THP-1 mononuclear cell culture medium and culture method thereof
By optimizing the formula and culture method of THP-1 monocyte culture medium, and adjusting cell density by rehydration and semi-replacement methods, the problem of slow growth and easy death of THP-1 cells was solved, and good cell growth and freezing effect was achieved.
Patent Information
- Application Number
- CN202510439218.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-07-08
AI Technical Summary
The culture method of THP-1 monocytes in the prior art leads to slow growth of cells and poor state, prone to death during passage and freezing, and is sensitive to serum quality and insufficient optimization process.
The THP-1 monocyte culture medium formula, including complete culture medium, fetal bovine serum, HEPES, sodium pyruvate and antibiotics, combined with the platelet-derived growth factor PDGF, regulates cell density through rehydration and semi-replacement, and optimizes the culture and passage process.
It improves the growth status and activity of THP-1 cells, reduces cell death, improves the resuscitation effect of frozen cells, reduces sensitivity to serum quality, and obtains uniform and good active cells.
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Figure CN120272419A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell culture, and particularly relates to a THP-1 monocyte culture medium and a culture method thereof. Background Art
[0002] The growth of human myeloid leukemia monocyte (THP-1) not only depends on cell concentration, but also is an eosinophilic suspension cell. In cell culture, using conventional cell culture methods will result in slow growth of THP-1 and poor cell growth states (such as cell adhesion, irregular cell morphology, and non-smooth cell edges). Therefore, if only simple centrifugation and medium replacement are performed and the cell number does not decrease, the cells will quickly deplete nutrients and die; if the cells are diluted to a low density, the cells will enter a stationary phase and stop proliferating or die. Currently, there have been reports on cell passage by combining cell counting and centrifugation for medium replacement, but there are few optimization processes for the culture method, passage, and culture medium of this cell. Moreover, when cryopreserved THP-1 cells are resuscitated, problems such as cell death and fragility are likely to occur. Summary of the Invention
[0003] In view of this, the purpose of the present invention is to provide a THP-1 monocyte culture medium and a culture method thereof, which can improve the culture method of THP-1, help researchers improve work efficiency, and reduce experimental costs.
[0004] The technical solution of the present invention is realized as follows:
[0005] A THP-1 monocyte culture medium, comprising the following components: complete medium, 8 v / v% - 12 v / v% fetal bovine serum, 20 - 30 mM HEPES, 0.8 - 1.2 mM sodium pyruvate, and 0.8 v / v% - 1.2 v / v% antibiotic.
[0006] Further, the HEPES is configured into a 2 - 4 M HEPES stock solution using the complete medium, the sodium pyruvate is configured into a 2 - 4 M sodium pyruvate stock solution using the complete medium, the complete medium is RPMI 1640 medium, and the antibiotic is penicillin-streptomycin.
[0007] Further, the components further include 2 - 5 ng / mL platelet-derived growth factor (PDGF).
[0008] A method for culturing THP-1 monocytes using the above culture medium, comprising the following steps:
[0009] (1) Activation: Take the revived THP-1 monocytes, add them to the complete medium, pipette, centrifuge, discard the supernatant, resuspend with the complete medium, and then transfer to a culture dish containing the complete medium and pipette again for activation culture to obtain activated THP-1 monocytes;
[0010] (2) Subculture: Take the activated THP-1 monocytes and add them to the above-mentioned medium, and perform subculture by means of medium supplementation and / or semi-medium replacement. The medium supplementation method includes: during subculture, by adding the above-mentioned medium, maintaining the cell density of THP-1 monocytes in subculture at 6-8×10 5 cells / mL; the semi-medium replacement method includes: taking 1 / 3-2 / 3 of THP-1 monocytes and transferring them to a culture dish containing the new above-mentioned medium, and by adding the above-mentioned medium, maintaining the cell density of the transferred THP-1 monocytes at 6-8×10 5 cells / mL. Suspended cells THP-1 generally undergo subculture by means of medium supplementation or semi-medium replacement. When the cell density is relatively high, use a cell counting chamber to count and determine the subculture method.
[0011] A method for culturing THP-1 monocytes using the above-mentioned medium, comprising the following steps:
[0012] (1) Activation: Take the revived THP-1 monocytes, add them to the complete medium, pipette, centrifuge, discard the supernatant, resuspend with the complete medium, and then transfer to a culture dish containing the complete medium and pipette again for activation culture to obtain activated THP-1 monocytes;
[0013] (2) Subculture: Take the activated THP-1 monocytes and add them to the above-mentioned medium, and perform subculture by means of medium supplementation and / or semi-medium replacement. The medium supplementation method includes: during subculture, by adding the above-mentioned medium, maintaining the cell density of THP-1 monocytes in subculture at 6-8×10 5 cells / mL, adding platelet-derived growth factor PDGF to the medium after culturing for 3-5 h, and the final concentration of platelet-derived growth factor PDGF is 2-5 ng / mL; the semi-medium replacement method includes: taking 1 / 3-2 / 3 of THP-1 monocytes and transferring them to a culture dish containing the above-mentioned medium, and by adding the above-mentioned medium, maintaining the cell density of the transferred THP-1 monocytes at 6-8×10 5 cells / mL, adding platelet-derived growth factor PDGF to the medium after culturing for 3-5 h, and the final concentration of platelet-derived growth factor PDGF is 2-5 ng / mL.
[0014] A further solution is that in step (1), THP-1 monocytes are added to 3-5 mL of complete medium, pipetted, centrifuged, the supernatant is discarded, and then 1-3 mL of complete medium is added for resuspension, and then transferred to a culture dish containing 16-20 mL of complete medium and pipetted again.
[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0016] The present invention provides a series of optimized processes for THP-1 cell culture, passage, culture medium, etc., improves the THP-1 cell culture method, helps to improve the resuscitation state of cryopreserved THP-1 cells, and reduces phenomena such as cell death; in addition, the THP-1 cell culture medium provided by the present invention has an optimized formula and scientific proportioning, which helps to reduce the sensitivity of THP-1 cells to the quality of serum and further improve the good growth state of THP-1 cells.
[0017] By optimizing the components of the THP-1 monocyte culture medium, culture containers, and selecting fluid addition or semi-fluid replacement to adjust the cell density, the THP-1 cells obtained by culture are round and bright spherical, with smooth cell edges, relatively uniform cell sizes, good cell growth states, fast cell growth, and good maintenance of cell activity; combined with the cryopreservation solution, the cryopreservation activity of the cells can be prolonged, and the cells have good activity after resuscitation. Description of the Drawings
[0018] Figure 1 It is a morphological diagram of THP-1 cells under the culture conditions of Example 1 of the present invention under an optical microscope;
[0019] Figure 2 It is a Calcein AM / PI live-dead cell staining diagram of THP-1 cells under the culture conditions of Example 1 of the present invention. Note: PI staining is for dead cells (red), and Calcein AM stains live cells (green);
[0020] Figure 3 It is the proliferation of THP-1 cells at different cell densities;
[0021] Figure 4 It is the influence of different culture methods on the viability of THP-1 cells;
[0022] Figure 5 It is the influence of different cryopreservation solution formulations on the resuscitation of THP-1 cells. Detailed Embodiments
[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0024] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.
[0025] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can all be obtained through commercial channels.
[0026] 4-Hydroxyethylpiperazineethanesulfonic acid (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, HEPES);
[0027] Dimethyl sulfoxide (DMSO).
[0028] Example 1
[0029] When resuscitating the suspension cells THP-1, use a pipette to transfer the cells (10 7 cells / mL) into a centrifuge tube containing 4 mL of complete medium, pipette and mix well. After centrifuging at 1000 rpm for 3 min, discard the supernatant, add 2 mL of medium to resuspend, and transfer to a petri dish containing 18 mL of complete medium and pipette evenly. Observe once every 2-3 days (the culture conditions are the same as the previous treatment. When observing, observe the cell state under the microscope. As long as the cells are not contaminated and in normal state), the cells should not be taken out of the incubator frequently.
[0030] The suspension cells THP-1 are generally passaged by replenishing the liquid or semi-changing the liquid. When the cell density is relatively high, use a cell counting plate to count and determine the passaging method. Adopt the form of replenishing the liquid to maintain the cell density at 6-8×10 5 cells per milliliter. When there is too much cell culture medium, adopt the form of semi-changing the liquid and splitting the plate. Take out 1 / 2 of the cells and transfer them to a new petri dish. Then, count the cells separately, and adopt the form of replenishing the liquid to maintain the cell density at 6-8×10 5 cells / mL.
[0031] For the cryopreservation of THP-1 cells, first pipette and mix the cell solution, take out an appropriate amount of cells and transfer them to a centrifuge tube. After centrifuging at 1000 rpm for 3 min, discard the supernatant, add cryopreservation solution to resuspend and then count. Then freeze the THP-1 cells at a density of 10 7 cells per tube. Finally, transfer the cryopreservation tube to a cryopreservation box and place it in an -80°C refrigerator. After overnight, transfer it to a liquid nitrogen tank.
[0032] Configure THP-1 cell culture medium: RPMI 1640 medium + 10% fetal bovine serum + 25 mM HEPES + 1 mM sodium pyruvate + 1% penicillin-streptomycin mixture. Among them, HEPES and sodium pyruvate are separately used to prepare 2.5 mol HEPEs (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) stock solution and 100 mM sodium pyruvate stock solution with complete medium, and stored in the refrigerator at 4 °C in the dark.
[0033] Cryopreservation solution formula: 90% complete medium + 10% DMSO.
[0034] As Figure 1 can be seen, the THP-1 cells obtained by culturing in Example 1 grew well. The cells were translucent, round and bright spherical, a few aggregated into grape-like clusters, the cell edges were smooth, the cell bodies were plump, and the cell sizes were relatively uniform.
[0035] As Figure 2 can be seen, under the fluorescence microscope, the PI staining of the cells was negative, and the live cells were stained green by Calcein AM, that is, there were fewer dead cells, indicating that the culture effect of Example 1 on THP-1 cells was good and the cells maintained good activity.
[0036] Example 2
[0037] The conditions for resuscitation and subculture of THP-1 cells in this example were slightly the same as those in Example 1, but the THP-1 cell culture medium was adjusted as follows:
[0038] When resuscitating suspension cells of THP-1, use a pipette gun to transfer the cells (10 7 cells / mL) into a centrifuge tube containing 4 mL of complete medium, pipette and mix well. After centrifuging at 1000 rpm for 3 min, discard the supernatant and then resuspend with 2 mL of medium, and transfer to a culture dish containing 18 mL of complete medium and pipette evenly. Observe once every 2-3 days (the culture conditions are the same as the previous treatment. When observing, observe the cell state under the microscope. As long as the cells are not contaminated and the state is normal), the cells should not be taken out of the incubator frequently.
[0039] Suspension cells of THP-1 are generally subcultured by replenishing the liquid or semi-changing the liquid. When the cell density is high, use a cell counting plate to count and determine the subculture method. Adopt the form of replenishing the liquid to maintain the cell density at 6-8×10 5 cells per milliliter. When there is too much cell culture medium, adopt the form of semi-changing the liquid and dividing the plate. Take out 1 / 2 of the cells and transfer them to a new culture dish. Then, count the cells separately, and adopt the form of replenishing the liquid to maintain the cell density at 6-8×10 5 cells / mL.
[0040] For the cryopreservation of THP-1 cells, first pipette and mix the cell suspension, then transfer an appropriate amount of cells into a centrifuge tube. After centrifuging at 1000 rpm for 3 min, discard the supernatant, resuspend with cryopreservation medium and then count. Then cryopreserve the THP-1 cells at a density of 10 7 cells per tube. Finally, transfer the cryotubes into a cryobox and place it in an -80 °C refrigerator. After overnight, transfer it into a liquid nitrogen tank.
[0041] Prepare THP-1 cell culture medium: RPMI 1640 medium + 10% fetal bovine serum + 25 mM HEPES + 1 mM sodium pyruvate + 1% penicillin-streptomycin mixture + 3 ng / mL PDGF. Among them, HEPES and sodium pyruvate are separately used to prepare 2.5 mol HEPEs (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) stock solution and 100 mM sodium pyruvate stock solution with complete medium, and store them in the dark at 4 °C in the refrigerator. (In subculture, add platelet-derived growth factor PDGF to the medium 3 - 5 h after culturing, and the final concentration of platelet-derived growth factor PDGF is 3 ng / mL).
[0042] Cryopreservation medium formula: 90% complete medium + 10% DMSO.
[0043] The THP-1 cells obtained by culturing in Example 2 grew well. The cells were bright, round and spherical, with smooth cell edges, plump cell bodies, uniform cell sizes. Occasionally, a small number of loose small cell clusters were seen, but the cells at the edge of the clusters still maintained a complete morphology without obvious fragmentation or erosion.
[0044] Under a fluorescence microscope, the PI staining of the cells was negative, and the live cells were stained green by Calcein AM. Compared with Example 1, there were fewer dead cells in Example 2, indicating that the culture effect of Example 2 on THP-1 cells was excellent and the cells maintained better viability.
[0045] Control experiment 1: Effect of different cell densities on cell proliferation
[0046] Transfer the suspension cells THP-1 with good growth status into four 6-cm culture dishes respectively, and the initial cell densities are 0.5×10 6 , 1×10 6 , 5×10 6 , 10×10 6 cells / mL. The medium components and other culture conditions are kept the same. Perform cell counting at the same time point every day after subculture until the cell density in the dish reaches logarithmic phase. A. Record the number of days for different cell densities to reach the logarithmic phase; B. Detect the LDH situation of different cell densities at the logarithmic phase; C. Detect the cell viability of different cell densities at the logarithmic phase; D. Detect the live-dead cell staining of different cell densities at the logarithmic phase;
[0047] It can be seen from Figure 3 that the experimental results of this part show that as the cell density gradually increases, the number of days to reach the logarithmic phase significantly decreases, the LDH expression significantly decreases, and the cell viability significantly increases.
[0048] In addition, under a fluorescence microscope ( Figure 4 ), the cells were negative for PI staining, and the live cells were stained green by Calcein AM, indicating that there were fewer dead cells and the culture effect was good.
[0049] Comparative Experiment 2: Effects of Different Culture Methods on Cell Viability
[0050] Centrifugal culture method: When the cell density is relatively high, transfer all the cell suspension to a centrifuge tube; centrifuge at 1000 rpm for 3 min; discard the supernatant, add an appropriate amount of fresh medium and gently resuspend the cell pellet, then inoculate the cell suspension back into the culture dish, mix the cells evenly, and place them in the incubator for continued culture.
[0051] Semi-medium replacement culture method: When the cell density is relatively high, use a cell counting chamber to count and determine the passage method. Maintain the cell density at 6 - 8×10 5 cells per milliliter by adding medium. When there is too much cell culture medium, take the form of semi-medium replacement and subculture. Take out 1 / 2 of the cells and transfer them to a new culture dish. Then, count the cells separately and maintain the cell density at 6 - 8×10 5 cells / mL by adding medium.
[0052] Operation: Passage the cells at a density of 10 7 cells (F2) with the same cell state. After passage, culture the cells according to methods A and B respectively. Statistically analyze the cell viability at 0 h, 24 h, 48 h, and 72 h after passage, and observe the cell morphology and density of the two culture methods under an optical microscope 72 h after passage.
[0053] It can be seen from Figure 5 that the experimental results of this part show that when the initial cell concentration is the same, the cell viability is detected at 0 h, 24 h, 48 h, and 72 h after passage. It can be seen that within the same time after passage, the cell viability of the semi-medium replacement culture method is significantly higher than that of the centrifugal method. In addition, 72 h after passage, under an optical microscope, the cell density of the semi-medium replacement culture method is significantly higher than that of the centrifugal culture method, and the cell size of the semi-medium replacement culture is relatively uniform and the cell bodies are plump.
[0054] Comparative Experiment 3: Effects of Different Cryopreservation Solution Formulations on Cell Recovery
[0055] Cryopreservation solution formulation 1: 95% complete medium + 5% DMSO
[0056] Cryopreservation solution formulation 2: 92% complete medium + 8% DMSO
[0057] Cryopreservation solution formulation 3: 90% complete medium + 10% DMSO
[0058] Operation: Freeze THP-1 cells at a density of 10^7 cells per tube. Freeze them according to the above cryopreservation solution formulation ratios respectively. Finally, transfer the cryotubes to a cryobox and place them in an -80°C refrigerator. After overnight, transfer them to a liquid nitrogen tank. After resuscitation, detect the dead cell staining of the cells.
[0059] It can be seen from Figure 5 that the experimental results of this part show that the cryopreservation solution formulation with 10% DMSO has a better effect.
[0060] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.
Claims
1. A THP-1 monocyte medium, characterized in that, It includes the following components: complete medium, 8 v / v% - 12 v / v% fetal bovine serum, 20 - 30 mM HEPES, 0.8 - 1.2 mM sodium pyruvate, and 0.8 v / v% - 1.2 v / v% antibiotics.
2. The THP-1 monocyte medium according to claim 1, characterized in that, The HEPES is prepared into a 2 - 4 M HEPES stock solution using the complete medium, the sodium pyruvate is prepared into a 2 - 4 M sodium pyruvate stock solution using the complete medium, the complete medium is RPMI 1640 medium, and the antibiotics are penicillin - streptomycin.
3. A THP-1 monocyte culture medium according to claim 1, characterized in that, The components further include 2 - 5 ng / mL platelet - derived growth factor.
4. A method for culturing THP-1 monocytes using the culture medium according to claim 1 or 2, characterized in that, It includes the following steps: (1) Activation: Take the revived THP - 1 monocytes, add them to the complete medium, pipette, centrifuge, discard the supernatant, then add the complete medium for resuspension, and then transfer to a culture dish containing the complete medium and pipette again for activation culture to obtain activated THP - 1 monocytes; (2) Subculture: Take the activated THP-1 monocytes and add them to the culture medium described in claim 1 or 2, and perform subculture by means of replenishing the medium and / or semi-changing the medium. The method of replenishing the medium includes: during subculture, by adding the culture medium described in claim 1 or 2, maintaining the cell density of THP-1 monocytes in the subculture at 6-8×10 5 cells / mL; The method of semi-changing the medium includes: transferring 1 / 3-2 / 3 of the THP-1 monocytes to a new culture medium described in claim 1 or 2, and by adding the culture medium described in claim 1 or 2, maintaining the cell density of the transferred THP-1 monocytes at 6-8×10 5 cells / mL.
5. A method for culturing THP-1 monocytes using the culture medium described in claim 3, characterized in that, It includes the following steps: (1) Activation: Take the revived THP - 1 monocytes, add them to the complete medium, pipette, centrifuge, discard the supernatant, then add the complete medium for resuspension, and then transfer to a culture dish containing the complete medium and pipette again for activation culture to obtain activated THP - 1 monocytes; (2) Subculture: Take the activated THP-1 monocytes and add them to the culture medium described in claim 1 or 2. Subculture is carried out by the method of replenishing the medium and / or semi-changing the medium. The method of replenishing the medium includes: during subculture, by adding the culture medium described in claim 1 or 2, maintaining the cell density of THP-1 monocytes in subculture at 6-8×10 5 cells / mL, adding platelet-derived growth factor to the culture medium after culturing for 3-5 h, and the final concentration of platelet-derived growth factor is 2-5 ng / mL; The method of semi-changing the medium includes: transferring 1 / 3-2 / 3 of the THP-1 monocytes to a new culture medium described in claim 1 or 2, and by adding the culture medium described in claim 1 or 2, maintaining the cell density of the transferred THP-1 monocytes at 6-8×10 5 cells / mL, adding platelet-derived growth factor to the culture medium after culturing for 3-5 h, and the final concentration of platelet-derived growth factor is 2-5 ng / mL.
6. The cultivation method according to claim 4 or 5, characterized in that, In step (1), the THP - 1 monocytes are added to 3 - 5 mL of the complete medium, pipetted, centrifuged, the supernatant is discarded, then 1 - 3 mL of the complete medium is added for resuspension, and then transferred to a culture dish containing 16 - 20 mL of the complete medium and pipetted again.