An improved serum-reduced cell culture medium supplemented with Chinese medicinal materials

Abstract

The invention discloses a kind of improved serum-reduced cell culture medium adding Chinese medicinal materials, and the preparation method is to fully dissolve the serum-free basal medium in ultrapure water, so that the final concentration of the basal medium is 13.7g / L, then add 5-20μg / mL hawthorn and hawthorn leaf extract, stir to mix the components evenly, then add 4% animal serum and stir, so that the components are fully mixed, and finally the solution is sterilized by filtering with a 0.22μm filter membrane. A kind of improved serum-reduced cell culture medium adding Chinese medicinal materials provided by the present invention can replace high serum culture medium, achieve the effect of efficient cell culture, and the optimum dose is 10μg / mL; greatly reduce the amount of animal serum, not only can reduce production costs, but also can reduce the potential danger of animal serum culture medium, and the culture effect is stable; in addition, the application of hawthorn and hawthorn leaves plays an important role in promoting the development of rural economy in my country, increasing farmers' income and protecting the environment.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, in particular to an improved serum-reduced cell culture medium added with traditional Chinese medicine. Background Art

[0002] Hawthorn, also known as red fruit and mountain red, belongs to the genus Crataegus in the Rosaceae family. my country is one of the origin centers of the genus Crataegus. There are 18 species and 1 variety of the genus Crataegus in my country. Among them, the large-fruited hawthorn is one of the unique varieties in my country and is also the main cultivated variety in the northern hawthorn producing areas of my country. At present, the cultivated area of ​​hawthorn in northern my country is 86,700 hectares, with an annual output of more than 1.5 million tons.

[0003] Hawthorn fruit has high nutritional value and is a top-grade tonic that can be used as both medicine and food. It contains a rich variety of amino acids, including 8 essential amino acids; the protein content is 17 times that of apples of the same weight; the vitamins it contains are mainly vitamins A, C, D, B1, B2, and B3, and a variety of minerals, among which the calcium content ranks first among fruits; it also contains more than 30 active substances such as chlorogenic acid, epicatechin, and choline.

[0004] Modern research shows that hawthorn kernels are rich in lignans, flavonoids and triterpenoids, and have a variety of pharmacological activities such as antibacterial, anti-inflammatory, and hypolipidemic. E Combining the UNIFI data analysis platform and multivariate statistical methods, a total of 58 chemical components were analyzed and identified from hawthorn leaves, including 6 organic acids, 16 flavonoids, 24 triterpenoids, and 12 monoterpenes and sesquiterpenes.

[0005] At present, the culture medium for culturing 293T17 cells, ST cells, Vero cells, BHK21 cells, and SF9 cells all contain animal serum. The components in animal serum may form cytotoxic substances with other components and affect cell growth and reproduction. They may also become a source of infection for blood-borne pathogens and xenoantigens.

[0006] In addition, the added animal serum is fetal bovine serum or newborn bovine serum, which is difficult to obtain, expensive, and has batch-to-batch differences, making cell culture effects unstable. Culture media with reduced serum content are prone to problems such as poor cell proliferation and changes in cell morphology and structure. Therefore, it is very necessary to develop a culture medium with low serum content, high safety, and good culture effects. Summary of the invention

[0007] The purpose of the present invention is to provide an improved reduced serum cell culture medium with added Chinese medicinal materials to solve the problems that the conventional culture medium for 293T17 cells, ST cells, Vero cells, BHK21 cells and SF9 cells currently has a high animal serum content, may produce bacterial toxins, carry pathogens and has a high production cost, while the culture effect of the reduced serum culture medium is not ideal.

[0008] To achieve the above-mentioned purpose, the present invention provides an improved reduced serum cell culture medium with added Chinese medicinal materials. The preparation method comprises the following steps: fully dissolving a serum-free basal culture medium in ultrapure water to make the final concentration of the basal culture medium be 13.7 g / L; then adding 5 to 20 μg / mL of hawthorn and hawthorn leaf extracts, stirring to uniformly mix the components; then adding 4% animal serum and stirring to fully mix the components; and finally filtering the solution with a 0.22 μm filter membrane for sterilization.

[0009] Preferably, the extraction method of the hawthorn and hawthorn leaf extract is:

[0010] S1. Remove diseased fruits, diseased leaves, and surface dust, bacteria, and insect eggs from hawthorn and hawthorn leaves;

[0011] S2. Grind the hawthorn and hawthorn leaves with a grinder to make the powder particles uniform and fine, and then pass through an 80-mesh sieve;

[0012] S3, adding water to the mixture powder obtained in step S2 and boiling, filtering the residue, sterilizing the filtrate and sealing it at 4°C.

[0013] Preferably, in step S1, the kernel and stalk of hawthorn are retained; in step S2, hawthorn and hawthorn leaves are mixed in a mass ratio of 1:1; hawthorn and hawthorn leaves are ground in a grinder for 25 to 30 minutes, and then sieved, and the large particle mixture remaining on the sieve is placed in the grinder for grinding again, and then sieved and ground again, until the hawthorn and hawthorn leaves are all sieved.

[0014] Preferably, in step S3, the water added to the mixed powder obtained in step S2 is ultrapure water, and the mass volume ratio of the mixed powder to the ultrapure water is 1 g:10 mL.

[0015] Preferably, in step S3, the mixed powder and ultrapure water are added to a clean beaker with a lid, placed in a water bath and boiled, the residue is filtered out after boiling, and the filtrate is then placed in a water bath and boiled, and this process is repeated until no residue is seen, and the boiling time is 0.5 to 1 h in total. During the boiling process, the lid of the beaker is slightly tightened to control the evaporation rate of the ultrapure water in the beaker to 8 to 12%.

[0016] Preferably, in step S3, the hawthorn and hawthorn leaf extracts are placed at 4° C. for not less than 4 hours, and then the upper extracts are aspirated to prepare the culture medium, and the bottom precipitate is removed.

[0017] The invention discloses an application of the improved reduced serum cell culture medium supplemented with Chinese medicinal materials in culturing 293T17, ST, vero, BHK21 and SF9 cells.

[0018] Therefore, the improved serum-reduced cell culture medium with added Chinese medicinal materials provided by the present invention has the following specific technical effects:

[0019] (1) The reduced serum cell culture medium of the present invention greatly reduces the amount of animal serum by adding hawthorn and hawthorn leaf extracts, but can well ensure the morphology of cultured cells, and is even slightly better than the high-concentration fetal bovine serum culture medium in promoting cell proliferation, and the culture effect is stable;

[0020] (2) The reduced serum cell culture medium of the present invention replaces animal serum with low-cost raw materials, greatly reducing production costs, and can also avoid the problems of batch-to-batch instability and cytotoxicity of the culture medium caused by adding high-concentration serum;

[0021] (3) The reduced serum cell culture medium of the present invention makes full use of hawthorn, hawthorn leaves and hawthorn seeds, which plays an important role in promoting the development of my country's rural economy and increasing farmers' income; it is of great significance to reducing agricultural waste, protecting the environment and sustainable economic development.

[0022] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The cell confluence diagrams in Comparative Example 1, Comparative Example 2, and Examples 5 to 8 of the present invention;

[0024] Figure 2 The cell proliferation multiples in Comparative Example 1, Comparative Example 2, and Examples 5 to 8 of the present invention are shown;

[0025] Figure 3 It is a trend diagram of the confluence of cells of different generations in Comparative Example 1 and Example 6 of the present invention;

[0026] Figure 4 The proliferation multiple trend diagram of cells of different generations in Comparative Example 1 and Example 6 of the present invention;

[0027] Figure 5 It is a trend diagram of the cell multilayer situation in Comparative Example 1, Comparative Example 2, and Examples 5 to 8 of the present invention;

[0028] Figure 6 It is a trend diagram of the light transmission of cells in Comparative Example 1, Comparative Example 2, and Examples 5 to 8 of the present invention;

[0029] Figure 7 The diagram is of 293T17 cells cultured in the culture medium of Comparative Example 1, Comparative Example 2, and Examples 5 to 8 of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0031] Embodiment 1

[0032] A modified reduced serum cell culture medium supplemented with Chinese medicinal materials is prepared, comprising the steps of:

[0033] (1) Accurately weigh 50 g of hawthorn and 50 g of hawthorn leaves, remove diseased fruits and leaves, dust on the surface, bacteria and eggs from the hawthorn and hawthorn leaves. Then add the hawthorn and hawthorn leaves to a grinder and grind for 25 min, then sieve, and grind the remaining large particles on the sieve again in the grinder, then sieve again, and grind again until the hawthorn and hawthorn leaves are completely sieved.

[0034] (2) Add the mixture powder of hawthorn and hawthorn leaves obtained in step (1) into a clean beaker, then add 1L of ultrapure water into the beaker, tighten the lid of the beaker slightly according to previous experimental results, place it in a water bath and boil it for 5 minutes, then filter out the residue, boil the filtrate again for 15 minutes, then filter out the residue, boil the filtrate again, filter it after boiling for 15 minutes, and finally place the boiled filtrate at 4°C for later use.

[0035] (3) After the hawthorn and hawthorn leaf extracts obtained in step (2) are placed at 4°C for 4 hours, 13.7 g of DMEM basal medium (produced by Gibco) dry powder and 1 L of ultrapure water are accurately weighed, a small amount of ultrapure water is added to the basal medium and stirred to fully dissolve the medium, and then 50 μL of the hawthorn and hawthorn leaf extracts obtained in step (2) are drawn from the upper part and added to the solution, that is, the final concentration of the hawthorn and hawthorn leaf mixture is calculated to be 5 μg / mL. A small amount of ultrapure water is then added to the solution and stirred to mix the components evenly.

[0036] Slowly pour 40mL of fetal bovine serum into the solution, stirring while pouring, and finally add ultrapure water to make up the volume of the solution to 1L. In the clean bench, filter and sterilize with a 0.22μm filter membrane.

[0037] Embodiment 2

[0038] A modified reduced serum cell culture medium supplemented with Chinese medicinal materials was prepared in the same manner as in Example 1, except that 100 μL of hawthorn and hawthorn leaf extract was added to the solution, i.e., the final concentration of the hawthorn and hawthorn leaf mixture was 10 μg / mL.

[0039] Embodiment 3

[0040] A modified reduced serum cell culture medium supplemented with Chinese medicinal materials was prepared in the same manner as in Example 1, except that 200 μL of hawthorn and hawthorn leaf extract was added to the solution, i.e., the final concentration of the hawthorn and hawthorn leaf mixture was 20 μg / mL.

[0041] Embodiment 4

[0042] A modified reduced serum cell culture medium supplemented with Chinese medicinal materials was prepared in the same manner as in Example 1, except that 400 μL of hawthorn and hawthorn leaf extract was added to the solution, i.e., the final concentration of the hawthorn and hawthorn leaf mixture was 40 μg / mL.

[0043] Embodiment 5

[0044] The steps for subculturing 293T17 cells are as follows:

[0045] (1) Turn on the ultraviolet light of the clean bench in advance to sterilize the clean bench for 30 minutes. Add 20 mL of the culture medium prepared in Example 1 into a 50 mL disposable sterile culture bottle in the clean bench and seal it for later use.

[0046] (2) The 293T17 cell cryopreservation tube was taken out from the liquid nitrogen tank and quickly placed in a 37°C water bath. After the cell fluid was completely thawed, it was completely aspirated into a sterile centrifuge tube containing 10 mL of the culture medium prepared in Example 1 in an ultra-clean workbench and centrifuged at 1000 r for 5 min.

[0047] (3) After centrifugation in step (2), the supernatant was discarded from the sterile centrifuge tube in the clean bench, and the cell pellet was resuspended and counted in the culture medium prepared in Example 1, and then 3×10 5 293T17 cells were inoculated into the culture flask prepared in step (1) at a density of 10 cells / mL, and the flask was sealed and cultured at 37°C.

[0048] (4) After 72 h, the cells were digested with 0.06% trypsin for 30 s and then the number of cells was counted. The cells were then serially subcultured as described above.

[0049] Embodiment 6

[0050] The steps for subculturing 293T17 cells are exactly the same as those in Example 5, except that the culture medium is replaced with the culture medium prepared in Example 2.

[0051] Embodiment 7

[0052] The steps for subculturing 293T17 cells are exactly the same as those in Example 5, except that the culture medium is replaced with the culture medium prepared in Example 3.

[0053] Embodiment 8

[0054] The steps for subculturing 293T17 cells are exactly the same as those in Example 5, except that the culture medium is replaced with the culture medium prepared in Example 4.

[0055] Embodiment 9

[0056] The steps of subculturing ST cells are exactly the same as those in Example 5.

[0057] Embodiment 10

[0058] The steps of subculturing ST cells are exactly the same as those in Example 6.

[0059] Embodiment 11

[0060] The steps of subculturing ST cells are exactly the same as those in Example 7.

[0061] Embodiment 12

[0062] The steps of subculturing ST cells are exactly the same as those in Example 8.

[0063] Embodiment 13

[0064] The steps of subculturing Vero cells are exactly the same as those in Example 5.

[0065] Embodiment 14

[0066] The steps of subculturing Vero cells are exactly the same as those in Example 6.

[0067] Embodiment 15

[0068] The steps of subculturing Vero cells are exactly the same as those in Example 7.

[0069] Embodiment 16

[0070] The steps of subculturing Vero cells are exactly the same as those in Example 8.

[0071] Embodiment 17

[0072] The steps of subculture of BHK21 cells are exactly the same as those in Example 5.

[0073] Embodiment 18

[0074] The steps of subculture of BHK21 cells are exactly the same as those in Example 6.

[0075] Embodiment 19

[0076] The steps of subculturing BHK21 cells are exactly the same as those in Example 7.

[0077] Embodiment 20

[0078] The steps of subculturing BHK21 cells are exactly the same as those in Example 8.

[0079] Embodiment 21

[0080] The steps of subculturing SF9 cells are exactly the same as those in Example 5.

[0081] Embodiment 22

[0082] The steps of subculturing SF9 cells are exactly the same as those in Example 6.

[0083] Embodiment 23

[0084] The steps of subculturing SF9 cells are exactly the same as those in Example 7.

[0085] Embodiment 24

[0086] The steps of subculturing SF9 cells are exactly the same as those in Example 8.

[0087] Comparative Example 1

[0088] A conventional culture medium was prepared as a positive control using the preparation method in Example 1, except that the components of the culture medium were 13.7 g / L DMEM basal culture medium and 8% fetal bovine serum, and then 293T17 cells were subcultured according to the method in Example 5.

[0089] Comparative Example 2

[0090] A blank control culture medium was prepared by the method in Example 1, except that hawthorn and hawthorn leaf extract were not added to the culture medium, and other components were exactly the same as in Example 1. Then, 293T17 cells were subcultured by the method in Example 5.

[0091] Comparative Example 3

[0092] The subculture method is exactly the same as that of Comparative Example 1, except that the cultured cells are replaced with ST cells.

[0093] Comparative Example 4

[0094] The subculture method is exactly the same as that of Comparative Example 2, except that the cultured cells are replaced with ST cells.

[0095] Comparative Example 5

[0096] The subculture method is exactly the same as that of Comparative Example 1, except that the culture cells are replaced with Vero cells.

[0097] Comparative Example 6

[0098] The subculture method is exactly the same as that of Comparative Example 2, except that the culture cells are replaced with Vero cells.

[0099] Comparative Example 7

[0100] The subculture method was exactly the same as that of Comparative Example 1, except that the cultured cells were replaced with BHK21 cells.

[0101] Comparative Example 8

[0102] The subculture method is exactly the same as that of Comparative Example 2, except that the cultured cells are replaced with BHK21 cells.

[0103] Comparative Example 9

[0104] The subculture method is exactly the same as that of Comparative Example 1, except that the cultured cells are replaced with SF9 cells.

[0105] Comparative Example 10

[0106] The subculture method is exactly the same as that of Comparative Example 2, except that the cultured cells are replaced with SF9 cells.

[0107] Results Analysis

[0108] After the second transmission and culture for 72 hours, the cells were digested with 0.06% trypsin for 30 seconds, and the cell numbers of the passaged cells in Examples 5 to 8 were counted, the confluence was calculated, and the cell morphology, distribution, stratification and light transmittance were observed.

[0109] The cell concentration was 3×10 5 / mL, inoculated in T75 cell culture flasks, observed the cells after 72 hours, counted the cell confluence, and then digested with trypsin and counted, and calculated the proliferation rate. Cultured for 6 generations continuously and recorded.

[0110] The confluence of the cultured cells was plotted with the amounts of hawthorn and hawthorn leaves added in Comparative Example 1, Comparative Example 2, and Examples 5 to 8 as the horizontal axis and the confluence % as the vertical axis, wherein Comparative Example 1 was recorded as "no addition", Comparative Example 2 was recorded as "0 μL / mL", and Examples 5 to 8 were recorded as "5 μL / mL, 10 μL / mL, 20 μL / mL, 40 μL / mL", respectively. The results are shown in FIG. Figure 1 shown.

[0111] Depend on Figure 1 It can be seen that there is no significant difference in the proliferation multiples of cells cultured in Example 6 and Comparative Example 1, and the confluence of cells cultured in Example 6 is even better than that in Comparative Example 1, while the other comparative examples and examples are inferior.

[0112] The cell proliferation times were plotted with the amount of hawthorn and hawthorn leaves added in Comparative Example 1, Comparative Example 2, and Examples 5 to 8 as the horizontal axis and the proliferation times as the vertical axis, wherein Comparative Example 1 was recorded as "no addition", Comparative Example 2 was recorded as "0 μL / mL", and Examples 5 to 8 were recorded as "5 μL / mL, 10 μL / mL, 20 μL / mL, 40 μL / mL" respectively. Figure 2 The results show that there is no significant difference in the proliferation times of cells cultured in Example 6 and Comparative Example 1, and the proliferation times of cells cultured in Example 6 are even better than that in Comparative Example 1, reaching about 6 times, while other comparative examples and examples are inferior.

[0113] The statistical results were plotted with the passage number as the horizontal axis and the confluence percentage of the comparative example 1 and the embodiment 6 as the vertical axis to draw a trend graph of the confluence of cells of different generations. The results are as follows: Figure 3 As shown, there is no significant difference in the confluence of cells cultured in Example 6 and Comparative Example 1, and the confluence of cells cultured in Example 6 is even slightly higher than that in Comparative Example 1.

[0114] The proliferation times trend graph was plotted with the number of passages as the abscissa and the proliferation times of Comparative Example 1 and Example 6 as the ordinate. The results are shown in Figure 4 As shown, there is no significant difference in cell proliferation multiples between Comparative Example 1 and Example 6.

[0115] The cell multilayer condition trend graph was drawn with the amount of hawthorn and hawthorn leaves added in Comparative Example 1, Comparative Example 2, and Examples 5 to 8 as the horizontal axis and the multilayer condition as the vertical axis ( Figure 5 ), where Comparative Example 1 is recorded as "no addition", Comparative Example 2 is recorded as "0 μL / mL", and Examples 5 to 8 are recorded as "5 μL / mL, 10 μL / mL, 20 μL / mL, 40 μL / mL", respectively. Where 1 represents the presence of a multilayer, and 0 represents the absence of a multilayer. The results are shown in Figure 5 As shown, the cells cultured in Comparative Example 2, Example 5 and Example 8 all have a multilayer, while the cells cultured in Example 6, Example 7 and Comparative Example 1 all have no multilayer.

[0116] The cell light transmittance trend chart was drawn with the amount of hawthorn and hawthorn leaves added in Comparative Example 1, Comparative Example 2, and Examples 5 to 8 as the horizontal axis and the light transmittance as the vertical axis, wherein Comparative Example 1 was recorded as "no addition", Comparative Example 2 was recorded as "0 μL / mL", and Examples 5 to 8 were recorded as "5 μL / mL, 10 μL / mL, 20 μL / mL, 40 μL / mL" respectively. Figure 6 1 represents good light transmission, and 0 represents poor light transmission. The results are as follows Figure 6 As shown, the cells cultured in Comparative Example 1, Comparative Example 2, Example 5, and Example 6 all have good light transmittance, while the cells cultured in Example 7 and Example 8 have poor light transmittance.

[0117] Figure 7 Figures 293T17 cells cultured in culture medium with different amounts of hawthorn and hawthorn leaf additives in Comparative Example 1, Comparative Example 2, and Examples 5 to 8 of the present invention, wherein Figure A shows cells cultured in Comparative Example 1, Figure B shows cells cultured in Comparative Example 2, Figure C shows cells cultured in Example 5, Figure D shows cells cultured in Example 6, Figure E shows cells cultured in Example 7, and Figure F shows cells cultured in Example 8.

[0118] From the pictures, it can be found that the cells cultured in Example 6 have the same degree of confluence, multilayering, and light transmittance as those cultured in Comparative Example 1, or even better than Comparative Example 1, and the cells in the example are inferior. Therefore, the optimal additive dosage of hawthorn and hawthorn leaves is 10 μg / mL.

[0119] The conditions of ST, vero, BHK21 and SF9 cells in other examples are the same as those of 293T17 cells.

[0120] Therefore, the improved reduced serum cell culture medium with added Chinese medicinal materials provided by the present invention can replace the high serum culture medium to achieve the effect of efficient cell culture, and the optimal dose is 10 μg / mL; the amount of animal serum used can be greatly reduced, which can not only reduce the production cost, but also reduce the potential risks of the animal serum culture medium, and the culture effect is stable; in addition, the application of hawthorn and hawthorn leaves plays an important role in promoting the development of my country's rural economy, increasing farmers' income and protecting the environment.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. An improved reduced serum cell culture medium supplemented with Chinese medicinal materials, characterized in that: The preparation method is to fully dissolve the serum-free basal medium in ultrapure water to make the final concentration of the basal medium 13.7 g / L, then add 10 μg / mL hawthorn and hawthorn leaf extract, stir to mix the components evenly, then add 4% animal serum and stir to mix the components thoroughly, and finally filter the solution with a 0.22 μm filter membrane for sterilization; The extraction method of the hawthorn and hawthorn leaf extract is: S1. Remove diseased fruits, diseased leaves, and surface dust, bacteria, and insect eggs from hawthorn and hawthorn leaves; S2. Grind the hawthorn and hawthorn leaves with a grinder to make the powder particles uniform and fine, and then pass through an 80-mesh sieve; S3, boiling the mixture powder obtained in step S2 with water, filtering the residue, sterilizing the filtrate, and sealing it at 4°C; The hawthorn and hawthorn leaves are purchased dried hawthorn and hawthorn leaves; in step S1, the core and stalk of the hawthorn are retained; in step S2, the hawthorn and hawthorn leaves are mixed in a mass ratio of 1:1; the hawthorn and hawthorn leaves are ground in a grinder for 25 to 30 minutes, and then sieved, and the large particles remaining on the sieve are placed in the grinder for grinding again, and then sieved and ground again, until the hawthorn and hawthorn leaves are all sieved; In step S3, the water added to the mixed powder obtained in step S2 is ultrapure water, and the mass volume ratio of the mixed powder to the ultrapure water is 1 g:10 mL; In step S3, the mixed powder and ultrapure water are added to a clean beaker with a lid, and the mixture is placed in a water bath for boiling. After boiling, the residue is filtered out, and the filtrate is placed in a water bath for boiling again. This is repeated until no residue is seen. The boiling time is 0.5 to 1 hour in total. During the boiling process, the lid of the beaker is slightly tightened to control the evaporation rate of the ultrapure water in the beaker to 8 to 12%.

2. The improved reduced serum cell culture medium with added Chinese medicinal materials according to claim 1, characterized in that: In step S3, the hawthorn and hawthorn leaf extracts are placed at 4° C. for not less than 4 hours, and then the upper extracts are aspirated to prepare the culture medium, and the bottom precipitate is removed.

3. Use of the improved reduced serum cell culture medium supplemented with Chinese medicinal materials as claimed in claim 1 or 2 in culturing 293T17, ST, vero, BHK21, and SF9 cells.

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