Condition optimization method for integrated cell culture and detection and application thereof

Abstract

The invention discloses an integrated cell culture and detection condition optimization method, which comprises the following steps of: establishing an oxygen consumption and culture solution pH value prediction model for cell culture in a closed system; the conditions such as cell inoculation amount, cell culture liquid volume, cell culture time and initial sodium bicarbonate addition amount required by closed system cell culture are optimized according to the model prediction trend, and the oxygen partial pressure in the gas phase of the closed headspace sample bottle is maintained at the physiological level of human tissues and organs within the culture time range; the pH value of the culture solution is maintained near the physiological level (pH = 7.4), the growth curve of the cells cultured under the closed system under the optimized culture condition is consistent with that of the cells cultured under the traditional air-permeable cell culture, the method provided by the invention can realize in-situ detection of the cell growth activity in the closed system, realizes integrated cell culture and detection, does not need to damage the closed state of the cells, and is suitable for large-scale production. The cell culture environment is free of pollution risk and steady-state damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a method for optimizing the conditions for integrated cell culture and detection and its application. Background Art

[0002] As an important basic means of modern biomedical research, in vitro cell culture technology has wide application value in fields such as drug development, toxicity testing, and basic biological research. In related technologies, in conventional open or breathable cell culture, adding sodium bicarbonate to the culture medium in advance and maintaining 5% carbon dioxide in the gas phase can better maintain the pH value of the culture medium suitable for cell growth during cell culture. And the low-cost cell culture system based on a closed culture bottle has attracted attention due to its low pollution risk and high space utilization rate. However, the conventional breathable or open cell culture method requires frequent opening of the culture container for sampling and detection, increasing the pollution risk and disrupting the homeostasis of the culture environment; in the closed cell culture system, the CO2 produced by cell respiration continuously accumulates in the closed space, while the irreversible consumption of dissolved oxygen cannot be effectively replenished, causing the pH value of the culture solution to drop, leading to cell metabolic disorders and seriously affecting cell proliferation activity and survival rate. Summary of the Invention

[0003] Based on the technical problems proposed in the above background art, an embodiment of the present invention provides a cell culture method based on optimizing culture conditions with a mathematical model, which can achieve low pollution, high throughput, and easy operation, and can realize integrated cell culture and in-situ detection of cell growth under a closed system.

[0004] An embodiment of the present invention provides a method for optimizing the conditions for integrated cell culture and detection, including the following steps:

[0005] (1) Establish a prediction model for oxygen consumption in closed-system cell culture;

[0006] (2) Establish a prediction model for the pH value of the culture solution in closed-system cell culture;

[0007] (3) Use the oxygen consumption prediction model and the initial oxygen content in the closed system to obtain the optimized volume of the cell culture solution, cell seeding amount, and cell culture time;

[0008] (4) Use the pH value prediction model to obtain the initial amount of sodium bicarbonate added to the optimized cell culture solution;

[0009] (5) Use the above optimized volume of the cell culture solution, cell seeding amount, cell culture time, and initial amount of sodium bicarbonate added to culture cells in a closed headspace sample bottle;

[0010] (6) After the culture is completed, use a headspace gas chromatography system to in-situ detect the content of CO2 generated during cell culture.

[0011] Further, in step (1), the oxygen consumption prediction model is the model of oxygen consumption varying with culture time in closed-system cell culture; the oxygen consumption prediction model is as follows:

[0012]

[0013] where O 2cons is the oxygen consumption in closed-system cell culture; OCR is the oxygen consumption per single cell per unit time of the cultured cells, mmol; N0 is the initial cell seeding amount; k = 1 / 24 h -1 , which is the cell growth rate; t is the cell culture time, h.

[0014] Further, in step (2), the pH value prediction model is the model of the pH value of the culture medium varying with time in closed-system cell culture; the pH value prediction model is as follows:

[0015]

[0016] where pH (t) is the pH value of the cell culture medium at time t; [HCO3 - 0 is the initial addition amount of sodium bicarbonate in the cell culture medium, mM; m (t) is the amount of CO2 generated at time t during cell culture, mmol; H = 0.642, which is the dimensionless Henry's constant of CO2 at 37°C; V l is the volume of the culture medium in the headspace sample bottle, mL; pKa = 6.15, which is the dissociation constant of carbonic acid at 37°C.

[0017] Further, in step (3), the initial oxygen content in the headspace sample bottle under a closed system is calculated by formula (3);

[0018] n T = -7.55V l + 167 (3)

[0019] where n T is the initial oxygen content in the headspace sample bottle under a closed system, μmol; V l is the volume of the culture medium in the headspace sample bottle, mL; the volume of the headspace sample bottle is 21.6 mL.

[0020] Further, according to the cell seeding amount, culture time, and culture medium volume, the amount of CO2 generated m (t) during cell culture under a closed system is measured by a headspace gas chromatography system at time t in a medium without sodium bicarbonate; the generation amount of CO2 is calibrated by the reaction of reference sodium carbonate with excessive hydrochloric acid in a closed headspace bottle.

[0021] Further, in the step (6), after the culture is completed, the headspace sample culture bottle is immediately placed in a headspace sampler for in-situ headspace gas chromatography analysis to collect CO2 chromatographic peak information; the area of the CO2 chromatographic peak is integrated in the gas chromatogram, and cell growth viability data is obtained through the area of the CO2 chromatographic peak.

[0022] An embodiment of the present invention also provides an application of a method for optimizing the conditions of integrated cell culture and detection, and the method is applied to the optimization of the conditions of integrated cell culture and the in-situ detection of cell growth viability under a closed system.

[0023] An embodiment of the present invention also provides an application of a method for optimizing the conditions of integrated cell culture and detection, and the oxygen consumption prediction model in the closed-system cell culture is applied to optimize the conditions of cell culture medium volume, cell seeding amount, and cell culture time in the closed-system cell culture.

[0024] An embodiment of the present invention also provides an application of a method for optimizing the conditions of integrated cell culture and detection, and the pH value prediction model in the closed-system cell culture is applied to optimize the initial sodium bicarbonate addition amount in the cell culture medium.

[0025] The beneficial effects brought by the technical solution provided by the embodiment of the present invention are as follows: The present invention provides a method for optimizing the conditions of integrated cell culture and detection, establishes an oxygen consumption prediction model and a culture medium pH value prediction model for cell culture under a closed system, and optimizes the conditions for culturing cells under the closed system according to the prediction models, including cell seeding amount, cell culture medium volume, culture time, initial sodium bicarbonate addition amount, etc. Culturing cells according to the optimized conditions can keep the oxygen amount and the culture medium pH value within the range suitable for cell growth during the cell culture process within a certain culture time range under the closed system, and in-situ detection can be carried out to realize integrated cell culture and detection. The method of the present invention can be applied to optimize the conditions of integrated cell culture and realize the in-situ detection of cell growth viability. The method of the present invention can realize in-situ detection without opening the container, has low cost for culturing cells under the closed system of the present invention, is easy to operate, and has no pollution risk to the cell growth environment. Description of the Drawings

[0026] Figure 1 It is a schematic flow chart of the method for optimizing the conditions of integrated cell culture and detection in Embodiment 1 of the present invention.

[0027] Figure 2 It is a graph showing the change of CO2 production amount with time during the culture of HepG2 cells measured in Embodiment 2 of the present invention.

[0028] Figure 3 It is a prediction graph of the change trend of the pH value of the HepG2 cell culture medium with time in Embodiment 2 of the present invention.

[0029] Figure 4 It is a comparison graph of the growth vitality of HepG2 cells cultured by the method of the embodiment of the present invention and the traditional cell culture method (SRB method). Specific embodiments

[0030] To make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be further described below in conjunction with the accompanying drawings.

[0031] As a mature detection means for volatile substances, headspace gas analysis technology has shown unique advantages in the field of microbial culture detection. By analyzing the changes in the gas composition of the headspace part of a sealed container, this technology can indirectly reflect the metabolic activity characteristics of the culture system. Existing research shows that during the cultivation of bacteria, the growth index can be automatically detected by headspace gas chromatography (HS-GC) technology. However, the application of this technology in the field of cell culture with a greater oxygen consumption rate and a faster accumulation rate of carbon dioxide has not yet formed a systematic solution. How to construct an integrated system for culture and in-situ detection based on headspace gas analysis, and synchronously solve the problems of contamination in an open system and the dynamic balance of pH regulation and oxygen supply in a closed system has become a technical problem that urgently needs to be broken through in this field.

[0032] Example 1

[0033] Referring to the attached Figure 1 , the embodiment of the present invention provides an optimization method for the conditions of integrated cell culture and detection, including the following steps:

[0034] (1) Establish a prediction model for oxygen consumption in closed-system cell culture;

[0035] (2) Establish a prediction model for the pH value of the culture medium in closed-system cell culture;

[0036] (3) Use the oxygen consumption prediction model and the initial oxygen content in the closed system to obtain the optimized volume of cell culture medium, cell seeding amount, and cell culture time;

[0037] (4) Use the pH value prediction model to obtain the initial sodium bicarbonate addition amount in the optimized cell culture medium;

[0038] (5) Use the above optimized volume of cell culture medium, cell seeding amount, cell culture time, and initial sodium bicarbonate addition amount to culture cells in a closed headspace sample bottle;

[0039] (6) After the culture is completed, use a headspace gas chromatography system to in-situ detect the amount of CO2 generated during cell culture.

[0040] In an embodiment of the present invention, the optimized conditions for cell culture in a closed system are obtained according to the prediction model, and a headspace gas chromatography system is used to realize in-situ detection of cell growth viability during cell culture in a closed system, so as to realize integrated cell culture and in-situ detection of growth in a closed system; the cell growth state in the closed system can be obtained without any impact on the cell culture environment.

[0041] Preferably, in step (1), the oxygen consumption prediction model is a model of the change of oxygen consumption with culture time in cell culture in a closed system; the oxygen consumption prediction model is:

[0042]

[0043] wherein, O 2cons is the oxygen consumption in cell culture in a closed system; OCR is the oxygen consumption per single cell of the cultured cells per unit time, mmol; N0 is the initial cell seeding amount; k = 1 / 24h -1 , which is the cell growth rate; t is the cell culture time, h. According to OCR, the initial seeding amount and culture time of the cells cultured in the closed system are optimized by using the oxygen consumption prediction model. Based on the ratio of the oxygen consumption to the total initial amount of oxygen (the corresponding initial partial pressure is 21%), it is ensured that the oxygen partial pressure in the gas phase of the closed headspace sample bottle is maintained at the physiological level of human tissues and organs (i.e., 4 - 14%) within the culture time range. Therefore, according to the oxygen consumption prediction model of the embodiment of the present invention, the change of the oxygen amount in the closed system over time can be known, and the influence of the consumption of dissolved oxygen on cell proliferation activity and survival rate in the closed system can be avoided.

[0044] Preferably, in step (2), the pH value prediction model is a model of the change of the pH value of the culture medium with time in cell culture in a closed system; the pH value prediction model is:

[0045]

[0046] wherein, pH (t) is the pH value of the cell culture medium at time t; [HCO3 - 0 is the initial addition amount of sodium bicarbonate in the cell culture medium, mM; m (t) is the amount of CO2 generated at time t during cell culture, mmol; H = 0.642, which is the dimensionless Henry's constant of CO2 at 37°C; V l is the volume of the culture medium in the headspace sample bottle, mL; pKa = 6.15, which is the dissociation constant of carbonic acid at 37°C. The CO2 produced by cell respiration continuously accumulates in the closed space, causing the pH value of the culture medium to decrease and leading to cell metabolic disorders; according to the pH value prediction model of the embodiment of the invention, the change of the pH value of the culture medium in the closed system with the amount of CO2 generated can be known, ensuring that the cells grow at an appropriate pH value.

[0047] Preferably, in step (3), the initial oxygen content in the headspace vial under a closed system is calculated by formula (3);

[0048] n T = -7.55V l +167 (3)

[0049] wherein, n T is the initial oxygen content in the headspace vial under a closed system, μmol; V l is the volume of the culture medium in the headspace vial, mL; the volume of the headspace vial is 21.6 mL.

[0050] Preferably, according to the cell seeding amount, culture time, and volume of the culture medium, when the culture time in the medium without sodium bicarbonate is t, the CO2 generation amount m during the cell culture process under a closed system is measured by a headspace gas chromatography system; the generation amount of CO2 is calibrated by the reaction of reference sodium carbonate with excessive hydrochloric acid in a closed headspace bottle. According to the pH value prediction model and formula (3), the initial addition amount of sodium bicarbonate in the cell culture medium can be optimized to ensure that the pH value of the culture medium is between 6 and 8 within the culture time range to adapt to cell growth. (t) ; the generation amount of CO2 is calibrated by the reaction of reference sodium carbonate with excessive hydrochloric acid in a closed headspace bottle. According to the pH value prediction model and formula (3), the initial addition amount of sodium bicarbonate in the cell culture medium can be optimized to ensure that the pH value of the culture medium is between 6 and 8 within the culture time range to adapt to cell growth.

[0051] Preferably, in step (6), after the culture is completed, the headspace sample culture bottle is immediately placed in a headspace sampler for in-situ headspace gas chromatography analysis to collect CO2 chromatographic peak information; the area of the CO2 chromatographic peak is integrated in the gas chromatogram, and cell growth viability data is obtained through the area of the CO2 chromatographic peak.

[0052] The embodiment of the present invention also provides an application of a method for optimizing the conditions of integrated cell culture and detection, and the method is applied to the optimization of the conditions of integrated cell culture and the in-situ detection of cell growth viability under a closed system.

[0053] The embodiment of the present invention also provides an application of a method for optimizing the conditions of integrated cell culture and detection, and the oxygen consumption prediction model in the closed system cell culture is applied to optimize the conditions of cell culture medium volume, cell seeding amount, and cell culture time in the closed system cell culture.

[0054] The embodiment of the present invention also provides an application of a method for optimizing the conditions of integrated cell culture and detection, and the pH value prediction model in the closed system cell culture is applied to optimize the initial addition amount of sodium bicarbonate in the cell culture medium.

[0055] Example 2

[0056] Using the integrated cell culture condition optimization method described in Example 1, the cell culture conditions in a closed system were optimized. In the embodiment of the present invention, human hepatoma cells HepG2 were cultured in a closed system, namely a headspace sample bottle.

[0057] Refer to the appendix Figure 2 , according to the oxygen consumption prediction model (1), the change trend of the oxygen consumption of HepG2 cells over time at different cell seeding densities (N0) was obtained. Among them, the oxygen consumption rate per unit time of a single cell OCR is 1.1×10 -16 mol. According to the conventional estimation of the culture volume in cell culture, that is: 0.2 - 0.5 mL of culture medium per cm 2 bottom area, the bottom area of the headspace sample bottle used in the embodiment of the present invention is 3 cm 2 , and the culture medium volume used is 1 mL. According to formula (3), when the culture medium volume (V l ) is 1 mL, the initial oxygen content in the headspace sample bottle (21.6 mL) is 160 μmol, and the partial pressure of gaseous oxygen is 21%. If the partial pressure of gaseous oxygen in the headspace sample bottle is maintained above the physiological level of human tissues (>4%), the remaining sample content in the headspace sample bottle needs to be above 30.5 μmol, that is, the oxygen content available for cell consumption is 129.5 μmol. From Figure 2 , it can be seen that when the cell seeding density is 10 5 or less, when the cell culture duration exceeds 96 h, the cumulative oxygen consumption of the cells is still much lower than 129.5 μmol, that is, the partial pressure of gaseous oxygen is still above the physiological level of human tissues; when the cell seeding density is 10 6 , after the cell culture duration is 81 h, the partial pressure of gaseous oxygen will be lower than the physiological level, and the cell culture conditions will be unfavorable for cell growth. According to the above optimization, the cell seeding density used in this example is 1.5×10 4 , and this seeding density can ensure that within 96 h of cell culture, the partial pressure of gaseous oxygen in the headspace bottle is still much higher than the physiological level of human tissues. Therefore, the cell culture durations used in the embodiments of the present invention are 6, 12, 24, 30, 36, 42, 48, 54, 60, 66, and 72 h.

[0058] Refer to the appendix Figure 3 , under the optimized culture medium volume, cell seeding density, and cell culture time, the amount of CO2 (m (t) ) generated by HepG2 cells cultured in a medium without sodium bicarbonate was measured in this example. Among them, the amount of CO2 was calibrated by the reaction of reference sodium carbonate with excessive hydrochloric acid. According to the experimental measurement data of m (t) , the pH change at different initial sodium bicarbonate addition amounts was calculated using the culture medium pH prediction model. When HepG2 cells were cultured in the headspace sample bottle for 72 h at different initial sodium bicarbonate addition amounts, the pH value of the culture medium was still in the range of 6 - 8, such asFigure 3 As shown, the sodium bicarbonate level was selected according to the predicted value, taking 10 mmol. Within 72 h of cultivation, the pH value of the culture medium was maintained near the physiological level of human tissues (pH = 7.4), that is, 6.8 - 7.6.

[0059] According to the optimized culture medium volume (1 mL), cell seeding amount (1.5×10 4 ), cell culture time (6, 12, 24, 30, 36, 42, 48, 54, 60, 66, and 72 h), and initial sodium bicarbonate addition amount (10 mM), in this example, HepG2 cells were cultured, and the amount of CO2 produced by cell metabolism was detected at the above-mentioned culture time points, which was used to represent cell growth activity. At the same time, in this example, HepG2 cells were cultured using the traditional breathable type at the same culture time points, and the cell growth activity was detected by the sulforhodamine B method (SRB). The results were consistent with the optimized integrated cell culture and detection method of the present invention. As Figure 4 shown, the correlation coefficient R 2 = 0.98. The results show that the cell culture conditions optimized by the method of this example of the present invention not only have the same trend of cell viability as the traditional scheme, but also solve the problems of cross-contamination and cumbersome sampling operations existing in traditional breathable cell culture.

[0060] In this article, the front, back, up, down, and other orientation words are defined based on the positions of the components in the drawings and the positions of the components relative to each other, only for the sake of clarity and convenience in expressing the technical solution. It should be understood that the use of the orientation words should not limit the scope of protection requested by this application.

[0061] Without conflict, the above-mentioned embodiments and the features in the embodiments in this article can be combined with each other.

[0062] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An optimization method for the conditions of integrated cell culture and detection, characterized in that, It includes the following steps: (1) Establish a prediction model for oxygen consumption in closed-system cell culture; (2) Establish a prediction model for the pH value of the culture medium in closed-system cell culture; (3) Use the oxygen consumption prediction model and the initial oxygen content in the closed system to obtain the optimized cell culture medium volume, cell seeding amount, and cell culture time; (4) Use the pH value prediction model to obtain the initial sodium bicarbonate addition amount in the optimized cell culture medium; (5) Use the above-optimized cell culture medium volume, cell seeding amount, cell culture time, and initial sodium bicarbonate addition amount to culture cells in a closed headspace sample bottle; (6) After the culture is completed, use a headspace gas chromatography system to in-situ detect the CO2 content generated during cell culture.

2. The method for optimizing conditions for integrated cell culture and detection according to claim 1, characterized in that, In step (1), the oxygen consumption prediction model is a model of the change in oxygen consumption with culture time in closed-system cell culture; the oxygen consumption prediction model is: Among them, O 2cons is the oxygen consumption in closed-system cell culture; OCR is the oxygen consumption per single cell of the cultured cells per unit time, mmol; N0 is the initial cell seeding amount; k = 1 / 24 h -1 , is the cell growth rate; t is the cell culture time, h.

3. The optimized method for the conditions of an integrated cell culture and detection according to claim 1, characterized in that, In step (2), the pH value prediction model is a model of the change in the pH value of the culture medium with time in closed-system cell culture; the pH value prediction model is: Among them, pH (t) is the pH value of the cell culture medium at time t; [HCO3 - 0 is the initial addition amount of sodium bicarbonate in the cell culture medium, mM; m (t) is the amount of CO2 generated at time t during cell culture, mmol; H = 0.642, which is the dimensionless Henry constant of CO2 at 37°C; V l is the volume of the culture medium in the headspace sample bottle, mL; pKa = 6.15, which is the dissociation constant of carbonic acid at 37°C.

4. An optimization method for the conditions of integrated cell culture and detection according to claim 1, characterized in that, In step (3), the initial oxygen content in the headspace sample bottle under the closed system is calculated by formula (3); n T = -7.55 V l +167 (3) where n T is the initial total amount of oxygen in the headspace sample bottle of the closed system, in μmol; V l is the volume of the culture medium in the headspace sample bottle, in mL; the volume of the headspace sample bottle is 21.6 mL.

5. The method for optimizing conditions for integrated cell culture and detection according to claim 3, characterized in that, According to the cell seeding density, culture time, and culture medium volume, the amount of CO2 generated m during the cell culture process in a closed system is measured by a headspace gas chromatography system at time t in a culture medium without sodium bicarbonate. (t) The amount of generated CO2 is calibrated by the reaction of reference sodium carbonate with excessive hydrochloric acid in a closed headspace bottle.

6. The method for optimizing conditions for integrated cell culture and detection according to claim 1, characterized in that In step (6), after the culture is completed, immediately place the headspace sample culture bottle in a headspace sampler for in-situ headspace gas chromatography analysis, collect CO2 chromatographic peak information; integrate the CO2 chromatographic peak area in the gas chromatogram, and obtain cell growth viability data through the CO2 chromatographic peak area.

7. Use of the method for optimizing conditions for integrated cell culture and detection according to claim 1, characterized in that, The method is applied to the condition optimization of integrated cell culture and the in-situ detection of cell growth viability.

8. Use of the method for optimizing conditions for integrated cell culture and detection according to claim 1, characterized in that The oxygen consumption prediction model in the closed-system cell culture is applied to optimize the cell culture medium volume, cell seeding amount, and cell culture time conditions in the closed-system cell culture.

9. Use of the method for optimizing conditions for integrated cell culture and detection according to claim 1, characterized in that, The pH value prediction model in the closed-system cell culture is applied to optimize the initial sodium bicarbonate addition amount in the cell culture medium.

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