CN107446892A - A kind of cultural method of LMH cells - Google Patents
A kind of cultural method of LMH cells Download PDFInfo
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- CN107446892A CN107446892A CN201710737213.6A CN201710737213A CN107446892A CN 107446892 A CN107446892 A CN 107446892A CN 201710737213 A CN201710737213 A CN 201710737213A CN 107446892 A CN107446892 A CN 107446892A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0693—Tumour cells; Cancer cells
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/34—Sugars
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10251—Methods of production or purification of viral material
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Abstract
The invention provides a kind of method of free serum culture LMH cells, first passes through and sets serum content gradient in DMEM in high glucose culture medium to LMH cells tame by generation, reuses the DMEM in high glucose culture medium without serum and scattered Secondary Culture is carried out to LMH cells.The large-scale culture of specific virus can be carried out using the LMH cells of the method for the invention culture, effectively improve the technical matters of LMH cell attachment cultures, the potency of virus can be ensured while production cost is significantly reduced, there is potential advantage and huge application value in actual production.
Description
Technical field
The invention belongs to biological product technical field, more particularly to a kind of cultural method of LMH cells.
Background technology
With the progress and research of cell culture technology and the needs of production, chicken gizzard cancer cell (LMH cells) gradually into
For one of most frequently used cell line.At present, LMH cells can be used successfully to avian infectious laryngotracheitis virus
(Infectious laryngotracheitis virus, ILTV), birds adenovirus (Fowl Adenovirus, FADV) etc.
The culture of totivirus and the expression of multiple protein.Popularization and biological products quality safety mark with concentration cultivation container
Accurate raising, the culture of LMH cells are with a wide range of applications.
However, due in cell cultivation process culture medium involve great expense, in particular for adding higher percent
Serum so that the production cost of this kind of training method always not under, amplification and popularization to LMH cell culture cause certain resistance
Hinder.
The content of the invention
The present invention is directed to the defects of cost is higher in LMH cell cultivation process, there is provided a kind of cheap LMH cells of cost
Cultural method.
To achieve the above object, the present invention provides a kind of cultural method of LMH cells, first passes through and sets DMEM in high glucose culture
Serum content gradient to LMH cells tame by generation in base, reuses the DMEM in high glucose culture medium without serum to LMH cells
Carry out scattered Secondary Culture.
The serum content gradient is：10%CS, 8%CS, 5%CS, 3%CS, 1%CS, 0%CS.It is of the present invention
Serum content gradient is that inventor pays a large amount of creative works, is determined through lot of experiment validation；Find under study for action, serum
Concentration gradients are set unreasonable, and cost can be caused too high, or obtained LMH cell qualities decline.
It is described to be by the parameter for domestication：37 DEG C, CO2Content is 3%.
In order to ensure cell can preferably adherent growth, during being tamed by generation, use 1 ‰ gelatin before domestication every time
Solution handles culture vessel, and treatment conditions are：37 DEG C of processing more than 12h.
During being tamed by generation, the sugared content in sampling palpus measure culture medium per 24h；Should when sugared content is less than 3.0g/L
Carry out changing liquid.
As the preferred embodiment of the present invention, the cultural method of the LMH cells comprises the following steps：
(1) LMH cells are cultivated using serum content 10%CS DMEM in high glucose culture medium, to cell growth extremely
80%-90% density, cell is digested and disperseed using pancreatin；
(2) scattered passage is carried out using in serum content 10%CS DMEM in high glucose culture medium and after removing pancreatin, thereafter
Fluid infusion culture is carried out using the DMEM in high glucose culture medium containing serum 8%CS, to cell growth to 80%-90% density；
(3) attached cell obtained by the DMEM in high glucose medium culture containing serum 8%CS is carried out after disperseing, used thereafter
DMEM in high glucose culture medium containing serum 5%CS carries out fluid infusion culture, to cell growth to 80%-90% density；
(4) attached cell obtained by the DMEM in high glucose medium culture containing serum 5%CS is carried out after disperseing, used thereafter
DMEM in high glucose culture medium containing serum 3%CS carries out fluid infusion culture, to cell growth to 80%-90% density；
(5) attached cell obtained by the DMEM in high glucose medium culture containing serum 3%CS is carried out after disperseing, used thereafter
DMEM in high glucose culture medium containing serum 1%CS carries out fluid infusion culture, to cell growth to 80%-90% density；
(6) attached cell obtained by 1%CS DMEM in high glucose medium culture is carried out after disperseing, used thereafter without serum
DMEM in high glucose culture medium carries out fluid infusion culture, to cell growth to 80%-90% density；
(7) scattered Secondary Culture is carried out to the LMH cells after domestication using the DMEM in high glucose culture medium without serum.
Above-mentioned steps (3) need first pancreatin to digest into step (6) before disperseing every time, then neutralize removal pancreatin, again finally
Dispersion culture.
The present invention also provides the LMH cells obtained by above-mentioned cultural method.
The present invention also provides a kind of virus inoculation method, comprises the following steps：
(1) before connecing poison, serum-free DMEM in high glucose culture medium is preheated 37 DEG C, while melt the viral solution frozen；
(2) original serum-free DMEM in high glucose medium culture base in the adhere-wall culture container equipped with LMH cells is removed, and
The dead cell to come off and other compositions (such as metabolite) are washed away using the fresh serum free DMEM in high glucose culture medium of above-mentioned preheating；
(3) viral solution is added into serum-free DMEM in high glucose culture medium according to the ratio of culture medium cumulative volume 2%, it is fully mixed
It is even；
(4) the Virus culture based sols of mixing are added in LMH cells, 35 DEG C of cultures, when cytopathy and coming off reaches
Virus-culturing fluid is harvested during to 90%.
In the virus inoculation method, the culture parameters of step (4) are：35 DEG C, CO2Content is 5%.
The large-scale culture of specific virus can be carried out using the LMH cells of the method for the invention culture, is effectively improved
The technical matters of LMH cell attachment cultures, the potency of virus can be ensured while production cost is significantly reduced,
There is potential advantage and huge application value in actual production.In addition, the LMH obtained using the method for the invention culture
Cell virus inoculation, TCID50≤7.0 of gained viral solution.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
1st, aviadenovirus pathological material of disease is taken, conventionally virus is isolated and purified, chicken gizzard cancer cell (LMH) is carried out
Secondary Culture, including domestication and dispersion culture, comprise the following steps that：
(1) when cultivating LMH cells first, serum, concentration 10%CS are added in DMEM in high glucose culture medium；Given birth to cell
Length is digested and disperseed to cell using pancreatin to 80%-90% density；
(2) scattered passage is carried out using in the DMEM in high glucose culture medium containing serum 10%CS and after removing pancreatin, thereafter
Fluid infusion culture is carried out using the DMEM in high glucose culture medium containing serum 8%CS, to cell growth to 80%-90% density.
(3) by attached cell obtained by DMEM in high glucose medium culture containing serum 8%CS carry out it is scattered after, using containing
Serum 5%CS DMEM in high glucose culture medium carries out fluid infusion culture, to cell growth to 80%-90% density.
(4) by attached cell obtained by DMEM in high glucose medium culture containing serum 5%CS carry out it is scattered after, using containing
Serum 3%CS DMEM in high glucose culture medium carries out fluid infusion culture, to cell growth to 80%-90% density.
(5) by attached cell obtained by DMEM in high glucose medium culture containing serum 3%CS carry out it is scattered after, using containing
Serum 1%CS DMEM in high glucose culture medium carries out fluid infusion culture, to cell growth to 80%-90% density.
(6) by attached cell obtained by DMEM in high glucose medium culture containing serum 1%CS carry out it is scattered after, using without
The DMEM in high glucose culture medium of serum carries out fluid infusion culture, until LMH cells can be in the adherent life of serum-free DMEM in high glucose culture medium
Grow to 80%-90% density.
During the above-mentioned domestication by generation, per 24h, sampling is surveyed culture medium sugar and consumed, and is changed when sugared content is less than 3.0g/L
Liquid.
2nd, the LMH cells that above-mentioned free serum culture obtains are carried out connecing poison, specific method is as follows：
(1) old liquid is discarded before connecing poison, and dead cell and other are gently washed away with the serum-free DMEM in high glucose culture medium of preheating
Metabolite, it is repeated 3 times.
(2) aviadenovirus (FADV) liquid of Secondary Culture is taken, serum-free DMEM in high glucose culture medium is added according to volume ratio 2%
In, after fully mixing, the serum-free DMEM in high glucose culture medium containing virus liquid is added in cell culture container, 35 DEG C of cultures,
Observation cytopathy degree daily, when cytopathy and viral solution is harvested when coming off and reaching 90%.
Take the viral solution of harvest to carry out gradient dilution, its cell median infective dose TCID is determined using 96 orifice plates50。
Repeat above-mentioned experiment twice.
Three batches of LMH cells of above-mentioned serum-free DMEM in high glucose medium culture are inoculated with obtained aviadenovirus liquid, respectively
Carry out cell median infective dose TCID50Measure, as a result such as table 1.
Table 1
| Batch | Virus titer (TCID50/0.2ml) |
| 001 | 7.0 |
| 002 | 7.5 |
| 003 | 7.2 |
As shown in Table 1, three batches of LMH cells inoculation gained virus liquid TCID of serum-free DMEM in high glucose medium culture50≧
7.0。
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. a kind of cultural method of LMH cells, it is characterised in that first pass through and serum content gradient in DMEM in high glucose culture medium is set
LMH cells tamed by generation, the DMEM in high glucose culture medium without serum is reused and scattered passage training is carried out to LMH cells
Support.
2. the cultural method of LMH cells according to claim 1, it is characterised in that the serum content gradient is：10%
CS, 8%CS, 5%CS, 3%CS, 1%CS, 0%CS.
3. the cultural method of LMH cells according to claim 1 or 2, it is characterised in that the parameter by generation domestication
For：37 DEG C, CO2Content is 3%.
4. according to the cultural method of any described LMH cells of claim 1-3, it is characterised in that during being tamed by generation,
It is using 1 ‰ gelatin solutions processing culture vessel, treatment conditions before domestication every time：37 DEG C of processing more than 12h.
5. according to the cultural method of any described LMH cells of claim 1-4, it is characterised in that during being tamed by generation,
Per 24h, sampling must determine sugared content in culture medium；It should carry out changing liquid when sugared content is less than 3.0g/L.
6. the cultural method of LMH cells according to claim 1, it is characterised in that the cultural method bag of the LMH cells
Include following steps：
(1) LMH cells are cultivated using serum content 10%CS DMEM in high glucose culture medium, to cell growth to 80%-
90% density, cell is digested and disperseed using pancreatin；
(2) scattered passage is carried out using in serum content 10%CS DMEM in high glucose culture medium and after removing pancreatin, used thereafter
DMEM in high glucose culture medium containing serum 8%CS carries out fluid infusion culture, to cell growth to 80%-90% density；
(3) by attached cell obtained by DMEM in high glucose medium culture containing serum 8%CS carry out it is scattered after, thereafter using containing
Serum 5%CS DMEM in high glucose culture medium carries out fluid infusion culture, to cell growth to 80%-90% density；
(4) by attached cell obtained by DMEM in high glucose medium culture containing serum 5%CS carry out it is scattered after, thereafter using containing
Serum 3%CS DMEM in high glucose culture medium carries out fluid infusion culture, to cell growth to 80%-90% density；
(5) by attached cell obtained by DMEM in high glucose medium culture containing serum 3%CS carry out it is scattered after, thereafter using containing
Serum 1%CS DMEM in high glucose culture medium carries out fluid infusion culture, to cell growth to 80%-90% density；
(6) attached cell obtained by 1%CS DMEM in high glucose medium culture is carried out after disperseing, thereafter using the height sugar without serum
DMEM culture mediums carry out fluid infusion culture, to cell growth to 80%-90% density；
(7) scattered Secondary Culture is carried out to the LMH cells after domestication using the DMEM in high glucose culture medium without serum.
7. the LMH cells that any cultural methods of claim 1-6 obtain.
A kind of 8. virus inoculation method, it is characterised in that comprise the following steps：
(1) before connecing poison, serum-free DMEM in high glucose culture medium is preheated 37 DEG C, while melt the viral solution frozen；
(2) viral solution is added into serum-free DMEM in high glucose culture medium according to the ratio of culture medium cumulative volume 2%, fully mixed；
(3) the Virus culture based sols of mixing are added in LMH cells described in claim 7,35 DEG C of cultures, work as cytopathy
And come off and virus-culturing fluid is harvested when reaching 90%.
9. virus inoculation method according to claim 8, it is characterised in that the culture parameters of step (4) are：35 DEG C, CO2
Content is 5%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111518768A (en) * | 2020-04-14 | 2020-08-11 | 四川百诺吉科技有限公司 | Low-serum culture medium suitable for LMH cell adherent culture and preparation method thereof |
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