CN116135971A - Human cytomegalovirus culture method - Google Patents

Human cytomegalovirus culture method Download PDF

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CN116135971A
CN116135971A CN202310340064.5A CN202310340064A CN116135971A CN 116135971 A CN116135971 A CN 116135971A CN 202310340064 A CN202310340064 A CN 202310340064A CN 116135971 A CN116135971 A CN 116135971A
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virus
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human cytomegalovirus
culturing
hcmv
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钱兴乐
邓坤
刘健
陈昱宏
洪福星
刘畅
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Tonrol Biopharmaceutical Co ltd
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Abstract

The invention provides a method for culturing human cytomegalovirus, which relates to the field of biotechnology and comprises the following steps: s1: culturing sensitive cells MRC-5 to compact monolayer cells; s2: determining the virus titer of the CMV virus seed; s3: according to the formula
Figure DDA0004157925670000011
Accurately calculating the inoculation amount of HCMV virus liquid, and inoculating CMV virus in MRC-5 cells; s4: harvesting viruses and freezing; the invention adopts original formula
Figure DDA0004157925670000012
Figure DDA0004157925670000013
To accurately calculate the virus inoculation amount, and the obtained virus titer is stable and higher than that of the traditional blind transmission mode by 1The titer of HCMV cultured by the invention can reach 10 after more than 0 times 6 CCID 50 And/0.05 ml, which can improve the stability of the preparation.

Description

Human cytomegalovirus culture method
Technical Field
The invention relates to the technical field of biology, in particular to a method for culturing human cytomegalovirus.
Background
Human cytomegalovirus (human cytomegalovirus, HCMV) is a DNA virus of the genus β of the family herpesviridae, consisting of 162 capsomers, positive 20-sided facets; HCMV is common in global infections, with adult HCMV infection rates of 52% -100%, and in china, pre-school children have HCMV seropositivity rates of over 90%. HCMV infection has strict species specificity, human is the only host of HCMV, HCMV establishes long-term latent infection after primary infection, HCMV primary infection is usually clinical symptom-free infection for immunocompetent patients, and can be continuously or even permanently hidden in certain cells or tissues to form latent infection, reactivation can occur when the physiological environment of host organisms is changed, and for patients with low immune function, the primary infection and reactivation can cause symptoms, and at present, effective vaccine is still lacking to prevent HCMV infection, intravenous injection cytomegalovirus human immunoglobulin is a medicament with relatively definite curative effect for clinically treating HCMV-related diseases, and clinical experiments show that the incidence rate of CMV-related diseases can be reduced from 60% to 21% by using HCMV-IVIG for kidney transplanted patients; the use of HCMV-IVIG in liver transplant patients can reduce the incidence of severe CMV disease from 26% to 12%; prophylactic use of HCMV-IVIG in pregnant women can reduce CMV infection rate of newborn infants from 40% to 16%;
the human immunoglobulin of the static injection cytomegalovirus is a blood product prepared by purifying high-titer HCMV antibody plasma through a low-temperature ethanol process, the raw material plasma is mainly obtained by screening the high-titer HCMV antibody plasma from plasma donors by adopting a neutralization test method, the neutralization test has the characteristics of complicated test, long test period, large fluctuation of detection data among different batches of CMV viruses and the like, the large-scale screening method can effectively improve the screening efficiency and the stability of the screened high-titer HCMV plasma, but the titer requirement on the HCMV virus is higher, the obtained virus titer is lower than 105CCID50/0.1ml by adopting a blind transfer method in most of the current institutions, and the large-scale screening of the high-titer HCMV antibody plasma by adopting the neutralization test method is greatly influenced.
Disclosure of Invention
The present invention provides a method for culturing human cytomegalovirus, which adopts original formula
Figure BDA0004157925650000021
The virus inoculation amount can be accurately calculated, and compared with the traditional blind transmission mode, the virus titer obtained is stable and is 10 times higher than that obtained by the traditional mode.
In order to achieve the purpose of the invention, the invention is realized by the following technical scheme: a method for culturing human cytomegalovirus, comprising the steps of:
s1: culturing sensitive cells MRC-5 to compact monolayer cells;
s2: determining the virus titer of the CMV virus seed;
s3: according to the formula
Figure BDA0004157925650000022
Accurately calculating the inoculation amount of HCMV virus liquid, and inoculating CMV virus in MRC-5 cells; />
S4: viruses were harvested and frozen.
The further improvement is that: the S1 comprises the following steps:
s11: taking MRC-5 cells stored in liquid nitrogen, and carrying out resuscitating operation;
s12: culturing the cells in a cell culture solution until the cells are compact and have a single layer for later use.
The further improvement is that: in the step S12, the formula of the cell culture solution is as follows: 10% FBS, MEM, pH was adjusted to 7.0 with 7.5% sodium bicarbonate solution.
The further improvement is that: the step S2 comprises the following steps:
s21: the virus titer of CMV virus seeds was determined using a microcytosis neutralization assay.
The further improvement is that: in S21, the HCMV virus titer unit is CCID 50 /0.05ml。
The further improvement is that: the step S3 comprises the following steps:
s31: taking a MRC-5 cell 1 bottle growing to a compact monolayer, and discarding a cell culture solution;
s32: according to the formula
Figure BDA0004157925650000031
Accurately calculating the inoculation amount of the HCMV virus liquid, and adding the HCMV virus liquid into a cell bottle for adsorption for 2 hours;
s33: removing adsorbed virus solution, adding cell maintenance solution into each bottle, and placing CO at 37deg.C 2 Culturing in an incubator.
The further improvement is that: in S33, the formula of the cell maintenance solution is: 2% FBS, MEM, pH was adjusted to 7.0 with 7.5% sodium bicarbonate solution.
The further improvement is that: the step S4 comprises the following steps:
s41: observing cytopathic CPE condition every day, and harvesting viruses 2 days after the CPE reaches 80-90%;
s42: freezing and thawing the culture flask below-70 ℃ for three times, and collecting the lysed cells and the culture solution;
s43: taking supernatant after low-temperature high-speed centrifugation, and adding fetal bovine serum FBS as a protective agent to ensure that the concentration of the FBS is 9-11%.
S44: small volume packaging, and freezing below-70deg.C.
The beneficial effects of the invention are as follows:
1. the invention adopts original formula
Figure BDA0004157925650000032
The virus titer obtained by the method is stable and 10 times higher than that obtained by the traditional blind transmission method, and the HCMV virus titer cultured by the method can reach 10 6 CCID 50 And/0.05 ml, can promote the stability of the intravenous cytomegalovirus human immunoglobulin product prepared by adopting a neutralization test method to screen plasma.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a schematic representation of a dense monolayer MRC-5 cell of the invention;
FIG. 3 is a schematic representation of MRC-5 cells of the invention beginning to develop lesions after virus inoculation;
FIG. 4 is a schematic representation of 50% MRC-5 cytopathic effect of the invention after virus inoculation.
Detailed Description
The present invention will be further described in detail with reference to the following examples, which are only for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
Example 1
According to FIGS. 1, 2, 3 and 4, the present embodiment provides a method for culturing human cytomegalovirus, comprising the following steps:
s1: culturing sensitive cells MRC-5 to compact monolayer cells;
s11: taking MRC-5 cells stored in liquid nitrogen, and carrying out resuscitating operation;
s12: culturing the cell culture solution to compact single-layer cells for later use, wherein the formula of the cell culture solution is as follows:
10% FBS, MEM, pH adjusted to 7.0 with 7.5% sodium bicarbonate solution
S2: determining the virus titer of the CMV virus seed;
s21: the virus titer of CMV virus seeds is determined by adopting a micro cytopathy neutralization test method, and the HCMV virus titer unit is CCID 50 /0.05ml。
S3: according to the formula
Figure BDA0004157925650000041
Accurately calculating the inoculation amount of HCMV virus liquid, and inoculating CMV virus in MRC-5 cells;
s31: taking a MRC-5 cell 1 bottle growing to a compact monolayer, and discarding a cell culture solution;
s32: according to the formula
Figure BDA0004157925650000042
Accurate HC calculationThe MV virus liquid inoculation amount is added into a cell bottle to be adsorbed for 2 hours;
s33: removing adsorbed virus solution, adding cell maintenance solution into each bottle, and placing CO at 37deg.C 2 Culturing in an incubator, wherein the formula of the cell maintenance solution is as follows: 2% FBS, MEM, pH was adjusted to 7.0 with 7.5% sodium bicarbonate solution.
S4: viruses were harvested and frozen.
S41: observing cytopathic CPE condition every day, and harvesting viruses 2 days after the CPE reaches 80-90%;
s42: freezing and thawing the culture flask below-70 ℃ for three times, and collecting the lysed cells and the culture solution;
s43: taking supernatant after low-temperature high-speed centrifugation, and adding fetal bovine serum FBS as a protective agent to ensure that the concentration of the FBS is 9-11%.
S44: small volume packaging, and freezing below-70deg.C.
Example two
According to FIGS. 1, 2, 3 and 4, the present embodiment provides a method for culturing human cytomegalovirus, comprising the following steps:
1. culture of sensitive cells MRC-5
Frozen MRC-5 cells were removed from liquid nitrogen, immediately placed in 37℃warm water, and shaken well to allow rapid thawing.
In an ultra-clean workbench, transferring the cell suspension in the freezing tube into a centrifuge tube containing 5ml of culture solution, centrifuging at 1000r/min for 5min, discarding the supernatant, adding the culture solution for rinsing, adding a proper amount of culture solution after centrifuging, and lightly blowing with a suction tube to prepare the cell suspension.
Transferring the cell suspension into T25 cell culture flask, and placing CO at 37deg.C 2 Culturing in an incubator. After the culture solution is changed once in the next day, the culture is continued until a compact monolayer is obtained for later use.
2. CMV virus seed titer assay
Taking 1 branch of frozen virus suspension, quickly thawing by flowing water, taking 100 mu l of virus liquid, adding into 900 mu l of cell maintenance liquid, and fully and uniformly mixing. Serial dilution of 10 times to 10 -8 。
Mu.l of each dilution of virus was added to a 96-well cell plate, 10 wells were set in parallel for each dilution, and a control 10 well was set, and only 50. Mu.l of cell maintenance solution was added.
Mu.l of cell maintenance solution was added to each well and the mixture was placed in 5% CO at 37 ℃ 2 Incubate in incubator for 1 hour.
50 μl of 2×10 concentration was added to each well 5 MRC-5 cell suspension at 37℃and 5% CO per ml 2 Culturing in an incubator for 9 days.
Cell growth was observed daily, cytopathic (CPE) was observed on day seven, the results were finally judged on day nine, and virus titers were calculated.
3. CMV Virus Vaccination
1 bottle of dense monolayer MRC-5 cells (T25, cell number 1X 10) 6 Individual/bottle), the culture broth was discarded.
According to the formula
Figure BDA0004157925650000061
And (3) calculating the inoculation volume of the HCMV virus liquid, adding the HCMV virus liquid with the corresponding calculated volume and 1ml of cell maintenance liquid, and placing the mixture in a CO2 incubator at 37 ℃ for adsorption for 2 hours.
Removing the adsorbed virus liquid, adding 5ml of cell maintenance liquid into each bottle, and culturing in a CO2 incubator at 37 ℃.
4. Toxin collection
The cytopathic effect is observed every day, the cell maintenance solution is changed every two days, and the virus is harvested 2 days after the cytopathic effect reaches 80-90%.
The flask was frozen and thawed three times below-70℃and the lysed cells and culture medium were collected.
After centrifugation at 8000r/min for 10 min at 5℃the supernatant was taken and 0.5ml Fetal Bovine Serum (FBS) was added as a protective agent.
200 μl/sub-packaging, and freezing below-70deg.C.
Verification example: preparation of 3 batches of HCMV Virus titres Using the method of the present invention
Sequence number HCMV viral lot number HCMV viral titers
1 20220201 10 6.3 CCID 50 /0.05ml
2 20220202 10 6.4 CCID 50 /0.05ml
3 20220303 10 6.1 CCID 50 /0.05ml
The culture method of the human cytomegalovirus adopts an original formula
Figure BDA0004157925650000071
The virus titer obtained by the method is stable and 10 times higher than that obtained by the traditional blind transmission method, and the HCMV virus titer cultured by the method can reach 10 6 CCID 50 And/0.05 ml, can promote the stability of the intravenous cytomegalovirus human immunoglobulin product prepared by adopting a neutralization test method to screen plasma.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (8)

1. A method for culturing human cytomegalovirus, comprising the steps of:
s1: culturing sensitive cells MRC-5 to compact monolayer cells;
s2: determining the virus titer of the CMV virus seed;
s3: according to the formula
Figure FDA0004157925640000011
Accurately calculating the inoculation amount of HCMV virus liquid, and inoculating CMV virus in MRC-5 cells;
s4: viruses were harvested and frozen.
2. The method for culturing human cytomegalovirus according to claim 1, wherein: the S1 comprises the following steps:
s11: taking MRC-5 cells stored in liquid nitrogen, and carrying out resuscitating operation;
s12: culturing the cells in a cell culture solution until the cells are compact and have a single layer for later use.
3. The method for culturing human cytomegalovirus according to claim 2, wherein: in the step S12, the formula of the cell culture solution is as follows: 10% FBS, MEM, pH was adjusted to 7.0 with 7.5% sodium bicarbonate solution.
4. The method for culturing human cytomegalovirus according to claim 2, wherein: the step S2 comprises the following steps:
s21: the virus titer of CMV virus seeds was determined using a microcytosis neutralization assay.
5. The method for culturing human cytomegalovirus of claim 4, wherein: in S21, the HCMV virus titer unit is CCID 50 /0.05ml。
6. The method for culturing human cytomegalovirus of claim 4, wherein: the step S3 comprises the following steps:
s31: taking a MRC-5 cell 1 bottle growing to a compact monolayer, and discarding a cell culture solution;
s32: according to the formula
Figure FDA0004157925640000012
Accurately calculating the inoculation amount of the HCMV virus liquid, and adding the HCMV virus liquid into a cell bottle for adsorption for 2 hours;
s33: removing adsorbed virus solution, adding cell maintenance solution into each bottle, and placing CO at 37deg.C 2 Culturing in an incubator.
7. The method for culturing human cytomegalovirus of claim 6, wherein: in S33, the formula of the cell maintenance solution is: 2% FBS, MEM, pH was adjusted to 7.0 with 7.5% sodium bicarbonate solution.
8. The method for culturing human cytomegalovirus of claim 6, wherein: the step S4 comprises the following steps:
s41: observing cytopathic CPE condition every day, and harvesting viruses 2 days after the CPE reaches 80-90%;
s42: freezing and thawing the culture flask below-70 ℃ for three times, and collecting the lysed cells and the culture solution;
s43: taking supernatant after low-temperature high-speed centrifugation, and adding fetal bovine serum FBS as a protective agent to ensure that the concentration of the FBS is 9-11%.
S44: small volume packaging, and freezing below-70deg.C.
CN202310340064.5A 2023-03-30 2023-03-30 Human cytomegalovirus culture method Pending CN116135971A (en)

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