CN115161284A - LMH suspension cell recovery culture method and application thereof - Google Patents
LMH suspension cell recovery culture method and application thereof Download PDFInfo
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- 239000000725 suspension Substances 0.000 title claims abstract description 56
- 238000011084 recovery Methods 0.000 title claims abstract description 40
- 238000012136 culture method Methods 0.000 title claims abstract description 20
- 239000006143 cell culture medium Substances 0.000 claims abstract description 30
- 239000007788 liquid Substances 0.000 claims abstract description 23
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000007710 freezing Methods 0.000 claims abstract description 12
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- 238000004113 cell culture Methods 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 7
- 238000005070 sampling Methods 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000010257 thawing Methods 0.000 claims description 5
- 230000004083 survival effect Effects 0.000 abstract description 6
- 238000010792 warming Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 74
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 21
- 238000000034 method Methods 0.000 description 16
- 241000287828 Gallus gallus Species 0.000 description 11
- 235000013330 chicken meat Nutrition 0.000 description 11
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
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- 235000013594 poultry meat Nutrition 0.000 description 4
- 241000701161 unidentified adenovirus Species 0.000 description 4
- 241000271566 Aves Species 0.000 description 3
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- 241000711450 Infectious bronchitis virus Species 0.000 description 1
- WBNQDOYYEUMPFS-UHFFFAOYSA-N N-nitrosodiethylamine Chemical compound CCN(CC)N=O WBNQDOYYEUMPFS-UHFFFAOYSA-N 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention discloses a recovery culture method and application of LMH suspension cells, S1, taking out a freezing tube storing LMH suspension cells from liquid nitrogen, placing the freezing tube into a constant-temperature water bath kettle at 35-39 ℃, and vibrating and warming for 2-3min; s2, mixing the cell liquid and the LMH cell culture medium, and adding the mixture into a centrifuge tube; s3, centrifuging the liquid in the S2 for 3min to remove a supernatant; s4, preheating the LMH cell culture medium for 5min, suspending by using 5ml of LMH cell culture medium, transferring to a shake flask, and adding 15ml of LMH cell culture medium; s5, sampling and counting to ensure the density of the recovered LMH suspension cells; s6, placing the shake flask in a shaking table for two days for culture, counting, and completing cell resuscitation. The invention finds the lowest cell culture density required by LMH suspension cell recovery, and more effectively improves the recovery success rate, the cell survival rate and the cell state compared with the conventional recovery operation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recovery culture method and application of LMH suspension cells.
Background
The avian group I adenovirus is a common infectious pathogen of poultry and is distributed worldwide, and poultry at all ages are found to be susceptible at present, but the pathogenicity of the poultry is different according to the variety and the age of the poultry. At present, the avian group I adenovirus has strong pathogenicity on chickens, the main pathological changes are inclusion body hepatitis and pericardial effusion-hepatitis syndrome, the disease can directly cause the death rate of 20-40 days old chickens in a chicken farm to be more than 30%, and in addition, the disease is clinically often infected with infectious bronchitis virus, reovirus, H9 subtype avian influenza virus and the like simultaneously, so that the problems of obvious respiratory disease, arthritis, serious egg laying reduction, abnormal egg and the like of laying hens are caused. Therefore, the disease is one of the major epidemic diseases which seriously affect the production performance of the chicken farm at present.
In the prior art, 4 primary cells including Chicken Embryo Fibroblast (CEF), chicken embryo kidney Cell (CEK), chicken chick kidney Cell (CK) and chicken embryo liver Cell (CEL) are mainly used for preparing the inactivated vaccine of the avian group I adenovirus in the prior art, and viruses are separated and propagated. However, primary hepatorenal cells are complex to prepare, easy to pollute and difficult to transfect, and CEF is easy to mix in the process of preparing the cells, which is not beneficial to experimental operation. The LMH cell line is cultured by Kitagawa et al, japan scientists in 1981 by using liver cells of Laiyaho chicken suffering from hepatocellular carcinoma and performing mutation by using diethylnitrosamine, and is a chicken liver cancer cell line capable of continuous passage. With the progress of cell culture technology and the need for development, LMH cells have gradually become one of the cell lines that are frequently used. LMH cells have had a transition process from adherent to suspension cell culture. LMH presents typical epithelial cell characteristics, the cell line keeps a large number of phenotype characteristics of chicken hepatocyte differentiation, is mainly used for construction of recombinant adenovirus, and has wide application prospect in preparation of avian adenovirus vaccines.
However, recovery culture of LMH suspension cell lines has been a problem, and conventional recovery methods have difficulty in obtaining stable LMH suspension cells with excellent cell status, and often the cells begin to die after being maintained in a state of few days. So that the popularization and the application of the LMH suspension cell culture are hindered.
Disclosure of Invention
The invention provides an LMH suspension cell recovery culture method and application thereof, which aim to solve the problems in the prior art.
The scheme of the invention is as follows:
a LMH suspension cell recovery culture method comprises the following steps:
s1, taking out a freezing tube storing LMH suspension cells from liquid nitrogen, and putting the freezing tube into a constant-temperature water bath kettle at 35-39 ℃ to vibrate and warm for 2-3min;
s2, mixing the cell liquid and the LMH cell culture medium and adding the mixture into a centrifuge tube;
s3, centrifuging the liquid in the S2 for 3min to remove a supernatant;
s4, preheating the LMH cell culture medium for 5min, suspending by using 5ml of LMH cell culture medium, transferring to a shake flask, and adding 15ml of LMH cell culture medium;
s5, sampling and counting to ensure the density of the recovered LMH suspension cells;
s6, placing the shake flask in a shaking table for two days for culture, counting, and finishing cell resuscitation.
Preferably, the number of the cryopreservation tubes for thawing the thawed LMH suspension cells in step S1 is 3.
Preferably, the recovery temperature in step S1 is 37 ℃.
Preferably, the mixing ratio of the cell liquid and the LMH cell culture medium in the step S2 is 1:3.
Preferably, the centrifugation conditions in step S3 are 1000rpm.
Preferably, the LMH cell culture medium preheating condition in step S4 is 37 ℃.
As a preferable technical scheme, the density of the recovered LMH suspension cells in the step S5 is more than or equal to 2E6. The invention finds that the lowest recovery density of LMH suspension cells is 2E6, the recovery cannot be completed when the recovery density of the cells is lower than 2E6, and the recovery of the LMH suspension cells has density dependence.
Preferably, the density of the recovered LMH suspension cells in the step S5 is more than 2E7.
As a preferred embodiment, the cell culture conditions in step S6, in which the shake flask is placed on a shaker, are 37 ℃,8% CO 2 ，120rpm。
An application of LMH suspension cell recovery culture method.
S1, taking out the frozen tube storing the LMH suspension cells from liquid nitrogen, and putting the frozen tube into a constant-temperature water bath kettle at 35-39 ℃ to shake and warm for 2-3min; s2, mixing the cell liquid and the LMH cell culture medium, and adding the mixture into a centrifuge tube; s3, centrifuging the liquid in the S2 for 3min to remove a supernatant; s4, preheating the LMH cell culture medium for 5min, suspending by using 5ml of LMH cell culture medium, transferring to a shake flask, and adding 15ml of LMH cell culture medium; s5, sampling and counting to ensure the density of the recovered LMH suspension cells; s6, placing the shake flask in a shaking table for two days for culture, counting, and completing cell resuscitation.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention finds the lowest cell culture density required by LMH suspension cell recovery, and more effectively improves the recovery success rate, the cell survival rate and the cell state compared with the conventional recovery operation.
2. The invention discovers that LMH suspension cells are very sensitive to DMSO, and how to remove DMSO without influencing cell recovery is a key step of operation.
The centrifugation conditions were 1000rpm,3min. The most common and stable cell cryopreservation protective agent at present is DMSO, and the cytotoxicity is negligible and the cell recovery state is not influenced when the DMSO concentration is less than 0.1% in the conventional way, but LMH suspension cells are very sensitive to DMSO, and the removal of DMSO is very important for LMH cell recovery in research. Meanwhile, the centrifugal rotating speed of the cells which are just recovered is related to whether the cells are damaged or not, and the centrifugal time also influences the cell state. Centrifugation at 1000rpm for 3min to remove DMSO is most suitable for recovery of LMH suspension cells.
Drawings
FIG. 1 is a graph of resuscitation effects using a conventional resuscitation method;
FIG. 2 is a graph of resuscitation effects using the resuscitation method of the present invention;
FIG. 3 is a graph comparing cell density using the present invention and conventional resuscitation methods;
FIG. 4 is a graph comparing the cell viability using the present invention and a conventional resuscitation method.
Detailed Description
In order to make up for the above deficiencies, the invention provides an LMH suspension cell recovery culture method and an application thereof to solve the problems in the background art.
A LMH suspension cell recovery culture method comprises the following steps:
s1, taking out a freezing tube storing LMH suspension cells from liquid nitrogen, and putting the freezing tube into a constant-temperature water bath kettle at 35-39 ℃ to vibrate and warm for 2-3min;
s2, mixing the cell liquid and the LMH cell culture medium and adding the mixture into a centrifuge tube;
s3, centrifuging the liquid in the S2 for 3min to remove a supernatant;
s4, preheating the LMH cell culture medium for 5min, suspending by using 5ml of LMH cell culture medium, transferring to a shake flask, and adding 15ml of LMH cell culture medium;
s5, sampling and counting to ensure the density of the recovered LMH suspension cells;
s6, placing the shake flask in a shaking table for two days for culture, counting, and completing cell resuscitation.
Preferably, the number of the cryopreservation tubes for thawing the thawed LMH suspension cells in step S1 is 3.
The recovery temperature in step S1 was 37 ℃.
The mixing ratio of the cell liquid and the LMH cell culture medium in the step S2 is 1:3.
The centrifugation condition in step S3 was 1000rpm.
The LMH cell culture medium preheating condition in the step S4 is 37 ℃.
And the density of the recovered LMH suspension cells in the step S5 is more than or equal to 2E6.
And (5) the density of the recovered LMH suspension cells is more than 2E7.
The cell culture conditions in step S6 in which the shake flask was placed in a shaker were 37 ℃ and 8% CO 2 ，120rpm。
An application of LMH suspension cell recovery culture method.
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1:
a method for an LMH suspension cell recovery culture process comprises the following steps:
s1, taking 3 freezing tubes storing LMH suspension cells out of liquid nitrogen, placing the freezing tubes in a 37 ℃ constant-temperature water bath kettle, and vibrating and warming for 2-3min;
s2, adding the cell liquid and an LMH cell culture medium 1:3 into a centrifuge tube in proportion;
s3, centrifuging the liquid in the S2 at 1000rpm for 3min to remove supernatant;
s4, placing the LMH cell culture medium into a tank to be preheated at 37 ℃ for 5min, carrying out basic suspension by using 5ml of LMH cell culture medium, transferring the basic suspension to a shake flask, and adding 15ml of LMH cell culture medium;
s5, sampling and counting to ensure that the density of the recovered LMH suspension cells is not lower than 2E7;
s6, placing the flask at 37 ℃,8% CO 2 Shaking at 120rpm for two days, counting, and recovering the cells.
Example 2:
the traditional resuscitation method comprises the following steps: taking out a freezing tube of LMH cells from a liquid nitrogen tank, rapidly thawing and thawing in 37 ℃ water bath to obtain 1ml of cell liquid, adding 2ml of PBS, mixing uniformly, centrifuging at 800-1200rpm speed of a centrifuge for 5min, discarding supernatant, adding 15ml of culture medium for re-suspension, transferring to a shake flask, sampling, counting, and placing in a cell culture box for culture.
Example 3:
comparison of resuscitating cell capacity and viability:
1. the results of comparing the proliferation capacities of the cells recovered in example 1 and example 2 according to the cytometry method are shown in fig. 3, and it can be seen that the cells recovered according to the method begin to recover in Day3 and rapidly recover normal multiplication, thus proving that the density increase of the cells recovered by the method is stable.
2. The results of comparing the survival rates of the cells recovered in example 1 and example 2 according to the counting method of a cell counter are shown in fig. 4, and it can be seen from the figure that the cells recovered according to the method maintain 90% or more of the survival rate after recovery and quickly recover 95% or more of the survival rate, which proves that the survival rate of the cells recovered by the method is better.
The foregoing shows and describes the general principles, essential 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, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A LMH suspension cell recovery culture method is characterized by comprising the following steps:
s1, taking out a freezing tube storing LMH suspension cells from liquid nitrogen, and putting the freezing tube into a constant-temperature water bath kettle at 35-39 ℃ to vibrate and warm for 2-3min;
s2, mixing the cell liquid and the LMH cell culture medium, and adding the mixture into a centrifuge tube;
s3, centrifuging the liquid in the S2 for 3min to remove a supernatant;
s4, preheating the LMH cell culture medium for 5min, suspending by using 5ml of LMH cell culture medium, transferring to a shake flask, and adding 15ml of LMH cell culture medium;
s5, sampling and counting to ensure the density of the recovered LMH suspension cells;
s6, placing the shake flask in a shaking table for two days for culture, counting, and finishing cell resuscitation.
2. The LMH suspension cell recovery culture method of claim 1, wherein: the number of the freezing tubes for thawing the thawed LMH suspension cells in the step S1 is 3.
3. The LMH suspension cell recovery culture method of claim 1, characterized in that: the recovery temperature in step S1 was 37 ℃.
4. The LMH suspension cell recovery culture method of claim 2, wherein: the mixing ratio of the cell liquid and the LMH cell culture medium in the step S2 is 1:3.
5. The LMH suspension cell recovery culture method of claim 1, wherein: the centrifugation condition in step S3 was 1000rpm.
6. The LMH suspension cell recovery culture method of claim 1, wherein: the LMH cell culture medium preheating condition in the step S4 is 37 ℃.
7. The LMH suspension cell recovery culture method of claim 1, wherein: and the density of the recovered LMH suspension cells in the step S5 is more than or equal to 2E6.
8. The LMH suspension cell recovery culture method of claim 7, wherein: and (5) the density of the recovered LMH suspension cells is more than 2E7.
9. The LMH suspension cell recovery culture method of claim 1, wherein: the cell culture conditions in step S6 in which the shake flask was placed in a shaker were 37 ℃ and 8% CO 2 ，120rpm。
10. Use of the LMH suspension cell resuscitation culture method of claim 1.
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