Composition for culturing kupffer cells and application thereof
By adding M-CSF, HGF, and EGF to Kupffer cell culture medium to create a serum-free medium, the problems of variable interference and potential contamination caused by serum were solved, achieving stable cell culture and functional maintenance, and improving the accuracy and reliability of experiments.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MILECELL BIOLOGICAL SCIENCE & TECHNOLOGY CO LTD
- Filing Date
- 2026-01-07
- Publication Date
- 2026-04-10
AI Technical Summary
Existing Kupffer cell culture media have problems such as large batch-to-batch variability in serum, potential pathogen contamination risk, and inconsistent experimental results. Furthermore, commercially available culture media are not optimized for the characteristics of Kupffer cells, resulting in decreased cell viability and poor experimental reproducibility.
A serum-free culture medium composition containing M-CSF, HGF, and EGF, combined with basal culture medium, bovine pancreatic insulin, human transferrin, sodium selenite, L-alanyl-L-glutamine solution, and bovine serum albumin, was used to replace serum function and ensure cell function maintenance.
This approach enables efficient and stable culture of Kupffer cells, reduces animal-derived risks, improves experimental consistency and cell viability, meets regulatory requirements for cell therapy products, and provides a reliable research platform.
Smart Images
Figure CN121825879A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of animal cell culture medium technology, and more particularly to a composition for culturing Kupffer cells and its application. Background Technology
[0002] Kupffer cells are a type of specialized immune cell in the liver, primarily distributed among sinusoidal endothelial cells, and are an important component of the mononuclear phagocytic system. These cells play a crucial role in clearing harmful substances from the blood, regulating immune responses, and maintaining liver microenvironment homeostasis. Furthermore, Kupffer cells are closely associated with the occurrence and development of various liver diseases, and may serve as an effective therapeutic target for these diseases. Constructing 3D cell models and organoids related to liver diseases using Kupffer cells can provide a more efficient in vitro research model for understanding liver disease mechanisms and screening drugs.
[0003] Kupffer cells comprise 20% of the non-parenchymal cells in the liver and play a crucial role in immune defense, inflammation regulation, and metabolic homeostasis. Serum is always added during Kupffer cell in vitro culture, and serum concentrations may even be increased to maintain growth when cell condition is poor. However, batch-to-batch variability in serum can introduce variables and affect the consistency of experimental results. Furthermore, serum has significant drawbacks such as unclear composition and potential pathogen contamination, severely limiting experimental reproducibility and clinical translational value. Moreover, currently available Kupffer cell culture media are not optimized for Kupffer cell characteristics; arbitrary changes or mixing of culture media can easily lead to cell death or increased fragmentation. Additionally, the aforementioned culture media are prone to precipitation after freeze-thaw cycles, requiring heating to dissolve; repeated handling may reduce the activity of nutrients, all of which can significantly reduce the viability of Kupffer cells.
[0004] Therefore, there is an urgent need for a serum-free culture medium for Kupffer cells to reduce the impact of serum on cells, thereby improving the culture stability, consistency, and cell viability of Kupffer cells. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides a composition for culturing Kupffer cells and its application, aiming to achieve efficient and stable Kupffer cell culture.
[0006] To achieve this objective, the present invention adopts the following technical solution:
[0007] In a first aspect, the present invention provides a composition for culturing Kupffer cells, the composition comprising M-CSF (macrophage colony-stimulating factor), HGF (hepatocyte growth factor) and EGF (epidermal growth factor).
[0008] In this invention, a specific composition is designed and added to serum-free culture medium for Kupffer cells. This composition can replace the function of serum, achieving a breakthrough in serum-free culture of Kupffer cells and ensuring the maintenance of cell function.
[0009] Preferably, the composition for culturing Kupffer cells comprises 0.01-0.5 ng / μL M-CSF, 5-20 ng / mL HGF, and 5-15 ng / mL EGF. The 0.01-0.5 ng / μL can be, for example, 0.01 ng / μL, 0.05 ng / μL, 0.1 ng / μL, 0.15 ng / μL, 0.2 ng / μL, 0.25 ng / μL, 0.3 ng / μL, 0.35 ng / μL, 0.4 ng / μL, 0.45 ng / μL, or 0.5 ng / μL. The 5-20 ng / mL can be, for example, 5 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL, or 20 ng / mL. The 5-15 ng / mL can be, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL or 15 ng / mL, etc.
[0010] In a second aspect, the present invention provides a serum-free culture medium for Kupffer cells, the culture medium comprising basal culture medium, bovine pancreatic insulin, human transferrin, sodium selenite, L-alanyl-L-glutamine solution, 4-hydroxyethylpiperazine ethanesulfonic acid, bovine serum albumin, and the composition for culturing Kupffer cells described in the first aspect.
[0011] The culture medium in this invention has a clearly defined and controllable composition, allowing for the precise addition of essential factors such as M-CSF and transferrin, eliminating interference from serum-related variables. Secondly, it avoids animal-derived risks, meeting regulatory requirements for cell therapy products. Finally, the standardized formulation ensures experimental consistency, providing a more reliable technical platform for liver disease mechanism research, drug toxicity testing, and cell therapy.
[0012] This invention incorporates specific cytokines: M-CSF, which specifically induces the differentiation, proliferation, and survival of macrophages; HGF, which promotes cell proliferation, migration, and morphogenesis; EGF, which broadly stimulates the proliferation and differentiation of epithelial cells and other cell types; bovine pancreatic insulin, which promotes the uptake and utilization of glucose and amino acids and enhances anabolism; human transferrin, which safely and efficiently transports essential iron ions to cells; and sodium selenite, which acts as a cofactor for antioxidant enzymes, protecting cells from oxidative damage.
[0013] Preferably, the basal culture medium comprises DMEM / F12.
[0014] Preferably, the culture medium comprises basal culture medium, 0.1 μg / mL-1 mg / mL bovine pancreatic insulin, 0.1 μg / mL-10 mg / mL human transferrin, 0.1 ng / mL-10 mg / mL sodium selenite, 0.2-10 mM L-alanyl-L-glutamine solution, 10-20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 0.05%-0.3% bovine serum albumin, 0.01-0.5 ng / μL M-CSF, 5-20 ng / mL HGF, and 10 ng / mL EGF. The 10-20 mM concentration can be, for example, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, or 20 mM. The 0.05-0.3% concentration can be, for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, or 0.3%. The 0.01-0.5 ng / μL can be, for example, 0.01 ng / μL, 0.05 ng / μL, 0.1 ng / μL, 0.15 ng / μL, 0.2 ng / μL, 0.25 ng / μL, 0.3 ng / μL, 0.35 ng / μL, 0.4 ng / μL, 0.45 ng / μL or 0.5 ng / μL, etc. The 5-20 ng / mL can be, for example, 5 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL or 20 ng / mL, etc. The 5-15 ng / mL concentration can be, for example, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, or 15 ng / mL. The 0.1 μg / mL-1 mg / mL concentration can be, for example, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 50 μg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, or 1 mg / mL. The 0.1 μg / mL-10 mg / mL can be, for example, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 50 μg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, or 10 mg / mL. The 0.2-10 mM can be, for example, 0.2 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM.The 0.1 ng / mL-10 mg / mL can be, for example, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL, 50 μg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, or 10 mg / mL, etc.
[0015] Thirdly, the present invention provides the application of the serum-free culture medium for Kupffer cells described in the second aspect in the culture of Kupffer cells.
[0016] Fourthly, the present invention provides a culture method comprising culturing Kupffer cells in the serum-free Kupffer cell culture medium described in the second aspect.
[0017] Preferably, the culture temperature is 35℃-40℃, the CO2 concentration is 3%-8%, and the culture time is 3-7 days. The 35℃-40℃ can be, for example, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃. The 3%-8% can be, for example, 3%, 4%, 5%, 6%, 7%, or 8%. The 3-7 days can be, for example, 3 days, 4 days, 5 days, 6 days, or 7 days.
[0018] The present invention uses the above-mentioned serum-free Kupffer cell culture medium, which can obtain Kupffer cells with high activity and is simple to operate. Because the serum-free culture medium has high stability, it can be used for batch cell culture, and can obtain cells with high consistency, which can significantly improve the accuracy of experiments.
[0019] Fifthly, the present invention provides Kupffer cells prepared according to the preparation method described in the fourth aspect.
[0020] In a sixth aspect, the present invention provides the use of Kupffer cells according to the fifth aspect in the preparation of immunomodulatory drugs.
[0021] Compared with the prior art, the present invention has at least the following beneficial effects:
[0022] 1. This invention adds M-CSF, HGF, and EGF to the serum-free culture medium for Kupffer cells, which can replace the function of serum, achieving a breakthrough in serum-free culture of Kupffer cells while ensuring the maintenance of cell function. Reducing the addition of serum avoids animal-derived risks and complies with regulatory requirements for cell therapy products. Standardized formulation ensures experimental consistency and provides a more reliable technical platform for liver disease mechanism research, drug toxicity testing, and cell therapy.
[0023] 2. The present invention uses serum-free culture medium to culture Kupffer cells. The method is simple to operate, does not require highly skilled personnel, can be used for batch cell culture, and can obtain cells with high uniformity, which can significantly improve the accuracy of experiments. Attached Figure Description
[0024] Figure 1 The image shows bright-field images of cell culture at different times in Example 1.
[0025] Figure 2 This is a bright-field image of cells cultured at different times for Comparative Example 1.
[0026] Figure 3 Bright field images of cells cultured at different times for Comparative Example 5.
[0027] Figure 4 This is a detection image of the CD68 protein by immunofluorescence.
[0028] Figure 5 This is a negative control image showing the detection of the CD68 protein using immunofluorescence. Detailed Implementation
[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.
[0030] The sources of reagents used in the following examples are:
[0031] M-CSF: Dongkang Biotechnology Y01501.
[0032] HGF: Nearshore protein CJ72.
[0033] EGF: Nearshore protein C029.
[0034] ITS: Biyuntian C0341.
[0035] Example 1
[0036] This embodiment provides a serum-free culture medium for Kupffer cells.
[0037] In this embodiment, the serum-free culture medium for Kupffer cells was prepared by adding 1.0 mg / L bovine pancreatic insulin, 0.55 mg / L human transferrin, 0.67 μg / L sodium selenite, 2 mM L-alanyl-L-glutamine solution, 15 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 0.1% bovine serum albumin, 0.1 ng / μL M-CSF, 10 ng / mL HGF, and 10 ng / mLEGF to DMEM / F12.
[0038] Example 2
[0039] This embodiment provides a serum-free culture medium for Kupffer cells.
[0040] In this embodiment, the serum-free culture medium for Kupffer cells was prepared by adding 0.5 mg / L bovine pancreatic insulin, 0.25 mg / L human transferrin, 0.33 μg / L sodium selenite, 1 mM L-alanyl-L-glutamine solution, 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 0.05% bovine serum albumin, 0.5 ng / μL M-CSF, 5 ng / mL HGF, and 15 ng / mLEGF to DMEM / F12.
[0041] Example 3
[0042] This embodiment provides a serum-free culture medium for Kupffer cells.
[0043] In this embodiment, the serum-free culture medium for Kupffer cells consisted of DMEM / F12 containing 2 mg / L bovine pancreatic insulin, 1 mg / L human transferrin, 1 μg / L sodium selenite, 4 mM L-alanyl-L-glutamine solution, 10 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 0.3% bovine serum albumin, 0.01 ng / μL M-CSF, 20 ng / mL HGF, and 5 ng / mL EGF.
[0044] Example 4
[0045] This embodiment provides a serum-free culture medium for Kupffer cells, which differs from Example 1 only in that the concentration of M-CSF in the serum-free culture medium for Kupffer cells is 0.005 ng / μL, the concentration of HGF is 25 ng / mL, and the concentration of EGF is 3 ng / mL. All other aspects are the same as in Example 1.
[0046] Example 5
[0047] This embodiment provides a serum-free culture medium for Kupffer cells, which differs from Example 1 only in that the concentration of M-CSF in the serum-free culture medium for Kupffer cells is 0.7 ng / μL, the concentration of HGF is 3 ng / mL, and the concentration of EGF is 17 ng / mL. All other aspects are the same as in Example 1.
[0048] Comparative Example 1
[0049] This comparative example provides a serum-free culture medium for Kupffer cells, which differs from Example 1 only in that HGF and EGF are not added to the serum-free culture medium for Kupffer cells; otherwise, it is the same as Example 1.
[0050] Comparative Example 2
[0051] This comparative example provides a serum-free culture medium for Kupffer cells, which differs from Example 1 only in that HGF is not added to the serum-free culture medium for Kupffer cells; otherwise, it is the same as Example 1.
[0052] Comparative Example 3
[0053] This comparative example provides a serum-free culture medium for Kupffer cells, which differs from Example 1 only in that EGF is not added to the serum-free culture medium for Kupffer cells; otherwise, it is the same as Example 1.
[0054] Comparative Example 4
[0055] This comparative example provides a serum-free culture medium for Kupffer cells, which differs from Example 1 only in that M-CSF is not added to the serum-free culture medium for Kupffer cells; otherwise, it is the same as Example 1.
[0056] Comparative Example 5
[0057] This comparative example provides a Kupffer cell culture medium, which differs from Example 1 only in that HGF, EGF, and M-CSF are not added to the Kupffer cell culture medium, and 5% fetal bovine serum is added instead; otherwise, it is the same as Example 1.
[0058] Test Example 1
[0059] This test case detects IL6 expression.
[0060] Kupffer cells were added to 48-well plates, with a cell count of 4 × 10⁶ cells. 5The culture medium added consisted of 1640, 10% FBS, 1.0 mg / L bovine pancreatic insulin, 0.55 mg / L human transferrin, 0.67 μg / L sodium selenite, 20 mM fructose, 0.2 ng / μL M-CSF, and 1% phosphatidylserine. The culture was incubated statically at 37°C in a 5% CO2 incubator.
[0061] After cell adhesion, the culture medium was aspirated, and serum-free culture medium (without serum, M-CSF, HGF, or EGF) was added. The cells were then incubated statically at 37°C with 5% CO2 for 2 days. The medium was then changed, the culture medium was aspirated, and the culture medium from the above examples and comparative examples was added again. The cells were then incubated statically at 37°C with 5% CO2 for 3 days.
[0062] LPS stimulation was performed by adding LPS to the culture medium of the above-described examples and comparative examples to a final concentration of 1 μg / mL. After culturing for 1 day following LPS stimulation, the cells were fixed, immunofluorescence was performed, and the culture medium was collected for IL-6 secretion detection. The specific detection results are shown in Table 1.
[0063] Table 1
[0064]
[0065] The results above show that:
[0066] (1) The serum-free culture medium of the present invention can effectively support the growth of Kupffer cells. M-CSF, HGF and EGF can replace serum and are of great significance for cell growth and cell function maintenance.
[0067] (2) By comparing Example 1 with Examples 4-5, it can be found that the concentration of each component in the culture medium outside the range will lead to a significant reduction in cell growth capacity and in terms of cell function, after LPS stimulation, the expression of IL6 in Examples 4-5 outside the component concentration range is significantly reduced, indicating that the factor concentration recommended in this invention can effectively support cell proliferation and function maintenance.
[0068] (3) By comparing Example 1 with Comparative Examples 1-4, it can be seen that the absence of any one of HGF, EGF and M-CSF will lead to cell proliferation and reduced cell function, indicating that the three factor components are indispensable in the serum-free culture process of Kupffer in vitro.
[0069] (4) By comparing Example 1 with Comparative Example 5, it can be seen that HGF, EGF and M-CSF can partially replace the function of serum, achieving a breakthrough in serum-free culture of Kupffer cells and ensuring the maintenance of cell function.
[0070] Test Example 2
[0071] This test case was used to detect the activity of Kupffer cells.
[0072] This test example examines cells cultured in the culture media prepared in Example 1, Comparative Example 1, and Comparative Example 5 of Test Example 1. Cells were cultured on days 1, 2, 5, 6, and 7, respectively. Specific results are as follows: Figure 1 , Figure 2 and Figure 3 As shown, there was no significant difference in cell morphology between Example 1 and Comparative Example 5, which contained serum. This indicates that the serum-free culture medium in Example 1 can maintain normal cell growth and cell state. In the comparative example, HGF and EGF were removed. During long-term culture, slow cell growth, low cell number, and poor cell state were observed. This shows that HGF and EGF have a very significant impact on the growth of Kupffer cells and are irreplaceable components in serum-free culture medium.
[0073] Test Example 3
[0074] This test case involved immunofluorescence detection of guanosine CD68.
[0075] This test case examines cells cultured for 3 days in the culture media prepared in Example 1, Comparative Example 1, and Comparative Example 5 of Test Example 1. The specific testing steps are as follows: Cells were divided into experimental and control groups. Cell culture supernatant was removed. After fixation, perforation, and blocking, the experimental group was incubated with CD68 antibody overnight at 4°C, while the control group was incubated without antibody overnight at 4°C. The next day, cells were incubated with fluorescent secondary antibody and nuclear dye. Finally, CD68 protein expression was observed under a fluorescence microscope. The fluorescence staining results show that the red fluorescence expression of CD68 in Example 1 is similar to that in Comparative Example 5, indicating that the expression of cell marker proteins in the serum-free culture medium proposed in this patent is consistent with the results of culture containing serum. This further demonstrates the effectiveness of the serum-free culture medium of this patent. Furthermore, comparing Example 1 and Comparative Example 1, it can be found that when HGF and EGF are removed from the serum-free culture medium, the CD68 staining expression decreases accordingly, indicating that EGF and HGF are indispensable components of the serum-free culture medium. Specific phenomena are as follows: Figure 4 and Figure 5 As shown.
[0076] In summary, this invention adds M-CSF, HGF, and EGF to the serum-free culture medium of Kupffer cells, which can replace the function of serum, achieving a breakthrough in the serum-free culture method of Kupffer cells and ensuring the maintenance of cell function.
[0077] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.
Claims
1. A composition for culturing Kupffer cells, characterized in that, The composition for culturing Kupffer cells includes M-CSF, HGF, and EGF.
2. The composition for culturing Kupffer cells according to claim 1, characterized in that, The composition for culturing Kupffer cells comprises 0.01-0.5 ng / μL M-CSF, 5-20 ng / mL HGF, and 5-15 ng / mL EGF.
3. A serum-free culture medium for Kupffer cells, characterized in that, The culture medium comprises basal medium, bovine pancreatic insulin, human transferrin, sodium selenite, L-alanyl-L-glutamine solution, 4-hydroxyethylpiperazine ethanesulfonic acid, bovine serum albumin, and the composition for culturing Kupffer cells as described in claim 1 or 2.
4. The serum-free Kupffer cell culture medium according to claim 3, characterized in that, The basal culture medium includes DMEM / F12.
5. The serum-free Kupffer cell culture medium according to claim 3 or 4, characterized in that, The culture medium includes basal medium, 0.1 μg / mL-1 mg / mL bovine pancreatic insulin, 0.1 μg / mL-10 mg / mL human transferrin, 0.1 ng / mL-10 mg / mL sodium selenite, 0.2-10 mM L-alanyl-L-glutamine solution, 10-20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 0.05%-0.3% bovine serum albumin, 0.01-0.5 ng / μL M-CSF, 5-20 ng / mL HGF, and 5-15 ng / mLEGF.
6. The use of serum-free Kupffer cell culture medium as described in any one of claims 3-5 in culturing Kupffer cells.
7. A method for culturing Kupffer cells, characterized in that, The culture method includes culturing Kupffer cells using the serum-free Kupffer cell culture medium according to any one of claims 3-5.
8. The method for culturing Kupffer cells according to claim 7, characterized in that, The culture temperature is 35℃-40℃, the CO2 concentration is 3%-8%, and the time is 3-7 days.
9. A Kupffer cell culture method according to claim 7 or 8.
10. The use of Kupffer cells as described in claim 9 in the preparation of immunomodulatory drugs.