Monoclonal screening culture method for suspension culture cells

Abstract

The invention discloses a suspension culture cell monoclonal screening culture method. The method comprises the following steps: selecting cells under suspension culture conditions and inducing the cells to recover adherence; separating the single cells developed into obvious clones, and carrying out radioactive ray irradiation on the remaining cells, so that the remaining cells lose the multiplication capacity and become feeder layer cells; culturing the separated monoclonal cells, recording the culture time and culture environment, establishing a high-density cell bank, and establishing a growth time prediction formula for the multiple monoclonal cells according to the culture time; and calculating the specific time for culturing the required number of cells according to the growth time prediction formula established in the step S3. According to the method, the remaining cells are subjected to radioactive ray irradiation and are converted into feeder layer cells with important effects, so that the waste of cell resources is avoided, the flexible utilization of the cell resources is realized, and the treatment cost of the remaining cells is also remarkably reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of suspension culture cell monoclonal screening culture, in particular to a suspension culture cell monoclonal screening culture method. BACKGROUND

[0002] Cell culture technology as a key supporting technology in modern biological research and biopharmaceutical field, plays an indispensable role in many aspects. In particular, the screening and culture of suspension culture cell monoclonal, for the production of high purity, consistency of biological products, and in-depth study of cell biology mechanism, etc. have extremely important significance;

[0003] In terms of cell resource utilization, after the separation operation of monoclonal cells is completed, a large number of remaining cells will not be used and need to be handled separately. This practice not only causes waste of resources, but also increases the subsequent processing cost. On the other hand, in terms of cell culture efficiency, due to the lack of effective prediction means for the growth time of monoclonal cells, researchers are difficult to accurately plan the experimental process, resulting in low overall experimental efficiency. Moreover, the cell cycle states of the cells separated at the same time are different, making it difficult to uniformly control the growth time of different monoclonal cells, which brings many inconveniences to the subsequent cell culture work. Therefore, a suspension culture cell monoclonal screening culture method needs to be proposed.

[0004] In view of the problems in the related art, an effective solution has not been proposed so far. SUMMARY

[0005] In view of the problems in the related art, the present application proposes a suspension culture cell monoclonal screening culture method to overcome the above technical problems existing in the prior art.

[0006] To this end, the specific technical solutions adopted by the present application are as follows:

[0007] A suspension culture cell monoclonal screening culture method, the method comprising the following steps:

[0008] S1, selecting cells under suspension culture conditions and inducing cells to restore adhesion;

[0009] S2, separating single cells developed into obvious clones, and irradiating the remaining cells with radiation to make the remaining cells lose the ability to proliferate and become feeder layer cells;

[0010] S3, culturing the separated monoclonal cells, recording the culture time and culture environment, and establishing a high-density cell bank and a growth time prediction formula for a plurality of monoclonal cells according to the culture time;

[0011] S4, calculate the specific time for culturing to the required number of cells according to the growth time prediction formula established in S3, and use feeder layer cells for culturing cells with long culturing time, so that cells with long culturing time and cells with short culturing time can be cultured to completion at the same time.

[0012] As a preferred embodiment, the separating the single cells which develop into obvious clones and collecting and processing the remaining cells into feeder layer cells comprises the following steps:

[0013] S21, treat the clone culture with a digestive enzyme to prepare a single cell suspension, and determine the cell concentration using a cell counter;

[0014] The cell counter is an instrument that can quickly and accurately determine the cell concentration in a cell suspension. It counts and analyzes the size of cells by optical or electrical principles to determine the number of cells per unit volume.

[0015] S22, calculate the dilution ratio to gradually dilute the cell suspension to a concentration of 1-10 cells per microliter, and the specific formula is:

[0016] C1V1=C2V2;

[0017] Wherein, C1 is the concentration of the suspension before dilution, V1 is the volume of the sample taken from the suspension, C2 is the target concentration after dilution, and V2 is the total volume of the final dilution;

[0018] S23, collect the remaining cells and process the remaining cells into feeder layer cells.

[0019] As a preferred embodiment, the collecting the remaining cells and processing the remaining cells into feeder layer cells comprises the following steps:

[0020] S231, collect the remaining cells, transfer the remaining cell suspension to a centrifuge tube for centrifugation, after centrifugation, remove the supernatant and leave the cell pellet;

[0021] S232, add an appropriate amount of preheated culture medium to the centrifuge tube, gently blow the bottom of the centrifuge tube to resuspend the cells, and avoid the formation of bubbles;

[0022] S233, transfer the resuspended cell suspension to a container and place the container in a radiation irradiation device, set the irradiation time and dose for irradiation;

[0023] S234, transfer the irradiated cell suspension to a sterile culture dish, and then place the culture dish in a preheated carbon dioxide incubator;

[0024] S235, monitor the cell growth, when the cells are attached and grown and formed a complete monolayer, indicating that the feeder layer cell growth is complete.

[0025] When performing radiation exposure, it is necessary to ensure that the cells receive uniform exposure, and only when all cells receive sufficient radiation dose can the proliferation ability be effectively inhibited, and the unexposed cells continue to divide and grow.

[0026] As a preferred embodiment, the growth time prediction formula for each of the plurality of monoclonal cells is established by the following steps:

[0027] S341, according to the initial density, final density and growth time of the plurality of monoclonal cells, a growth time prediction formula is established, and the specific formula is:

[0028]

[0029] Where a and b are model parameters, N0 is the initial density, N1 is the final density, and t is the growth time.

[0030] As a preferred embodiment, the specific time for culturing the required number of cells is calculated according to the growth time prediction formula established in S3, and the feeder layer cell culture is used for cells with long culture time, so that cells with long culture time and cells with short culture time can be cultured to completion at the same time, including the following steps:

[0031] S41, monoclonal cells are taken out from the high-density cell bank, the specific number and density value of the monoclonal cells to be grown are determined, and the initial density of each in the high-density cell bank is obtained;

[0032] S42, the known density value data is substituted into the prediction formula to predict the specific growth time of each monoclonal cell;

[0033] S43, according to the calculated time, set the time range t1, the monoclonal cells that grow within t1 are monoclonal cells that are cultured to completion at the same time, and the monoclonal cells with growth time exceeding the maximum value of t1 are considered as monoclonal cells with long growth time;

[0034] S44, for monoclonal cells with long growth time, add them to the feeder layer cells that have grown into a monolayer for culture;

[0035] S45, monoclonal cells with growth time within t1 are cultured normally;

[0036] It should be noted that the specific steps of normal culture are as follows:

[0037] First, the cells taken out from the high-density cell bank are thawed in water bath;

[0038] Prepare the bioreactor for culture, and directly place the thawed cells in the bioreactor for scale-up culture.

[0039] It should be noted that by establishing a high-density cell bank, the efficiency of cell resource storage and management can be significantly improved. The high-density cell bank can store a large number of cells, including normal cells and cells in specific states or after processing, which can be easily accessed at any time, reducing the time and resource consumption of primary culture or re-induction.

[0040] S46. During the culture, the growth of the monoclonal cells is observed regularly, and the cell viability, cell morphology, and growth rate are monitored.

[0041] S47. When all the monoclonal cells reach the desired growth state, the cells are digested with trypsin and harvested, and stored.

[0042] As a preferred embodiment, the step of culturing the monoclonal cells with a long growth time in the feeder layer cells grown into a monolayer includes the following steps:

[0043] S441. The cells taken from the high-density cell bank are thawed in a water bath.

[0044] S442. The cells are evenly inoculated on the feeder layer cells grown into a monolayer.

[0045] S443. The culture environment and temperature are controlled to ensure that the cells are within the optimal environmental temperature.

[0046] The beneficial effects of the present application are:

[0047] 1. The present application separates individual cells that have developed into obvious clones, and irradiates the remaining cells with radiation, rendering the remaining cells unable to proliferate, thereby serving as feeder layer cells. This not only effectively utilizes the remaining cells, avoids wasting the remaining cells, saves cell resources, and reduces unnecessary cell expansion and processing costs.

[0048] 2. The present application records the culture time and culture environment by culturing the separated monoclonal cells, and establishes a high-density cell bank. The growth time prediction formula for multiple monoclonal cells based on the culture time can effectively improve the efficiency of cell culture and provide a strong basis for subsequent cell growth time prediction. The establishment of a high-density cell bank can quickly expand subsequent cell culture and speed up the efficiency of cell culture. In addition, even if the cells are separated at the same time, their cell cycle states may be different. Therefore, the establishment of a growth time prediction formula for different monoclonal cells can predict the growth time of different cells, thereby providing a strong basis for subsequent unified cell growth time.

[0049] 3. This invention calculates the expected growth time by establishing a growth time prediction formula and performs different treatments on cells with different growth times, which can improve the overall efficiency of the experiment. The growth time range is set according to the calculation results. When the cell growth time exceeds the maximum value of the range, it is considered that the culture time is too long. For cells with long culture times, feeder cells are used to shorten the culture time, thereby achieving the effects of unifying the culture time, optimizing resource utilization, and improving experimental efficiency. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 This is a flowchart of a method for screening and culturing single-clone cells in suspension culture according to an embodiment of the present invention. Detailed Implementation

[0052] To further illustrate the various embodiments, the present invention provides accompanying drawings, which are part of the disclosure of the present invention. These drawings are mainly used to illustrate the embodiments and can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. With reference to these drawings, those skilled in the art should be able to understand other possible implementation methods and the advantages of the present invention. The components in the drawings are not drawn to scale, and similar component symbols are generally used to represent similar components.

[0053] According to an embodiment of the present invention, a method for screening and culturing single-clone cells in suspension culture is provided.

[0054] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments, such as... Figure 1 As shown, a method for screening and culturing single clonal cells in suspension culture according to an embodiment of the present invention includes the following steps:

[0055] S1. Select cells under suspension culture conditions and induce cells to regain their adherence;

[0056] It should be noted that suspension culture enables large-scale, high-density cell expansion, meeting the needs of industrial production and the biopharmaceutical field, and significantly increasing cell yield. Secondly, by inducing cells to regain their adherence, specific cell functions or phenotypes can be regained. Since some adherent cells exhibit stronger differentiation capacity or functional activity in the adherent state, inducing their restoration of adherence is very important.

[0057] S2, separate the single cell which develops into obvious clone, and irradiate the remaining cells to make the remaining cells lose the ability of proliferation, and become feeder layer cells;

[0058] Further, the single cell which develops into obvious clone is separated, and the remaining cells are collected and treated to become feeder layer cells, including the following steps:

[0059] S21, treat the clone culture with digestive enzyme to prepare single cell suspension, and determine the cell concentration by using a cell counter;

[0060] S22, calculate the dilution ratio, and gradually dilute the cell suspension to a concentration of 1-10 cells per microliter, and the specific formula is:

[0061] C1V1=C2V2;

[0062] Wherein, C1 is the concentration of the suspension before dilution, V1 is the volume of the sample taken from the suspension, C2 is the target concentration after dilution, and V2 is the total volume of the final dilution;

[0063] S23, collect the remaining cells, and treat the remaining cells to become feeder layer cells

[0064] Further, the remaining cells are collected, and the remaining cells are treated to become feeder layer cells, including the following steps:

[0065] S231, collect the remaining cells, and transfer the remaining cell suspension to a centrifuge tube for centrifugation, after centrifugation, remove the supernatant and leave the cell precipitate;

[0066] S232, add an appropriate amount of preheated culture medium to the centrifuge tube, gently blow the bottom of the centrifuge tube to resuspend the cells, and avoid the generation of bubbles;

[0067] S233, transfer the resuspended cell suspension to a container, and place the container in a radiation irradiation device, set the irradiation time and dose for irradiation;

[0068] S234, transfer the irradiated cell suspension to a sterile culture dish, and place the culture dish in a preheated carbon dioxide incubator;

[0069] S235, monitor the cell growth, and when the cells adhere and grow to form a complete monolayer, it indicates that the feeder layer cells have grown.

[0070] It should be noted that the main purpose of irradiating the feeder layer cells is to eliminate their proliferative ability, so that the cells focus on providing support and nutrition without interfering with the growth of experimental cells. This treatment not only effectively inhibits the excessive proliferation of feeder layer cells, ensuring the accuracy of experimental results, but also maintains the normal function of cells, thereby optimizing the growth and differentiation conditions of experimental cells.

[0071] S3, culturing the isolated monoclonal cells, recording the culture time and culture environment, and establishing a high-density cell bank and a growth time prediction formula for the multiple monoclonal cells according to the culture time;

[0072] Further, the isolated monoclonal cells are cultured, the culture time and culture environment are recorded, and a high-density cell bank is established and a growth time prediction formula is established for the multiple monoclonal cells according to the culture time, the following steps:

[0073] S31, transferring the selected clones to larger culture containers and gradually increasing the culture volume to increase the cell density;

[0074] S32, during the culture process, the growth rate, morphology and density of the cells are regularly detected;

[0075] S33, presetting a density standard value, when the density reaches the preset value, recording the growth time of the multiple monoclonal cells, and storing the cells that have completed growth in liquid nitrogen to establish a high-density cell bank;

[0076] S34, establishing a growth time prediction formula for the cells grown by the multiple monoclonal cells.

[0077] It should be noted that even if the cells are isolated at the same time, their cell cycle states may be different, and the different synchronization of the cell cycle will cause some cells to grow and divide faster, while others will take longer to enter the next stage, in addition, there may be random fluctuations in the gene expression of individual cells, which will also affect their metabolic activity and growth rate.

[0078] Further, S341, establishing a growth time prediction formula according to the initial density, final density and growth time of the cells grown by the multiple monoclonal cells, the specific formula is:

[0079]

[0080] Where a and b are model parameters, N0 is the initial density, N1 is the final density, and t is the growth time;

[0081] It should be noted that during the cell culture experiment, the growth time of multiple monoclonal cells is significantly different due to their respective differences. Therefore, considering the uniqueness of the growth process of each monoclonal cell, the parameters of each model based on them are naturally different. Therefore, multiple models are established based on multiple monoclonal cells, which can more accurately predict the cell growth time.

[0082] S4, calculate the specific time to culture to the required number of cells according to the growth time prediction formula established in S3, and use feeder layer cells to culture cells with long culture time, so that cells with long culture time and cells with short culture time can be cultured to completion at the same time.

[0083] Feeder layer cells are a type of cells that lose their proliferation ability after treatment, but can provide support for the growth of other cells. In cell culture, feeder layer cells can secrete various growth factors such as epidermal growth factor and insulin-like growth factor, providing growth signals for target cells, like injecting power for cell growth, and promoting better proliferation. At the same time, it can also secrete nutrients such as transferrin to create a nutrient-rich environment for target cells and ensure that cells have sufficient nutrients to maintain normal metabolism.

[0084] It should be noted that this cell culture method can effectively optimize the experimental process, reduce unnecessary waiting time, and improve overall experimental efficiency. For cells with long culture time, using feeder layer cells for assistance can accelerate the growth rate of cells with long culture time, allowing them to reach the required number in a shorter time, thereby eliminating the problem of different steps caused by different cell growth rates and ensuring that all cells are cultured at the same time. point, it is convenient to process and subsequent experimental operation.

[0085] Further, according to the growth time prediction formula established in S3, the specific time to culture to the required number of cells is calculated, and feeder layer cells are used to culture cells with long culture time, so that cells with long culture time and cells with short culture time can be cultured to completion at the same time, including the following steps:

[0086] S41, monoclonal cells are taken out from the high-density cell bank, the specific number and density value of the monoclonal cells required for growth are determined, and the initial density of each in the high-density cell bank is obtained.

[0087] S42, substitute the known density value data into the prediction formula to predict the specific growth time of each monoclonal cell;

[0088] S43, according to the calculated time, set the time range t1, the monoclonal cells that grow within t1 are monoclonal cells that are cultured to completion at the same time, and the monoclonal cells with a growth time exceeding the maximum value of t1 are considered as monoclonal cells with long growth time.

[0089] S44, the long time growth of monoclonal cells, which are added to the feeder layer cells have been grown into a single layer of culture;

[0090] Further, the long time growth of monoclonal cells, which are added to the feeder layer cells have been grown into a single layer of culture includes the following steps:

[0091] S441, the cells from the high-density cell bank to water bath thawing;

[0092] S442, the cells are evenly seeded in the feeder layer cells have been grown into a single layer;

[0093] S443, control the culture environment and temperature, to ensure that the cells are in the optimum temperature

[0094] S45, the normal culture of monoclonal cells in the range of t1 time growth;

[0095] It should be noted that the specific steps of normal culture are:

[0096] First, the cells from the high-density cell bank to water bath thawing;

[0097] Preparation of the bioreactor for culture, the thawed cells are placed directly in the reaction of biological reactor for amplification culture.

[0098] It should be noted that by establishing a high-density cell bank, the efficiency of cell resource storage and management can be significantly improved. The high-density cell bank can store a large number of cells, including normal cells and cells in specific states or after treatment, which can be easily taken at any time, reducing the time and resource consumption from primary culture or reinduction.

[0099] S46, during the culture, the growth of monoclonal cells is observed regularly, and the cell viability, cell morphology and growth rate are monitored;

[0100] S47, when all the monoclonal cells reach the expected growth state, the cells are digested and harvested using trypsin, and are stored.

[0101] It should be noted that during the culture of monoclonal cells, metabolic waste such as lactic acid, ammonia and other toxic metabolites will gradually accumulate with the proliferation of cells. If not removed, these waste will be toxic to cells, inhibiting their growth, differentiation and even leading to cell death. Regular removal of metabolic waste can maintain the health of the culture environment and promote the normal growth and functional expression of cells.

[0102] In summary, the present application separates single cells that develop into obvious clones, and irradiates the remaining cells with radiation, so that the remaining cells lose the ability to proliferate and become feeder layer cells. Thus, not only can the remaining cells be effectively utilized, but the remaining cells are also prevented from being wasted, cell resources are saved, and unnecessary cell expansion and processing costs are reduced. The present application records the culture time and culture environment of the separated single clones, and establishes a high-density cell bank. A growth time prediction formula is established for multiple single clone cells according to the culture time, which can effectively improve the efficiency of cell culture and provide a strong basis for subsequent cell growth time prediction. The establishment of a high-density cell bank can quickly expand subsequent cell culture and speed up the efficiency of cell culture. In addition, even if the cells are separated at the same time, their cell cycle states may be different. Therefore, a growth time prediction formula is established for different single clone cells to predict the growth time of different cells, thereby providing a strong basis for subsequent unified cell growth time. The present application calculates the predicted growth time by establishing a growth time prediction formula, and different cells with different growth times are subjected to different treatments, which can improve the overall efficiency of the experiment. According to the calculation result, the growth time range is set. When the cell growth time exceeds the maximum value of the range, it is considered that the culture time is long. The use of feeder cells for culture shortens the culture time, thereby achieving the effect of unifying the culture time and optimizing the resource utilization to improve the experimental efficiency.

[0103] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for screening a monoclonal culture of cells in suspension culture, characterized by, The method comprises the following steps: S1, selecting cells in a suspension culture condition and inducing the cells to restore adhesion; S2, separating the single cells developed into obvious clones, and irradiating the remaining cells to make the remaining cells lose the ability to proliferate and become feeder cells; S3, culturing the separated single clones, recording the culture time and culture environment, and establishing a high-density cell bank and a growth time prediction formula for the multiple single clone cells according to the culture time; S4, calculating the specific time for culturing to the required number of cells according to the growth time prediction formula established in S3, and using feeder cells to culture the cells with long culture time, so that the cells with long culture time and the cells with short culture time can be cultured to completion at the same time.

2. The method for screening and culturing single-clone cells in suspension culture according to claim 1, characterized in that, The separating the single cells developed into obvious clones and collecting and processing the remaining cells to become feeder cells comprises the following steps: S21, treating the clone culture with a digestive enzyme to prepare a single cell suspension, and determining the cell concentration using a cell counter; S22, calculating a dilution ratio to gradually dilute the cell suspension to a concentration of 1-10 cells per microliter, and the specific formula is: C1V1=C2V2; Wherein, C1 is the concentration of the suspension before dilution, V1 is the volume of the sample taken from the suspension, C2 is the target concentration after dilution, and V2 is the total volume of the final diluent; S23, collecting the remaining cells and processing the remaining cells to become feeder cells.

3. The method of claim 2, wherein the cells are cultured in suspension.

3. The method of claim 2, wherein the cells are cultured in suspension. The collecting the remaining cells and processing the remaining cells to become feeder cells comprises the following steps: S231, collecting the remaining cells, transferring the remaining cell suspension to a centrifuge tube for centrifugation, removing the supernatant after centrifugation, and leaving the cell precipitate; S232, adding an appropriate amount of preheated culture medium to the centrifuge tube, gently blowing the bottom of the centrifuge tube to resuspend the cells, and avoiding the generation of air bubbles; S233, transferring the resuspended cell suspension to a container and placing the container in a radiation irradiation device, setting the irradiation time and dose for irradiation; S234, transferring the irradiated cell suspension to a sterile culture dish, and placing the culture dish in a preheated carbon dioxide incubator; S235, monitoring cell growth, and when the cells adhere and grow to form a complete monolayer, it indicates that the feeder cell growth is complete.

4. The method for screening and culturing single-clone cells in suspension culture according to claim 1, characterized in that, The culturing the separated single clones, recording the culture time and culture environment, and establishing a high-density cell bank and a growth time prediction formula for the multiple single clone cells according to the culture time comprises the following steps: S31, transferring the selected clones to larger culture containers, gradually increasing the culture volume to increase the cell density; S32, regularly detecting the growth rate, morphology and density of the cells during the culture process; S33, presetting a density standard value, recording the growth time of multiple single clones when the density reaches the preset value, and storing the cells that have grown to completion in liquid nitrogen to establish a high-density cell bank; S34, establishing a growth time prediction formula for the multiple single clone cells.

5. The method of claim 4, wherein the cells are cultured in suspension. 5 The specific steps for establishing a growth time prediction formula for the multiple single clone cells are as follows: S341, according to the initial density, final density and growth time of the plurality of monoclonal cells, a growth time prediction formula is established, and the specific formula is: Wherein, a and b are model parameters, N0 is the initial density, N1 is the final density, and t is the growth time.

6. The method of claim 1, wherein the cells are cultured in suspension. 5 The specific time for culturing the required number of cells is calculated according to the growth time prediction formula established in S3, and the cells with long culture time are cultured using feeder cells, so that the cells with long culture time and the cells with short culture time can be cultured to completion at the same time, including the following steps: S41, monoclonal cells are taken out from the high-density cell bank, the specific number and density value of the monoclonal cells required to grow are determined, and the initial density of each monoclonal cell in the high-density cell bank is obtained; S42, the known density value data is substituted into the prediction formula to predict the specific growth time of each monoclonal cell; S43, according to the calculated time, a time range t1 is set, and the monoclonal cells that grow within t1 are considered to be cultured to completion at the same time, and the monoclonal cells with a growth time exceeding the maximum value of t1 are considered to be monoclonal cells with long growth time; S44, the monoclonal cells with long growth time are added to the feeder cells that have grown into a monolayer for culture; S45, the monoclonal cells with growth time within t1 are normally cultured; S46, during the culture, the growth of the monoclonal cells is observed regularly, and the cell viability, cell morphology and growth rate are monitored; S47, when all the monoclonal cells reach the expected growth state, trypsin digestion is used to harvest the cells and store them.

7. The method of claim 6, wherein the cells are cultured in suspension. The monoclonal cells with long growth time are added to the feeder cells that have grown into a monolayer for culture, including the following steps: ​ S441, the cells taken out from the high-density cell bank are thawed in water bath; S442, the cells are evenly inoculated on the feeder cells that have grown into a monolayer; S443, the culture environment and temperature are controlled to ensure that the cells are in the best environment temperature.

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