Cell culture medium capable of efficiently delivering manganese ions

Abstract

The invention discloses a cell culture medium capable of efficiently delivering manganese ions. The cell culture medium contains amino acid, vitamins, inorganic salt, glucose, phenol red and 4-(2-ethoxyl)-1-piperazine ethanesulfonic acid and does not contain sodium bicarbonate, disodium hydrogen phosphate and calcium chloride, and the content of sodium chloride is increased in the inorganic salt; when Mn < 2 + > is delivered under the cell culture medium, more than 98% of manganese elements can stably exist in the form of free-state free manganese ions, and the free-state free manganese ions have a higher cell transmembrane transport rate, so that the manganese ions can quickly enter cells and directly act on signal molecules in the cells; signal transduction pathways responding to manganese ions in the cells are more quickly activated, so that the research process of manganese related signal pathways in the cells is greatly promoted; therefore, the improved cell culture medium provided by the invention has a good application prospect.

Description

Technical Field

[0001] The invention relates to a cell culture medium for efficiently delivering manganese ions, belonging to the technical field of cell culture. Background Art

[0002] In recent years, the research on manganese in the field of immunology has increased, and how to effectively study the effect of manganese ions on signaling pathways in cells has become a major problem limiting this field. In common cell culture and buffer systems, 80%-90% of the free manganese ions added will be rapidly converted into chemical manganese elements and exist in the form of solid manganese-containing compounds, which greatly affects the efficiency of manganese entering cells. Therefore, constructing a cell culture system that can efficiently deliver manganese ions has become an urgent problem to be solved. Summary of the invention

[0003] The purpose of the present invention is to provide a cell culture medium for efficiently delivering manganese ions in view of the deficiencies of the prior art.

[0004] In order to achieve the above object, the present invention provides a cell culture medium for efficiently delivering manganese ions, wherein the cell culture medium contains amino acids, vitamins, inorganic salts, glucose, phenol red and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, but does not contain sodium bicarbonate, disodium hydrogen phosphate and calcium chloride, and the content of sodium chloride is increased in the inorganic salts.

[0005] Preferably, the amino acids include glycine, L-arginine hydrochloride, L-cystine dihydrochloride, L-glutamine, L-histidine hydrochloride hydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt dihydrate and L-valine.

[0006] Preferably, the microorganism comprises choline chloride, D-calcium pantothenate, folic acid, nicotinamide, pyridoxine hydrochloride (vitamin B6), riboflavin (vitamin B2), thiamine hydrochloride (vitamin B1) and inositol (vitamin B8).

[0007] Preferably, the inorganic salt comprises Fe(NO3)3·9H2O, anhydrous MgSO4, KCl and NaCl; and the increased sodium chloride content in the inorganic salt is 45 mM.

[0008] Preferably, the glucose is D-anhydrous glucose.

[0009] Preferably, the formula of the cell culture medium is as shown in Table 1 of the specification.

[0010] The present invention also provides the use of the cell culture medium as described above in delivering manganese ions, wherein the use is selected from the following group:

[0011] a) Application in delivering manganese ions;

[0012] b) Application in studying the effect of manganese ions on cell signaling pathways.

[0013] Preferably, the application comprises: culturing cells in the cell culture medium as described above, adding divalent manganese ions, and observing the effect of manganese ions on signal pathways in the cells.

[0014] Preferably, the divalent manganese ions are derived from at least one of manganese chloride, manganese sulfate and manganese nitrate.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The cell culture medium provided by the present invention for efficiently delivering manganese ions does not contain 2+ The combined components of sodium bicarbonate and disodium hydrogen phosphate are adjusted to maintain the conditions required for cell culture and ensure the normal and effective culture of cells. 2+ When delivered, more than 98% of the manganese elements can stably exist in the form of free manganese ions, and free manganese ions have a higher cell transmembrane transport rate, so that manganese ions can quickly enter cells and directly act on signal molecules in cells, and more quickly activate signal transduction pathways in cells that respond to manganese ions, thereby greatly promoting the research progress of intracellular manganese-related signal pathways; therefore, the improved cell culture medium provided by the present invention has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Mn in M.DMEM cell culture system 2+ A: Using 0.5 mM concentration of manganese chloride (Mn 2+ ) or manganese hydrogen phosphate (MnHPO4) for 0 to 3 hours, and then the manganese content in the cells was detected by inductively coupled plasma mass spectrometry (ICP-MS); B: 0.5 mM manganese chloride (Mn 2+ ) or manganese phosphate (MnHPO4) were used to treat human cervical epithelial HeLa cells for 1 to 6 hours, and then the activation expression of transcription factor IRF3 in the cells was detected by immunoblotting (activated expression formed phosphorylated IRF3, i.e. pIRF3); C: Using 16 to 1024 μM manganese chloride (Mn2+ ) or manganese phosphate (MnHPO4) for 1-24 hours, the manganese-containing medium was removed by centrifugation and fresh RPMI-1640 medium was added again. After culturing for another 12-24 hours, the interferon concentration in the above cell culture supernatant was directly detected using the interferon stimulated response element (ISRE)-luciferase method. DETAILED DESCRIPTION

[0018] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0019] Example

[0020] In order to construct a cell culture system for efficiently delivering manganese ions, this example used a phenol red-free high-sugar DMEM (dulbecco's modified eagle medium) culture medium as a model in the early basic test of culture medium components, and determined the components in 31 components that can bind to free manganese ions and form chemical manganese-containing compounds. The results showed that sodium bicarbonate and disodium hydrogen phosphate in the DMEM culture medium system are the main components that bind to free manganese ions; while other amino acids, vitamins and inorganic salts have almost no effect on free manganese ions.

[0021] On this basis, the cell culture medium for efficient delivery of manganese ions of the present invention is adjusted based on the high-glucose DMEM culture medium (containing phenol red): 1) sodium bicarbonate, disodium hydrogen phosphate and calcium chloride components are removed; 2) 45mM sodium chloride is additionally added to adjust the osmotic pressure; 3) 25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is added to provide acid-base buffering capacity under cell culture conditions. Thus, a modified DMEM cell culture medium (modified dulbecco's modified eagle medium, M.DMEM) is finalized, as shown in Table 1 below:

[0022] Table 1M.DMEM medium formula

[0023]

[0024]

[0025] It has been determined that in this improved cell culture system for efficient delivery of manganese ions, more than 98% of the manganese element can stably exist in the form of free manganese ions. Free manganese ions have a higher cell transmembrane transport rate, allowing manganese ions to quickly enter cells and directly act on signal molecules within cells, activating signal transduction pathways in cells that respond to manganese ions more quickly, thereby greatly promoting the research progress on intracellular manganese-related signaling pathways.

[0026] In M.DMEM culture medium, manganese can exist in two forms. If a soluble manganese chloride solution is added, most of the manganese is in the form of free manganese ions; correspondingly, if a slightly soluble manganese-containing compound with very low solubility, such as manganese hydrogen phosphate, is added, most of the manganese exists in the form of a compound. The latter can be used to simulate the effect of adding manganese chloride to ordinary culture medium, that is, in ordinary culture medium, free manganese ions will form a compound of manganese hydrogen phosphate with disodium hydrogen phosphate in the culture medium. Using a 0.5mM concentration of free manganese ions (manganese chloride, Mn 2+ ) or manganese salt (manganese hydrogen phosphate, MnHPO4) was used to treat human cervical epithelial HeLa cells cultured in the M.DMEM medium of the present invention, and the manganese content in the cells was detected after 0 to 3 hours. The results showed that the use of free manganese ions (Mn 2+ ) was significantly higher than that after treatment with combined manganese salts (MnHPO4). Figure 1 As shown in A; 0.5 mM manganese chloride (Mn 2+ ) or manganese hydrogen phosphate (MnHPO4) treated human cervical epithelial HeLa cells cultured in the M.DMEM medium of the present invention for 1 to 6 hours, and then the activation expression of the transcription factor IRF3 in the cells was detected by immunoblotting (activated expression formed phosphorylated IRF3, i.e., pIRF3). The results showed that consistent with the transmembrane transport rate of manganese elements, free manganese ions (Mn 2+ ) can activate the cGAS-STING signal transduction pathway in cells that responds to manganese ions more quickly than combined manganese salts (MnHPO4). Only one hour of free manganese ion treatment is required for the phosphorylated transcription factor IRF3 to be detected in the cells. Correspondingly, it takes more than 4 hours for the combined manganese salt treatment to show the same level of changes in the cells, such as Figure 1 As shown in B; using 16~1024μM concentration of manganese chloride (Mn 2+) or manganese hydrogen phosphate (MnHPO4) treated human mononuclear THP1 cells cultured in the M.DMEM medium of the present invention for 1-24 hours, centrifuged to remove the manganese-containing medium and re-added with fresh RPMI-1640 medium, and then cultured for another 12-24 hours, and then the interferon concentration in the above cell culture supernatant was directly detected using the interferon stimulated response element (ISRE)-luciferase method. The results showed that compared with the combined manganese salt (MnHPO4) treatment, the free manganese ions (Mn) at a lower concentration and for a shorter time could 2+ ) treatment can induce the production of type I interferon, such as Figure 1 The above results show that in the M.DMEM cell culture system of the present invention, manganese ions (Mn 2+ ) has a higher transmembrane transport rate and can activate the signal transduction pathway in cells that responds to manganese ions more quickly.

[0027] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A cell culture medium for efficiently delivering manganese ions, characterized in that: The cell culture medium contains amino acids, vitamins, inorganic salts, glucose, phenol red and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, but does not contain sodium bicarbonate, disodium hydrogen phosphate and calcium chloride. The content of sodium chloride is increased in the inorganic salts.

2. The cell culture medium according to claim 1, characterized in that The amino acids include glycine, L-arginine hydrochloride, L-cystine dihydrochloride, L-glutamine, L-histidine hydrochloride hydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt dihydrate and L-valine.

3. The cell culture medium according to claim 1, characterized in that The vitamins include choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride (vitamin B6), riboflavin (vitamin B2), thiamine hydrochloride (vitamin B1), and inositol (vitamin B8).

4. The cell culture medium according to claim 1, characterized in that The inorganic salts include Fe(NO3)3·9H2O, anhydrous MgSO4, KCl and NaCl; the increased sodium chloride content in the inorganic salts is 45 mM.

5. The cell culture medium according to claim 1, characterized in that The glucose is D-anhydrous glucose.

6. The cell culture medium according to claim 1, characterized in that The formula of the cell culture medium is shown in Table 1 of the instruction manual.

7. Use of the cell culture medium according to any one of claims 1 to 6, characterized in that: The application is selected from the group consisting of: a) Application in delivering manganese ions; b) Application in studying the effect of manganese ions on cell signaling pathways.

8. The use according to claim 7, characterized in that The application comprises: culturing cells in the cell culture medium according to any one of claims 1 to 6, adding divalent manganese ions, and observing the action mode of manganese ions on the signal pathway in the cells.

9. The use according to claim 8, characterized in that The divalent manganese ions are derived from at least one of manganese chloride, manganese sulfate and manganese nitrate.

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