A complete medium for culturing DF-1 cells

Abstract

The present invention discloses a complete medium for culturing DF-1 cells, including DMEM basal medium 13.7g / L, insulin, non-essential amino acids, transferrin, glutathione, ascorbyl phosphate, sodium selenite, ethanolamine, hydrocortisone, and newborn calf serum. Wherein non-essential amino acids include glycine, alanine, proline, serine, and asparagine. A complete medium for culturing DF-1 cells provided by the present invention can well promote the proliferation of DF-1 cells, can achieve the same proliferation effect as high-concentration fetal bovine serum, and is suitable for the adherent culture of DF-1 cells; using low-concentration newborn calf serum, it is possible to greatly avoid the batch differences, unstable culture results, cytotoxicity, and high risk of carrying pathogens brought by the culture medium with high-concentration serum, narrow application range, expensive fetal bovine serum, and high culture cost; the components are simple, the culture medium preparation process is short in time consumption, the culture efficiency is high, the repeatability is good, and it is suitable for batch production.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, in particular to a complete culture medium for culturing DF-1 cells. Background Art

[0002] DF-1 cells are a substitutable chicken fibroblast cell line. DF-1 cells are derived from ELL chicken embryos. The cells do not have endogenous genes for avian leukosis virus and sarcoma virus, and are fibrous in morphology. The DF-1 cell line is a stable, tumor gene-free, spontaneously infinitely proliferating cell line. It is widely used in animal virus research, vaccine development, cancer research and many other fields. It is an important biomaterial for virus transfection and culture in the field of life sciences.

[0003] The conventional culture system of DF-1 cells requires the addition of 10% to 15% animal serum, usually fetal bovine serum. Serum is a complex mixture of many biological molecules of different sizes. Although most of its components are known, some are still unclear.

[0004] The main functions of serum in cell culture are as follows: (1) providing cell survival and proliferation; (3) containing some matrix components that allow adherent cells to grow; (4) providing carrier proteins that can bind vitamins, lipids, metal ions, etc.; (5) in some cases, neutralizing toxic substances to protect cells from damage; (6) providing a good buffer system for the culture medium; (7) providing protease inhibitors to protect cells from damage by proteases released by dead cells.

[0005] However, serum-supplemented culture media also have many defects in cell culture, such as: (1) serum also contains some substances that are toxic to cells, such as polyamine oxidase, which can react with polyamines (such as spermine and spermidine) from highly reproductive cells to form cytotoxic substances; (2) serum may promote the growth of some cells while inhibiting the growth of other cells; (3) there may be known or unknown pathogens carried by animals, posing a threat to future clinical applications, and there are batch differences between each batch of serum, resulting in differences in cell culture results; (4) serum is becoming increasingly difficult to obtain and expensive, and is one of the main components of the production cost of animal cell culture.

[0006] For the above reasons, researchers are actively developing DF-1 cell culture media that do not contain animal serum. However, DF-1 cell culture media that do not contain animal serum generally have the problem of a wide variety of components and a long preparation time. For example, the culture medium in patent CN112210542A requires 55 components of different concentrations. In addition, serum-free culture media also generally have the problem of slow growth of DF-1 cells and even stagnant proliferation. This will directly affect the stable passage of DF-1 cells, the accuracy and reliability of the results of cell experiments, and the production efficiency of cell-related products.

[0007] Therefore, it is necessary to develop a complete culture medium with low serum concentration to overcome the problems of the current DF-1 cell culture medium, such as numerous components, poor repeatability, unsatisfactory culture effect and high cost. Summary of the invention

[0008] The purpose of the present invention is to provide a complete culture medium for culturing DF-1 cells, so as to solve the problems that a large amount of animal serum is added to the conventional culture medium for DF-1 cells, so that the cultured cells and cell products contain bacterial toxins and have a high risk of carrying pathogens; the serum is expensive, resulting in high production costs; there are differences between serum batches, resulting in unstable culture effects; and the serum-free culture medium has many components, DF-1 cells grow slowly, and proliferation stagnation occurs.

[0009] To achieve the above object, the present invention provides a complete culture medium for culturing DF-1 cells, comprising 13.7 g / L DMEM basal culture medium, insulin, non-essential amino acids, transferrin, glutathione, ascorbic acid phosphate, sodium selenite, ethanolamine, hydrocortisone, and newborn calf serum.

[0010] Preferably, the non-essential amino acids include glycine, alanine, proline, serine, and asparagine.

[0011] Preferably, it includes 1-15 mg / L insulin, 20-45 mg / L glycine, 5-25 mg / L alanine, 2-20 mg / L proline, 40-60 mg / L serine, 5-25 mg / L asparagine, 1-20 mg / L transferrin, 0.1-10 mg / L glutathione, 0.1-6 mg / L ascorbic acid phosphate, 0.1-8 ug / L sodium selenite, 0.1-6 mg / L ethanolamine, 0.01-6 mg / L hydrocortisone, and 10-60 mL / L newborn calf serum.

[0012] Preferably, it includes 15 mg / L insulin, 45 mg / L glycine, 25 mg / L alanine, 20 mg / L proline, 60 mg / L serine, 25 mg / L asparagine, 20 mg / L transferrin, 10 mg / L glutathione, 6 mg / L ascorbic acid phosphate, 8 ug / L sodium selenite, 6 mg / L ethanolamine, 6 mg / L hydrocortisone, and 60 mL / L newborn calf serum.

[0013] Preferably, it includes 1 mg / L insulin, 20 mg / L glycine, 5 mg / L alanine, 2 mg / L proline, 40 mg / L serine, 5 mg / L asparagine, 1 mg / L transferrin, 0.1 mg / L glutathione, 0.1 mg / L ascorbic acid phosphate, 0.1 ug / L sodium selenite, 0.1 mg / L ethanolamine, 0.01 mg / L hydrocortisone, and 10 mL / L newborn calf serum.

[0014] Preferably, it includes 7 mg / L insulin, 34 mg / L glycine, 10 mg / L alanine, 9 mg / L proline, 50 mg / L serine, 15 mg / L asparagine, 6 mg / L transferrin, 2 mg / L glutathione, 2 mg / L ascorbic acid phosphate, 3 ug / L sodium selenite, 2 mg / L ethanolamine, 0.03 mg / L hydrocortisone, and 20 mL / L newborn calf serum.

[0015] Preferably, after each component is added with water and stirred to dissolve, the solution is filtered and sterilized using a 0.22 um filter membrane.

[0016] A use of the above-mentioned DF-1 cell complete culture medium in culturing DF-1 cells.

[0017] Therefore, the complete medium for culturing DF-1 cells provided by the present invention has the following specific technical effects:

[0018] (1) The complete medium of the present invention is compounded with low-concentration newborn calf serum and other added components, which can well promote the proliferation of DF-1 cells and can achieve the same effect as high-concentration fetal bovine serum in promoting cell proliferation, and is suitable for the adherent culture of DF-1 cells;

[0019] (2) The complete culture medium of the present invention uses low-concentration newborn calf serum, which can greatly avoid the problems of batch-to-batch differences, unstable culture results, high cytotoxicity and pathogen-carrying risks, narrow application range, expensive fetal bovine serum, and high culture costs caused by culture medium with high-concentration serum;

[0020] (3) The complete culture medium of the present invention has simple components, a short preparation process, high culture efficiency, good repeatability, and has prospects for industrialization and commercialization and is suitable for mass production.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the growth curve of P3 generation DF-1 cells in Example 4 of the present invention and the comparative experiment;

[0023] Figure 2 This is a diagram of the subculture of DF-1 cells in the comparative experiment of the present invention;

[0024] Figure 3 This is a diagram of the subculture of DF-1 cells in Example 4 of the present invention. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0026] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and complete, the technical scheme in the embodiments of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0027] Embodiment 1

[0028] A complete medium for culturing DF-1 cells is prepared, comprising the steps of:

[0029] (1) Accurately weigh 13.7 g of DMEM basal medium (DMEM dry powder medium produced by Yixing Saier Biotechnology Co., Ltd.), 15 mg of insulin, 45 mg of glycine, 25 mg of alanine, 20 mg of proline, 60 mg of serine, 25 mg of asparagine, 20 mg of transferrin, 10 mg of glutathione, 6 mg of ascorbic acid phosphate, 8 ug of sodium selenite, 6 mg of ethanolamine, and 6 mg of hydrocortisone.

[0030] (2) Add a small amount of water for injection to each component of step (1) and stir to fully dissolve each component, then add 60 mL of newborn calf serum and stir to mix the components evenly, and add water for injection to 1 L.

[0031] (3) Filter the prepared culture medium with a 0.22 μm sterile filter (produced by BLOFIL) in a clean bench. The culture medium should be prepared immediately before use, or prepared in advance and stored aseptically at 4°C.

[0032] Embodiment 2

[0033] A complete medium for culturing DF-1 cells is prepared, comprising the steps of:

[0034] (1) Accurately weigh 13.7 g of DMEM basal medium powder, 1 mg of insulin, 20 mg of glycine, 5 mg of alanine, 2 mg of proline, 40 mg of serine, 5 mg of asparagine, 1 mg of transferrin, 0.1 mg of glutathione, 0.1 mg of ascorbic acid phosphate, 0.1 μg of sodium selenite, 0.1 mg of ethanolamine, and 0.01 mg of hydrocortisone.

[0035] (2) Add a small amount of water for injection to each component of step (1) and stir to fully dissolve each component, then add 10 mL of newborn calf serum and stir to mix the components evenly, and add water for injection to 1 L.

[0036] (3) Filter the prepared culture medium with a 0.22 μm sterile filter (produced by BLOFIL) in a clean bench. The culture medium should be prepared immediately before use, or prepared in advance and stored aseptically at 4°C.

[0037] Embodiment 3

[0038] A complete medium for culturing DF-1 cells is prepared, comprising the steps of:

[0039] (1) Accurately weigh 13.7 g of DMEM basal medium powder, 7 mg of insulin, 34 mg of glycine, 10 mg of alanine, 9 mg of proline, 50 mg of serine, 15 mg of asparagine, 6 mg of transferrin, 2 mg of glutathione, 2 mg of ascorbic acid phosphate, 3 μg of sodium selenite, 2 mg of ethanolamine, and 0.03 mg of hydrocortisone.

[0040] (2) Add a small amount of water for injection to each component of step (1) and stir to fully dissolve each component, then add 20 mL of newborn calf serum and stir to mix the components evenly, and add water for injection to 1 L.

[0041] (3) Filter the prepared culture medium with a 0.22 μm sterile filter (produced by BLOFIL) in a clean bench. The culture medium should be prepared immediately before use, or prepared in advance and stored aseptically at 4°C.

[0042] Embodiment 4

[0043] Subculture of DF-1 cells

[0044] (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 DF-1 cell culture medium prepared in Example 3 to a 50 mL disposable sterile culture bottle (produced by NEST) in the clean bench, and seal it for later use. The DF-1 cell culture medium prepared in Example 3 is dispensed into sterile disposable cell culture bottles of appropriate specifications on the sterile operating table as needed for later use.

[0045] (2) The P3 generation DF-1 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 3 in an ultra-clean workbench and centrifuged at 1000 r / min for 5 min.

[0046] (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 2×10 4 The P3 generation DF-1 cells were inoculated into the culture flask prepared in step (1) at a density of 10 cells / mL. The culture flask was sealed and placed in a CO 2 The culture conditions were set at 39°C and CO 2 The concentration is 5% and the humidity is 100%.

[0047] (4) After 3 days, DF-1 cells were subcultured and digested with 0.25% trypsin and 0.02% EDTA for 2-3 min. The cells were counted and then subcultured continuously as described above.

[0048] Comparative experiment

[0049] A culture medium containing 13.7 g / L DMEM basal culture medium and 10% FBS was prepared by the method in Example 1, and then DF-1 cells were subcultured according to the DF-1 cell subculture method in Example 4.

[0050] Results Analysis

[0051] The P3 generation DF-1 cells cultured in Example 4 and the comparative experiment were respectively taken to prepare single cell suspensions, and the cells were counted.

[0052] Adjust the cell concentration to 22 × 10 4 / mL, inoculated in a 24-well cell culture plate, 24 hours later, three wells in the plate were digested with trypsin and counted, three wells were taken every day for 8 consecutive days, digested with trypsin and counted, and then the average number of 8 days was calculated.

[0053] The growth curve is plotted with time (d) as the horizontal axis and the number of cells as the vertical axis. The results are as follows: Figure 1As shown, series 1 is the growth curve of DF-1 cells cultured in the comparative experiment, and series 2 is the growth curve of DF-1 cells cultured in Example 4 of the present invention.

[0054] Depend on Figure 1 It can be seen that the growth curves of the P3 generation DF-1 cells cultured in the complete medium of the present invention and the medium containing high concentration of fetal bovine serum are almost completely consistent. It can also be seen that the cell number does not change much 1 to 2 days after inoculation, and from the 3rd day onwards, the cells proliferate in large quantities, and the cell number reaches a peak on the 6th day, and the cell doubling time is 19.6h.

[0055] The results of DF-1 cell passage in the comparative experiment are shown in Figure 2 , where Figure A is a picture of DF-1 cells at generation P1 (i.e. before subculture), Figure B is a picture of DF-1 cells at generation P6, and Figure C is a picture of DF-1 cells at generation P12.

[0056] Figure 3 The DF-1 cell passage results in Example 4 of the present invention are shown in Figure A, where Figure A is a P1 generation (i.e., before passage) DF-1 cell picture, Figure B is a P6 generation DF-1 cell picture, and Figure C is a P12 generation DF-1 cell picture.

[0057] Depend on Figure 1 and Figure 2 It can be seen that the complete medium of the present invention and the medium containing high concentration of fetal bovine serum have the same culture effect on DF-1 cells, the cells are in normal state, the cells are transparent, the cell density is high, and the confluence is about 90%.

[0058] Therefore, the complete culture medium for culturing DF-1 cells provided by the present invention can well promote the proliferation of DF-1 cells, can achieve the same effect as high-concentration fetal bovine serum in promoting cell proliferation, and is suitable for the adherent culture of DF-1 cells; the use of low-concentration newborn calf serum can greatly avoid the problems of batch differences, unstable culture results, high cytotoxicity and risk of carrying pathogens, narrow application range, expensive fetal bovine serum, high culture cost, etc. brought about by the culture medium with high-concentration serum; the composition is simple, the culture medium preparation process is short, the culture efficiency is high, the repeatability is good, it has the prospect of industrialization and commercialization, and is suitable for mass production.

[0059] 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. A complete medium for culturing DF-1 cells, Features: Includes DMEM basal medium 13.7 g / L, including insulin 7 mg / L, glycine 34 mg / L, alanine 10 mg / L, proline 9 mg / L, serine 50 mg / L, asparagine 15 mg / L, transferrin 6 mg / L, glutathione 2 mg / L, ascorbyl phosphate 2 mg / L, sodium selenite 3 μg / L, ethanolamine 2 mg / L, hydrocortisone 0.03 mg / L, and newborn calf serum 20 mL / L.

2. A complete medium for culturing DF-1 cells according to claim 1, Features: After each component was dissolved by adding water and stirring, the solution was sterilized by filtering with a 0.22 μm filter membrane.

3. Use of the DF-1 cell complete culture medium as claimed in claim 1 or 2 in culturing DF-1 cells.

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