CN106318900A - Method for rapidly preparing large number of chick embryo fibroblasts - Google Patents

Method for rapidly preparing large number of chick embryo fibroblasts Download PDF

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Publication number
CN106318900A
CN106318900A CN201610762198.6A CN201610762198A CN106318900A CN 106318900 A CN106318900 A CN 106318900A CN 201610762198 A CN201610762198 A CN 201610762198A CN 106318900 A CN106318900 A CN 106318900A
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embryo
gallus domesticus
tissue
piece
embryo gallus
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陈瑞爱
徐家华
王新秋
孙贝贝
任常宝
谢小雨
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Guangdong Wens Dahuanong Biotechnology Co Ltd
Zhaoqing Dahuanong Biological Pharmaceutical Co Ltd
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Guangdong Wens Dahuanong Biotechnology Co Ltd
Zhaoqing Dahuanong Biological Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

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  • Developmental Biology & Embryology (AREA)
  • Reproductive Health (AREA)
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  • General Health & Medical Sciences (AREA)
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Abstract

The invention discloses a method for rapidly preparing a large number of chick embryo fibroblasts. The method comprises steps as follows: 1) taking of chick embryos; 2) pancreatin washing; 3) homogenization; 4) digestion; 5) dispersion; 6) subculture, and obtaining of the chick embryo fibroblasts. The yield of the chick embryo fibroblasts obtained with the method is more than 20 times that of chick embryo fibroblasts obtained with traditional methods, and the operation time is short. Practices prove that each chick embryo can be transferred in 10-20 T225 cell bottles.

Description

A kind of quick method preparing a large amount of chick embryo fibroblast
Technical field
The present invention relates to a kind of quick method preparing a large amount of chick embryo fibroblast, belong to technical field of cell culture.
Background technology
Chick embryo fibroblast (CEF), except being applied to the correlational study of field of virology, is also widely used in live vaccine Produce, such as infectious bursal disease, Marek’s disease, newcastle etc., it is also possible to for expressing some genetic engineered product etc..
The method of the chick embryo fibroblast of report separation at present is varied, is mainly suitable for being applied to laboratory preparation a small amount of Chick embryo fibroblast, it generally includes and aseptic takes Embryo Gallus domesticus, decaptitates, extremity, internal organs etc., shreds, PBS or Hank ' s solution is washed Wash, trypsinization, dispel, filtered through gauze, the step such as centrifugal recovery cell, cell counting, cultivation.Its operating procedure is various easily Polluting, operating time length typically requires about 3 hours, and the chick embryo fibroblast gathered in the crops is considerably less, is completely unsuitable for A large amount of productions.Therefore, the present invention specifically addresses the problems referred to above, quickly prepare substantial amounts of CEF cell.
Summary of the invention
In order to overcome the deficiencies in the prior art, first purpose of the present invention is to provide a kind of cell harvesting amount height, behaviour The method quickly preparing a large amount of chick embryo fibroblast that time of making is short.
Realize the purpose of the present invention to reach by adopting the following technical scheme that:
A kind of quick method preparing a large amount of chick embryo fibroblast, comprises the following steps:
1) taking Embryo Gallus domesticus: take 9-11 age in days SPF Embryo Gallus domesticus, sterilization, sterile working takes out Embryo Gallus domesticus；
2) pancreatin rinses: by step 1) Embryo Gallus domesticus that takes out is cut into a diameter of 0.5-1cm massive texture；It is subsequently adding 0.25wt% trypsin solution rinses, and removes erythrocyte；
3) homogenate: under aseptic condition use refiner by step 2) process after Embryo Gallus domesticus pulverize homogenate straight to chicken embryo tissue block The little even 1mm in footpath；
4) digestion: to step 3) piece of tissue after homogenate adds 0.25wt% trypsin solution, light shake mixing and make to disappear Change；
5) dispersion: aspiration step 4) supernatant that obtains after digestion, it is transferred to cell bottle, blows and beats 1-3min, make cell divide Dissipate；
6) cultivate: to step 5) cell bottle in add contain hyclone culture medium, at 37 DEG C, 5%CO2Incubator Middle cultivation, changes culture medium, Secondary Culture, obtains chick embryo fibroblast after 24-32h.
As preferably, step 1) in, concrete sterilisation step is: uses 75vt% alcohol swab wiping Embryo Gallus domesticus, uses flame disinfection Embryo egg.
As preferably, step 2) in, the volume of trypsin solution is 1-10 times of the volume of piece of tissue.
As preferably, step 2) in, the volume of trypsin solution is 3-5 times of the volume of piece of tissue.
As preferably, step 4) in, the volume of trypsin solution is 1-10 times of the volume of piece of tissue.
As preferably, step 4) in, the volume of trypsin solution is 3-5 times of the volume of piece of tissue.
As preferably, step 4) in, gently shake 3-5min.
As preferably, step 5) in, use pipet to shift.
As preferably, step 6) in, the concentration of hyclone is 10wt%.
As preferably, step 6) in, described culture medium is 1640 culture medium.
The formulation Design Principle of the present invention is as follows:
Compared to existing technology, the beneficial effects of the present invention is: the CEF cell harvesting amount that the method that the present invention provides obtains Compared to traditional method, harvest yield improves more than 20 times and the operating time is short.Being proven, each Embryo Gallus domesticus can pass 10- The cell bottle of 20 T225.
Detailed description of the invention
Below, in conjunction with detailed description of the invention, the present invention is described further:
The present invention provides a kind of quick method preparing a large amount of chick embryo fibroblast, comprises the following steps:
1) taking Embryo Gallus domesticus: take 9-11 age in days SPF Embryo Gallus domesticus, sterilization, sterile working takes out Embryo Gallus domesticus；
2) pancreatin rinses: by step 1) Embryo Gallus domesticus that takes out is cut into a diameter of 0.5-1cm massive texture；It is subsequently adding 0.25wt% trypsin solution rinses；In this step, the purpose that pancreatin rinses is to remove a large amount of erythrocyte, to reduce interference；
3) homogenate: under aseptic condition use refiner by step 2) process after Embryo Gallus domesticus pulverize homogenate straight to chicken embryo tissue block The little even 1mm in footpath；Refiner is used can quickly to obtain the preferable chicken embryo tissue of volume homogeneity；The diameter of piece of tissue is less than 1mm, group The area knitting block contact pancreatin is big, it is easier to digestion, is conducive to collecting more homogeneous cell；
4) digestion: to step 3) piece of tissue after homogenate adds 0.25wt% trypsin solution, light shake mixing and make to disappear Change；Rinsed by pancreatin and combine digestion, can be effectively improved the efficiency of digestion, be conducive to simultaneously the most in large quantities obtain homogeneous thin Born of the same parents；And the too low digestion process of concentration of pancreatin will be slow, too high then cell manipulation is excessive；
5) dispersion: aspiration step 4) supernatant that obtains after digestion, it is transferred to cell bottle, is transferred to cell bottle, blow and beat 1- 3min, makes cell disperse；
6) cultivate: to step 5) cell bottle in add contain hyclone culture medium, at 37 DEG C, 5%CO2Incubator Middle cultivation, changes culture medium, Secondary Culture after 24-32h；Replacing culture medium, to remove the most adherent cells such as a small amount of cell, subtracts Few impurity, to improve the purity of chick embryo fibroblast.
Use said method obtain CEF cell harvesting amount compared to traditional method, gather in the crops improve more than 20 times and Operating time is short.Being proven, each Embryo Gallus domesticus can pass the cell bottle of 10-20 T225.
Embodiment 1:
A kind of quick method preparing a large amount of chick embryo fibroblast, comprises the following steps:
1) Embryo Gallus domesticus is taken: take 11 age in days SPF Embryo Gallus domesticus, put into super-clean bench, after 75% alcohol swab cleaning disinfection, use flame disinfection Embryo egg, the position of air chamber upward, is knocked against eggshell hard with aseptic nipper, is used new aseptic nipper instead and take out Embryo Gallus domesticus；
2) pancreatin rinses: by step 1) Embryo Gallus domesticus that takes out is cut into a diameter of 0.5-1cm massive texture, it is subsequently adding 0.25wt% trypsin solution rinses 2 times, and the volume of trypsin solution is 5 times of the volume of piece of tissue；
3) homogenate: under aseptic condition use refiner by step 2) process after Embryo Gallus domesticus pulverize homogenate straight to chicken embryo tissue block The little even 1mm in footpath；
4) digestion: to step 3) homogenate after piece of tissue in add 0.25wt% trypsin solution, trypsin solution and piece of tissue Volume ratio be 3:1, the light 4min that shakes makes to digest；
5) dispersion: by step 4) the supernatant pipet that obtains is transferred to T225 cell bottle after digestion, and blow and beat 2min, make Cell disperses；
6) cultivate: to step 5) T225 cell bottle in add contain 10wt% hyclone 1640 culture medium, 37 DEG C, 5%CO2Incubator is cultivated, changes culture medium, Secondary Culture after 24h, obtain chick embryo fibroblast.
In the present embodiment 1, just can obtain in a large number containing thin through " pancreatin rinses, is homogenized and digests " carried out the most successively The supernatant of born of the same parents, such that it is able to carry out mass propgation.
In the present embodiment, the fibroblast that each Embryo Gallus domesticus is separated to can pass the cell bottle of 15 T225.
Embodiment 2:
A kind of quick method preparing a large amount of chick embryo fibroblast, comprises the following steps:
1) Embryo Gallus domesticus is taken: take 9 age in days SPF Embryo Gallus domesticus, put into super-clean bench, after 75% alcohol swab cleaning disinfection, use flame disinfection Embryo egg, the position of air chamber upward, is knocked against eggshell hard with aseptic nipper, is used new aseptic nipper instead and take out Embryo Gallus domesticus；
2) pancreatin rinses: by step 1) Embryo Gallus domesticus that takes out is cut into a diameter of 0.5-1cm massive texture, it is subsequently adding 0.25wt% trypsin solution rinses 3 times, and the volume of trypsin solution is 3 times of the volume of piece of tissue；
3) homogenate: under aseptic condition use refiner by step 2) process after Embryo Gallus domesticus pulverize homogenate straight to chicken embryo tissue block The little even 1mm in footpath；
4) digestion: to step 3) homogenate after piece of tissue in add 0.25wt% trypsin solution, trypsin solution and piece of tissue Volume ratio be 5:1, the light 4min that shakes makes to digest；
5) dispersion: by step 4) the supernatant pipet that obtains is transferred to T225 cell bottle after digestion, and blow and beat 2min, make Cell disperses；
6) cultivate: to step 5) T225 cell bottle in add contain 10wt% hyclone 1640 culture medium, 37 DEG C, 5%CO2Incubator is cultivated, changes culture medium, Secondary Culture after 24h, obtain chick embryo fibroblast.
In the present embodiment, the fibroblast that each Embryo Gallus domesticus is separated to can pass the cell bottle of 13 T225.
Embodiment 3:
A kind of quick method preparing a large amount of chick embryo fibroblast, comprises the following steps:
1) Embryo Gallus domesticus is taken: take 10 age in days SPF Embryo Gallus domesticus, put into super-clean bench, after 75% alcohol swab cleaning disinfection, use flame disinfection Embryo egg, the position of air chamber upward, is knocked against eggshell hard with aseptic nipper, is used new aseptic nipper instead and take out Embryo Gallus domesticus；
2) pancreatin rinses: by step 1) Embryo Gallus domesticus that takes out is cut into a diameter of 0.5-1cm massive texture, it is subsequently adding 0.25wt% trypsin solution rinses 3 times, and the volume of trypsin solution is equal with the volume of piece of tissue；
3) homogenate: under aseptic condition use refiner by step 2) process after Embryo Gallus domesticus pulverize homogenate straight to chicken embryo tissue block The little even 1mm in footpath；
4) digestion: to step 3) homogenate after piece of tissue in add 0.25wt% trypsin solution, trypsin solution and piece of tissue Volume ratio be 10:1, the light 5min that shakes makes to digest；
5) dispersion: by step 4) the supernatant pipet that obtains is transferred to T225 cell bottle after digestion, and blow and beat 3min, make Cell disperses；
6) cultivate: to step 5) T225 cell bottle in add contain 10wt% hyclone 1640 culture medium, 37 DEG C, 5%CO2Incubator is cultivated, changes culture medium, Secondary Culture after 24h, obtain chick embryo fibroblast.
In the present embodiment, the fibroblast that each Embryo Gallus domesticus is separated to can pass the cell bottle of 19 T225.
Comparative example 1:
A kind of method preparing chick embryo fibroblast, comprises the following steps:
1) Embryo Gallus domesticus is taken: take 10 age in days SPF Embryo Gallus domesticus, put into super-clean bench, after 75% alcohol swab cleaning disinfection, use flame disinfection Embryo egg, the position of air chamber upward, is knocked against eggshell hard with aseptic nipper, is used new aseptic nipper instead and take out Embryo Gallus domesticus；
2) pancreatin rinses: by step 1) Embryo Gallus domesticus that takes out is cut into a diameter of 0.5-1cm massive texture, it is subsequently adding 0.25wt% trypsin solution rinses 3 times, and the volume of trypsin solution is 5 times of the volume of piece of tissue；
3) homogenate: under aseptic condition use refiner by step 2) process after Embryo Gallus domesticus pulverize homogenate straight to chicken embryo tissue block The little even 1mm in footpath；
4) digestion: to step 3) homogenate after piece of tissue in add 0.25wt% trypsin solution, trypsin solution and piece of tissue Volume ratio be 10:1, the light 5min that shakes makes to digest；
5) dispersion: by 5 layers of filtered through gauze collecting step 4) postdigestive supernatant；Then use 1500 revs/min from Heart 15min collects cell, is transferred to T225 cell bottle, blows and beats 3min, makes cell disperse；
6) cultivate: to step 5) T225 cell bottle in add contain 10wt% hyclone 1640 culture medium, 37 DEG C, 5%CO2Incubator is cultivated, changes culture medium, Secondary Culture after 24h, obtain chick embryo fibroblast.
In the present embodiment, the fibroblast that each Embryo Gallus domesticus obtains can pass the cell bottle of 3 T225.
Comparative example 2:
A kind of method preparing chick embryo fibroblast, comprises the following steps:
1) Embryo Gallus domesticus is taken: take 11 age in days SPF Embryo Gallus domesticus, put into super-clean bench, after 75% alcohol swab cleaning disinfection, use flame disinfection Embryo egg, the position of air chamber upward, is knocked against eggshell hard with aseptic nipper, is used new aseptic nipper instead and take out Embryo Gallus domesticus；
2) rinsing: by step 1) Embryo Gallus domesticus that takes out is cut into a diameter of 0.5-1cm massive texture, is subsequently adding PBS solution punching Wash 2 times, to remove a large amount of erythrocyte；
3) digestion: to step 2) piece of tissue in add 0.25wt% trypsin solution, the volume of trypsin solution and piece of tissue Make to digest than for 3:1, the light 4min that shakes；
4) dispersion: by step 3) the supernatant pipet that obtains is transferred to T225 cell bottle after digestion, and blow and beat 3min, make Cell disperses；
5) cultivate: to step 4) T225 cell bottle in add contain 10wt% hyclone 1640 culture medium, 37 DEG C, 5%CO2Incubator is cultivated, changes culture medium, Secondary Culture after 24h, obtain chick embryo fibroblast.
In the present embodiment, the fibroblast that each Embryo Gallus domesticus obtains can pass the cell bottle of 1 T225.
Comparative example 3:
A kind of method preparing chick embryo fibroblast, comprises the following steps:
1) Embryo Gallus domesticus is taken: take 11 age in days SPF Embryo Gallus domesticus, put into super-clean bench, after 75% alcohol swab cleaning disinfection, use flame disinfection Embryo egg, the position of air chamber upward, is knocked against eggshell hard with aseptic nipper, is used new aseptic nipper instead and take out Embryo Gallus domesticus；
2) rinsing: by step 1) Embryo Gallus domesticus that takes out is cut into a diameter of 0.5-1cm massive texture, is subsequently adding 0.25wt% pancreas Enzymatic solution rinses 2 times, to remove a large amount of erythrocyte；
3) digestion: to step 2) piece of tissue in add 0.25wt% trypsin solution, the volume of trypsin solution and piece of tissue Make to digest than for 3:1, the light 4min that shakes；
4) dispersion: by 5 layers of filtered through gauze collecting step 3) postdigestive supernatant.Then use 1500 revs/min from Heart 15min collects cell, is transferred to T225 cell bottle, blows and beats 3min, makes cell disperse；
5) cultivate: to step 4) T225 cell bottle in add contain 10wt% hyclone 1640 culture medium, 37 DEG C, 5%CO2Incubator is cultivated, changes culture medium, Secondary Culture after 24h, obtain chick embryo fibroblast.
In the present embodiment, the fibroblast that each Embryo Gallus domesticus obtains can pass the cell bottle of 1 T225.
From the comparison of embodiment 1-3 Yu comparative example 1,3, use traditional filtered through gauze, the side of centrifugal cell harvesting Formula, is suppressed by bigger the vigor of embryo fibroblast.From the comparison of embodiment 1-3 Yu comparative example 2,3, relative to Use PBS to wash except erythrocyte mode, first use pancreatin to use the mode of trypsinization preferably to protect again after rinsing homogenate Deposit the vigor of chick embryo fibroblast.
It will be apparent to those skilled in the art that can technical scheme as described above and design, make other various Corresponding change and deformation, and all these change and deformation all should belong to the protection domain of the claims in the present invention Within.

Claims (10)

1. the quick method preparing a large amount of chick embryo fibroblast, comprises the following steps:
1) taking Embryo Gallus domesticus: take 9-11 age in days SPF Embryo Gallus domesticus, sterilization, sterile working takes out Embryo Gallus domesticus；
2) pancreatin rinses: by step 1) Embryo Gallus domesticus that takes out is cut into a diameter of 0.5-1cm massive texture；It is subsequently adding 0.25wt% pancreas Enzymatic solution rinses, and removes erythrocyte；
3) homogenate: under aseptic condition use refiner by step 2) process after Embryo Gallus domesticus pulverize homogenate little to chicken embryo tissue block diameter Even 1mm；
4) digestion: to step 3) piece of tissue after homogenate adds 0.25wt% trypsin solution, light shake mixing and make to digest；
5) dispersion: aspiration step 4) supernatant that obtains after digestion, it is transferred to cell bottle, blows and beats 1-3min, make cell disperse；
6) cultivate: to step 5) cell bottle in add contain hyclone culture medium, at 37 DEG C, 5%CO2Incubator is trained Support, change culture medium, Secondary Culture after 24-32h, obtain chick embryo fibroblast.
Method the most according to claim 1, it is characterised in that step 1) in, concrete sterilisation step is: use 75vt% wine Essence cotton rub wipes Embryo Gallus domesticus, with flame disinfection embryo egg.
Method the most according to claim 1, it is characterised in that step 2) in, the volume of trypsin solution is the body of piece of tissue Long-pending 1-10 times.
Method the most according to claim 1, it is characterised in that step 2) in, the volume of trypsin solution is the body of piece of tissue Long-pending 3-5 times.
Method the most according to claim 1, it is characterised in that: step 4) in, the volume of trypsin solution is the body of piece of tissue Long-pending 1-10 times.
Method the most according to claim 1, it is characterised in that: step 4) in, the volume of trypsin solution is the body of piece of tissue Long-pending 3-5 times.
Method the most according to claim 1, it is characterised in that: step 4) in, gently shake 3-5min.
Method the most according to claim 1, it is characterised in that: step 5) in, use pipet to shift.
Method the most according to claim 1, it is characterised in that: step 6) in, the concentration of hyclone is 10wt%.
Method the most according to claim 9, it is characterised in that: step 6) in, described culture medium is 1640 culture medium.
CN201610762198.6A 2016-08-29 2016-08-29 Method for rapidly preparing large number of chick embryo fibroblasts Pending CN106318900A (en)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN107446880A (en) * 2017-09-01 2017-12-08 铜仁职业技术学院 A kind of method for preparing chicken embryo fibroblasts
CN113789297A (en) * 2021-09-09 2021-12-14 豪威生物科技有限公司 Method for preparing chicken embryo fibroblasts in large scale

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107446880A (en) * 2017-09-01 2017-12-08 铜仁职业技术学院 A kind of method for preparing chicken embryo fibroblasts
CN113789297A (en) * 2021-09-09 2021-12-14 豪威生物科技有限公司 Method for preparing chicken embryo fibroblasts in large scale

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