CN108707578A - One boar list placenta in vitro culture method - Google Patents
One boar list placenta in vitro culture method Download PDFInfo
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Abstract
The invention discloses a boar list placenta in vitro culture method.Belong to agricultural-animal and veterinary field.Specially co-cultured with single embryo using feeder cells droplet, obtain preferable development result, single Embryo Culture is consistent with more Embryo Culture developmental processes used in tradition, moreover, single embryo with oolemma attachment or zona-free can get pig parthenogenetic development blastaea in 7 days.The feeder cells droplet provided in the present invention prepares simple, reliable original material can be provided to study the development of single embryo according to the method provided in the present invention, the needs observed the single embryonic development Process Tracking of pig and further studied can be met completely.The single Embryo Culture method provided in the present invention can be widely applied in single Embryo Culture of a variety of species.
Description
Technical field
The invention belongs to agricultural-animal and veterinary fields, specifically, being related to outside the lonely female activation embryo list embryoid body of a boar
Cultural method.
Background technology
In recent years, produced in vitro embryo, which is widely used in production, animal, disease model and the biology of economic value
Reactor etc., meanwhile, technology (IVF) in vitro fertilization, intracytoplasmic sperm injection technology (ICSI), embryo transfer technology, embryo separating skill
The application of art and embryonic stem cell engineering etc. has greatly facilitated the development of animal husbandry, however, these new technologies development with
In, the lower developmental potency of produced in vitro embryo and early embryonic development block the hair for significantly limiting these new technologies
Exhibition and application.Although numerous researchs in the culture solution of Mammalian Embryo in vitro culture by adding or reducing the factor
Improve the developmental potency of embryo, still, compared with the embryo developed in vivo, the embryo of produced in vitro develops in quality and blastaea
Rate still differs greatly, and this requires us, and research is continued deeper into it.
With the rapid development in the fields such as social economy and animal husbandry, there is an urgent need to the more and better animals of external preparation
Embryo, however, in real production process, ectogenetic embryo is arrested in a certain stage and is particularly acute, and wastes a large amount of moneys
Source.
Mammalian Embryo ectogenesis some retardation refer to fertilized eggs from the development of zygote period to blastaea period this
During a, the blocking of generation prevents embryo to rest on some period from continuing the phenomenon that development is gone down, early embryonic development resistance
The stagnant time often joins with the time correlation of developmental regulation transition with the different and different of species, and goat and sheep betide
The 8-16 cell stages of embryo, the embryo of people are generally easy to be arrested in vitro the 4-8 cell stages of embryo in growth course, and pig
It is serious that embryo is arrested in 4 cell stages in growth course in vitro.Therefore, related Mammalian Embryo developmental arrest generates
Molecular mechanism be when one of progenesising with the hot spot of Developmental Biology research.
Research embryonic development some retardation first has to obtain research object, while to ensure that studied object is individually to deposit
.Currently, the research for single embryo is concentrated mainly on mouse fertilized egg and each stage of development embryo, and mice embryonic obtains
It obtains and is mainly obtained from internal punching to therefore, mice embryonic acquisition is relatively easy, at low cost.And for outside mouse list embryoid body
Culture selects calcium alginate gel embedding culture method mainly after zona-free.Due to the difference of species, embryo's size, embryo
Condition of in vitro culture (including culture medium, cultivation temperature etc.) and fostering requirement are different, so, mouse list In vitro culture
Method is not suitable for big mammal --- single In vitro culture of pig.
Therefore, it is badly in need of establishing single placenta in vitro culture method suitable for pig, to meet the single embryonic development to pig
The needs that Process Tracking is observed and further studied.
Invention content
Of the existing technology in order to solve the problems, such as, the present invention carries out total training using feeder cells droplet and single embryo
Foster method is used for single In vitro culture of pig, obtains preferable development as a result, can meet completely to the single embryo of pig
Developmental process Follow-up observation and the needs further studied.
The present invention is achieved through the following technical solutions goal of the invention：
In a first aspect, the present invention provides a boar list placenta in vitro culture method, include the following steps：
1) prepared by pig Oocytes Maturation In vitro：
Acquire sow ovary；Ovary tissue first with 75% ethanol wash 1min, then with sterilizing and preheating physiology salt
Water uses the asepsis injector (inside having the DPBS after a small amount of balance) equipped with No. 20 syringe needles of standard to extract Ovarian surface after cleaning 3 times
The ovarian follicle of a diameter of 3-8mm, extract are injected in 50mL conical centrifuge tubes, are placed in the conical centrifuge tube in 39 DEG C of water-baths；
Wait for cumulus oocytes complesxes (COCS) after precipitation, examined under stereomicroscope after washing 2 times with DPBS (being purchased from GIBCO)
Ovum.The COCs that form is normal, cytoplasm is uniform and the granular cell that at least haves three layers wraps up is picked, the COCs of detection is used and contains 5% tire ox
The DPBS of serum (FBS) is washed 3 times, is then washed 3 times with In-vitro maturation liquid, is moved into and is contained 500 μ L In-vitro maturations
Liquid is simultaneously covered in five porocyte culture plates of paraffin oil；Often drop puts COCs100-150 pieces；Liquid is in advance in CO2In incubator
Balance 2h or more；In-vitro maturation 42-44h, 39 DEG C of condition of culture, 5%CO2, saturated humidity；
In-vitro maturation liquid is+0.1% polyvinyl alcohol+3.05mM glucose+0.91mM acetone of TCM-199 (GIBCO)
+ 10% (V/V) liquor folliculi+10IU/mL of+100 μ g/mL streptomysins of sour sodium+0.57mM L-cysteine+100IU/mL penicillin
PMSG (Ningbo hormone preparations factory)+10IU/mL hCG (Ningbo hormone preparations factory)；
2) the lonely female activation of pig Oocytes Maturation In vitro：
The COCs of In-vitro maturation 42-44h is moved into the DPBS containing 0.1% hyaluronidase and digests and uses liquid-transfering gun
Slight piping and druming is washed 3 times with removing cumulus cell with the activation liquid preheated in advance repeatedly, integration slot of the merging with activation liquid
In, it is electrically activated with BTX-2001 fusion instruments, activation parameter：30 electric pulses of μ s, 1.1kv/cm, 1 times；Ovum after activation
Mother cell is spare after being washed three times with the embryo medium NCSU-23 preheated in advance；
Activate formula of liquid：0.25mol/L mannitol+0.5mol/L calcium chloride+0.5mol/L magnesium sulfate+0.5mol/L
HEPES+0.01%PVA；
3) feeder cells droplet is co-cultured to prepare：
(1) prepared by Epithelium Cells feeder layer droplet
A, the separation of Epithelium Cells and original cuiture：It is dual anti-it to be put into addition after slaughterhouse acquisition sow fallopian tubal
In taking back laboratory in 2h in 30-37 DEG C of sterile saline, extra mesosalpinx is cut off with sterilizing scissors, and by defeated ovum
Pipe is cut into the segment of length 2cm, is washed 3 times with 35 DEG C of dual anti-sterile salines of addition, is placed in the disposable of diameter 35mm
It in plastics capsule, is rushed while observing under stereomicroscope and takes epithelial cell, left hand is clamped defeated by clock and watch tweezers when operation
Oviduct side incision position, the right hand are drawn 0.25% pancreatin of working concentration with 1 milliliter of disposable syringe and are directly clamped from tweezers
Fallopian tubal incision position be slowly injected into, Epithelium Cells are taken with punching, collects in capsule and contains on fallopian tubal after repeating 2-3 time
The pancreatin liquid of chrotoplast is rapidly added isometric DMEM containing 10%FBS to terminate digestion, avoids digestion excessive, carefully
Born of the same parents are dead, and time control of the pancreatin in fallopian tubal is within 5min, to ensure the purity of Epithelium Cells.By collection
Liquid 3000r/min centrifuges 5min, abandons supernatant, and the DMEM repeated centrifugations containing 10%FBS are added and abandon supernatant 2 times, obtain
Precipitation merging is cultivated in the 6 orifice plates containing 2 milliliters of addition 10%FBS DMEM；Culture replaces fresh culture after 3 days, waits for
Cell can digest spare when covering with 6 orifice plates board bottom；
B, prepared by feeder layer co-cultured cell droplet：48h is digested with 0.25% pancreatin before co-cultivation has covered with 6 orifice plates
Epithelium Cells, time 2min is added DMEM containing 10%FBS and terminates digestion, is that 1 milliliter of pipettor is light with range
Featheriness breaks into single cell suspension, and cell density is adjusted to 104A/ml simultaneously ensures that cell is uniformly existing, profit in cell suspension
It is to draw 10 μ L cells with the pipettor that range is 10 μ L to be prepared with this cell suspension and co-culture feeder layer list Embryo Culture droplet
Suspension does cell droplet on the disposable capsule of 35mm；10 μ L of each droplet size cover paraffin oil, are placed in 39 DEG C, and 5%
CO2, cultivate in the incubator of saturated humidity, co-cultured for cell after 48h；
Alternatively,
(2) prepared by cumulus granulosa cells feeder layer droplet：
The ripe egg mother cell of culture is placed in ripe liquid, blows and beats the ovarian cumulus removed outside egg mother cell repeatedly with liquid-transfering gun
Cell, piping and druming is clean and picks egg mother cell, and remaining cumulus granulosa cells stay in a liquid, and adjustment cell density is 104
A/ml simultaneously ensures that cell is uniformly existing in cell suspension, is prepared using this cell suspension and co-cultures feeder layer list embryo training
It is to draw 10 μ L cell suspensions with the pipettor that range is 10 μ L to support droplet, and cell droplet is done on the disposable capsule of 35mm；
10 μ L of each droplet size cover paraffin oil, are placed in 39 DEG C, 5%CO2, cultivate in the incubator of saturated humidity, for thin after 48h
Born of the same parents co-culture；
Alternatively,
(3) prepared by porcine fetus fibroblasts feeder layer droplet
A, prepared by porcine fetus fibroblasts：Concrete operation method is with reference to ZL201310381274.5《Hubei white pig is at fibre
Tie up cell line》Disclosed in method prepare；
B, prepared by feeder layer co-cultured cell droplet：48h is digested with 0.25% pancreatin before co-cultivation has covered with 6 orifice plates
Porcine fetus fibroblasts, time 2min is added DMEM containing 10%FBS and terminates digestion, is 1 milliliter of pipettor with range
Single cell suspension is gently blown and beaten into, cell density is adjusted to 104A/ml simultaneously ensures that cell is uniformly existing in cell suspension,
It is thin with the 10 μ L of pipettor absorption that range is 10 μ L to co-culture feeder layer list Embryo Culture droplet using the preparation of this cell suspension
Born of the same parents' suspension does cell droplet on the disposable capsule of 35mm；10 μ L of each droplet size cover paraffin oil, are placed in 39 DEG C, and 5%
CO2, cultivate in the incubator of saturated humidity, co-cultured for cell after 48h；
4) single embryo co-cultures with feeder cells：
(1) egg mother cell with oolemma attachment is co-cultured with feeder cells
2h changes the cell culture medium DMEM in droplet into fresh embryo culture mediums before egg mother cell is placed in droplet
NCSU-23 sets spare in incubator；
Selecting step 2) obtain with first polar body, cytoplasm uniform Oocytes Maturation In vitro in balance 2h in advance
In embryo culture medium NCSU-23, total training is carried out in the unicellular droplet of co-cultivation feeder layer that after washing 3 times prepared by merging step 3)
It supports, each droplet sets 1 piece of egg mother cell；48h carries out half amount and changes liquid；
Alternatively,
(2) egg mother cell of zona-free is co-cultured with feeder cells
2h changes the cell culture medium DMEM in droplet into fresh embryo culture mediums before egg mother cell is placed in droplet
NCSU-23 sets spare in incubator；
After the lonely female activation of Oocytes Maturation In vitro, choose with first polar body, cytoplasm is uniform, after birth completely in vitro at
The pronase digestion of ripe egg mother cell working concentration 0.25% removes oolemma, by lensless Fourier transform holography egg mother cell in advance
It is micro- to balance the co-cultivation feeder layer list Embryo Culture that after being washed 3 times in the embryo culture medium NCSU-23 of 2h prepared by merging step 3)
It is co-cultured in drop, each droplet sets 1 piece of egg mother cell, and 48h carries out half amount and changes liquid；
5) pig parthenogenetic development blastaea is obtained within 7 days：Embryo's cleavage rates are counted in 36h, after co-culturing 7d, are observed under the microscope
Embryonic development situation, and count blastocyst rate.
Preferably, single embryo that step 4) co-cultures in the above method is IVF Embryos, intracytoplasmic sperm injection embryo, body
Any one of nuclear transplantation embryo.
The present invention has the following advantages：
The present invention uses the method that feeder cells droplet is co-cultured with single embryo for single embryo of pig for the first time
In vitro culture.Preferable development is obtained as a result, single Embryo Culture and more Embryo Culture developmental processes used in tradition are consistent
, moreover, single embryo with oolemma attachment or zona-free can get pig parthenogenetic development blastaea in 7 days.It is provided in the present invention
Feeder cells droplet prepare simple, be provided according to the development that the method provided in the present invention can be the single embryo of research can
The original material leaned on can meet the needs observed the single embryonic development Process Tracking of pig and further studied completely.This hair
Single Embryo Culture method of bright middle offer can be widely applied in single Embryo Culture of a variety of species.
Description of the drawings
Fig. 1 is porcine oviductal epithelial cells schematic diagram in the present invention；
Fig. 2 is pig cumulus granulosa cells schematic diagram in the present invention；
Fig. 3 is porcine fetus fibroblasts schematic diagram in the present invention；
Fig. 4 is that feeder cells co-culture single embryonic development blastaea schematic diagram in the present invention；
A. the Oocyte Development with oolemma attachment is to blastaea；B. the Oocyte Development of zona-free is to blastaea；
Specific implementation mode
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.【Embodiment 1】Pig body
It is prepared by outer mature oocyte
After Meat processing factory of China Oil and Food Import and Export Corporation of Wuhan City acquires sow ovary, it is immediately placed in and adds 35 DEG C of dual anti-sterile physiologicals
Send laboratory in brine in 2-4h back to.Ovary tissue first with 75% ethanol wash 1min, then with sterilizing and preheating physiology
Brine uses the asepsis injector (inside having the DPBS after a small amount of balance) equipped with No. 20 syringe needles of standard to extract ovary table after cleaning 3 times
The ovarian follicle of a diameter of 3-8mm in face, extract are injected in 50mL conical centrifuge tubes, the conical centrifuge tube being placed in 39 DEG C of water-baths
In.Wait for cumulus oocytes complesxes (COCS) after precipitation, washed 2 times with DPBS (GIBCO), after examined under stereoscope
Ovum.The COCs that form is normal, cytoplasm is uniform and the granular cell that at least haves three layers wraps up is picked, the COCs of detection is used and contains 5% tire ox
The DPBS of serum (FBS) is washed 3 times, is then washed 3 times with maturation culture solution, is moved into containing 500 μ L maturation culture solutions and is covered
Have in five porocyte culture plates of paraffin oil.Often drop puts COCs100-150 pieces.Liquid is in advance in CO2In incubator balance 2h with
On.In-vitro maturation 42-44h, 39 DEG C of condition of culture, 5%CO2, saturated humidity.
In-vitro maturation liquid is+0.1% polyvinyl alcohol+3.05mM glucose+0.91mM acetone of TCM-199 (GIBCO)
+ 10% (V/V) liquor folliculi+10IU/mL of+100 μ g/mL streptomysins of sour sodium+0.57mM L-cysteine+100IU/mL penicillin
PMSG (Ningbo hormone preparations factory)+10IU/mL hCG (Ningbo hormone preparations factory).
【Embodiment 2】The lonely female activation of pig Oocytes Maturation In vitro
The COCs of In-vitro maturation 42-44h is moved into the DPBS containing 0.1% hyaluronidase and digests and uses liquid-transfering gun
Slight piping and druming is washed 3 times with removing cumulus cell with the activation liquid preheated in advance repeatedly, integration slot of the merging with activation liquid
In, it is electrically activated with BTX-2001 fusion instruments, activation parameter：30 electric pulses of μ s, 1.1kv/cm, 1 times.Ovum after activation
Mother cell is spare after being washed three times with the embryo medium NCSU-23 preheated in advance.
Activate formula of liquid：0.25mol/L mannitol+0.5mol/L calcium chloride+0.5mol/L magnesium sulfate+0.5mol/
LHEPES+0.01%PVA.
【Embodiment 3】Feeder cells droplet is co-cultured to prepare
(1) prepared by Epithelium Cells feeder layer droplet
A, the separation of Epithelium Cells and original cuiture：It is dual anti-it to be put into addition after slaughterhouse acquisition sow fallopian tubal
In taking back laboratory in 2h in 30-37 DEG C of sterile saline, extra mesosalpinx is cut off with sterilizing scissors, and by defeated ovum
Pipe is cut into the segment of length 2cm, is washed 3 times with 35 DEG C of dual anti-sterile salines of addition, is placed in the disposable of diameter 35mm
It in plastics capsule, is rushed while observing under Stereo microscope and takes epithelial cell, left hand is clamped defeated by clock and watch tweezers when operation
Oviduct side incision position, the right hand are drawn 0.25% pancreatin of working concentration with 1 milliliter of disposable syringe and are directly clamped from tweezers
Fallopian tubal incision position be slowly injected into, Epithelium Cells are taken with punching, collects in capsule and contains on fallopian tubal after repeating 2-3 time
The pancreatin liquid of chrotoplast is rapidly added isometric DMEM containing 10%FBS to terminate digestion, avoids digestion excessive, carefully
Born of the same parents are dead, and time control of the pancreatin in fallopian tubal is within 5min, to ensure the purity of Epithelium Cells.By collection
Liquid 3000r/min centrifuges 5min, abandons supernatant, and the DMEM repeated centrifugations containing 10%FBS are added and abandon supernatant 2 times, obtain
Precipitation merging is cultivated in the 6 orifice plates containing 2 milliliters of addition 10%FBS DMEM.Culture replaces fresh culture after 3 days, waits for
Cell can digest spare when covering with 6 orifice plates board bottom.
B, prepared by feeder layer co-cultured cell droplet：48h is digested with 0.25% pancreatin before co-cultivation has covered with 6 orifice plates
Epithelium Cells, time 2min is added DMEM containing 10%FBS and terminates digestion, is that 1 milliliter of pipettor is light with range
Featheriness breaks into single cell suspension, and cell density is adjusted to 104A/ml simultaneously ensures that cell is uniformly existing, profit in cell suspension
It is prepared with this cell suspension and co-cultures feeder layer list Embryo Culture droplet, that is, the pipettor that range is 10 μ L is used to draw 10 μ L cells
Suspension, does cell droplet on the disposable capsule of 35mm, 10 μ L of each droplet size, covers paraffin oil, is placed in 39 DEG C, and 5%
CO2, cultivate in the incubator of saturated humidity, co-cultured for cell after 48h.
(2) prepared by cumulus granulosa cells feeder layer droplet：The ripe egg mother cell of culture is placed in ripe liquid, liquid relief is used
Rifle blows and beats the cumulus cell removed outside egg mother cell repeatedly, and piping and druming is clean and picks egg mother cell, remaining cumulus granulosa cells
It stays in a liquid, adjustment cell density is 104A/ml simultaneously ensures that cell is uniformly existing in cell suspension, thin using this
It is to draw 10 μ L cell suspensions with the pipettor that range is 10 μ L that born of the same parents' suspension, which prepares and co-cultures feeder layer list Embryo Culture droplet,
Cell droplet is done on the disposable capsule of 35mm, 10 μ L of each droplet size cover paraffin oil, are placed in 39 DEG C, 5%CO2, saturation
It cultivates in the incubator of humidity, is co-cultured for cell after 48h.
(3) prepared by porcine fetus fibroblasts feeder layer droplet
A, prepared by porcine fetus fibroblasts：Concrete operation method is with reference to Chinese patent《Hubei white pig fibroblast》
Disclosed in method prepare.
B, prepared by feeder layer co-cultured cell droplet：48h is digested with 0.25% pancreatin before co-cultivation has covered with 6 orifice plates
Porcine fetus fibroblasts, time 2min is added DMEM containing 10%FBS and terminates digestion, is 1 milliliter of pipettor with range
Single cell suspension is gently blown and beaten into, cell density is adjusted to 104A/ml simultaneously ensures that cell is uniformly existing in cell suspension,
It is prepared using this cell suspension and co-cultures feeder layer list Embryo Culture droplet, that is, use the pipettor that range is 10 μ L to draw 10 μ L thin
Born of the same parents' suspension does cell droplet on the disposable capsule of 35mm.10 μ L of each droplet size cover paraffin oil, are placed in 39 DEG C, and 5%
CO2, cultivate in the incubator of saturated humidity, co-cultured for cell after 48h.
【Embodiment 4】Cell co-cultures
(1) egg mother cell with oolemma attachment is co-cultured with feeder cells
2h changes the cell culture medium DMEM in droplet into fresh embryo culture mediums before egg mother cell is placed in droplet
NCSU-23 sets spare in incubator.
After the lonely female activation of Oocytes Maturation In vitro, choose with first polar body, cytoplasm is uniform, after birth completely in vitro at
The co-cultivation that after ripe egg mother cell washs 3 times in the embryo culture medium NCSU-23 of balance 2h in advance prepared by merging embodiment 3 is raised
It supports and is co-cultured in layer list Embryo Culture droplet, each droplet sets 1 piece of egg mother cell.48h carries out half amount and changes liquid.It unites in 36h
Embryo's cleavage rates are counted, 7d counts blastocyst rate.
(2) egg mother cell of zona-free is co-cultured with feeder cells.
2h changes the cell culture medium DMEM in droplet into fresh embryo culture mediums before egg mother cell is placed in droplet
NCSU-23 sets spare in incubator.
After the lonely female activation of Oocytes Maturation In vitro, choose with first polar body, cytoplasm is uniform, after birth completely in vitro at
The pronase digestion of ripe egg mother cell working concentration 0.25% removes oolemma, by lensless Fourier transform holography egg mother cell in advance
It is micro- to balance the co-cultivation feeder layer list Embryo Culture that after being washed 3 times in the embryo culture medium NCSU-23 of 2h prepared by merging embodiment 3
It is co-cultured in drop, each droplet sets 1 piece of egg mother cell.48h carries out half amount and changes liquid.Embryo's cleavage rates, 7d systems are counted in 36h
Count blastocyst rate.
【Embodiment 5】Single In vitro culture experiment is carried out using traditional Microdrop culture
This experiment prepares droplet on the disposable capsules of 35mm and carries out single In vitro culture, has been substantially carried out and has compareed
Experiment：A：Droplet size is divided into three groups：5 μ L/ drops, 10 μ L/ drops, 50 μ L/ drops, 100 μ L/ drops；B：In the base of droplet size grouping
Single In vitro culture is carried out using different culture mediums on plinth：1. being carried out using fresh Pig embryos culture medium NCSU-23 single
In vitro culture；2. the NCSU-23 for having turned out blastaea in five orifice plates is taken directly to carry out single In vitro culture；3. taking five
25% NCSU-23 for having turned out blastaea adds 75% fresh NCSU-23 directly to carry out single In vitro culture in orifice plate；④
Take 50% NCSU-23 for having turned out blastaea in five orifice plates that 50% fresh NCSU-23 is added directly to carry out training outside single embryoid body
It supports；5. taking 75% NCSU-23 for having turned out blastaea in five orifice plates that 25% fresh NCSU-23 is added directly to carry out single embryoid body
Outer culture；6. fresh embryo culture medium NCSU-23 adds 10%FBS；7. fresh NCSU-23 additions 1% must amino acid
With 0.5% nonessential amino acid；8. fresh TCM199 adds 10%FBS；9. fresh TCM199 additions 1% must amino acid
With 0.5% nonessential amino acid.
Every group of experiment at least in triplicate more than, every group of single control group of experiment is no less than 100 pieces of embryos.
As a result：Droplet size is 10 μ L/ drops, preferable using TCM199 additions 10%FBS culture effects, embryo's cleavage rates
60%, it is developed to 16 cells 10%, no mulberry body and blastaea, embryo's major retardation is in 4 cell stages.It is carried out using NCSU-23
When culture, cell development to 8 cell deaths, cleavage rates 50%.
【Embodiment 6】Feeder cells are added to carry out single In vitro culture experiment using traditional micro drop method
This experiment prepares feeder layer droplet on the disposable capsules of 35mm and carries out single In vitro culture, 10 μ of droplet size
L/ drops.It has been substantially carried out control experiment：Each feeder cells is replaced by the culture medium in B.A：Feeder layer is thin
Born of the same parents include cumulus granulosa cells, porcine fetus fibroblasts and Epithelium Cells；B：Culture medium includes following several：1. adopting
Single In vitro culture is carried out with fresh Pig embryos culture medium NCSU-23；2. taking in five orifice plates and having turned out blastaea
NCSU-23 directly carries out single In vitro culture；3. 25% NCSU-23 for having turned out blastaea in five orifice plates is taken to add 75%
Fresh NCSU-23 directly carries out single In vitro culture；4. 50% NCSU-23 for having turned out blastaea in five orifice plates is taken to add
50% fresh NCSU-23 directly carries out single In vitro culture；5. taking in five orifice plates 75% NCSU- for having turned out blastaea
23 add 25% fresh NCSU-23 directly to carry out single In vitro culture；6. fresh embryo culture medium NCSU-23 additions 10%
FBS；7. fresh NCSU-23 additions 1% must amino acid and 0.5% nonessential amino acid；8. fresh TCM199 additions
10%FBS；9. fresh TCM199 additions 1% must amino acid and 0.5% nonessential amino acid.
Every group of experiment at least in triplicate more than, every group of single control group of experiment is no less than 100 pieces of embryos.
As a result：After adding feeder cells, single In vitro culture effect will be got well compared with feeder cells are not added with.Using new
Fresh Pig embryos culture medium NCSU-23 adds feeder cells to be got well compared with TCM-199 additions 10%FBS effects, and uterine tubal epithelium
It is relatively high that cell does single layer list In vitro culture blastocyst rate, up to 8.3%.Porcine fetus fibroblasts and cumulus cell are raised
There is blastaea in layer, but relatively low compared with Epithelium Cells.
【Embodiment 7】Single embryonic feeder layer droplet is co-cultured co-cultures developmental process check experiment with traditional polyembryony tire
Egg mother cell after maturation in vitro activation is grouped culture：A, it is co-cultured in feeder cells droplet, each
Droplet sets one piece of egg mother cell；B, covering paraffin oil culture in five orifice plates, 100-150 pieces/500 μ L cultures are traditionally placed in
Base；C, traditional microfarad culture, 10-15 pieces/μ L culture mediums are pressed.
The single embryonic feeder layer droplet of table 1 is co-cultured co-cultures developmental process check experiment with traditional polyembryony tire
According to table 1, single Embryo Culture is consistent with more Embryo Culture developmental processes used in tradition.
【Embodiment 8】Blastocyst rate counts
Obtain pig parthenogenetic development blastaea within 7 days.After co-culturing 7d, in the microscopically observation embryonic development situation of 400X, and
Count blastocyst rate.See Fig. 4.
After measured and check experiment, the lonely female activation embryo list placenta in vitro culture method of pig in above example,
To obtain normal development to the embryo of blastaea.
In conclusion by the present invention in that with feeder cells droplet and purpose embryo co-cultivation, can normally be sent out
The feeder cells droplet educated single embryo, and provided in the present invention is prepared simply, can be according to the method provided in the present invention
The development for studying single embryo provides reliable original material, and the single Embryo Culture method provided in the present invention can be widely applied to
In single Embryo Culture of a variety of species.
The present invention although an embodiment of the present invention has been shown and described, for those of ordinary skill in the art and
Speech, it is possible to understand that can these embodiments be carried out with a variety of variations without departing from the principles and spirit of the present invention, repaiied
Change, replace and modification, the scope of the present invention is defined by the appended.
Claims (2)
1. a boar list placenta in vitro culture method, which is characterized in that include the following steps：
1）It is prepared by pig Oocytes Maturation In vitro：
Acquire sow ovary；Ovary tissue first with 75% 1 min of ethanol wash, then with sterilizing and preheating physiological saline it is clear
The asepsis injector equipped with No. 20 syringe needles of standard is used after washing 3 times, is had the DPBS after a small amount of balance in asepsis injector, is extracted ovum
Nest surface diameter is the ovarian follicle of 3-8 mm, and extract is injected in 50 mL conical centrifuge tubes, and the sharp bottom in 39 DEG C of water-baths is placed in
In centrifuge tube；Wait for cumulus oocytes complesxes（COCS) after precipitation, after washing 2 times with DPBS under stereomicroscope ovoscopy；
The COCs that form is normal, cytoplasm is uniform and the granular cell that at least haves three layers wraps up is picked, the COCs of detection is used and contains 5% fetal calf serum
DPBS wash 3 times, then wash 3 times with In-vitro maturation liquid, immigration, which contains, 500 μ L In-vitro maturations liquid and to be covered with
In five porocyte culture plates of paraffin oil；Often drop puts COCs100-150 pieces；Liquid is in advance in CO2 Balanced in incubator 2 h with
On；In-vitro maturation 42-44 h, 39 DEG C of condition of culture, 5% CO2, saturated humidity；
In-vitro maturation liquid is：+ 3.05 mM glucose+0.91 of TCM-199+0.1% polyvinyl alcohol
+ 0.57+100 IU of mM L-cysteines of mM Sodium Pyruvates/+ 100 μ g/mL streptomysins+10% of mL penicillin（V/V）Ovum
Steep+10 IU/mL PMSG+10 IU/mL hCG of liquid；
2）The lonely female activation of pig Oocytes Maturation In vitro：
Will In-vitro maturation 42-44h COCs move into containing 0.1% hyaluronidase DPBS in digest and with liquid-transfering gun repeatedly
Slight piping and druming is washed 3 times with removing cumulus cell with the activation liquid preheated in advance, in integration slot of the merging with activation liquid, is used
BTX-2001 fusion instruments are electrically activated, activation parameter：30 electric pulses of μ s, 1.1kv/cm, 1 times；Ovum after activation is female thin
Born of the same parents are spare after being washed three times with the embryo medium NCSU-23 preheated in advance；
Activate formula of liquid：0.25mol/L mannitol+0.5mol/L calcium chloride+0.5mol/L magnesium sulfate+0.5mol/L HEPES+
0.01%（V/V）PVA；
3）Feeder cells droplet is co-cultured to prepare：
（1）It is prepared by Epithelium Cells feeder layer droplet
A, the separation of Epithelium Cells and original cuiture：Slaughterhouse is put into the dual anti-30- of addition after obtaining sow fallopian tubal
In taking back laboratory in 2h in 37 DEG C of sterile salines, extra mesosalpinx is cut off with sterilizing scissors, and by fallopian tubal
It is cut into the segment of length 2cm, is washed 3 times with 35 DEG C of dual anti-sterile salines of addition, is placed in the disposable modeling of diameter 35mm
Expect in capsule, rushed while observing under stereomicroscope and take epithelial cell, left hand clamps defeated ovum by clock and watch tweezers when operation
Pipe side incision position, the right hand are directly clamped from tweezers defeated with 1 milliliter of disposable syringe absorption 0.25% pancreatin of working concentration
Oviduct incision position is slowly injected into, and Epithelium Cells are taken with punching, is collected after repeating 2-3 times thin containing uterine tubal epithelium in capsule
The pancreatin liquid of born of the same parents is rapidly added isometric DMEM containing 10%FBS to terminate digestion, avoids digestion excessive, cell is dead
It dies, time control of the pancreatin in fallopian tubal is within 5min, to ensure the purity of Epithelium Cells；By the liquid of collection
3000r/min centrifuges 5min, abandons supernatant, and the DMEM repeated centrifugations containing 10%FBS are added and abandon supernatant 2 times, obtained precipitation is set
Enter and is cultivated in the 6 orifice plates containing 2 milliliters of addition 10%FBS DMEM；Culture replaces fresh culture after 3 days, waits for that cell is long
It can be digested when full 6 orifice plates board bottom spare；
B, prepared by feeder layer co-cultured cell droplet：48h is digested with 0.25% pancreatin before co-cultivation has covered with the defeated of 6 orifice plates
Oviduct epithelial cell, time 2min are added the DMEM containing 10%FBS and terminate digestion, are that 1 milliliter of pipettor is gently blown with range
Single cell suspension is broken into, cell density is adjusted to 104A/ml simultaneously ensures that cell is uniformly existing in cell suspension, utilizes this
It is to draw 10 μ L cell suspensions with the pipettor that range is 10 μ L that cell suspension, which prepares and co-cultures feeder layer list Embryo Culture droplet,
Cell droplet is done on the disposable capsule of 35mm；10 μ L of each droplet size cover paraffin oil, are placed in 39 DEG C, 5% CO2, satisfy
It cultivates in the incubator of humidity, is co-cultured for cell after 48h；
Alternatively,
（2）It is prepared by cumulus granulosa cells feeder layer droplet：
The ripe egg mother cell of culture is placed in ripe liquid, it is thin to blow and beat the ovarian cumulus removed outside egg mother cell repeatedly with liquid-transfering gun
Born of the same parents, piping and druming is clean and picks egg mother cell, and remaining cumulus granulosa cells stay in a liquid, and adjustment cell density is 104A/
Ml simultaneously ensures that cell is uniformly existing in cell suspension, and it is micro- to prepare co-cultivation feeder layer list Embryo Culture using this cell suspension
Drop draws 10 μ L cell suspensions with the pipettor that range is 10 μ L, and cell droplet is done on the disposable capsule of 35mm；Each
10 μ L of droplet size cover paraffin oil, are placed in 39 DEG C, 5% CO2, it cultivates in the incubator of saturated humidity, it is total for cell after 48h
Culture；
Alternatively,
（3）It is prepared by porcine fetus fibroblasts feeder layer droplet
A, prepared by porcine fetus fibroblasts：Concrete operation method is with reference to ZL201310381274.5《Hubei white pig is at fiber finer
Born of the same parents system》Disclosed in method prepare；
B, prepared by feeder layer co-cultured cell droplet：48h digests the pig for having covered with 6 orifice plates with 0.25% pancreatin before co-cultivation
Fetal fibroblast, time 2min are added DMEM containing 10%FBS and terminate digestion, with range be 1 milliliter of pipettor gently
Single cell suspension is blown and beaten into, cell density is adjusted to 104A/ml simultaneously ensures that cell is uniformly existing in cell suspension, is utilized
It is to draw 10 μ L cells with the pipettor that range is 10 μ L to hang that this cell suspension, which prepares co-cultivation feeder layer list Embryo Culture droplet,
Liquid does cell droplet on the disposable capsule of 35mm；10 μ L of each droplet size cover paraffin oil, are placed in 39 DEG C, 5% CO2,
It cultivates in the incubator of saturated humidity, is co-cultured for cell after 48h；
4）Single embryo co-cultures with feeder cells：
（1）Egg mother cell with oolemma attachment is co-cultured with feeder cells
The cell culture medium DMEM in droplet is changed into fresh embryo culture medium NCSU-23 by 2h before egg mother cell is placed in droplet
It sets spare in incubator；
Selecting step 2）What is obtained carries first polar body, the uniform Oocytes Maturation In vitro of cytoplasm in the embryo for balancing 2h in advance
In culture medium NCSU-23, step 3 is placed in after washing 3 times）It is co-cultured in the unicellular droplet of co-cultivation feeder layer of preparation,
Each droplet sets 1 piece of egg mother cell；48h carries out half amount and changes liquid；
Alternatively,
（2）The egg mother cell of zona-free is co-cultured with feeder cells
The cell culture medium DMEM in droplet is changed into fresh embryo culture medium NCSU-23 by 2h before egg mother cell is placed in droplet
It sets spare in incubator；
It after the lonely female activation of Oocytes Maturation In vitro, chooses with first polar body, cytoplasm is uniform, the complete maturation in vitro ovum of after birth
The pronase digestion of mother cell working concentration 0.25% removes oolemma, and lensless Fourier transform holography egg mother cell is balanced in advance
Step 3 is placed in after being washed 3 times in the embryo culture medium NCSU-23 of 2h）In the co-cultivation feeder layer list Embryo Culture droplet of preparation
It is co-cultured, each droplet sets 1 piece of egg mother cell, and 48h carries out half amount and changes liquid；
5）Obtain pig parthenogenetic development blastaea within 7 days：Embryo's cleavage rates are counted in 36h, after co-culturing 7d, observe embryo under the microscope
Developmental state, and count blastocyst rate.
2. pig list placenta in vitro culture method according to claim 1, which is characterized in that the step 4）It co-cultures
Single embryo is any one of IVF Embryos, intracytoplasmic sperm injection embryo, body-cell neucleus transplanting embryo.
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