Raw264.7 mouse monocyte macrophage leukemia cell special complete culture medium and application

Abstract

The application belongs to the technical field of biology, and provides a special complete culture medium for Raw264.7 mouse monocyte macrophage leukemia cells and application, so as to solve the problem that the cell morphology of Raw264.7 is obviously changed during the culture process, and the polygonal, long-cleaved and pseudopod cells are easily formed, prevent the cell from being polarized during the culture process, and ensure that the cell has the induced polarization potential. The special complete culture medium contains 83-89% DMEM, 10-15% fetal bovine serum and 1-2% double antibodies according to the volume percentage, and further contains various supplementary additives. The culture medium of the application can make the cell morphology stable and present the best monocyte morphology of the round and transparent shape.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture, and in particular relates to a complete culture medium specifically for Raw264.7 mouse mononuclear macrophage leukemia cells and its application, so as to solve the problem that Raw264.7 cells have obvious morphological variations during culture and are prone to forming polygonal, long spindle-shaped, and pseudopod-type cells, thereby preventing the cells from deforming and polarizing during culture and ensuring that the cells have the potential to induce polarization. Background Art

[0002] Raw264.7 (mouse mononuclear phagocyte leukemia) cells are a cell line derived from the Salk Institute in California by inducing tumors in BALB / c mice using Abelson murine leukemia virus. As a mouse macrophage cell line, Raw264.7 cells have become a popular model cell in immunology and cell biology due to their diverse applications and research. However, the variable morphology and differentiation state of Raw264.7 cells make them difficult to control in practice, presenting challenges for scientific research.

[0003] While there are various opinions on the ideal morphology of these cells, the mainstream view and numerous studies indicate that "the optimal morphology of Raw264.7 cells is a round, translucent mononuclear cell. Polymorphism or the growth of pseudopodia are signs of aging and differentiation. Experiments using cells at this stage can lead to a series of problems, such as insensitivity to drug stimulation and excessive adhesion to the cell wall, making digestion and passage difficult."

[0004] The American Type Culture Collection (ATCC) provides a standard culture protocol for these cells: DMEM + 10% fetal bovine serum + 1% double-stranded antibody. However, in actual scientific research, many researchers have found these cells difficult to culture, manifested by significant morphological variation (e.g., prone to polygonal, spindle-shaped, and pseudopodia-like cells). Researchers have differing opinions on evaluating the cell state. Many researchers believe that the optimal morphology of Raw264.7 cells is round, translucent mononuclear cells. Polymorphism or the growth of pseudopodia indicate aging and differentiation. Experiments using cells at this stage can lead to a range of problems, including insensitivity to drug stimulation and excessive adherence to the surface, making digestion and passaging difficult. For example, according to the description of the American type culture collection (ATCC), the morphology of Raw264.7 cells is diverse, including round, quasi-round, long spindle-shaped and polygonal with pseudopodia, and can be mononuclear or multinuclear (the cell morphology shown in the photo of cells on the ATCC official website is polymorphic with antennae); researcher Fang Yao (2012) believes that polygonal and spindle-shaped cell morphology may indicate poor cell growth, causing changes in various physiological states of the cells, which will greatly affect subsequent experiments; researcher Xu Dan (2016) believes that the optimal morphology of Raw264.7 cells should be smaller mononuclear round cells, and that if the cells become long spindle-shaped or polygonal with pseudopodia, it will have an adverse effect on subsequent experimental results (osteoclast induction); Li Yongjun et al. (2006) showed that newly isolated primary mouse peritoneal macrophages are mostly round after attachment, and only a few are deformed into spindle or star-shaped. After the stimulated macrophages adhered to the wall, almost all deformed cells became significantly larger and extended pseudopodia, indicating that their ability to extend outward was significantly enhanced, which is a morphological sign of macrophage activation.

[0005] In addition to the standard protocol provided by ATCC, other media used for Raw264.7 culture reported in the literature include MEM + 10% fetal bovine serum + 1% double-antibody and MEMα + 10% fetal bovine serum + 1% double-antibody. However, the commonality is "basal medium + fetal bovine serum + double-antibody."

[0006] When activated by foreign molecules, immune cells undergo changes in gene expression and metabolism. While these changes are short-lived in response to acute infection, they can persist long-term in chronic inflammation. This suggests that the morphological changes in Raw264.7 cells are related to their sensitivity as immune cells and to certain molecules in traditional complete culture media. If the effects of these stimulatory molecules on Raw264.7 cells can be reduced, there is hope that the cells will exhibit the well-defined "round, translucent, classic monocyte morphology." However, no authoritative and professional reports, research, related products or related information on this issue have been found so far. There are many different opinions on the cause of this problem, and the solutions are varied but difficult to stably and effectively eliminate the problem: for example, each time the cells are passaged, the deformed cells adhere firmly to the wall and are discarded, while the round cells that do not adhere to the wall continue to be used. However, when the round cells are inoculated to prepare for the experiment, a certain proportion of deformed cells will still appear, that is, "deformed cells are always lost and there are always more"; for example, when cells are passaged, cells are scraped or gently blown with a pipette, and trypsin is not used for digestion to reduce irritation. This solution can alleviate the trend of further deterioration of cell morphology to a certain extent, but it cannot solve the problem of how to improve the initial morphology of cells; for example, the morphology of Raw264.7 cells is greatly affected by the quality of serum. Before culturing 264.7, researchers usually need to test different brands and batches of serum, and select high-quality serum to alleviate the cell deformation phenomenon. However, even so, it cannot guarantee that the problem will be completely solved. Affected by the differences between serum batches and brands, researchers usually need to conduct frequent screening tests, which undoubtedly adds a lot of unnecessary workload to scientific research, and the results obtained by different researchers are also not uniform. Summary of the Invention

[0007] The present invention provides a complete culture medium specifically for Raw264.7 (mouse mononuclear macrophage leukemia cells), aiming to solve the problem of significant cell morphological variation (easily forming polygonal, long spindle-shaped, and pseudopod-type cells) during the culture process of Raw264.7; it overcomes the technical defects of existing traditional Raw264.7 complete culture medium, such as polymorphic, non-uniform, and non-standard cell morphology, high requirements for serum quality, cumbersome operation methods, and high cost.

[0008] The complete culture medium for Raw264.7 mouse mononuclear macrophage leukemia cells provided by the present invention is used to prevent cell deformation and polarization during culture and ensure that the cells have the potential to induce polarization. The complete culture medium contains 83-89% DMEM, 10-15% fetal bovine serum, and 1-2% double-antibody according to volume percentage;

[0009] The dedicated complete culture medium further contains supplementary additives, the components and contents of which are as follows:

[0010] Recombinant human insulin 5-25 mg / L, recombinant human transferrin 5-50 mg / L, recombinant human serum albumin 50-500 mg / L, L-ascorbic acid 2-10 mg / L, ethanolamine 1-5 mg / L, reduced glutathione 1-5 mg / L, sodium selenite pentahydrate 0.003-0.01 mg / L, ammonium metavanadate 0.0002-0.0004 mg / L, and manganese chloride 0.00003-0.00009 mg / L.

[0011] Preferably, the concentration of the recombinant human transferrin is 1 to 2 times the concentration of the recombinant human insulin.

[0012] Preferably, the concentration of the recombinant human serum albumin is 10 times the concentration of the recombinant human transferrin.

[0013] Preferably, the concentration of the ethanolamine is 1 to 3 mg / L.

[0014] Preferably, the concentration of the ammonium metavanadate is 0.0002 to 0.0003 mg / L.

[0015] The present invention also provides a cell culture method used in conjunction with the above-mentioned complete culture medium, in which the cell passage step does not require the use of cell scrapers or digestion, and can be completed by pipetting only.

[0016] The effects of each component in the present invention are as follows:

[0017] (1) Recombinant human serum albumin has multiple protective functions. It can bind to toxic and harmful substances to protect cells. It has anti-inflammatory effects that can reduce the inflammatory response of cells. It can transport potential toxins to the treatment site to render them harmless. It plays a major role in the antioxidant capacity of human serum. The morphological changes and polarization of Raw264.7 cells are caused by external stimuli. Albumin can protect cells from stimulation by various substances and maintain a good resting state. In addition, unlike animal-derived albumin, recombinant sources can prevent exogenous substances from stimulating Raw264.7 cells and causing polarization.

[0018] (2) Recombinant human insulin has multiple effects. It can significantly increase glucose uptake by activating the PI3K / Akt pathway. This process provides sufficient energy for Raw264.7 cells (doubling time is about 15-18 hours), which not only ensures DNA synthesis and organelle assembly, but also regulates actin remodeling through the mTORC1 pathway, ultimately maintaining a good cell state and morphology. More and more studies have shown that insulin has a series of non-glucogenic effects. The expression of insulin and Nrf2 has a mutually promoting positive correlation. As a key molecule for cells to resist oxidative stress, Nrf2 plays an important role in maintaining cellular redox balance. It regulates oxidative / exogenous substance stress response, blocks the transcription of pro-inflammatory cytokines, and inhibits macrophage inflammatory response. Therefore, in a resting state, insulin helps regulate the inflammatory response of Raw264.7 cells and keeps them in a healthy balance.

[0019] (3) Recombinant human transferrin can reversibly bind to trivalent iron ions. It plays a vital role in the transport and metabolism of iron ions and is also an important extracellular antioxidant. Transferrin maintains iron homeostasis in mononuclear macrophages and helps the cells maintain a stable, inactive state.

[0020] (4) L-ascorbic acid, reduced glutathione, and sodium selenite pentahydrate are important antioxidants used to maintain low levels of reactive oxygen species in cells. When excessive reactive oxygen species (ROS) act on polyunsaturated fatty acids (PUFAs) on biological membranes, they trigger lipid peroxidation, thereby changing the permeability and fluidity of the intracellular biological membranes, leading to changes in cell structure and functional disorders. Selenium plays a physiological role in animal cells in the form of a family of selenoproteins. Currently, 11 forms have been confirmed. According to research, these proteins are all involved in antioxidant activities, such as glutathione peroxidase and thioredoxin.

[0021] (5) Ethanolamine is an important compound that stimulates cell growth and is a precursor for the biosynthesis of phospholipids. Phospholipids play an important role in the structure and function of cell membranes and organelles and are the structural basis for the stability of cell morphology.

[0022] (6) Vanadate exhibits unique cell signaling intervention capabilities through broad-spectrum receptor-ligand homeostasis regulation across cell types. In lymphocytes, it inhibits the lysosomal degradation of the insulin receptor (IR) and growth hormone receptor (GHR) in a dose-dependent manner, prolonging the retention time of the ligand-receptor complex on the cell membrane. In fibroblast models, vanadate specifically blocks the transport of LDL receptor (LDLR) to lysosomes. In adipocytes, vanadate improves the recycling efficiency of transferrin receptor (TfR) by inhibiting its ubiquitination, while maintaining the structural integrity of the ligand (Tf)-receptor complex. This regulation by vanadium leads to the sustained activation of insulin / IGF-1 signaling, iron metabolism homeostasis, and LDLR-mediated cholesterol uptake balance, ultimately forming a multi-pathway coordinated cell homeostasis network. The homeostasis of cellular pathways contributes to the stability of cell morphology, resulting in intact morphology of Raw264.7 cells.

[0023] The beneficial effects achieved by the present invention are:

[0024] The supplements used in this invention mitigate the effects of external stimuli on Raw264.7 cells, effectively maintaining their optimal health and morphology, and maintaining this stability across multiple passages. This study demonstrates for the first time that using a specific ratio of supplements in Raw264.7 macrophages can target and reshape the cytoskeleton, resulting in polarized and semi-polarized phenotypes distinct from those found in traditional culture conditions. This approach overcomes the stringent fetal bovine serum (FBS) requirement of traditional culture, eliminating the need for repeated testing of serum products. This approach provides a morphologically and functionally stable cell tool for macrophage-related research, such as tumor microenvironment modeling and nanoparticle phagocytosis assessment, addressing the technical challenge of high morphological heterogeneity (CV > 25%) in conventional Raw264.7 cell passages. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The cell culture and morphological identification (photographing) after passage of the embodiment of the present invention; Figure 1 A corresponds to Example 1, Figure 1 B corresponds to Example 2, Figure 1 C corresponds to Example 3, Figure 1 D corresponds to Example 4, Figure 1 E corresponds to Example 5.

[0026] Figure 2 The morphological identification (photographing) of cells after culture and passage in each comparison ratio is shown; Figure 2 A corresponds to the proportion 1, Figure 2 B corresponds to the proportion 2, Figure 2 C corresponds to the proportion 3, Figure 2 D corresponds to the ratio 4, Figure 2 E corresponds to a ratio of 5.

[0027] Figure 3 1 is a graph showing the test results of cell proliferation detection experiments in the embodiments of the present invention and various comparative examples.

[0028] Figure 4 1 is a graph showing the test results of the M1 polarization induction and its signature indicator IL-6 in the embodiments of the present invention and comparative examples.

[0029] Figure 5 1 is a graph showing the test results of the M2 polarization induction and its signature indicator IL-10 in the embodiments of the present invention and comparative examples. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] Example 1

[0032] The specific steps are as follows:

[0033] (1) Prepare a complete culture medium: Prepare a complete culture medium according to the following dosages of the following substances.

[0034] DMEM (Pnosai PM150210): 89%

[0035] Fetal bovine serum (Gibco A5256701): 10%

[0036] Double antibody (Punosai PB180120): 1%

[0037] Recombinant human insulin (Pnosai PB180432): 5 mg / L

[0038] Recombinant human transferrin (Solebol IR4170): 5 mg / L

[0039] Recombinant human serum albumin (Heyuan Biotechnology HYC002M01): 50 mg / L

[0040] L-ascorbic acid (McLean A800295): 2 mg / L

[0041] Ethanolamine (McLean E808764): 1 mg / L

[0042] Reduced glutathione (McLean R917465): 1 mg / L

[0043] Sodium selenite pentahydrate (Aladdin S337199): 0.003 mg / L

[0044] NH4VO3 ammonium metavanadate (McLean A800972): 0.0002 mg / L

[0045] MnCl2*4H2O manganese chloride (McLean M813685): 0.00003 mg / L

[0046] (2) Cell culture and morphological identification after passage (photographing):

[0047] After thawing the Raw264.7 cells, add dedicated complete culture medium for cell culture.

[0048] When the cell density reaches 80%~100%, the cells are passaged.

[0049] When passaging cells, discard the old supernatant medium and add fresh complete medium. Gently pipette the Raw264.7 cells at the bottom of the culture dish until a cell suspension forms. Collect the cell suspension and centrifuge at 1200 rpm for 3 minutes. Discard the supernatant. Add fresh complete medium to the cell pellet, resuspend the cell pellet, and passage the cells at a 1 / 5 ratio.

[0050] Continue to subculture the cells until the cell density reaches 80%-100% and then perform subculturing again.

[0051] The cells were cultured and passaged continuously for 6 times.

[0052] After the cell culture passaging is completed, the cell morphology is observed and recorded under a microscope and photos are taken.

[0053] (3) Cell proliferation assay:

[0054] After the cell culture and subculture are completed, cells are collected to prepare a cell suspension for later use (refer to (2) Cell culture and morphological identification after subculture (photographing)).

[0055] The cell suspension was counted and inoculated into 96-well cell culture plates at a density of 5000 cells / well, for a total of 7 plates.

[0056] One plate was taken daily for CCK8 assay. For the assay, CCK8 solution (purchased from Wuhan Elerite E-CK-A362) was added to each well at a 10% volume ratio, incubated at 37°C for 2 h, and the absorbance OD450 was measured.

[0057] After 7 consecutive days of testing, the cell growth curve was drawn.

[0058] (4) Examination of M1 polarization induction and its hallmark indicator IL-6:

[0059] After the cell culture and passaging is completed, the cells are collected to prepare a cell suspension (refer to (2) Cell culture and passaging) for later use.

[0060] The cell suspension was counted and seeded into 6-well cell culture plates at 1*10^5 cells / well and cultured overnight.

[0061] 2.5 ng / mL IFN-γ (purchased from Pronocell, PCK064) and 200 ng / mL LPS (purchased from Aladdin, L386714) were added together for 12 hours to induce polarization towards the M1 type.

[0062] Subsequently, the IL-6 expression level was detected according to the operating procedures of the IL-6 detection kit (purchased from Elabscience, E-EL-M0044).

[0063] (5) Examination of M2 polarization induction and its hallmark indicator IL-10:

[0064] After the cell culture and passaging is completed, the cells are collected to prepare a cell suspension (refer to (2) Cell culture and passaging) for later use.

[0065] The cell suspension was counted and seeded into 6-well cell culture plates at 1*10^5 cells / well and cultured overnight.

[0066] The cells were then polarized to the M2 type by adding 10 ng / mL IL-4 (purchased from Pronocell, PCK033) for 12 hours.

[0067] Subsequently, the IL-10 expression level was detected according to the operating procedures of the IL-10 detection kit (purchased from Elabscience, E-EL-M0046).

[0068] Example 2

[0069] The specific steps are as follows:

[0070] (1) Prepare a complete culture medium: Prepare a complete culture medium according to the following dosages of the following substances.

[0071] DMEM (Pnosai PM150210): 89%

[0072] Fetal bovine serum (Gibco A5256701): 10%

[0073] Double antibody (Punosai PB180120): 1.5%

[0074] Recombinant human insulin (Pnosai PB180432): 15 mg / L

[0075] Recombinant human transferrin (Solabio IR4170): 27.55 mg / L

[0076] Recombinant human serum albumin (Heyuan Biology HYC002M01): 275 mg / L

[0077] L-ascorbic acid (Macklin A800295): 6 mg / L

[0078] Ethanolamine (Macklin E808764): 3 mg / L

[0079] Reduced glutathione (Macklin R917465): 3 mg / L

[0080] Sodium selenite pentahydrate (Arladdin S337199): 0.0065 mg / L

[0081] NH4VO3ammonium metavanadate (Macklin A800972): 0.0003 mg / L

[0082] MnCl2*4H2O manganese chloride (Macklin M813685): 0.00006 mg / L

[0083] (2) Cell morphology identification (photograph) after cell culture and passage, same as Example 1.

[0084] (3) Cell proliferation detection: same as Example 1.

[0085] (4) M1 polarization induction and its marker IL-6 detection: same as Example 1.

[0086] (5) M2 polarization induction and its marker IL-10 detection: same as Example 1.

[0087] Example 3

[0088] The specific steps are as follows:

[0089] (1) Preparation of special complete medium: prepare complete medium according to the following amount of each substance.

[0090] DMEM (Pronova PM150210): 83%

[0091] Fetal bovine serum (Gibco A5256701): 15%

[0092] Double antibody (Pronova PB180120): 2%

[0093] Recombinant human insulin (Pronova PB180432): 25 mg / L

[0094] Recombinant human transferrin (Solabio IR4170): 50 mg / L

[0095] Recombinant human serum albumin (HYC002M01 from Heyuan Biotechnology): 500 mg / L

[0096] L-ascorbic acid (McLean A800295): 10 mg / L

[0097] Ethanolamine (McLean E808764): 5 mg / L

[0098] Reduced glutathione (McLean R917465): 5 mg / L

[0099] Sodium selenite pentahydrate (Aladdin S337199): 0.01 mg / L

[0100] NH4VO3 ammonium metavanadate (McLean A800972): 0.0004 mg / L

[0101] MnCl2*4H2O Manganese chloride (McLean M813685): 0.00009 mg / L

[0102] (2) Cell culture and morphological identification (photographing) after passage were the same as in Example 1.

[0103] (3) Cell proliferation detection: Same as Example 1.

[0104] (4) Examination of M1 polarization induction and its hallmark indicator IL-6: Same as Example 1.

[0105] (5) Examination of M2 polarization induction and its hallmark indicator IL-10: Same as Example 1.

[0106] Example 4

[0107] The specific steps are as follows:

[0108] (1) Prepare a complete culture medium: Prepare a complete culture medium according to the following dosages of the following substances.

[0109] DMEM (Pnosai PM150210): 89%

[0110] Fetal bovine serum (FSP500): 10%

[0111] Double antibody (Punosai PB180120): 1%

[0112] Recombinant human insulin (Pnosai PB180432): 15 mg / L

[0113] Recombinant human transferrin (Solebol IR4170): 27.55 mg / L

[0114] Recombinant human serum albumin (Heyuan Biotechnology HYC002M01): 275 mg / L

[0115] L-ascorbic acid (McLean A800295): 6 mg / L

[0116] Ethanolamine (McLean E808764): 3 mg / L

[0117] Reduced glutathione (McLean R917465): 3 mg / L

[0118] Sodium selenite pentahydrate (Aladdin S337199): 0.0065 mg / L

[0119] NH4VO3 ammonium metavanadate (McLean A800972): 0.0003 mg / L

[0120] MnCl2*4H2O Manganese chloride (McLean M813685): 0.00006 mg / L

[0121] (2) Cell culture and morphological identification (photographing) after passage were the same as in Example 1.

[0122] (3) Cell proliferation detection: Same as Example 1.

[0123] (4) Examination of M1 polarization induction and its hallmark indicator IL-6: Same as Example 1.

[0124] (5) Examination of M2 polarization induction and its hallmark indicator IL-10: Same as Example 1.

[0125] Example 5

[0126] The specific steps are as follows:

[0127] (1) Prepare a complete culture medium: Prepare a complete culture medium according to the following dosages of the following substances.

[0128] DMEM (Pnosai PM150210): 89%

[0129] Fetal bovine serum (Global Health F800820): 10%

[0130] Double antibody (Punosai PB180120): 1%

[0131] Recombinant human insulin (Pnosai PB180432): 15 mg / L

[0132] Recombinant human transferrin (Solebol IR4170): 27.55 mg / L

[0133] Recombinant human serum albumin (Heyuan Biotechnology HYC002M01): 275 mg / L

[0134] L-ascorbic acid (McLean A800295): 6 mg / L

[0135] Ethanolamine (McLean E808764): 3 mg / L

[0136] Reduced glutathione (McLean R917465): 3 mg / L

[0137] Sodium selenite pentahydrate (Aladdin S337199): 0.0065 mg / L

[0138] NH4VO3 ammonium metavanadate (McLean A800972): 0.0003 mg / L

[0139] MnCl2*4H2O Manganese chloride (McLean M813685): 0.00006 mg / L

[0140] (2) Cell culture and morphological identification (photographing) after passage were the same as in Example 1.

[0141] (3) Photographing cell morphology: Same as in Example 1.

[0142] (4) Cell proliferation detection: Same as Example 1.

[0143] (5) Examination of M1 polarization induction and its hallmark indicator IL-6: Same as Example 1.

[0144] (6) Examination of M2 polarization induction and its hallmark indicator IL-10: Same as Example 1.

[0145] Comparative Example 1

[0146] The specific steps are as follows:

[0147] (1) Prepare a complete culture medium: Prepare a complete culture medium according to the following dosages of the following substances.

[0148] DMEM (Pnosai PM150210): 89%

[0149] Fetal bovine serum (Gibco A5256701): 10%

[0150] Double antibody (Punosai PB180120): 1%

[0151] Recombinant human transferrin (Solebol IR4170): 27.55 mg / L

[0152] Recombinant human serum albumin (Heyuan Biotechnology HYC002M01): 275 mg / L

[0153] L-ascorbic acid (McLean A800295): 6 mg / L

[0154] Ethanolamine (McLean E808764): 3 mg / L

[0155] Reduced glutathione (McLean R917465): 3 mg / L

[0156] Sodium selenite pentahydrate (Aladdin S337199): 0.0065 mg / L

[0157] NH4VO3 ammonium metavanadate (McLean A800972): 0.0003 mg / L

[0158] MnCl2*4H2O Manganese chloride (McLean M813685): 0.00006 mg / L

[0159] (2) Cell culture and morphological identification (photographing) after passage were the same as in Example 1.

[0160] (3) Cell proliferation detection: Same as Example 1.

[0161] (4) Examination of M1 polarization induction and its hallmark indicator IL-6: Same as Example 1.

[0162] (5) Examination of M2 polarization induction and its hallmark indicator IL-10: Same as Example 1.

[0163] Comparative Example 2

[0164] The specific steps are as follows:

[0165] (1) Prepare a complete culture medium: Prepare a complete culture medium according to the following dosages of the following substances.

[0166] DMEM (Pnosai PM150210): 89%

[0167] Fetal bovine serum (Gibco A5256701): 10%

[0168] Double antibody (Punosai PB180120): 1%

[0169] Recombinant human insulin (Pnosai PB180432): 15 mg / L

[0170] Recombinant human serum albumin (Heyuan Biotechnology HYC002M01): 275 mg / L

[0171] L-ascorbic acid (McLean A800295): 6 mg / L

[0172] Ethanolamine (McLean E808764): 3 mg / L

[0173] Reduced glutathione (McLean R917465): 3 mg / L

[0174] Sodium selenite pentahydrate (Aladdin S337199): 0.0065 mg / L

[0175] NH4VO3 ammonium metavanadate (McLean A800972): 0.0003 mg / L

[0176] MnCl2*4H2O Manganese chloride (McLean M813685): 0.00006 mg / L

[0177] (2) Cell culture and morphological identification (photographing) after passage were the same as in Example 1.

[0178] (3) Cell proliferation detection: Same as Example 1.

[0179] (4) Examination of M1 polarization induction and its hallmark indicator IL-6: Same as Example 1.

[0180] (5) Examination of M2 polarization induction and its hallmark indicator IL-10: Same as Example 1.

[0181] Comparative Example 3

[0182] The specific steps are as follows:

[0183] (1) Prepare a complete culture medium: Prepare a complete culture medium according to the following dosages of the following substances.

[0184] DMEM (Pnosai PM150210): 89%

[0185] Fetal bovine serum (Gibco A5256701): 10%

[0186] Double antibody (Punosai PB180120): 1%

[0187] Recombinant human insulin (Pnosai PB180432): 15 mg / L

[0188] Recombinant human transferrin (Solebol IR4170): 27.55 mg / L

[0189] L-ascorbic acid (McLean A800295): 6 mg / L

[0190] Ethanolamine (McLean E808764): 3 mg / L

[0191] Reduced glutathione (McLean R917465): 3 mg / L

[0192] Sodium selenite pentahydrate (Aladdin S337199): 0.0065 mg / L

[0193] NH4VO3 ammonium metavanadate (McLean A800972): 0.0003 mg / L

[0194] MnCl2*4H2O Manganese chloride (McLean M813685): 0.00006 mg / L

[0195] (2) Cell culture and morphological identification (photographing) after passage were the same as in Example 1.

[0196] (3) Cell proliferation detection: Same as Example 1.

[0197] (4) Examination of M1 polarization induction and its hallmark indicator IL-6: Same as Example 1.

[0198] (5) Examination of M2 polarization induction and its hallmark indicator IL-10: Same as Example 1.

[0199] Comparative Example 4

[0200] The specific steps are as follows:

[0201] (1) Prepare a complete culture medium: Prepare a complete culture medium according to the following dosages of the following substances.

[0202] DMEM (Pnosai PM150210): 89%

[0203] Fetal bovine serum (Gibco A5256701): 10%

[0204] Double antibody (Punosai PB180120): 1%

[0205] Recombinant human insulin (Pnosai PB180432): 15 mg / L

[0206] Recombinant human transferrin (Solebol IR4170): 27.55 mg / L

[0207] Recombinant human serum albumin (Heyuan Biotechnology HYC002M01): 275 mg / L

[0208] (2) Cell culture and morphological identification (photographing) after passage were the same as in Example 1.

[0209] (3) Cell proliferation detection: Same as Example 1.

[0210] (4) Examination of M1 polarization induction and its hallmark indicator IL-6: Same as Example 1.

[0211] (5) Examination of M2 polarization induction and its hallmark indicator IL-10: Same as Example 1.

[0212] Comparative Example 5

[0213] The specific steps are as follows:

[0214] (1) Prepare a complete culture medium: Prepare a complete culture medium according to the following dosages of the following substances.

[0215] DMEM (Pnosai PM150210): 89%

[0216] Fetal bovine serum (Gibco A5256701): 10%

[0217] Double antibody (Punosai PB180120): 1%

[0218] (2) Cell culture and morphological identification (photographing) after passage were the same as in Example 1.

[0219] (3) Cell proliferation detection: Same as Example 1.

[0220] (4) Examination of M1 polarization induction and its hallmark indicator IL-6: Same as Example 1.

[0221] (5) Examination of M2 polarization induction and its hallmark indicator IL-10: Same as Example 1.

[0222] The culture medium components of each embodiment and comparative example are shown in Table 1 and Table 2, respectively.

[0223] Result Analysis

[0224] (1) Cell culture and morphological identification after subculture (photographing)

[0225] Through various embodiments (Appendix Figure 1 )、Comparative Example(Attached Figure 2 ) experimental results show that after six passages (P6), Raw264.7 cells in the Raw264.7 supplementation groups (Examples 1, 2, 3, 4, and 5) exhibited a typical plump, translucent, uniform, and regularly rounded cell morphology. Compared to the control group (Comparative Examples 1, 2, 3, 4, and 5), the proportion of pseudopods decreased from 30% (±10%) to 5% (±5%). After six passages (P6), no significant changes in morphological parameters were observed (p>0.05, n=3 independent experiments).

[0226] (2) Cell proliferation assay.

[0227] The experimental results of various embodiments and comparative examples (see Appendix Figure 3) It can be seen that in the cell proliferation test, the 7-day growth curve of the cells showed that the proliferation rate of the Raw264.7 special supplement additive intervention group (Examples 1, 2, 3, 4, and 5) was faster, and the proliferation rate of the control group (Comparative Examples 1, 2, 3, 4, and 5) was significantly slower.

[0228] (3) Examination of M1 polarization induction and its signature indicator IL-6.

[0229] The experimental results of various embodiments and comparative examples (see Appendix Figure 4 ) As can be seen from the results of the IL-6 expression test after cell polarization induction, the Raw264.7 special supplementation intervention groups (Examples 1, 2, 3, 4, and 5) showed higher expression levels, indicating that the cells responded well to the induced stimulation and rapidly underwent M1 polarization. In contrast, the control groups (Comparative Examples 1, 2, 3, 4, and 5) showed lower expression levels, indicating that the cells responded poorly to the induced stimulation.

[0230] (4) Examination of M2 polarization induction and its signature indicator IL-10.

[0231] The experimental results of various embodiments and comparative examples (see Appendix Figure 5 ) As can be seen from the results of the IL-10 expression test after cell polarization induction, the Raw264.7 special supplementation intervention groups (Examples 1, 2, 3, 4, and 5) showed higher expression levels, indicating that the cells responded well to the induced stimulation and rapidly underwent M2 polarization. In contrast, the control groups (Comparative Examples 1, 2, 3, 4, and 5) showed lower expression levels, indicating that the cells responded poorly to the induced stimulation.

[0232] (5) In summary

[0233] The Raw264.7 special supplementary additive intervention group, i.e., the present invention, can solve the problem that Raw264.7 has obvious cell morphological variation during culture, and is prone to forming polygonal, long spindle-shaped, and pseudopod-type cells.

[0234] It enables cells to exhibit higher proliferation activity while ensuring that cells remain highly sensitive to polarization induction.

[0235] The present invention makes the culture of RAW264.7 no longer restricted by serum, is compatible with serum of different brands and achieves good results (Examples 1, 4, and 5).

[0236] Table 1 Example culture medium preparation table

[0237]

[0238] Table 2 Comparative culture medium preparation

[0239]

Claims

1. A complete culture medium for Raw264.7 mouse mononuclear macrophage leukemia cells, used to prevent cell deformation and polarization during culture and ensure that the cells have the potential to induce polarization, characterized in that: The dedicated complete culture medium contains 83-89% DMEM, 10-15% fetal bovine serum, and 1-2% double antibody by volume; The dedicated complete culture medium further contains supplementary additives, the components and contents of which are as follows: Recombinant human insulin 5-25 mg / L, recombinant human transferrin 5-50 mg / L, recombinant human serum albumin 50-500 mg / L, L-ascorbic acid 2-10 mg / L, ethanolamine 1-5 mg / L, reduced glutathione 1-5 mg / L, sodium selenite pentahydrate 0.003-0.01 mg / L, ammonium metavanadate 0.0002-0.0004 mg / L, and manganese chloride 0.00003-0.00009 mg / L.

2. The complete culture medium for Raw264.7 mouse mononuclear macrophage leukemia cells according to claim 1, characterized in that: The concentration of the recombinant human transferrin is 1 to 2 times the concentration of the recombinant human insulin.

3. The complete culture medium for Raw264.7 mouse mononuclear macrophage leukemia cells according to claim 1, characterized in that: The concentration of the recombinant human serum albumin is 10 times that of the recombinant human transferrin.

4. The complete culture medium for Raw264.7 mouse mononuclear macrophage leukemia cells according to claim 1, characterized in that: The concentration of the ethanolamine is 1 to 3 mg / L.

5. The complete culture medium for Raw264.7 mouse mononuclear macrophage leukemia cells according to claim 1, characterized in that: The concentration of the ammonium metavanadate is 0.0002 ~ 0.0003 mg / L.

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