CN106754651B - Method for culturing effective passage of human diploid cell microcarrier - Google Patents
Method for culturing effective passage of human diploid cell microcarrier Download PDFInfo
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- CN106754651B CN106754651B CN201710112043.2A CN201710112043A CN106754651B CN 106754651 B CN106754651 B CN 106754651B CN 201710112043 A CN201710112043 A CN 201710112043A CN 106754651 B CN106754651 B CN 106754651B
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- C12N2531/00—Microcarriers
Abstract
The invention relates to the technical field of biology, in particular to a method for effectively passaging human diploid cells in a bioreactor microcarrier amplification culture process. The method comprises the steps of culturing human diploid cell seeds by using microcarriers, pre-balancing blank microcarriers in a seed culture system, digesting the seed cells by using pancreatin digestive juice containing sodium citrate, carrying out mild intermittent stirring culture in a seed reactor for 3-6 hours, transferring to an amplification reactor for amplification culture, and obtaining effective passage in the amplification culture process of the human diploid cell microcarriers. The invention has the advantages of improving the activity, the adhesion rate and the adhesion uniformity of cells in the passage process, obviously improving the passage efficiency and solving the problem that the human diploid cells are difficult to amplify and culture on a bioreactor and a microcarrier.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an effective passage method for human diploid cells in the process of amplification culture of microcarriers in a bioreactor.
Background
The diploid cells of MRC-5, KMB17, WI-38 and the like are in-vitro cultured cells derived from normal tissues of human bodies, have no potential carcinogenicity, no exogenous factor pollution and wide virus sensitivity spectrum, are considered to be the safest and most suitable cell matrix at present, and are widely applied to the production of vaccines.
Large-scale culture techniques of animal cells are widely used for the production of biologicals. Since the 80 s in the 20 th century, a culture technology integrating a bioreactor and a microcarrier is gradually established, the technology has the advantages of monolayer adherent culture and suspension culture, the specific surface area of the microcarrier is large, and high-density culture can be realized; meanwhile, the bioreactor is easy to measure and monitor process parameters, good in reproducibility and high in production efficiency, and can realize large-scale production. The production of biologicals using bioreactor microcarrier cultured animal cells is a necessary trend in the biopharmaceutical industry.
The amplification culture is the key and difficult point in the bioreactor microcarrier culture technology. The basic transfer modes of microcarrier culture to be amplified to the next-stage reactor are pancreatin digestion transfer and ball-to-ball transfer. The ball-to-ball transfer is that the previous stage culture (microcarrier full of cells) is directly used as a seed to be inoculated into the next stage bioreactor, and the cells are redistributed between the old ball and the new ball to realize amplification culture. However, for cells with low plate efficiency, the transfer efficiency of the cells is low, the empty sphere rate is high, and the growth states of the cells on the new carrier and the old carrier are asynchronous and different in generation, so that the amplification mode of sphere rotation cannot meet the requirement of large-scale drug production. Pancreatin digestion transfer is the most common digestion mode for anchorage-dependent cells, cells are digested from microcarriers by using pancreatin, and then cell suspension is added into a next-stage bioreactor and is adhered to a new microcarrier for culture.
The scale-up culture of the bioreactor of human diploid cells is always a difficult point in the scale production of vaccines. The culture difficulty of human diploid cells is high, the efficiency of a plate is low, the requirements on nutrient components such as a culture medium and bovine serum are high, and the diploid cells are sensitive to environmental stimuli such as pH, osmotic pressure, pancreatin action, shearing force and the like. In that
In the process of amplifying culture of the microcarrier of the bioreactor, particularly in the process of digestion, damage caused by pancreatin after digestion exceeds a certain time limit, shearing force caused by stirring and ventilation, mechanical extrusion to cells during precipitation of the microcarrier, and changes of pH and osmotic pressure during replacement of cleaning liquid, digestion liquid and culture liquid are easy to damage the cells, so that the cell activity is reduced, the adhesion efficiency is reduced, and the cells grow slowly after passage, thereby limiting the amplifying culture and hindering the improvement of the production efficiency. Therefore, the key of the amplification culture of the human diploid cell microcarrier is to find out a proper pancreatin digestion method and action time, a microcarrier and cell separation method and a proper initial culture condition after effective passage in the amplification culture process and improve the cell activity, the cell harvest rate and the cell adhesion rate, and no literature report exists at present.
Disclosure of Invention
The invention aims to solve the defects of low cell activity, low yield and adhesion rate and uneven adhesion in the existing digestion passage method for culturing human diploid cells by using a bioreactor and a microcarrier, and provides a method for effectively passing the human diploid cells in the process of amplification culture of the bioreactor and the microcarrier.
The method comprises the steps of culturing human diploid cell seeds by using microcarriers, pre-balancing blank microcarriers in a seed culture system, digesting the seed cells on the microcarriers by using pancreatin digestive juice containing sodium citrate, carrying out mild intermittent stirring culture in a seed reactor for 3-6 hours, transferring the seed cells to an amplification reactor for amplification culture, and obtaining effective passage in the amplification culture process of the human diploid cell microcarriers.
The invention provides a method for effectively subculturing diploid cell microcarrier, which is characterized in that human diploid cells are MRC-5 or KMB17 cells, microcarrier is cytodex1, human diploid cell seeds are cultured by microcarrier, when the human diploid cells cultured on the microcarrier in a seed reactor grow to reach the confluence of more than 80%, blank microcarrier which is well pretreated is added into the seed reactor to be cultured together with cell seed cultures for more than 6 hours or overnight, pancreatin digestive juice containing sodium citrate is used for digesting the seed cells on the microcarrier, complete culture medium is added for neutralization, then the mixture is subjected to moderate-temperature intermittent stirring in the seed reactor, initial culture is carried out for 3-6 hours, and initial culture is transferred from the seed reactor to an amplification reactor for amplification culture, so that effective subculturing of the human diploid cell microcarrier in the amplification culture process is obtained.
The concentration of the microcarrier is 1-10 g/l, and the pretreatment of the blank microcarrier means that the dry cytodex1 microcarrier is hydrated by Phosphate Buffer Solution (PBS) overnight, washed for 3-5 times, autoclaved at 121 ℃ and rinsed and replaced for 1-2 times by a basal medium preheated to 37 ℃; the ratio of the blank microcarrier to the original microcarrier in the seed reactor is 2:1-5: 1.
The method for digesting the seed cells on the microcarrier comprises the following steps:
① stopping stirring of the seed bioreactor, allowing the cell seed culture and newly added microcarrier to settle naturally, removing the supernatant culture solution, and washing the microcarrier and cells with washing solution preheated to 37 deg.C for 1-3 times, wherein the washing solution is D-Hanks containing 0.01% EDTA-2Na, the ratio of the washing solution to the microcarrier is 100 ml/g cytodex1, the stirring speed is 30-50rpm, and the washing time is 5-10 min.
② digesting cells with intermittent stirring by using digestive juice preheated to 37 ℃, sampling and microscopic examination to observe the digestion state of the cells, detecting the concentration and activity of the exfoliated cells to control the digestion time, wherein the digestive juice comprises 0.8% of sodium chloride, 0.02% of potassium chloride, 0.005% of disodium hydrogen phosphate dihydrate, 0.1% of glucose, 0.1% of sodium bicarbonate, 0.3% of sodium citrate dihydrate, 0.25% of pancreatin and 0.002% of phenol red, the ratio of the digestive juice to microcarriers is 20-30 ml/g of cytodex1, the digestion pH value is 7.4-8.0, the digestion stirring speed is 50-150rpm, stirring is performed for 1 minute and intermittent stirring every 5 minutes, the digestion temperature is 36-38 ℃, the total digestion time is 5-20 minutes, the digestion degree is observed every 5 minutes, more than 90% of the cells are removed from the microcarriers, the cells are added into a complete culture medium and stirred for 1-3 minutes after 50-100 rpm.
The initial culture of the invention is carried out in a seed reactor, and the gentle intermittent stirring is carried out according to the stirring frequency of 1-3 minutes every 15-45 minutes, the stirring speed is 20-50rpm, and the minimum speed and the minimum time for stirring all microcarriers are adopted. The culture parameters are 35-37 deg.C, pH7.2-7.4, and Dissolved Oxygen (DO) 30% -50%. The initial culture time is 3-6 hours, and the cell adhesion condition is observed by sampling every 3 hours; and transferring the initial culture from the seed reactor to an amplification reactor for amplification and effective subculture.
The specific technical scheme of the invention is as follows:
1. the method for effectively passaging the human diploid cells in the process of amplifying culture of the microcarriers in the bioreactor comprises the following steps:
⑴ culturing human diploid cells in a seed reactor with microcarriers;
⑵ pre-equilibrating microcarriers in a seed culture system;
⑶ washing and digesting the seed cells;
⑷ initiating culture in the seed reactor;
⑸ are cultured in an amplification reactor.
In the step ⑴, the human diploid cell is MRC-5 or KMB17 cell, the microcarrier in the seed culture is cytodex1, the concentration of the microcarrier is 1-10 g/L, and the confluency of the cell on the microcarrier is not less than 80%.
The pre-balancing of the step ⑵ in the technical scheme of the invention means that a blank microcarrier is hydrated by Phosphate Buffer Solution (PBS) for overnight, washed for 3-5 times and autoclaved at 121 ℃, then rinsed and replaced for 1-2 times by a basic culture medium preheated to 37 ℃, when cells of a seed reactor grow on the microcarrier to reach the confluency of more than 80%, the blank microcarrier is added into the seed reactor to be cultured together with a cell seed culture for more than 6 hours or overnight, and the ratio of the newly added blank microcarrier to the original microcarrier in the seed reactor is 2:1-5: 1.
The step ⑶ of washing the digested cells according to the present invention comprises the following steps:
① stopping stirring of the seed bioreactor, allowing the cell seed culture and newly added microcarrier to settle naturally, removing the supernatant culture solution, washing the microcarrier and cells with a washing solution preheated to 37 deg.C for 1-3 times, wherein the washing solution is D-Hanks containing 0.01% EDTA-2Na, the ratio of microcarrier to washing solution is 100 ml/g cytodex1, the stirring speed is 30-50rpm, and the washing time is 5-10 min.
② cells are digested by a digestive juice preheated to 37 ℃, which comprises 0.8% of sodium chloride, 0.02% of potassium chloride, 0.005% of disodium hydrogen phosphate dihydrate, 0.1% of glucose, 0.1% of sodium bicarbonate, 0.3% of sodium citrate dihydrate, 0.25% of pancreatin (1:250) and 0.002% of phenol red (pH 7.5-8.0), wherein the ratio of the digestive juice to the microcarriers is 20-30 ml/g of cytodex1, the digestion pH value is 7.4-8.0, the digestion stirring speed is 50-150rpm, the digestion is intermittently stirred for 1 minute every 4 minutes, the digestion temperature is 36-38 ℃, the total digestion time is 5-20 minutes, the digestion degree is observed every 5 minutes, more than 90% of the cells are removed from the microcarriers, the cells are added into a complete culture medium and are stirred for 1-3 minutes, the carriers and the cells are mixed uniformly, and the digestion state, the cell concentration and the cell viability are detected by microscopic examination.
In the step ⑷ of the present invention, the initial culture is performed in the seed reactor, wherein the complete culture medium is added to the culture volume of the seed reactor, the stirring is performed for 1-3 minutes every 15-45 minutes, the stirring speed is 20-50rpm, the minimum speed and the minimum time for stirring all microcarriers are adopted, the other culture parameters are 35-37 ℃, the pH value is 7.2-7.4, the Dissolved Oxygen (DO) is 30-50%, the initial culture time is 3-6 hours, and the cell adhesion condition is observed by sampling every 3 hours.
According to the amplification culture in the step ⑸, the amplification reactor is preheated by adding a complete culture medium in advance according to culture parameters (37 ℃, pH7.2-7.4, DO50-70%, 30-70 rpm), after the seeds are initially cultured for 3-6 hours and more than 95% of cells are adhered to microcarriers, the initial culture is transferred from the seed reactor to the amplification reactor for amplification culture, the growth and metabolism conditions of the cells are regularly detected, and the culture medium is replenished according to the detection result.
The beneficial effects of the invention are shown in the following aspects:
1. the new microcarrier is rinsed by using a culture medium and added into a seed culture for pre-balancing, so that the new microcarrier is balanced to a microenvironment most suitable for cell adhesion and growth in a culture solution containing adhesion molecules and cell growth factors at a proper pH and osmotic pressure, and the improvement of the adhesion efficiency and the shortening of the lag phase after inoculation are facilitated. Meanwhile, the newly added blank microcarrier and the original microcarrier are cultured in the same environment, so that the blank microcarrier and the original microcarrier tend to be consistent, and the improvement of the uniformity of adhesion is facilitated.
2. The components of the digestive juice, the sodium citrate and the potassium chloride replace EDTA, which is beneficial to improving the digestion efficiency,
increase cell dispersion degree, shorten digestion time, reduce damage to cells and improve cell activity.
3. The initial culture after pre-balancing, washing, digestion and passage is carried out in a seed reactor, so that multiple transfer of carriers and cells in different containers is avoided, the loss of the cells is reduced, and the risk of pollution is reduced; the original microcarrier in the seed reactor is continuously used after amplification culture, so that the utilization rate of the carrier is improved.
4. The initial culture is carried out in the seed reactor, so that the culture volume is reduced, and the concentration of the cells and the microcarriers is improved, thereby increasing the contact and adhesion chance of the cells and the microcarriers; for human diploid cells with low anchorage efficiency, the adhesion rate and the adhesion ratio of the cells will increase at an early stage, with intermittent gentle stirring, and with a certain period of stationary culture.
5. The invention has the advantages of improving the activity, the adhesion rate and the adhesion uniformity of cells in the passage process, improving the passage efficiency, solving the problem that the human diploid cells are difficult to amplify and culture on a bioreactor and a microcarrier, and being widely applied to the large-scale production of vaccines.
Drawings
FIG. 1 is a graph showing the results of adhesion of KMB17 cells of the present invention after 3 hours of initial culture in a 5-liter bioreactor;
FIG. 2 is a graph showing the results of adhesion of KMB17 cells of the present invention after initial culture in a 5-liter bioreactor for 6 hours.
Detailed description of the preferred embodiments
In order that the invention may be more clearly understood, the invention will now be further illustrated by way of example. The examples are intended only to illustrate the invention in further detail and are not intended to limit the invention in any way.
Efficient subculturing of microcarriers for implementation of 1 MRC-5 cells
⑴ microcarrier culture of MRC-5 cell seeds in a 1.5 liter bioreactor:
dry 2.0 g of cytodex1 microcarriers were added to a suitable siliconized glass bottle and 200 ml of Ca free2+、Mg2+The PBS was mixed well, hydrated overnight at room temperature, washed 3 times with the PBS, and sterilized by autoclaving at 121 ℃ for 30 minutes for further use.
The suspension was allowed to stand to allow the sterile microcarriers to settle, the supernatant was decanted, and the suspension was rinsed and replaced 2 times with warm MEM medium. The microcarriers were transferred to a 1.5 liter bioreactor and 500 ml MEM complete medium was added, setting the culture parameters: the temperature was 37 ℃, pH7.2, DO50%, and the speed 30rpm balance overnight.
After 7 MRC-5 cells in T225 cell bottles were digested, the cell suspension was added to the bioreactor, and the volume of the pre-conditioned medium in the bioreactor was adjusted to a total volume of 1000 ml based on the amount of cell suspension. Setting culture parameters: culturing at 37 deg.C, pH7.2, DO50%, and rotation speed of 30rpm twice per day
And (3) detecting the growth and metabolism of the cells, and replacing half volume of the culture medium when the concentration of the lactic acid is more than 1.5 g/L.
(2) Pre-equilibration of blank microcarriers:
dry 10 g of cytodex1 microcarriers were added to a suitable siliconized glass bottle and 800 ml of Ca free2 +、Mg2+The PBS was mixed well, hydrated overnight at room temperature, washed 3 times with the PBS, and sterilized by autoclaving at 121 ℃ for 30 minutes for further use. Allowing to stand to allow the sterile microcarriers to settle, decanting the supernatant, and incubating in warm MEM mediumRinse and replace 3 times.
When the cells in the seed reactor grow on the microcarriers to reach a confluence of more than 80%, adding the new carriers into the seed reactor to balance overnight in a culture system according to seed culture parameters.
(3) Washing and digesting the seed cells:
① stopping stirring of the seed bioreactor, naturally settling the microcarrier adhered with the cell and the newly added microcarrier in the reactor, removing the supernatant culture solution, washing the microcarrier and the cell with 1200 ml of washing solution preheated to 37 ℃, stirring and re-suspending the microcarrier and the cell at 30rpm for 10 minutes, stopping stirring, standing, precipitating, removing the supernatant, and washing for 3 times.
② cells were digested with 250 ml of a digest (containing 0.8% sodium chloride, 0.02% potassium chloride, 0.005% disodium hydrogen phosphate dihydrate, 0.1% glucose, 0.1% sodium bicarbonate, 0.3% sodium citrate dihydrate, 0.25% pancreatin (1:250), 0.002% phenol red) preheated to 37 ℃, stirred at 100rpm for 1 minute every 5 minutes, digested at 37 ℃ for 10-20 minutes, sampled every 5 minutes to observe the degree of digestion, after 10 minutes, more than 90% of the cells were detached from the microcarriers, neutralized by adding 300 ml of MEM complete medium, stirred at 60rpm for 1 minute to mix the carriers and cells, and sampled to detect the concentration and activity of the cells.
(4) Initiating culture in a seed reactor:
MEM complete medium preheated to 37 ℃ was added to 1000 ml, and gentle intermittent stirring was performed at a frequency of 2 minutes per 30 minutes at a stirring speed of 30 rpm. Other culture parameters were 37 ℃, ph7.2, DO50%, three-way air model, compressed air, oxygen, carbon dioxide (CO 2), during which samples were taken every 3 hours to observe cell adhesion.
(5) Amplification culture in a 5 liter bioreactor:
a5 liter bioreactor was started 5 days earlier and sterilized with MEM basal medium according to the culture parameters (37 ℃, pH7.2, DO 50%). After passing the sterility test, 3 liters of complete MEM medium at 37 ℃, pH7.2 and DO50% is added for pre-culture, after the culture in the seeding tank is initially cultured for 6 hours, most cells are uniformly adhered to the microcarriers, the initial culture is transferred to a 5 liter bioreactor, after stirring uniformly at 60rpm for 1 minute, the culture is stirred at 37 ℃, pH7.2 and DO50%, a three-way air mode, compressed air, oxygen and carbon dioxide (CO 2), stirring culture at 30-50rpm, and sampling and detecting the pH value, the glucose and the lactic acid concentration every day, and the microcrystallization is carried out
Observing the growth condition and shape of the cells on the microcarriers by using a mirror, and counting cell nucleuses after the cells are hypotonic. Adjusting culture parameters and filling fresh culture medium to supply nutrition according to the growth and metabolism condition of cells.
Example 2 microcarrier efficient subculture of KMB17 cells
(1) Microcarrier culture of KMB17 cell seeds in a 5.0 liter bioreactor:
dry 10.0 g of cytodex1 microcarriers were added to a suitable siliconized glass bottle and 1000 ml of Ca free2+、Mg2+The PBS was mixed well, hydrated overnight at room temperature, washed 5 times with the PBS, and sterilized by autoclaving at 121 ℃ for 30 minutes for further use.
The sterile microcarriers were allowed to settle by standing, the supernatant decanted and replaced 2 times with warm M199 medium. The microcarriers were transferred to a 1.5 liter bioreactor and 500 ml of M199 complete medium was added, setting the culture parameters: the temperature was 37 ℃, pH7.2, DO50%, and the speed 30rpm balance overnight.
5.0 liter bioreactor was started 5 days earlier and sterile tested by adding M199 basal medium according to the culture parameters (37 ℃, pH7.2, DO 50%). After the sterility test was passed, 3.0 liters of M199 complete medium was added at 37 ℃ and pH7.2, DO50% for preculture. After digesting KMB17 cells of a 10-layered cell factory, the cell suspension was added to the bioreactor, and the volume of the pre-conditioned medium in the bioreactor was adjusted to a total volume of 3.5 liters based on the amount of the cell suspension. Setting culture parameters (37 ℃, pH7.2, DO50% and rotation speed of 30 rpm) for culture, detecting the growth and metabolism condition of cells twice a day, and starting perfusion culture after 2 days, wherein the perfusion speed is based on the fact that the concentration of lactic acid can be maintained to be lower than 1.5 g/L.
(2) Pre-equilibration of blank microcarriers:
dry 20.0 g of cytodex1 microcarriers were added to a suitable siliconized glass bottle and 2000 ml Ca free2+、Mg2+The PBS is mixed evenly and hydrated overnight at room temperature, washed with the PBS for 5 times, and sterilized by moist heat under high pressure at 121 ℃ for 30min for standby. The sterile microcarriers were allowed to settle by standing, the supernatant decanted and replaced 3 times with warm M199 medium.
When the cells in the seed reactor grow on the microcarriers to reach a confluence of more than 80%, adding the new carriers into the seed reactor to balance overnight in a culture system according to seed culture parameters.
(3) Washing and digesting the seed cells:
① stopping stirring in the seed bioreactor, naturally settling the microcarrier adhered with the cell and the newly added microcarrier, removing the supernatant culture solution, washing the microcarrier and the cell with 3.0L washing solution preheated to 37 ℃, stirring and re-suspending the microcarrier and the cell at 30rpm, suspending the microcarrier and the cell, stopping stirring, standing, precipitating, removing the supernatant, and washing for 3 times.
② the digestion solution (containing 0.8% sodium chloride, 0.02% potassium chloride, 0.005% disodium hydrogen phosphate dihydrate, 0.1% glucose, 0.1% sodium bicarbonate, 0.3% sodium citrate dihydrate, 0.25% pancreatin) preheated at 37 deg.C is used in 600 ml
(1:250), 0.002% phenol red), stirred at 100rpm every 5 minutes for 1 minute, and digested at 37 ℃ for 10-20 minutes. Sampling every 5 minutes to observe the digestion degree, after 15 minutes, more than 90 percent of cells fall off from the microcarrier, adding 2.5 liters of M199 complete culture medium for neutralization, and stirring at 100rpm for 2 minutes to uniformly mix the carrier and the cells; and (5) sampling and microscopic-examining to observe the digestion state of the cells and detect the concentration and the survival rate of the cells.
(4) Initiating culture in a seed reactor:
the stirring was carried out at 30rpm with gentle intermittent stirring at a frequency of 2 minutes per 15 minutes. Other culture parameters were 37 ℃, ph7.2, DO50%, triple aeration mode (compressed air, O2, CO 2), during which cell adhesion was observed every 3 hours of sampling.
(5) Amplification culture in 14.0 liter bioreactor:
14.0 liter bioreactor 5 days earlier, 10.0 liters of M199 basal medium was added according to culture parameters: sterility testing was performed at 37 deg.C, pH7.2, Dissolved Oxygen (DO) 50%. After passing the sterility test, 6.5L of M199 complete medium was replaced at 37 ℃ with pH7.2 and DO50% for preculture. After the culture in the seeding tank is initially cultured for 6 hours, most cells are uniformly adhered to the microcarriers, the initial culture is transferred into a 14.0-liter bioreactor, after the culture is uniformly stirred at 60rpm for 2 minutes, the temperature is 37 ℃, the pH value is 7.2, the DO50 percent, the culture is stirred in a three-aeration mode (compressed air, oxygen and carbon dioxide) at 30-60rpm, the culture is sampled every day to detect the pH value and the concentrations of glucose and lactic acid, and the growth condition and the morphology of the cells on the microcarriers are observed by a microscope; adjusting culture parameters and filling fresh culture medium to supply nutrition according to the growth and metabolism condition of cells.
Example 3 determination and calculation of cell viability, percent cell shedding, cell adhesion and cell recovery during passaging
(1) Before the cell digestion step, 1 ml of the uniformly suspended culture is taken, centrifuged at 1000rpm for 5 minutes to settle the carrier cells, the supernatant is carefully removed, 0.1 mol/L citric acid solution containing 0.1% crystal violet is added to resuspend the precipitate, the precipitate is incubated at 37 ℃ for 10 minutes and then is shaken on a vortex mixer for 5 minutes, then a sample is taken and counted on a blood counting plate to count the stained released cell nuclei, and the cell nucleus concentration is measured and multiplied by the culture volume to obtain the total number of the cells before digestion;
(2) when sampling once every 5 minutes of digestion, sucking supernatant, adding a serum-containing complete culture medium and pancreatin, sampling at a ratio of 1:1, adding trypan blue staining solution, and detecting cell viability on a TC20 automatic cell counter after acting for 5 minutes;
(3) after 6 hours of initial culture, 2 ml of the uniformly suspended culture was taken, wherein 1 ml of the culture was centrifuged at 1000rpm for 5 minutes to settle the carrier cells, the cell nucleus concentration was counted by ⑴ in the same manner, and the cell nucleus concentration multiplied by the culture volume was the total number of cells after passage, and the other 1 ml was left to settle the carrier for about 5 minutes to carefully remove the supernatant, the cell nucleus concentration was counted by ⑴ in the same manner, and the cell nucleus concentration multiplied by the culture volume was the number of cells adhered to the microcarriers after passage.
Cell viability rate: the TC20 automatic cell counter directly displays the detection result;
cell adhesion rate: the number of cells adhering to the microcarriers after passage/total number of cells after passage x 100%;
and (3) cell recovery rate: total number of cells after passage/total number of cells before digestion × 100%.
TABLE 1 evaluation of passage Effect
| Results | Example 1 | Example 2 |
| Cells | MRC-5 | KMB17 |
| Microcarrier | Cytodex1 | Cytodex1 |
| Seed reactor volume/culture volume (liter) | 1.5/1.0 | 5.0/3.5 |
| Microcarrier concentration (g/l) in seed reactor | 2.0 | 2.9 |
| Amplification reactor volume/culture volume (liter) | 5.0/3.5 | 14.0/10.0 |
| Amplification reactor microcarrier concentration (g/l) | 3.4 | 3.0 |
| Cell viability (%) | 98 | 96 |
| Initial 6-hour cell adhesion (%) | 100 | 97 |
| Cell recovery (%) | 95 | 91 |
As can be seen from the results of table 1 and fig. 1 and 2, the cell viability after passage was above 95%, the cells were mostly uniformly adhered to the microcarriers after 3 hours of initial culture, only a very small amount of free cells were observed in the supernatant, the cells adhered to the microcarriers had spread after 6 hours of initial culture, the cell adhesion rate reached above 97%, and the total recovery rate of the cells during passage was above 90%. Aiming at the characteristics of low plate efficiency and sensitivity to environment of the human diploid cells, the invention improves the adhesion efficiency of the cells on microcarriers by changing or optimizing a blank microcarrier pre-balancing method before digestion, a digestion solution formula and an initial culture condition, reduces the damage to the cells in the passage process, realizes effective passage of the human diploid cells in the culture process of the microcarriers in a bioreactor, and lays a solid foundation for industrial amplification of high-density culture of the human diploid cells.
Claims (4)
1. A method for culturing the effective passage of human diploid cell by the microcarrier includes such steps as culturing the seeds of human diploid cell in the condition of cytodex1 as MRC-5 or KMB17 cell, adding the pretreated blank microcarrier to seed reactor, culturing for more than 6 hr or overnight, digesting the seed cells on microcarrier with the digestive liquid of sodium citrate, adding complete culture medium, stirring in seed reactor, initial culturing for 3-6 hr, transferring the initial culture from seed reactor to amplification reactor, and amplifying culture.
2. The method for efficient passaging in microcarrier culture of human diploid cells of claim 1 wherein said digesting of seed cells on microcarriers comprises the steps of:
① stopping stirring of the seed bioreactor to allow the cell seed culture and the newly added microcarrier to settle naturally, removing the supernatant culture solution, and washing the microcarrier and cells with washing solution preheated to 37 deg.C for 1-3 times;
② the digestion liquid containing pancreatin-sodium citrate as main component preheated to 37 deg.C is used to intermittently stir and digest cells, the digestion state of the cells is observed by sampling and microscopic examination, and the concentration and activity of the exfoliated cells are detected to control the digestion time.
3. The method for efficient passaging of microcarrier cultures of human diploid cells according to claim 1, wherein said initial culture is carried out in a seed reactor, gently and intermittently agitated at a frequency of 1-3 minutes per 15-45 minutes at a speed of 20-50rpm for a minimum speed and a minimum time to agitate all microcarriers, and wherein the culture parameters are temperature of 35-37 ℃, pH7.2-7.4, dissolved oxygen 30% -50%; the initial culture time is 3-6 hours, during which the cells are sampled every 3 hours to observe the adhesion condition, when most of the cells are uniformly adhered to the microcarriers, the initial culture is transferred to an amplification reactor for amplification culture.
4. The method for efficient passage of human diploid cell microcarrier culture of claim 2 wherein said digest of step ② comprises 0.8% sodium chloride, 0.02% potassium chloride, 0.005% disodium hydrogen phosphate dihydrate, 0.1% glucose, 0.1% sodium bicarbonate, 0.3% sodium citrate dihydrate, 0.25% pancreatin, 0.002% phenol red, the ratio of digest to microcarrier is 20-30 ml/g cytodex1, the digestion pH is 7.4-8.0, the digestion stirring speed is 50-150rpm, the intermittent stirring is performed every 5 minutes, the digestion temperature is 36-38 ℃, the total digestion time is 5-20 minutes, the digestion degree is observed every 5 minutes, more than 90% of cells are removed from microcarriers, the cells are added to complete culture medium for neutralization, the digestion is performed at 50-100rpm, and the mixture of carriers and cells are mixed by 1-3 minutes.
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| CN102559617A (en) * | 2010-12-20 | 2012-07-11 | 北京清大天一科技有限公司 | Method of bioreactor micro-carrier for cultivating human diploid cell to produce viral vaccine |
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| CN103289948A (en) * | 2013-05-30 | 2013-09-11 | 冯玉萍 | Application of GF (glass fiber) microcarrier for cell culture in adherent dependent cell culture |
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| CN1147833A (en) * | 1994-03-08 | 1997-04-16 | 麦克公司 | Hepatitis A virus culture process |
| CN102559617A (en) * | 2010-12-20 | 2012-07-11 | 北京清大天一科技有限公司 | Method of bioreactor micro-carrier for cultivating human diploid cell to produce viral vaccine |
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