Serum-free and ultra-low protein PK-15 cell culture medium and preparation method thereof

Abstract

The present invention discloses a serum-free ultra-low protein PK-15 cell culture medium and a preparation method thereof, wherein the PK-15 cell culture medium comprises a basal culture medium and an additive, wherein the basal culture medium comprises amino acids, vitamins, inorganic salts and auxiliary components; and the additive comprises 0.01-0.5 mg / L iron ion chelator and 0.1-2.5 μmol / L small molecule compound CHIR99021. The culture medium of the present invention has the characteristics of being serum-free, free of any animal-derived components, low in protein and having clear chemical components, and is low in cost and suitable for industrialized large-scale production.

Description

Technical Field

[0001] The invention relates to the field of PK-15 cell culture medium technology and cell engineering, and in particular to a serum-free and ultra-low-protein PK-15 cell culture medium and a preparation method thereof. Background Art

[0002] Serum provides cells with hormones, growth factors and other nutrients needed for growth and proliferation. However, due to batch differences between different batches of serum, it is expensive, and there is a risk of being easily infected by viruses and mycoplasma. It contains a large number of complex proteins, which brings great difficulties to the downstream separation and purification of cell products. Porcine PK-15 (Porcine kidney, porcine kidney epithelial cells) is an adherent culture cell line that can be continuously passaged. It is a vaccine production cell line approved by the World Health Organization and my country's biological product regulations. It has been used in the production of a variety of vaccines. However, many animal-derived components in traditional serum culture media are unclear, and the exogenous viruses they may carry pose serious safety risks. In recent years, commercial serum-free culture media have also developed from the initial research on serum substitutes to the research on chemically defined culture media. At present, the development of culture media commonly used in serum-free culture technology is classified as follows:

[0003] The first generation does not contain serum and adds biological materials that replace serum. The characteristic is that the protein content of the culture medium is high, which increases the difficulty and cost of purification.

[0004] The second generation, no animal-derived culture medium, adds genetic engineering and recombinant proteins or plant protein hydrolysates. It does not contain animal-derived protein derivatives, which can ensure product safety and cell growth, but the recombinant protein content is high and expensive, and the plant hydrolyzed protein composition is complex, which is not conducive to downstream protein purification.

[0005] The third generation, protein-free culture medium, contains no protein at all. The advantage is that it is easy to purify and separate, and the culture medium is relatively stable, but its application is limited.

[0006] The fourth generation, chemically defined culture medium. The components of the culture medium are all clear, which is conducive to the metabolic research of cell culture in vitro. The new culture medium with no serum, no protein, no animal origin and clear components is the most ideal and safest universal culture medium, which is widely used in the large-scale production of viral vaccines, recombinant proteins and other products in the field of cell engineering.

[0007] At present, the fourth-generation chemically defined medium has not been used in the adherent culture of PK-15 for vaccine production. Based on the current large-scale production of vaccines, there is an urgent need for a low-cost, animal-free, chemically defined, serum-free, efficient culture medium for PK-15 cells for the adherent process. Summary of the invention

[0008] In view of the above problems, the main purpose of the present invention is to provide a serum-free and ultra-low protein PK-15 cell culture medium and a preparation method thereof, which has the characteristics of being serum-free, animal albumin-free, free of any animal-derived ingredients, low protein and having clear chemical components, is low in cost, can support serum-free culture of PK-15 cells, is suitable for various PK-15 cell culture methods including culture bottles, cell culture roller bottles, and biological fermentation tanks, and can be used for laboratory research and industrial-scale production.

[0009] To achieve the above object, the technical solution of the present invention is as follows:

[0010] As a first aspect of the present invention, a serum-free and ultra-low protein PK-15 cell culture medium is provided, comprising a basal culture medium and additives, wherein the basal culture medium comprises amino acids, vitamins, inorganic salts and auxiliary components; the additives comprise 0.01-0.5 mg / L iron ion chelator and 0.1-2.5 μmol / L small molecule compound CHIR99021.

[0011] As a second aspect of the present invention, a method for preparing the serum-free ultra-low protein PK-15 cell culture medium as described above is provided, comprising: dissolving a basal culture medium and an additive in water to obtain a mixed solution; adjusting the pH value and osmotic pressure of the mixed solution to make the pH value within the range of 7.0 to 7.4 and the osmotic pressure within the range of 300 to 340 mOsm / kg; and filtering the mixed solution obtained after adjustment to obtain the PK-15 cell culture medium.

[0012] Based on the above technical scheme, the serum-free ultra-low protein PK-15 cell culture medium and the preparation method thereof provided by the present invention have at least one or part of the following beneficial effects:

[0013] The PK-15 cell culture medium provided by the present invention is a chemically defined, serum-free, animal-derived adherent culture medium developed for PK-15 cells, with small batch-to-batch variability. The PK-15 cell culture medium does not require cell acclimation, does not require the addition of transferrin and plant protein hydrolysate, has a fast cell proliferation rate, can be continuously and stably passaged, and maintains a high amplification rate.

[0014] In the PK-15 cell culture medium of the present invention, the basal culture medium (hereinafter referred to as PK-15 CD ) does not contain serum, hormones and defoaming agents. Only iron ion chelators with clear ingredients need to be added to the components of common basic culture medium. Small molecule compounds can maintain efficient and rapid growth of cells. It is suitable for large-scale culture in reactors and has good prospects for industrial application.

[0015] The invention is used for preparing PK-15 CDThe raw materials are cheap, the process is simple, and it is suitable for large-scale industrial production. Compared with foreign culture media, there is no obvious difference in cell growth and cell morphology, the growth rate is comparable, and the overall price of the culture medium is low, so the application prospects are bright.

[0016] The present invention establishes a serum-free culture system suitable for high-density cell culture. Since the PK-15 cell culture medium of the present invention is conducive to continuous cell culture and efficient expansion, the culture density of PK-15 cells can be increased, thereby improving the efficiency of vaccine preparation, saving precious time, and saving more lives. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1( a ) is a morphological diagram (4X) of PK-15 cells in the control group in Example 1 of the present invention;

[0018] FIG1( b ) is a diagram of the PK-15 cell morphology (4X) of the experimental group in Example 1 of the present invention;

[0019] Figure 2 is a graph showing the number and viability of PK-15 cells in the control group and the experimental group in Example 1 of the present invention;

[0020] Figure 3 It is a bar graph of the number of PK-15 cells obtained by using different combinations of additives in Example 2 of the present invention;

[0021] Figure 4 It is a bar graph of the expansion multiples of PK-15 cells with different additive combinations in Example 2 of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0023] Through the investigation and analysis of relevant technologies, it was found that PK-15 cells use commercial complete culture medium in most laboratories, and the serum composition and content vary greatly, and the culture efficiency is also uneven. In addition, commercial culture medium is expensive on the one hand, and the composition of the culture medium is unclear on the other hand, which has unpredictable effects on the efficacy of later vaccines. Commercial culture medium usually adds different concentrations of serum to ensure the cell proliferation effect. In addition to containing a large amount of cell growth factors, the transferrin contained in the serum plays a key role in the growth and division of PK. Cell growth factors can be added after synthesis of recombinant protein factors. However, since transferrin can play a good protective and supporting role for human and animal cells, serum-free culture medium must also add transferrin to achieve the same or similar cell culture performance as serum-containing culture medium, so as to achieve the regulation of cell iron ion balance and the maintenance of cell growth and proliferation. Transferrin plays a very important role in serum-free culture medium, including: a. Avoiding the generation of free radicals in the extracellular environment to prevent cell damage; b. Maintaining cell growth and proliferation; c. Maintaining intracellular iron balance.

[0024] In addition, in cell culture, the number of transferrin receptors is also closely related to the iron content in the culture environment. If there are more iron-containing media in the culture environment, the number of transferrin receptors on the cell surface will decrease, thereby avoiding excessive iron intake by cells. Therefore, the role of transferrin is not only to transport iron ions into cells, but also to maintain cell growth and proliferation through iron ions. However, most of the transferrin sold on the market is animal-derived ingredients, while human transferrin is scarce and very expensive, and there are individual differences. Therefore, some researchers have tried to add iron ion chelators to serum-free culture media, using iron chelators to replace the role of transferrin in the culture medium, on the one hand, to neutralize excess iron ions in the culture medium, on the other hand, to transport iron ions into cells, thereby providing the growth of cells, so that the use of transferrin can be avoided, thereby preparing a serum-free culture medium without protein components suitable for efficient cell culture.

[0025] According to the above analysis, in the process of realizing the present invention, it is attempted to add iron ion chelating agent in PK-15 basal medium, and it is found that the cell growth rate has obviously increased, and the cell survival rate has also improved. However, the PK-15 cell growth rate is not as good as the growth rate of cells maintained in serum medium. The reason for analyzing it is that iron ion chelating agent only has the effect of transferring iron, but can not realize the growth and proliferation effect of transferrin on cells.

[0026] Therefore, small molecule compounds that are beneficial to cell division were further investigated to study their effects on cell proliferation and division. Because the wnt signaling pathway is closely related to the proliferation, differentiation and apoptosis of various cells. Preliminary experimental screening showed that the Wnt / β-catenin signaling pathway activator Chir99021 (hereinafter referred to as CH) has an effect on the growth rate of PK-15 cells. Therefore, in the embodiments of the present invention, an attempt was made to add an appropriate concentration of CH to the PK-15 cell culture to observe the effect on PK-15 cell proliferation. Figure 3 and Figure 4 As shown, the results of the embodiment prove that CH activates the wnt signaling pathway to promote the proliferation of PK-15 cells. The reason for this is that activating the signaling pathway can start the signaling pathway proliferation target, promote the expression of division-related proteins, and thus cause cell proliferation.

[0027] In the embodiment of the present invention, an iron ion chelator is added to replace transferrin in a serum-free ultra-low protein culture medium and a small molecule compound CH is added to promote cell division, and PK-15 cells are cultured and passaged. It is found that the iron ion chelator and the small molecule compound CH not only improve cell survival, proliferation and expansion rate, but also have a synergistic effect when combined. At the same time, the cell growth rate is significantly increased after addition, and the cell morphology is not much different. Because the culture medium has clear components and low price, it is suitable for large-scale cell expansion and subsequent vaccine preparation.

[0028] Specifically, according to an embodiment of the present invention, a serum-free ultra-low protein PK-15 cell culture medium is provided, including PK-15 CD Basic culture medium and additives, including PK-15 CD The basic culture medium includes amino acids, vitamins, inorganic salts and auxiliary components; the additives include 0.01-0.5 mg / L iron ion chelator and 0.1-2.5 μmol / L small molecule compound CHIR99021.

[0029] According to an embodiment of the present invention, in one aspect, by CD The iron chelate additive replaces the transferrin component in the traditional culture medium, meeting its function of balancing iron ions in cell culture and protecting cells from excessive free radicals, thus achieving efficient culture of PK-15 cells in serum-free ultra-low protein medium. On the other hand, by using the effect of small molecule compounds on the cell division cycle, in PK-15 CD The addition of the wnt pathway activator CH can achieve rapid proliferation of PK-15 cells in serum-free ultra-low protein medium. In another aspect, the synergistic effect of the iron ion chelator and the small molecule compound CH further promotes the rapid expansion of PK-15 cells in serum-free ultra-low protein.

[0030] According to an embodiment of the present invention, the concentration of CH added to the additive is 0.1-2.5 μmol / L, preferably 0.2-1.0 μmol / L, for example, 0.2 μmol / L, 0.4 μmol / L, 0.6 μmol / L, 0.8 μmol / L or 1.0 μmol / L, etc. In the embodiment, the added concentration is 0.5 μmol / L, that is, the mass concentration is 0.23 mg / L. Through investigation and experimental analysis, it was found that CH has a dose-dependent effect on cell proliferation. If the concentration is too low, the corresponding signal pathway cannot be effectively activated. If the concentration is too high, the normal division of cells will be significantly inhibited due to excessive activation of cell-related receptors, thereby causing cell apoptosis.

[0031] According to an embodiment of the present invention, the iron ion chelator in the additive is preferably ammonium ferric citrate (FAC for short), which can synergize with the small molecule compound CH to significantly increase the number of cells cultured.

[0032] According to an embodiment of the present invention, the added concentration of the iron ion chelator is 0.01-0.5 mg / L, preferably 0.05-0.25 mg / L, for example, 0.05 mg / L, 0.08 mg / L, 0.10 mg / L, 0.15 mg / L or 0.2 mg / L, etc. In an embodiment, the added concentration is 0.2 mg / L. Because FAC is a physiological form of non-transferrin-bound iron, it is concentration-dependent on the growth and survival state of cells. If the concentration used in the culture medium without transferrin is too low, the balance demand of cells for iron ions and the anti-free radical oxidation self-use cannot be met. If the concentration used is too high, oxidative stress will be induced because of intracellular iron overload, thereby causing selective damage to cells and then inducing cell ion poisoning apoptosis.

[0033] According to an embodiment of the present invention, the PK-15 cell culture medium of the present invention does not contain protein and protein hydrolysate. More specifically, the protein and protein hydrolysate include serum albumin, transferrin and plant hydrolyzed protein, etc. Therefore, when used for PK-15 cell culture, it does not rely on the use of animal serum, and does not use animal-derived protein and any hormones, which greatly reduces the production cost, fully guarantees the stability of product quality, and can achieve continuous cell culture expansion and passage without increasing the proportion of cell apoptosis.

[0034] According to an embodiment of the present invention, the above amino acid components and concentrations are shown in Table 1 below:

[0035] Table 1

[0036]

[0037] According to an embodiment of the present invention, the above vitamin components and concentrations are shown in Table 2 below:

[0038] Table 2

[0039]

[0040] According to an embodiment of the present invention, the composition and concentration of the above-mentioned inorganic salt are shown in Table 3 below:

[0041] Table 3

[0042]

[0043] According to an embodiment of the present invention, the auxiliary components include growth factors, lipids, trace elements, etc. The specific components and concentrations are shown in Table 4 below:

[0044] Table 4

[0045]

[0046] According to an embodiment of the present invention, a method for preparing the above-mentioned serum-free and ultra-low protein PK-15 cell culture medium is also provided, comprising the following steps S1 to S3.

[0047] In step S1, the above-mentioned basic culture medium and additives are dissolved in water to obtain a mixed solution. Furthermore, the water used can be ultrapure deionized water.

[0048] In step S2, the pH value and osmotic pressure of the mixed solution obtained in step S1 are adjusted to a pH value in the range of 7.0 to 7.4 and an osmotic pressure in the range of 300 to 340 mOsm / kg. In some embodiments, the pH value is adjusted to a range of 7.0 to 7.4 by adding a 6 mol / L strong base NaOH solution and a 6 mol / L strong acid HCl solution; the osmotic pressure of the solution is adjusted to a value of 300 to 340 mOsm / kg by adding NaCl.

[0049] In step S3, the mixed solution obtained after adjustment is filtered to obtain PK-15 cell culture medium. Furthermore, the mixed solution can be filtered using a 0.22 μm filter membrane and stored for later use.

[0050] The technical scheme of the present invention is further described below by means of specific examples in combination with the accompanying drawings. It should be noted that the following specific examples are only used as examples, and the protection scope of the present invention is not limited thereto. The chemicals and raw materials used in the following examples are all commercially available or homemade by known preparation methods.

[0051] Example 1

[0052] Add amino acids, vitamins, inorganic salts and additives to ultrapure deionized water in accordance with the contents in the above ingredient table. Add 6 mol / L strong base NaOH solution and 6 mol / L strong acid HCl solution to adjust the pH value to 7.0-7.4. Add NaCl to adjust the osmotic pressure of the solution to 300-340 mOsm / kg. Finally, filter the solution with a 0.22 μm filter membrane and bottle it for later use. The above-configured serum-free ultra-low protein chemically defined basic medium is referred to as PK-15. CD This culture medium can be added with human epidermal growth factor (EGF) at a concentration of 10 ng / ml before use to ensure that the human epidermal growth factor is effective.

[0053] The experimental steps of PK-15 cell culture are as follows:

[0054] 1) Experimental group: Take a PK-15 cell cryopreservation tube from the liquid nitrogen tank, shake and dissolve it in a 37°C water bath, and add it to 10 mL of fresh PK-15 CD 15 mL centrifuge tube containing basal medium, mix well. Then centrifuge at 100 rpm for 5 min and discard the supernatant. CD The cells were resuspended in basal medium and inoculated into T75 flasks for culture. Epidermal growth factor was added before use. Three days later, the PK-15 cells that had been adjusted to a good state were digested with trypsin and resuspended for counting. The cells were counted according to 2*10 4 / cm 2 The density was 3 ml and the volume was inoculated into a 6-well plate. The culture conditions were: 37°C, 5% CO2. The culture was continued for 6 days. The cells were digested every 24 hours to detect the cell number. The cells were digested with trypsin, collected, counted, and their survival rate was detected. The cell morphology after 6 days is shown in Figure 1(b), and the total cell number and cell viability are shown in Figure 1(c). Figure 2 shown.

[0055] 2) At the same time, another control group culture medium was carried out as a control experiment, the only difference being that the traditional serum culture DMEM + 10% FBS was used. Among them, the DMEM basal culture medium used Gibico's commercial product, with the product number DMEM (10567-014), and the fetal bovine serum used German P∧N TM SERATECH (ST30-3302). The cell morphology after 6 days is shown in Figure 1(a), and the total number of cells and cell viability are shown in Figure 2 shown.

[0056] The above experimental results show that PK-15 of the present invention CD The basal culture medium is similar to the traditional classic culture medium DMEM + 10% FBS in terms of cell morphology. 2The total cell number after 6 days of density inoculation culture was significantly lower than that in the serum group, and the cell viability was relatively close but slightly lower, at 90%. The reason for this was found to be related to the lack of transferrin in the culture medium.

[0057] Example 2

[0058] The specific PK-15 cell culture experimental steps are as follows:

[0059] The adherent PK-15 cells were digested with trypsin and resuspended for counting. The following experimental and control culture media were used, and the cells were counted at 4*10 4 / cm 2 The density was 3 ml and the cells were inoculated into 6-well plates at 37°C and 5% CO2 for 3 consecutive days. After the culture, the cells were digested with trypsin and collected and counted. The total number of cells per unit area was obtained as shown in Figure 2. Figure 3 , cell proliferation times see Figure 4 .

[0060] Among them, the culture medium settings of the experimental and control groups were as follows: 1) Control group 1, Gibico DMEM + 10% FBS;

[0061] 2) Control group 2, serum-free and exogenous protein-free chemical composition medium PK-15 CD ;

[0062] 3) Control group 3 in PK-15 CD Only the iron ion chelator FAC was added;

[0063] 4) Control group 4 in PK-15 CD Only small molecule compounds CH are added;

[0064] 5) Experimental group in PK-15 CD FAC and CH were added simultaneously.

[0065] The concentration of CHIR99021 (CH for short) added to the control group 4 and the experimental group was 0.5 μmol / L, i.e., the mass concentration was 0.23 mg / L. The concentration of FAC added to the control group 3 and the experimental group was 0.2 mg / L.

[0066] Depend on Figure 3 It can be seen that in the cell PK-15 of the present invention CD Adding iron ion chelator (FAC) and wnt activator CH to the culture medium can promote the growth and survival of PK-15 cells. 2 The total number of cells per unit area after 3 days of density inoculation was significantly increased compared with the group without addition, and the total number of cells increased from 16.0E4 / cm2 Increased to 18.0E4 / cm 2 and 17.5E4 / cm 2 However, the cell density in the FBS group was 18.9E4 / cm 2 Compared with the experimental group, the cell growth rate is still slightly inferior, and the total cell expansion multiple is also slightly lower than that of the FBS group. Figure 4 However, in the experimental group with simultaneous addition of FAC and CH, the total number of cells per unit area increased significantly after 3 days, and the cell growth rate was significantly accelerated, with the total number of cells and cell expansion times increasing from 16E4 / cm to 26E4 / cm respectively. 2 and 4 times higher to 20.7E4 / cm 2 and 5.2 times, showing better cell growth efficiency compared with commercial culture medium.

[0067] The examples of the present invention demonstrate the synergistic effect of the iron ion chelator FAC and the small molecule compound CH on cell growth in PK-15 CD Adding FAC and CH to the basal medium can support the rapid growth of PK-15 and meet the growth metabolism of cells. In addition, since the medium provided by the present invention has clear chemical composition, no serum and no expensive blood-derived proteins such as transferrin, it is inexpensive and has great application prospects.

[0068] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A serum-free ultra-low protein PK-15 cell culture medium, comprising a basal culture medium and an additive, characterized in that: The basic culture medium includes amino acids, vitamins, inorganic salts and auxiliary components; The additive consists of 0.01-0.5 mg / L ammonium ferric citrate and 0.1-2.5 μmol / L small molecule compound CHIR99021; The PK-15 cell culture medium does not contain protein and protein hydrolysate, and the protein and protein hydrolysate include serum albumin, transferrin and plant hydrolyzed protein.

2. The PK-15 cell culture medium according to claim 1, characterized in that The concentration of the ammonium ferric citrate is 0.05-0.5 mg / L, and the concentration of the small molecule compound CHIR99021 is 0.2-1.0 μmol / L.

3. The PK-15 cell culture medium according to claim 1, characterized in that The amino acids include 10-30 mg / L glycine, 1-10 mg / L alanine, 100-300 mg / L arginine hydrochloride, 2-20 mg / L asparagine, 1-15 mg / L aspartic acid, 5-50 mg / L cysteine ​​hydrochloride hydrate, 5-50 mg / L L-cystine dihydrochloride, 5-30 mg / L L-glutamic acid, 100-1000 mg / L glutamine, 10-100 mg / L histidine hydrochloride hydrate, 10-500 mg / L isoleucine, 10-1000 mg / L leucine, 10-1000 mg / L lysine hydrochloride, 1-100 mg / L methionine, 1-100 mg / L phenylalanine, 5-100 mg / L proline, 1-100 mg / L serine, 10-500 mg / L threonine, 1-100 100 mg / L L-tryptophan, 5-100 mg / L L-tyrosine disodium salt dihydrate and 10-100 mg / L valine.

4. The PK-15 cell culture medium according to claim 1, characterized in that The vitamins include 0.001-0.1 mg / L biotin, 1-100 mg / L choline chloride, 1-50 mg / L D-calcium pantothenate, 1-5 mg / L folic acid, 1-25 mg / L nicotinamide, 1-25 mg / L pyridoxine hydrochloride, 0.05-0.5 mg / L riboflavin, 1-10 mg / L thiamine hydrochloride, 0.1-2.5 mg / L vitamin B12, 1-50 mg / L inositol and 1-500 mg / L vitamin C.

5. The PK-15 cell culture medium according to claim 1, characterized in that The inorganic salts include 100-200 mg / L calcium chloride, 0.001-0.002 mg / L copper sulfate, 0.01-0.1 mg / L ferric nitrate, 0.1-0.8 mg / L ferric sulfate, 10-50 mg / L magnesium chloride, 20-100 mg / L magnesium sulfate, 100-500 mg / L potassium chloride, 4000-7000 mg / L sodium chloride, 50-100 mg / L disodium hydrogen phosphate, 10-100 mg / L sodium dihydrogen phosphate monohydrate and 0.2-0.8 mg / L zinc sulfate.

6. The PK-15 cell culture medium according to claim 1, characterized in that The auxiliary components include 1000-5000 mg / L glucose, 1000-5000 mg / L 4-hydroxyethylpiperazineethanesulfonic acid, 1-10 mg / L sodium hypoxanthine, 0.005-0.05 mg / L sodium selenite, 0.01-0.1 mg / L linoleic acid, 1-10 mg / L linolenic acid, 0.05-0.5 mg / L lipoic acid, 1-40 mg / L phenol red, 0.01-0.2 mg / L putrescine, 1-100 mg / L sodium pyruvate, 0.1-0.55 mg / L thymidine, 1-20 mg / L carnitine, 1-50 mg / L carboxylic acid and 0.002-0.025 mg / L human epidermal growth factor.

7. A method for preparing a serum-free ultra-low protein PK-15 cell culture medium according to any one of claims 1 to 6, comprising: dissolving the basal culture medium and the additive in water to obtain a mixed solution; Adjusting the pH value and osmotic pressure of the mixed solution to a pH value within the range of 7.0 to 7.4 and an osmotic pressure within the range of 300 to 340 mOsm / kg; The mixed solution obtained after adjustment is filtered to obtain the PK-15 cell culture medium.

Images