Method for culturing Walker 256 cells
By adopting a fixed time and fixed proportion of passages, combined with optimized culture media and conditions, the problems of inconsistent quality and large batch differences in the existing Walker 256 cell culture method were solved, and the growth of high cell viability and consistency was achieved, which significantly improved the number of cells that can be used.
Patent Information
- Application Number
- CN202510036864.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-18
AI Technical Summary
The existing Walker 256 cell culture methods have problems such as inconsistent quality, irregular culture process, and large differences between cell batches, resulting in uneven quality of established animal disease models.
Cell culture method with fixed time and fixed proportion passages was used, combined with optimized culture medium and culture conditions, including the use of RPMI 1640 medium containing 10% FBS, 6-10 mM glutamine and 10-50 ng/mL EGF(a), ensuring high viability and consistency of cells over 12 generations.
It significantly improves the number of cells that can be used, ensures the high viability and consistent growth rate of Walker 256 cells, and the population doubling time is consistent, effectively ensuring cell quality.
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Figure CN119842620A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of in vitro cell culture, and in particular to a method for culturing Walker 256 cells. Background Art
[0002] Walker 256 cells are a rat ascites cancer cell line. After being injected into the rat peritoneal cavity, the cells can quickly form ascites tumors. They are excellent materials for studying in vivo models of anti-tumor drugs and are often used to establish animal disease models. Injecting Walker 256 into the rat peritoneal cavity can form rat ascites; injecting Walker 256 cells into the corresponding tissue sites can quickly form corresponding disease models, providing a basis for the study of various tumor diseases. As Walker 256 is used more and more widely, the requirements for cell quality are also getting higher and higher, so a good culture method for Walker 256 cells is of great significance.
[0003] The current Walker 256 cell culture methods are not very effective. The main reasons are as follows: 1) primary cells are used and the third generation of cells are mostly used; 2) adherent culture is mostly used, and subculture is performed after the confluence reaches more than 90%. However, the confluence is subjective and there is no unified standard. At the same time, digestive fluid needs to be added to digest the cells during cell subculture, which will cause cell damage. Therefore, the Walker 256 cells cultured by the existing methods have problems such as inconsistent quality, non-standardized culture process, and large differences between cell batches, which ultimately lead to uneven quality of established animal disease models and serious waste of manpower and materials.
[0004] Therefore, there is an urgent need to develop a culture method suitable for Walker 256 cells to provide high-quality Walker 256 cells for scientific research and production. Summary of the invention
[0005] In view of the technical problems existing in the background technology, the present invention provides a method for culturing Walker 256 cells, aiming to solve the technical problems of uneven quality and large differences between batches of Walker 256 cells.
[0006] In order to achieve the above object, the technical solution of the present invention is as follows: A method for culturing Walker 256 cells comprises the following steps: Walker 256 cells are inoculated into fresh culture medium at a fixed ratio and cultured for a fixed time, the supernatant is discarded after centrifugation, the cell pellet is resuspended in fresh culture medium, the obtained cell suspension is inoculated into fresh culture medium at the fixed ratio and cultured continuously; Repeat the above steps for passaging.
[0007] Preferably, in the above method, the culture medium contains fetal bovine serum (FBS), glutamine and EGF(a).
[0008] In some preferred embodiments of the present invention, the culture medium is specifically RPMI 1640 medium supplemented with 10% FBS, 6-10 mM glutamine and 10-50 ng / mL EGF(a).
[0009] In the above method, the culture time of each generation of Walker 256 cells is fixed, and preferably, the culture time is specifically 3-4 days.
[0010] In the above method, Walker 256 cells are passaged at a fixed ratio; preferably, the initial seeding density during each culture is (4.0-8.0)×10 5 cells / mL.
[0011] Preferably, in the above method, the number of passages is ≤ 12. Experimental data show that when cells are passaged according to the method of the present invention, the cell viability will gradually decrease after passage P12, and the cell viability can be maintained above 90% within 12 passages, and the cells can proliferate normally.
[0012] In the above method, when the Walker 256 cells used are frozen cells, the following pre-treatment steps are required: The frozen Walker 256 cells were thawed in a water bath, and the Walker 256 cells and the freezing solution were separated by centrifugation. The Walker 256 cell pellet was resuspended in a culture medium containing FBS, and then inoculated into the above culture medium for activation culture for 20-48 hours, and then replaced with fresh culture medium for subculture.
[0013] Preferably, in the above method, the culture conditions of Walker 256 cells are: 37±0.5°C, 5±1.0% CO2, 110±10 rpm.
[0014] Preferably, in the above method, the centrifugation condition of Walker 256 cells is: 200g centrifugation for 5 min.
[0015] Preferably, in the above method, the operation during centrifugation is specifically as follows: after the culture solution is transferred into a centrifuge tube, phosphate buffer is added to the culture container for rinsing, and the rinsing solution is transferred to the centrifuge tube, followed by centrifugation.
[0016] Compared with the prior art, the present invention has the following beneficial effects: The method of the present invention innovatively adopts a cell culture method of fixed time and fixed ratio passaging, avoids subjective factors in confluence judgment, and effectively reduces differences between cell batches. In combination with the above culture method, the present invention simultaneously optimizes the culture medium and culture conditions, so that Walker 256 cells can be passaged to 12 generations, significantly increasing the number of usable cell generations, and the obtained Walker 256 cells have a high viability, the same growth rate, and a consistent population doubling time, effectively ensuring cell quality. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.
[0018] Figure 1 Comparison of cell population doubling levels of total cells (A) and live cells (B) at different initial cell seeding densities in Example 3 of the present invention; Figure 2 It is a curve chart showing the changes of living cell density and total cell density with growth time in Comparative Example 3 of the present invention. DETAILED DESCRIPTION
[0019] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.
[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "including" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions.
[0021] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.
[0022] The detection method of cell concentration and viability in the following examples is: cell staining is performed using a trypan blue staining method, and then cell concentration and viability are measured using a cell counter; specifically, the cells are stained according to a ratio of cell suspension volume: 0.2% trypan blue = 1:1, and the stained sample is added to a cell counting plate, inserted into a cell counter, and measured.
[0023] If no specific techniques or conditions are specified in the following examples, the procedures were carried out according to the techniques or conditions described in the literature in the art or according to the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.
[0024] Example 1 This example provides a method for culturing Walker 256 cells, comprising the following steps: Cell recovery: Take the frozen Walker 256 cells out of liquid nitrogen, quickly put them into a 37°C water bath, thaw them within 3 minutes, disinfect the cryopreserved tubes with 75% medical alcohol, transfer them into a biosafety cabinet, transfer the cell suspension in the cryopreserved tubes into a centrifuge tube containing 10% FBS+RPMI 1640 medium for centrifugation (200g, 5min), discard the supernatant after centrifugation, add an appropriate amount of 10% FBS+RPMI 1640 medium to resuspend the cell pellet, and mix well. After mixing, inoculate the cell suspension into a culture bottle and culture it in an incubator (37°C, 5% CO2, 110rpm).
[0025] Cell medium replacement: After 24 hours of culture, transfer the cell suspension into a centrifuge tube for centrifugation (200g, 5min), discard the supernatant after centrifugation, resuspend the cell pellet in RPMI 1640 medium containing 10% FBS, 8mM glutamine and 20ng / mL EGF(a), and mix well. After mixing, inoculate the cell suspension (initial inoculation density is 4.00E+05 cells / mL) in a culture flask and culture it in an incubator (37℃, 5% CO2, 110rpm).
[0026] Cell passaging: After 4 days of cell culture, transfer the cell suspension into a centrifuge tube for centrifugation (200g, 5min). After completion, discard the supernatant, add RPMI 1640 medium containing 10% FBS, 8mM glutamine and 20ng / mL EGF(a) to resuspend the cell pellet and mix well. Inoculate the obtained cell suspension into a culture flask, add RPMI 1640 medium containing 10% FBS, 8mM glutamine and 20ng / mL EGF(a) to the culture volume (so that the initial cell seeding density is 4.00E+05 cells / mL), and culture in an incubator (37℃, 5% CO2, 110rpm).
[0027] Example 2 According to the method in Example 1, multiple passages were performed, and the cell viability of each generation was detected, and the test results are shown in Table 1.
[0028] Table 1 Cell viability at different generations
[0029] From the above test results, we can see that the cell viability of Walker 256 cells gradually decreased after passage to P12, but the cell viability was maintained above 90% within 12 passages, and the cells were able to proliferate normally.
[0030] Example 3 Referring to Example 1, in this example, the initial cell seeding density was set to 2.00E+05, 4.00E+05, 8.00E+05 and 1.00E+06 cells / mL, respectively, and the culture time was 48 h. Other operations were the same as in Example 1.
[0031] After 48 h of culture, the live cell density and total cell density of the cells were detected, and the cell population doubling levels of live cells and total cells were calculated respectively. Figure 1 As shown: Figure 1 A in the figure shows that the smaller the initial seeding density, the greater the total cell population doubling level. Figure 1 Figure B shows that the live cell population doubling level is the highest when the initial seeding density is 4.00E+05 cells / mL. The number of live cells is a key indicator for evaluating cell quality. In summary, the optimal initial seeding density is 4.00E+05 cells / mL.
[0032] Example 4 This example provides a method for culturing Walker 256 cells. Different from Example 1, in this example, in the cell medium replacement and cell passaging steps, the culture medium used is RPMI1640 culture medium containing 10% FBS, 6 mM glutamine and 30 ng / mL EGF(a).
[0033] Referring to Example 2, when Walker 256 cells were cultured using the culture method of this example and passaged to the P12 generation, the cell viability could still reach 90.40%.
[0034] Example 5 This example provides a method for culturing Walker 256 cells. Different from Example 1, the initial cell seeding density is set to 5.00E+05 cells / mL in this example.
[0035] Referring to Example 2, when Walker 256 cells were cultured using the culture method of this example and passaged to the P12 generation, the cell viability was 90.12%.
[0036] Comparative Example 1 This example provides a method for culturing Walker 256 cells. The difference from Example 1 is that in the cell medium replacement and cell passaging steps of this example, the RPMI 1640 medium containing 10% FBS is used to replace the RPMI 1640 medium containing 10% FBS, 8 mM glutamine and 20 ng / mL EGF(a) in Example 1, and other operations are the same as in Example 1.
[0037] Comparative Example 2 This example provides a method for culturing Walker 256 cells. The difference from Example 1 is that in the cell medium replacement and cell passaging steps of this example, the RPMI 1640 medium containing 10% FBS+8mM glutamine is used to replace the RPMI 1640 medium containing 10% FBS, 8mM glutamine and 20ng / mL EGF(a) in Example 1, and other operations are the same as Example 1.
[0038] The cell viability within 4 days of cell subculture in Example 1 and Comparative Examples 1-2 was detected, and the results are shown in Table 2.
[0039] Table 2 Comparison of cell viability under different culture treatments
[0040] From the results in Table 2, it can be seen that under the conditions of RPMI 1640 medium with 10% FBS, 8mM glutamine and 20ng / mL EGF(a), the cell viability increased from the initial 72.36% to 94.26%, and the cell viability increased significantly; under the conditions of RPMI 1640 medium with 10% FBS, the cell viability did not increase and was always significantly lower than the initial viability; in Comparative Example 2, the cell viability increased slightly in the early stage, but the cell viability decreased slightly after culturing the cells for 4 days.
[0041] At the same time, the 24h cell population doubling level (PDL) within 4 days of cell subculture in Example 1 and Comparative Examples 1-2 was also tested, and the test results are shown in Table 3: Table 3 Cell population doubling levels under different culture treatments
[0042] As can be seen from Table 3, when cultured in 10% FBS+RPMI 1640 medium, the average population doubling level of Walker 256 cells every 24 hours was 0.63; when cultured in 10% FBS, 8mM glutamine and 20ng / mL EGF(a) medium, the average population doubling level of Walker 256 cells every 24 hours was 1.23; when cultured in 10% FBS+RPMI 1640+8mM glutamine medium, the average population doubling level of Walker 256 cells every 24 hours was 1.01.
[0043] Comparative Example 3 Different from Example 1, the initial cell seeding density in this example is 3.00E+05 cells / mL, and the cell culture time is 72 hours. Other operations are the same as in Example 1.
[0044] The viable cell density and total cell density at different culture times were tested, and the results were as follows Figure 2 As shown. Figure 2 It can be seen that the cell density reached the maximum at 48 hours of cell culture, and the cell concentration began to decrease after 48 hours, and the cell growth rate was lower than the cell death rate.
[0045] The raw materials listed in the present invention, as well as the upper and lower limits and interval values of the raw materials of the present invention, and the upper and lower limits and interval values of the process parameters (such as temperature, time, etc.) can all realize the present invention, and the embodiments are not listed one by one here.
[0046] In summary, the method of the present invention overcomes the problems of uneven quality and large batch-to-batch differences in the existing Walker 256 cell culture methods, and cultured high-quality Walker 256 cells with the same growth rate, consistent population doubling time, and high viability, and the cell quality consistency is good, the batch-to-batch difference is small, and the generation number can reach 12, which is of great significance for the high-quality and large-scale production of Walker 256 cells.
[0047] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.
Claims
1. A method for culturing Walker 256 cells, characterized in that: The following steps are involved: Walker 256 cells are inoculated into fresh culture medium at a fixed ratio and cultured for a fixed time, the supernatant is discarded after centrifugation, the cell pellet is resuspended in fresh culture medium, the obtained cell suspension is inoculated into fresh culture medium at the fixed ratio and cultured continuously; Repeat the above steps for passaging.
2. The culture method according to claim 1, characterized in that The culture medium contains fetal bovine serum, glutamine and EGF(a).
3. The culture method according to claim 2, characterized in that The culture medium is RPMI 1640 medium supplemented with 10% fetal bovine serum, 6-10 mM glutamine and 10-50 ng / mL EGF(a).
4. The culture method according to claim 3, characterized in that The fixed time is selected from 3-4d.
5. The culture method according to claim 3, characterized in that The seeding density of the Walker 256 cells was (4.0-8.0)×10 5 cells / mL.
6. The culture method according to claim 4, characterized in that The number of passages was ≤12.
7. The culture method according to claim 1, characterized in that When the Walker 256 cells used are frozen cells, the following pre-treatment steps are also included: The frozen Walker 256 cells were thawed in a water bath, the cells and the freezing solution were separated by centrifugation, and the cells were inoculated into a culture medium containing fetal bovine serum and activated and cultured for 24 hours before replacing with fresh culture medium.
8. The culture method according to claim 1, characterized in that The culture conditions of the Walker 256 cells are: 37±0.5°C, 5±1.0% CO2, 110±10rpm.
9. The culture method according to claim 1, characterized in that: The centrifugal conditions are: 200 g centrifugation for 5 min.
10. The culture method according to claim 1, characterized in that: After the culture solution was transferred into the centrifuge tube, phosphate buffer was added to the culture container for rinsing, and the rinsing solution was transferred into the centrifuge tube, followed by centrifugation.