solution.

[0063] Experimental example

[0064] A comparative experiment was conducted using Example 1 as Experimental Example 1 and a conventional freezing solution on the market as Experimental Example 2. The conventional freezing solution comprised 80% basal culture medium + 10% fetal bovine serum + 10% DMSO.

[0065] 4T1 cells and CT26 cells were cultured with RPMI1640 complete medium and passaged every other day.

[0066] The experimental steps are as follows:

[0067] 1. Cell recovery:

[0068] Take out the frozen cell line, heat the water bath to 37℃ in advance, thaw the cells in the water bath as soon as possible, quickly transfer the thawed cells to the clean bench, add them to a clean culture bottle with a pipette, and place it in a 37℃ constant temperature cell culture incubator. When the cells reach the logarithmic phase, they are passaged.

[0069] 2. Cell passaging:

[0070] When the cells are in good growth condition, carefully remove the upper culture medium, add PBS buffer to wash two to three times, spread an appropriate amount of trypsin evenly on the plate to digest the cells. After about 3 minutes, aspirate an equal amount of culture medium and trypsin and add it to the bottle to stop digestion. Transfer the digested cells to a new centrifuge tube, centrifuge at 1200rpm for 5 minutes, aspirate the supernatant and retain the precipitate, add culture medium and blow to resuspend the cells, add pre-sterilized culture medium into the culture dish with a pipette in advance, add the suspended cells, put them into the incubator, and pass the cells to the third generation for cell freezing.

[0071] 3. Cell cryopreservation:

[0072] Cells in the logarithmic growth phase were digested, centrifuged (1200 rpm, 10 min), counted, and the corresponding freezing solution was added according to the number of cells to maintain the cell density at (3-5) x10 6 / mL, dispense 1-1.5mL of cell-containing freezing solution into each tube, gently mix the cells, tighten the cap, mark the tube, transfer the dispensed freezing tubes into a program cooling box and directly store at -80℃ overnight. The next day, transfer to liquid nitrogen for storage. Resuscitate the frozen cells after 3 months and observe them after 24 hours.

[0073] 4. 0.4% Trypan Blue Cell Staining:

[0074] Re-digest the cells and gently pipette the cell culture to be tested to make a single cell suspension. If the cell density is too high, dilute it appropriately with PBS or culture medium. The recommended cell concentration is (0.1-1) × 10 6 / mL, take 100μL of cell suspension and add it to the centrifuge tube, add 100μL of 0.4% trypan blue dye, and mix it gently by pipetting. The volume ratio of cell suspension to dye is 1:1. Incubate at room temperature or 37℃ for 3-5 minutes to avoid damage to living cells due to long staining time; take a clean blood cell counting plate, cover it with a coverslip, use a pipette to absorb the stained cell suspension, and slowly add it along the edge of the coverslip to allow the suspension to fill the counting chamber through capillary action to avoid bubbles or liquid overflow. Place the counting plate under a microscope, use a 10× objective lens to observe the counting chamber, and count the cells in 2 counting chambers, a total of 4 large squares, among which living cells: colorless and transparent, with complete morphology; dead cells: blue, enlarged or irregular in shape. The results are as follows Figure 1 and Figure 2 shown.

[0075] The number of viable cells per 100 cells is shown in Table 1:

[0076] Cell name Experimental Example 1 Experimental Example 2 4T1 93.9+6.4 92.4+5.6 CT26 96.6+3.8 94.5+4.7

[0077] Table 1

[0078] from Figure 1 It can be seen that compared with the traditional cell freezing solution formula, the serum-free cell freezing solution we developed did not significantly change the 4T1 cell adhesion and morphology;

[0079] from Figure 2 It can be seen that compared with the traditional cell freezing solution formula, the serum-free cell freezing solution we developed did not significantly change the adhesion and morphology of CT26 cells.

Claims

1. A serum-free cell freezing solution, characterized in that: The invention comprises the following components in percentage by mass: 70% to 80% of basic culture medium, 5% to 10% of dimethyl sulfoxide (DMSO), and 10% to 20% of skim milk powder.

2. The serum-free cell freezing solution according to claim 1, wherein: The basal culture medium includes amino acids, inorganic salts, sugars and vitamins.

3. The serum-free cell freezing solution according to claim 2, characterized in that: Amino acids include L-arginine 250-320mg / L, L-asparagine 30-70mg / L, L-aspartic acid 10-30mg / L, L-cystine dihydrochloride 50-70mg / L, L-glutamic acid 10-30mg / L, glycine 5-15mg / L, L-histidine 10-40mg / L, L-hydroxyproline 10-30mg / L, L-isoleucine 30-70mg / L, L-leucine L-amino acid 30-70mg / L, L-lysine hydrochloride 30-50mg / L, L-methionine 10-20mg / L, L-phenylalanine 10-20mg / L, L-proline 10-30mg / L, L-serine 20-40mg / L, L-threonine 10-30mg / L, L-tryptophan 3-4mg / L, L-tyrosine 22-24mg / L, L-valine 10-30mg / L.

4. The serum-free cell freezing solution according to claim 2, wherein: Inorganic salts include calcium nitrate 50-120 mg / L, anhydrous magnesium sulfate 44-50 mg / L, anhydrous sodium dihydrogen phosphate 500-700 mg / L, potassium chloride 300-500 mg / L, and sodium chloride 5000-7000 mg / L.

5. The serum-free cell freezing solution according to claim 2, wherein: Sugars include glucose 1500-3000 mg / L.

6. The serum-free cell freezing solution according to claim 2, wherein: Vitamins include reduced glutathione 0.5-1.5 mg / L, biotin 0.1-0.3 mg / L, D-calcium pantothenate 0.2-0.3 mg / L, folic acid 0.5-1.5 mg / L, inositol 30-40 mg / L, niacinamide 0.5-1.5 mg / L, pyridoxine hydrochloride 0.5-1.5 mg / L, choline chloride 2-4 mg / L, thiamine hydrochloride 0.5-1.5 mg / L, vitamin B 0.1-0.2 mg / L, and para-aminobenzoic acid 0.5-1.5 mg / L.

7. The serum-free cell freezing solution according to claim 1, wherein: Skimmed milk powder is 50-70g / L.

8. A method for preparing a serum-free cell freezing solution, characterized in that The method comprises the serum-free cell freezing solution according to claim 1, wherein the steps are as follows: S1: Raw material preparation S11: Prepare a basal culture medium according to a given formula, and uniformly dissolve and mix various amino acids, inorganic salts, sugars, and vitamins according to corresponding contents in a magnetic stirrer at a stirring speed of 300-500 rpm and a heating temperature of ≤40°C; S12: Prepare 60 g / L skim milk powder solution, dissolve the skim milk powder in an appropriate amount of water, and stir thoroughly in a magnetic stirrer at a stirring speed of 400-600 rpm and a heating temperature of 50-60°C; S2: Mixing preparation According to the ratio of 70% to 80% basal culture medium, 10% to 20% skim milk powder, and 5% to 10% DMSO, measure the corresponding volume of basal culture medium, skim milk powder solution, and DMSO, pour them into a magnetic stirrer, and mix at room temperature at a stirring speed of 200-300 rpm for 5 minutes. Stir thoroughly to prepare cell freezing solution.

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