DC cell culture medium and preparation method thereof

Abstract

The invention relates to the technical field of cell culture, and particularly provides a DC cell culture medium and a preparation method thereof, and the DC cell culture medium is prepared by mixing 36% by mass of a traditional Chinese medicine extract containing caulis spatholobi, radix zanthoxyli, radix linderae, lucid ganoderma, trametes robiniophila and ginseng with the balance of a complete culture medium. The traditional Chinese medicine extract is combined with the complete culture medium, so that the method focuses on promoting self-repairing of cells and secreting cell factors, reduces the pollution influence of foreign factors and bacteria, and improves the long-term survival rate of mature DC cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a DC cell culture medium and a preparation method thereof. Background Art

[0002] Human bone marrow dendritic cells, also known as DC cells, are induced by bone marrow monocytes; DC cells are divided into mature DC cells and immature DC cells. Mature DC cells are further induced by immature DC cells through TNFα, LPS, etc. Most of them grow in suspension and have a round cell morphology. The cells are highly differentiated cells and belong to the non-proliferating cell group. Under high magnification or electron microscope, a large number of dendrite-like protrusions of varying thickness can be observed on the surface. The surface of mature DC cells highly expresses major histocompatibility complex MHC and co-stimulatory molecules such as CD80 and CD86, which in turn activate T lymphocytes and induce immune responses. They are at the center of initiating, regulating, and maintaining immune responses. With the development of cell commercialization, mature DC cells no longer require experimenters to separate and induce them from peripheral blood and gradually culture them. It has become possible to directly purchase mature DC cells.

[0003] However, most of the existing DC cell culture medium technologies are still for promoting the induction culture of DC cell maturation, and do not pay special attention to the survival rate of mature DC cells, which is not conducive to the long-term use of mature DC cells. Therefore, it is of great practical significance to provide a culture medium for improving the long-term survival rate of mature DC cells and a preparation method thereof. Summary of the invention

[0004] In order to solve the above problems, on the one hand, the present invention provides a method for preparing a DC cell culture medium, the method for preparing a DC cell culture medium comprising the following steps: Step 1: Extracting Chinese herbal medicine extracts: taking 14-24% of Millettia reticulata, 10-19% of Zanthoxylum bungeanum, 7-15% of Linderae umbellatae, 4-10% of Ganoderma lucidum, 4-9% of Sophora japonicae, 2-5% of Ginseng and making up the rest of the mass fraction with pure water, chopping the above substances until they pass through a 20-mesh sieve, adding them to a colloid mill with a speed of 2800r / min and a discharge fineness of 300 mesh at a feeding rate of 50kg / h, grinding at 5°C at low temperature until the material is discharged to obtain the extract, adding 85% ethanol with a mass of 4 times the mass of the extract at 18°C, mixing and immersing for 15h, and filtering through 400 mesh to obtain a filtrate; distilling the filtrate under reduced pressure at 71°C and a vacuum degree of 800mbar until no liquid continuously flows out, collecting the remaining liquid and filtering through a 0.22μm film to obtain the Chinese herbal medicine extract; Step 2: Mix 36% of the Chinese herbal medicine extract by weight with the remaining complete culture medium to obtain the DC cell culture medium.

[0005] Furthermore, the mass fraction of Millettia spatholobi is 19%.

[0006] Furthermore, the mass fraction of the two-sided needle is 15%.

[0007] Furthermore, the mass fraction of the Linderae umbellatae is 11%.

[0008] Furthermore, the mass fraction of the Ganoderma lucidum is 7%.

[0009] Furthermore, the mass fraction of the Sophora japonica urn is 6%.

[0010] Furthermore, the mass fraction of the ginseng is 3%.

[0011] In another aspect, the present invention provides a DC cell culture medium prepared by the above-mentioned method for preparing a DC cell culture medium.

[0012] The present invention can bring the following beneficial effects: The present invention combines traditional Chinese medicine extracts with complete culture medium, focuses on promoting cell self-repair and cytokine secretion, reduces the contamination effects of external factors and bacteria, and improves the long-term survival rate of mature DC cells.

[0013] The active substances such as flavonoids and polyphenols of the extracts of Millettia spatholobi and Zanthoxylum bungeanum of the present invention have strong free radical scavenging ability, which may protect mature DC cells from oxidative damage, and may also inhibit the production of inflammatory mediators, and reduce the damage of inflammatory response to mature DC cells; the extract of Linderae spatholobi of the present invention may have antibacterial activity, and can maintain the sterile state of the mature DC cell culture environment for a long time, reducing the influence of bacteria and their metabolites on mature DC cells; the active substances such as polysaccharides of the extracts of Ganoderma lucidum and Sophora japonica of the present invention may promote the immune activity of mature DC cells, and help mature DC cells maintain a mature state; the extract of ginseng of the present invention may promote the secretion of various factors of mature DC cells, and help to improve cell activity; the above-mentioned Millettia spatholobi, Zanthoxylum bungeanum, Linderae spatholobi, Ganoderma lucidum, Sophora japonica and ginseng cooperate together, and improve the long-term survival rate of mature DC cells together under the preparation process of the present invention. It should be noted that the distribution ratio and process parameters of each group of the present invention will also affect the stability of the entire system and thus affect the effect. DETAILED DESCRIPTION

[0014] In order to more clearly explain the overall concept of the present invention, the overall scheme of the present invention is described in detail in the form of embodiments below; in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention; however, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details; in other examples, in order to avoid confusion with the present invention, some technical features known in the art are not described.

[0015] In the present invention, human bone marrow dendritic cells (mature DC cells) were purchased from Wuhan Pronocell Life Science Co., Ltd., Cat NO.: CP-H187B, with a live cell density of 5×10 5 / mL, loaded in T25 cell culture flask; complete culture medium was purchased from Wuhan Punosai Life Science Technology Co., Ltd., which is complete culture medium for human peripheral blood dendritic cells (mature DC cells), item number: CM-H179B; colloid mill was purchased from Zhejiang Hengdong Stainless Steel Co., Ltd., model: horizontal-100; vacuum distiller was purchased from Shanghai Yuhua Instrument Equipment Co., Ltd., model RE-501, speed 120rpm, maximum feed 1L each time; Millettia reticulata, Zanthoxylum bungeana, Linderae scutellariae, Ganoderma lucidum, Sophora japonica fungus and ginseng were purchased from Bozhou Qiaoyi Health Industry Co., Ltd., with a water content of 5%, and ginseng was raw sun-dried white ginseng; membrane filtration used TST-BLGL membrane filter.

[0016] Unless otherwise specified, all raw material components in the following examples can be purchased through commercial channels, the experimental instruments used are conventional laboratory experimental instruments, and the performance testing methods are the testing methods known in the art.

[0017] The preferred implementation is as follows: Embodiment 1: The DC cell culture medium was prepared by the following method: Step 1: Extracting Chinese herbal medicine extracts: Take 19% of Millettia spatholobi, 15% of Zanthoxylum bungeanum, 11% of Linderae rutaecarpa, 7% of Ganoderma lucidum, 6% of Sophora japonica, 3% of Ginseng and make up the rest of the mass fraction with pure water, chop the above substances until they pass through a 20-mesh sieve, add them into a colloid mill with a speed of 2800r / min and a discharge fineness of 300 mesh at a feeding rate of 50kg / h, grind at 5°C at low temperature until the material is discharged to obtain the extract, add 85% ethanol with a mass of 4 times the mass of the extract at 18°C, mix and immerse for 15h, and then filter through 400 mesh to obtain a filtrate; distill the filtrate under reduced pressure at 71°C and a vacuum degree of 800mbar until no liquid flows out continuously, collect the remaining liquid and filter through a 0.22μm film to obtain the Chinese herbal medicine extract; Step 2: Mix 36% of the Chinese herbal medicine extract by weight with the remaining complete culture medium to obtain the DC cell culture medium.

[0018] Embodiment 2-13: The only difference between Example 2 and Example 1 is that the mass fraction of Millettia reticulata is 14%; The only difference between Example 3 and Example 1 is that the mass fraction of Millettia reticulata is 24%; The only difference between Example 4 and Example 1 is that the mass fraction of Zanthoxylum bungeanum is 10%; The only difference between Example 5 and Example 1 is that the mass fraction of Zanthoxylum bungeanum is 19%; The only difference between Example 6 and Example 1 is that the mass fraction of Linderae obesa is 7%; The difference between Example 7 and Example 1 is that the mass fraction of Linderae obesa is 15%; The only difference between Example 8 and Example 1 is that the mass fraction of Ganoderma lucidum is 4%; The only difference between Example 9 and Example 1 is that the mass fraction of Ganoderma lucidum is 10%; The only difference between Example 10 and Example 1 is that the mass fraction of Sophora japonica urn is 4%; The only difference between Example 11 and Example 1 is that the mass fraction of Sophora japonica urn is 9%; The only difference between Example 12 and Example 1 is that the mass fraction of ginseng is 2%; The only difference between Example 13 and Example 1 is that the mass fraction of ginseng is 5%.

[0019] Comparative Examples 1-25: The only difference between Comparative Example 1 and Example 1 is that the mass fraction of Millettia spatholobi is 7%; The only difference between Comparative Example 2 and Example 1 is that the mass fraction of Millettia spatholobi is 40%; The difference between Comparative Example 3 and Example 1 is that the mass fraction of Zanthoxylum bungeanum is 5%; The difference between Comparative Example 4 and Example 1 is that the mass fraction of Zanthoxylum bungeanum is 30%; The difference between Comparative Example 5 and Example 1 is that the mass fraction of Linderae obesa is 3%; The difference between Comparative Example 6 and Example 1 is that the mass fraction of Linderae obesa is 30%; The only difference between Comparative Example 7 and Example 1 is that the mass fraction of Ganoderma lucidum is 2%; The only difference between Comparative Example 8 and Example 1 is that the mass fraction of Ganoderma lucidum is 20%; The only difference between Comparative Example 9 and Example 1 is that the mass fraction of Sophora japonica urn is 2%; The only difference between Comparative Example 10 and Example 1 is that the mass fraction of Sophora japonica urn is 20%; The only difference between Comparative Example 11 and Example 1 is that the mass fraction of ginseng is 1%; The only difference between Comparative Example 12 and Example 1 is that the mass fraction of ginseng is 10%; The only difference between Comparative Example 13 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 20%; The only difference between Comparative Example 14 and Example 1 is that the mass fraction of the Chinese herbal medicine extract is 50%; The only difference between Comparative Example 15 and Example 1 is that the grinding temperature of the colloid mill is 25°C; The difference between Comparative Example 16 and Example 1 is that the temperature of vacuum distillation is 85°C; The only difference between Comparative Example 17 and Example 1 is that Millettia reticulata is replaced with Zanthoxylum bungeanum, Linderae scutellariae, Ganoderma lucidum, Sophora japonica foetida and Ginseng of the same mass ratio as that of Example 1; The only difference between Comparative Example 18 and Example 1 is that the two-sided needles are replaced with Millettia reticulata, Lindera striata, Ganoderma lucidum, Sophora japonica fungus and ginseng of the same mass ratio as that of Example 1; The difference between Comparative Example 19 and Example 1 is that Linderae spatholobi, Zanthoxylum bungeanum, Ganoderma lucidum, Sophora japonica fungus and Ginseng are replaced with the same mass ratio as that of Example 1; The only difference between Comparative Example 20 and Example 1 is that Ganoderma lucidum is replaced by Millettia spatholobi, Zanthoxylum bungeanum, Linderae spatholobi, Sophora japonica fungus and Ginseng in the same mass ratio as that of Example 1; The only difference between Comparative Example 21 and Example 1 is that Sophora japonica is replaced with Millettia spatholobi, Zanthoxylum bungeanum, Linderae scutellariae, Ganoderma lucidum and Ginseng in the same mass ratio as in Example 1; The difference between Comparative Example 22 and Example 1 is that ginseng is replaced by Millettia reticulata, Zanthoxylum bungeanum, Linderae umbellatae, Ganoderma lucidum and Sophora japonicae in the same mass ratio as that in Example 1; The difference between Comparative Example 23 and Example 1 is that the Chinese herbal medicine extract is replaced with pure water of equal mass; The difference between Comparative Example 24 and Example 1 is that the Chinese herbal medicine extract is replaced with a complete medium of equal mass; The only difference between Comparative Example 25 and Example 1 is that the later experiments were conducted on Purnosai HS-27A human bone marrow cells.

[0020] The survival rate of mature DC cells was tested: 3 samples were prepared for each example for cell culture; the purchased mature DC cell T25 cell culture flask was taken out, the flask was disinfected with 75% alcohol, the sealing film was removed, and it was placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 4 hours. After the cells were stabilized, the culture medium in the T25 cell culture flask was collected into a 50mL centrifuge tube, PBS buffer was aspirated with a pipette, the cell culture flask was washed twice, and the washing solution was collected; after centrifugation at 1400rpm for 3min, the supernatant was discarded, and the cell precipitate was collected①; 1mL of 0.25% trypsin digestion solution was added to the T25 culture flask, and the culture flask was slightly rotated until the digestion solution covered the entire bottom of the culture flask, Aspirate excess trypsin digestion solution and incubate at 37°C for 2 minutes; observe under an inverted microscope, and after the cells shrink, add 5 mL of complete culture medium to terminate digestion; gently blow and mix with a pipette, and collect the cell suspension into a centrifuge tube; centrifuge at 1400 rpm for 3 minutes, discard the supernatant, and collect the cell pellet ②; draw 5 mL of the DC cell culture medium sample of the present invention, resuspend the cell pellet ① and the cell pellet ②, mix ① and ②, gently blow and mix with a pipette, disperse the cells, inoculate them into a new T75 culture flask, and then add 20 mL of the DC cell culture medium sample of the present invention, and place it in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for static culture; replace the DC cell culture medium sample of the present invention every 3 days.

[0021] After 21 days of culture, the survival rate was tested: the mature DC cells cultured in each sample were resuspended by blowing, and 2 drops of trypan blue dye were added to every 1 mL of cell suspension. The cells were stained at room temperature for 4 minutes. A drop of cell suspension was placed on a glass slide, and the cells were covered with a glass slide and observed under a high-power microscope. Dead cells were light blue and swollen, and had no luster. Living cells were not stained and maintained a normal morphology and were shiny. The number of live and dead cells was counted, and the cell viability was calculated according to the formula: cell viability (%) = number of unstained cells / total number of observed cells × 100%.

[0022] The mature DC cell survival rate test results of each example are shown in Table 1. In Table 1, the mature DC cell survival rate test results are the average of the results of 3 samples in each example, and are retained to the integer.

[0023] Table 1: Test results of mature DC cell survival rate of each example:

[0024] From the data in Table 1, it can be seen that compared with other examples, the survival rate of mature DC cells cultured in the present invention, especially in Example 1, is higher. The present invention, especially in Example 1, provides a culture medium and a preparation method thereof for improving the long-term survival rate of mature DC cells. Comparative Example 25 shows that the present invention is a preferred experiment for mature DC cells.

[0025] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a DC cell culture medium, characterized in that: The preparation method of the DC cell culture medium comprises the following steps: Step 1: Extracting Chinese herbal medicine extracts: taking 14-24% of Millettia reticulata, 10-19% of Zanthoxylum bungeanum, 7-15% of Linderae umbellatae, 4-10% of Ganoderma lucidum, 4-9% of Sophora japonicae, 2-5% of Ginseng and making up the rest of the mass fraction with pure water, chopping the above substances until they pass through a 20-mesh sieve, adding them to a colloid mill with a speed of 2800r / min and a discharge fineness of 300 mesh at a feeding rate of 50kg / h, grinding at 5°C at low temperature until the material is discharged to obtain the extract, adding 85% ethanol with a mass of 4 times the mass of the extract at 18°C, mixing and immersing for 15h, and filtering through 400 mesh to obtain a filtrate; distilling the filtrate under reduced pressure at 71°C and a vacuum degree of 800mbar until no liquid continuously flows out, collecting the remaining liquid and filtering through a 0.22μm film to obtain the Chinese herbal medicine extract; Step 2: Mix 36% of the Chinese herbal medicine extract by weight with the remaining complete culture medium to obtain the DC cell culture medium.

2. The method for preparing a DC cell culture medium according to claim 1, characterized in that: The mass fraction of the Millettia reticulata is 19%.

3. The method for preparing a DC cell culture medium according to claim 1, characterized in that: The mass fraction of the two-sided needle is 15%.

4. The method for preparing a DC cell culture medium according to claim 1, characterized in that: The mass fraction of the Linderae umbellatae is 11%.

5. The method for preparing a DC cell culture medium according to claim 1, characterized in that: The mass fraction of the Ganoderma lucidum is 7%.

6. The method for preparing a DC cell culture medium according to claim 1, characterized in that: The mass fraction of the Sophora japonica fungus is 6%.

7. The method for preparing a DC cell culture medium according to claim 1, characterized in that: The mass fraction of the ginseng is 3%.

8. A DC cell culture medium prepared by the method for preparing a DC cell culture medium according to any one of claims 1 to 7.

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