CN115572707A - Koi muscle cell line and in-vitro culture method and application thereof - Google Patents

Koi muscle cell line and in-vitro culture method and application thereof Download PDF

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CN115572707A
CN115572707A CN202211336761.5A CN202211336761A CN115572707A CN 115572707 A CN115572707 A CN 115572707A CN 202211336761 A CN202211336761 A CN 202211336761A CN 115572707 A CN115572707 A CN 115572707A
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cell line
koi
medium
muscle cell
virus
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CN115572707B (en
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景宏丽
陈冬杰
高隆英
孔玉方
王娜
张旻
吴绍强
吕继洲
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Chinese Academy of Inspection and Quarantine CAIQ
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Abstract

The disclosure relates to a koi muscle cell line and a method and application for in vitro culture of the koi muscle cell line. The disclosure particularly relates to a koi muscle cell line, and an in vitro culture method and application thereof.

Description

Koi muscle cell line and in-vitro culture method and application thereof
Technical Field
The invention belongs to the technical field of fish cell culture, and particularly relates to a koi muscle cell line, and an in-vitro culture method and application thereof.
Background
The brocade carp is also called original breed of Pycnopsis carpi, and is Cyprinus carpio. The reported fancy carp originates from Guangxi Longzhou fancy carp, jiangxi Xingguan red carp and Zhejiang Hangzhou golden carp in China, is imported into Japan from China at an early stage, and is artificially bred by Japanese people for a long time, so that more than 100 varieties exist. Nowadays, the fancy carp is a high-grade ornamental fish in the world, and is covered by the terms of 'living jewels in water', 'art articles capable of swimming' and the like. However, due to the outbreak of highly pathogenic and infectious virus diseases of aquatic animals in recent years, a large number of fancy carps are killed, which brings serious economic losses to the breeding industry.
Therefore, the isolation of the pathogen as soon as possible is the most critical issue for the prevention and control of the disease. However, it is well known that cell line isolation of viruses is the "gold standard" for the detection of viral diseases in aquatic animals. Thus, many scholars at home and abroad are studying fish cell lines. Cell lines derived from koi tissues are also established in succession. However, no koi muscle cell line capable of stable passage has been established at present.
Disclosure of Invention
The purpose of the present disclosure is to provide a koi muscle cell line capable of stable passage.
In a first aspect, the present disclosure provides a koi muscle cell line, which is preserved in the common microorganism center of the china committee for culture collection management (CGMCC) at 21.9/2022, with the preservation number of CGMCC No.45307, and the preservation address of CGMCC: beijing in China.
In a second aspect, the present disclosure provides a method of culturing the cell line provided in the first aspect in vitro.
In some embodiments, the method comprises culturing the cell line using a medium comprising 10% to 20% serum.
In some embodiments, the serum concentration is 10% to 15%, preferably 10%.
In some embodiments, the temperature of the culture is 20 to 30 ℃, preferably 25 to 30 ℃, more preferably 25 ℃.
In some embodiments, the culturing is for a period of 4 to 8 days, preferably 5 to 7 days, more preferably 5 to 6 days.
In some embodiments, the medium is selected from the group consisting of M199 medium, MEM medium, DMEM medium, and L-15 medium.
In some embodiments, the serum is selected from fetal bovine serum and neonatal bovine serum.
In some embodiments, the method further comprises digesting the cells with pancreatin digest after the culturing is complete.
In some embodiments, the pancreatin digest comprises the following components:
pancreatin 0.6 g/L;
0.2g/L EDTA;
2.3g/L disodium hydrogen phosphate;
0.1g/L potassium dihydrogen phosphate;
8.0g/L sodium chloride;
0.2g/L potassium chloride.
In a third aspect, the present disclosure provides the use of a koi muscle cell line of the first aspect in the isolation, propagation or viral vaccine development of an aquatic animal virus.
In some embodiments, the aquatic animal is selected from the group consisting of fish, shrimp, crab, and shellfish, and the like, preferably fish.
In some embodiments, the virus is selected from the group consisting of spring viraemia of carp virus, ictalurus punctatus virus, infectious pancreatic necrosis virus, grass carp reovirus (e.g., strain 873 grass carp reovirus), infectious hematopoietic necrosis virus, and the like.
The koi muscle cell line provided by the disclosure is stable in characteristics and uniform in components. It is a material for researching aquatic animal virus.
The koi muscle cell line provided by the present disclosure is sensitive to aquatic animal viruses.
The koi muscle cell line provided by the disclosure can be used as a host cell for researching aquatic animal viruses. The cell line can be used for separating and propagating aquatic animal viruses or developing virus vaccines. Provides a foundation for researching aquatic animal diseases.
Drawings
Figure 1 shows the morphology of koi muscle cell lines under phase contrast microscopy.
Figure 2 shows the effect of serum concentration on the culture of koi muscle cell lines.
Figure 3 shows the effect of temperature on the culture of koi muscle cell lines.
FIG. 4 shows the results of the doubling curve analysis of koi muscle cell lines.
FIG. 5 shows morphology of koi muscle cell lines after infection with infectious hematopoietic necrosis virus.
Detailed Description
The present disclosure is further illustrated by the following examples, and it is understood that these examples are included merely for purposes of illustration and are not intended to limit the disclosure, so that modifications of the disclosure that are obvious from the spirit of the disclosure are intended to be included within the scope of the claims.
Definition of
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. It is noted that the terms used herein should be interpreted as having a meaning that is consistent with the context of this specification and should not be interpreted in an idealized or overly formal sense.
The expression "about" as used herein is as understood by one of ordinary skill in the art and varies within certain limits depending on the context in which it is used. If one of ordinary skill in the art would not understand the use of this term based on the context of its use, then "about" would mean that the particular value is at most plus or minus 10%.
The term "cell line" as used herein refers to a population of cells propagated from a primary cell culture after successful initial passage, and also refers to cultured cells that can be serially passaged for a long period of time.
The term "aquatic animal" as used herein refers to an animal that lives primarily in water. In the present disclosure, aquatic animals are wild or farmed animals of some economic value to humans, including mollusks, echinoderms, coelenterates, aquatic arthropods such as fish, shrimp, crab, and shellfish.
The examples do not specify particular techniques or conditions, and are performed according to techniques or conditions described in literature in the art or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Examples
Example 1 isolation and Primary culture of muscle cell lines of Koi
1) Test materials and reagents
Experimental animals: 30 healthy red and white fancy carps purchased from a certain farm in Beijing, the body length of which is 10-15 cm;
experimental apparatus: scalpels, ophthalmic scissors, ophthalmic forceps, gauze, and the like.
Media and related reagents:
the concentration of the penicillin-streptomycin double-antibody solution is 10000U/mL;
culture medium: m199 medium (gibco) containing penicillin-streptomycin double antibody solution with concentration of 100U/mL and 20% special fetal bovine serum (Hangzhou Sijiqing bioengineering materials Co., ltd.);
pancreatin digestive juice: 2.3g of disodium hydrogen phosphate, 0.1g of monopotassium phosphate, 8.0g of sodium chloride, 0.2g of potassium chloride, 0.2g of EDTA (ethylene diamine tetraacetic acid), 0.6g of pancreatin and water with constant volume of 1000mL;
medical alcohol;
0.01M PBS buffer (NaCl 80g, na 2 HPO 4 ·12H 2 O 29g，KH 2 PO 4 2g, KCl 2g and water till the volume is 1000 mL).
2) Experimental method
The detected 30 healthy fancy carps are kept for 15 days in a laboratory, no other diseases exist, and the experiments are respectively started for all fancy carps. The method comprises the following specific steps:
(1) Soaking Cyprinus Carpio in medical alcohol for 1-5min, peeling skin layer and inner membrane of Cyprinus Carpio under aseptic condition, clipping muscle, placing in 1000U/mL penicillin-streptomycin double-resistant solution, and standing for 10-15min.
(2) Shearing muscle tissue of Cyprinus Carpio into several pieces of 1mm 3 Spreading 5 or 6 small pieces uniformly on the bottom surface of a culture bottle, wherein the number of the spread bottles for each brocade carp is 8-10 bottles, and standing upside down for 1-2h.
(3) The medium was slowly added to the flask and incubated in an incubator at 25 ℃.
(4) The next day, after medium was replenished to 5mL, fresh medium was changed every three days.
(5) When the state of cell elongation is observed at this time, some cells are elongated but no longer grow in the late stage. And finally, only 1 bottle of cells continuously grows, and after the cells are paved on the bottom surface of the culture bottle, pancreatin digestive juice is used for preliminary digestion, and then fresh culture medium is added for culture.
(6) After passage for 10 times, the cell line is frozen and stored, and can continue to stably proliferate and grow after recovery, and the cell line is named as KM. The morphology of koi muscle cell line KM is shown in fig. 1.
(7) Passage 1 every 7 days.
Example 2 subculture and cryopreservation of Cyprinus carpiod muscle cell line
1) Reagent
Pancreatin digestive juice: 2.3g of disodium hydrogen phosphate, 0.1g of monopotassium phosphate, 8.0g of sodium chloride, 0.2g of potassium chloride, 0.2g of EDTA, 0.6g of pancreatin and water with the constant volume of 1000mL.
Freezing and storing liquid: special grade fetal bovine serum containing 10% DMSO (Hangzhou ilex bioengineering materials Co., ltd.).
2) Experimental method
When the cells obtained by screening in example 1 grow to cover 90% of the bottom surface of the culture flask, the old culture medium is discarded, and 2mL of pancreatin digestive juice is added for digestion.
When the cells are in a white fog shape, 2mL of the cryopreservation solution is added to stop the action of the pancreatin, and the cells are mixed evenly by shaking slightly and counted.
Collecting the cells into a freezing tube, and filling the name, the number and the freezing date of the cells on the freezing tube.
After one month, cell recovery is carried out according to a conventional cell recovery method, the cell proliferation capacity is still very strong, and the state is good.
The obtained muscle cell line of koi has been preserved in China general microbiological culture Collection center (CGMCC) on 21.9.2022, with the preservation number of CGMCC No.45307. The koi muscle cell line was designated as KM.
When the koi muscle cell line is cultured for 1 day, the elongation growth is obviously generated; after 5 days, the bottom of the culture bottle is paved, and after digestion, the culture bottle has stronger reproductive capacity and presents a typical epithelial cell shape. The cells are continuously subcultured for 16 months and are transferred to 50 generations, and the morphological characteristics, growth and proliferation characteristics of the epithelial cells are still maintained.
Example 3 determination of optimal conditions for muscle cell lines of koi
1) Reagent
Pancreatin digestive juice: 2.3g of disodium hydrogen phosphate, 0.1g of monopotassium phosphate, 8.0g of sodium chloride, 0.2g of potassium chloride, 0.2g of EDTA, 0.6g of pancreatin and water with the constant volume of 1000mL.
Culture medium: m199 culture medium (gibco) of special fetal bovine serum (Hangzhou Sijiqing bioengineering materials Co., ltd.) at different concentrations.
2) Experimental method
(1) Determination of optimal serum concentration
Respectively preparing culture solution with special fetal calf serum concentration of 5%, 10%, 15% and 20%, and adjusting cell density to 2.5 × 10 5 mL -1 Four kinds of serum concentration media were inoculated in a 24-well plate at 1 mL/well, and cultured in an incubator at 25 ℃.3 wells of cells were taken out of each experimental group every day, and the cells were collected by digestion with pancreatin digest and counted, cultured for 7 days in total, counted 7 times in a row, and their growth curves were plotted.
(2) Determination of optimum temperature
Selecting four different culture temperatures of 15 deg.C, 20 deg.C, 25 deg.C and 30 deg.C, adding 15% special grade Tai bovine serum M199 culture solution, and adjustingThe whole cell density was 1.9X 10 5 mL -1 The cell suspension was inoculated into 24-well plates at 1 mL/well and placed in four incubators at different incubation temperatures. Cells were removed from 3 wells of each experimental group each day, collected and counted, cultured for 7 days, counted 7 times in a row, and a growth curve was plotted.
3) Results of the experiment
The determination of the optimal serum is shown in fig. 2, and the suitable serum concentration for culturing the koi muscle cell line is 10-20%, and the more suitable serum concentration is 10-15%. The optimal serum concentration is 10% depending on the cell growth state and the cost of the medium.
The determination of the optimum temperature is shown in figure 3, the culture of the koi muscle cell line is carried out at a suitable temperature of 20-30 ℃, more suitably at a temperature of 25-30 ℃, and the koi muscle cell line grows at 25 ℃ and has the best propagation.
Example 4 growth curves of koi muscle cell lines
1) Reagent
Pancreatin digestive juice: 2.3g of disodium hydrogen phosphate, 0.1g of monopotassium phosphate, 8.0g of sodium chloride, 0.2g of potassium chloride, 0.2g of EDTA, 0.6g of pancreatin and water with the constant volume of 1000mL.
Culture medium: 15% M199 medium of special grade Fetal Bovine Serum (FBS).
2) Experimental methods
Counting cells were collected from cells in the logarithmic growth phase, cultured using M199 containing 15% FBS, and adjusted to a cell concentration of 8.34X 10 4 mL -1 The cells were inoculated into 24-well plates at a concentration of 1 mL/well, and cultured in an incubator at 25 ℃. Taking 3 cells in a hole every day, digesting with pancreatin to prepare a cell suspension, uniformly mixing, counting by using a cell counting instrument, repeating the experiment for 3 times, continuously performing for 7 days, and drawing a growth curve by taking the culture time (days) as an abscissa and taking the cell concentration (cells/mL) as an ordinate. The population doubling time of the cells was calculated according to the formula T = T × lg2/lg (Nt/N0), where N0 is the number of seeded cells and Nt is the number of cells after time T.
3) Results of the experiment
As shown in fig. 4, the group doubling time of the koi muscle cell line was calculated to be 43.98 hours when the koi muscle cell line entered the 0-2 day delay phase, entered the logarithmic growth phase for 2-4 days and entered the stationary phase at 4 days.
Example 5 application of muscle cell line of koi
1) Reagent
Pancreatin digestive juice: 2.3g of disodium hydrogen phosphate, 0.1g of monopotassium phosphate, 8.0g of sodium chloride, 0.2g of potassium chloride, 0.2g of EDTA, 0.6g of pancreatin and water with the constant volume of 1000mL.
Culture medium: 10% M199 medium of extra-grade Fetal Bovine Serum (FBS).
Infectious hematopoietic necrosis virus (IHNV, ATCC VR-714) TM )
2) Experimental methods
The koi muscle cell line was digested with pancreatin digest, passaged 1 in M199 medium containing 10% special Fetal Bovine Serum (FBS), and cultured in an incubator at 25 ℃. Cell proliferation infectious hematopoietic necrosis virus was inoculated within 24h, placed in an incubator at 16 ℃ and observed for changes every day.
3) Results of the experiment
As shown in FIG. 5, after inoculation of the virus for 36h, obvious cytopathic effect (CPE) appears in the cells, which indicates that the cyprinus carpio muscle cell line can proliferate the virus IHNV. It can be seen that this cell line provides a powerful material for the study of IHNV.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations will not be further described in the present disclosure.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims (10)

1. The cryprinus carpiod muscle cell line is preserved in the China general microbiological culture Collection center on 21.9.2022 with the preservation number of CGMCC No.45307.
2. A method of culturing the cell line of claim 1 in vitro.
3. The method of claim 2, comprising culturing the cell line using a medium comprising 10% to 20% serum.
4. The method according to claim 3, wherein the serum concentration is 10% to 15%, preferably 10%.
5. The method according to claim 3, wherein the temperature of the cultivation is 20 to 30 ℃, preferably 25 to 30 ℃, more preferably 25 ℃.
6. The method according to claim 3, wherein the culturing is carried out for a period of 4 to 8 days, preferably 5 to 7 days, more preferably 5 to 6 days.
7. The method according to claim 3, wherein the medium is selected from the group consisting of M199 medium, MEM medium, DMEM medium, and L-15 medium.
8. The method of claim 3, wherein the serum is selected from fetal bovine serum and neonatal bovine serum.
9. Use of a koi muscle cell line according to claim 1 for the isolation, propagation or vaccine development of a virus in an aquatic animal.
10. Use according to claim 9, wherein the aquatic animal is selected from the group consisting of fish, shrimp, crab and shellfish, preferably fish;
preferably, the virus is selected from the group consisting of spring viraemia of carp virus, channel catfish virus, infectious pancreatic necrosis virus, grass carp reovirus and infectious hematopoietic necrosis virus.
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