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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- CN108707578A CN108707578A CN201810426299.5A CN201810426299A CN108707578A CN 108707578 A CN108707578 A CN 108707578A CN 201810426299 A CN201810426299 A CN 201810426299A CN 108707578 A CN108707578 A CN 108707578A
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- 238000002360 preparation method Methods 0.000 claims description 23
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- 210000002459 blastocyst Anatomy 0.000 claims description 18
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- 108010059712 Pronase Proteins 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
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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
- C12N5/0604—Whole embryos; Culture medium therefor
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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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
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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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
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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 the field of agriculture, animal husbandry and veterinary medicine, and particularly relates to an in vitro culture method of a swine parthenogenetic activated embryo single embryo.
Background
In recent years, in vitro embryo production is widely used for producing economically valuable animals, disease models, bioreactors and the like, and meanwhile, the development of animal husbandry is greatly promoted by the application of in vitro fertilization technology (IVF), single sperm injection technology (ICSI), embryo transfer technology, embryo segmentation technology, embryonic stem cell engineering and the like, however, in the development and application of the new technologies, the development and application of the new technologies are greatly limited by the low development capability and early embryonic development retardation of in vitro embryo production. Although many studies have been made to improve the development ability of embryos by adding or reducing factors to the culture medium of in vitro culture of early mammalian embryos, the quality and blastocyst development rate of embryos produced in vitro are still far from those of embryos developed in vivo, which requires further research.
With the rapid development of the fields of social economy, animal husbandry and the like, more and better animal embryos need to be prepared in vitro urgently, however, in the actual production process, the embryo retardation of in vitro development is particularly serious at a certain stage, and a large amount of resources are wasted.
The phenomenon of in vitro development retardation of mammalian early embryos refers to the phenomenon that embryos cannot continue to develop due to the occurrence of blocking in the process of fertilized egg development from zygote stage to blastula stage, the time of early embryo development retardation varies with species, but is often related to the time of development regulation transition, goats and sheep both occur at 8-16 cell stages of embryos, human embryos are generally easy to block at 4-8 cell stages of embryos in the in vitro development process, and pig embryos are severely blocked at 4 cell stages in the in vitro development process. Therefore, the molecular mechanism involved in the development of early embryonic development arrest in mammals is one of the hot spots in current reproductive and developmental biology research.
The embryo development retardation phenomenon is researched by firstly obtaining a research object and ensuring that the research object exists independently. Currently, research aiming at single embryo mainly focuses on mouse fertilized eggs and embryos at all development stages, and the mouse embryos are mainly obtained by in vivo washing, so that the mouse embryos are relatively simple to obtain and low in cost. And aiming at the in vitro culture of the mouse single embryo, the calcium alginate gel embedding culture method is mainly selected after the zona pellucida is removed. Because of the species difference, the embryo size, the embryo in vitro culture conditions (including culture medium, culture temperature, etc.) and the culture requirements are different, the mouse single embryo in vitro culture method is not suitable for the single embryo in vitro culture of the large mammal, namely the pig.
Therefore, it is urgently needed to establish a single embryo in vitro culture method suitable for pigs so as to meet the requirements of tracking and observing the development process of single embryos of the pigs and further researching the development process of the single embryos.
Disclosure of Invention
In order to solve the problems in the prior art, the method for co-culturing the feeder layer cell microdroplets and the single embryo is used for in-vitro culture of the single embryo of the pig, so that a better development result is obtained, and the requirements of tracking observation and further research on the development process of the single embryo of the pig can be completely met.
The invention achieves the purpose through the following technical scheme:
in a first aspect, the invention provides a method for culturing pig single embryos in vitro, which comprises the following steps:
1) preparing mature oocytes in vitro of pigs:
collecting ovaries of sows; washing ovarian tissue with 75% alcohol for 1min, cleaning with sterilized and preheated normal saline for 3 times, extracting ovarian follicle with diameter of 3-8mm with sterile syringe equipped with standard 20-gauge needle (containing small amount of balanced DPBS), injecting the extractive solution into 50mL pointed-bottom centrifuge tube, and placing into the pointed-bottom centrifuge tube in 39 deg.C water bath; cumulus-oocyte complex (COC)S) After precipitation, eggs were examined under a stereomicroscope after 2 washes with DPBS (purchased from GIBCO). Picking up COCs which are normal in shape, uniform in cytoplasm and wrapped by at least 3 layers of granular cells, washing the detected COCs for 3 times by using DPBS (DPBS) containing 5% Fetal Bovine Serum (FBS), then washing the COCs for 3 times by using in-vitro maturation culture solution, and transferring the COCs into a five-hole cell culture plate which contains 500 mu L of in-vitro maturation culture solution and is covered with paraffin oil; placing 100-150 COCs per drop; the liquid is pre-treated with CO2Balancing in an incubator for more than 2 hours; culturing in vitro at 39 deg.C for 42-44 hr with 5% CO2Saturated humidity;
the in vitro maturation culture solution is TCM-199(GIBCO) +0.1% polyvinyl alcohol +3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin +100 μ g/mL streptomycin +10% (V/V) follicular fluid +10IU/mLPMSG (Ningbo hormone products factory) +10IU/mL hCG (Ningbo hormone products factory);
2) parthenogenetic activation of porcine in vitro mature oocytes:
COCs matured in vitro for 42-44h were transferred into DPBS containing 0.1% hyaluronidase for digestion and gently blown with a pipette gun repeatedly to remove cumulus cells, washed 3 times with pre-warmed activation solution, placed in a fusion tank with activation solution, electrically activated with BTX-2001 fusion instrument with activation parameters: 1 direct current pulse of 30 mus and 1.1 kv/cm; the activated oocyte is washed for three times by embryo culture solution NCSU-23 preheated in advance for standby;
the formula of the activating liquid comprises: 0.25mol/L mannitol, 0.5mol/L calcium chloride, 0.5mol/L magnesium sulfate, 0.5mol/L LHEPES and 0.01 percent PVA;
3) co-culture feeder layer cell droplet preparation:
(1) preparation of microdroplets of oviduct epithelial cell feeder layer
A. Isolation and primary culture of oviduct epithelial cells: putting a sow oviduct after obtaining an oviduct of a sow, adding sterile physiological saline added with double antibodies at 30-37 ℃ into the sterile physiological saline to be brought back to a laboratory within 2h, shearing redundant oviduct membranes by using sterile scissors, shearing the oviduct into small sections with the length of 2cm, washing for 3 times by using sterile physiological saline added with double antibodies at 35 ℃, putting the small sections into a disposable plastic dish with the diameter of 35mm, flushing epithelial cells while observing under a stereoscopic microscope, clamping a fracture on one side of the oviduct by using forceps of a clock and a watch in the operation process, sucking pancreatin with the working concentration of 0.25 percent by using a disposable syringe with the volume of 1 ml in the right hand, directly and slowly injecting from the fracture of the oviduct clamped by the forceps to flush the oviduct epithelial cells, collecting pancreatin the dish after repeating for 2-3 times, quickly adding DMEM containing 10 percent of FBS with the same volume to stop digestion, and avoiding excessive digestion, the cell is dead, and the time of pancreatin in the oviduct is controlled within 5min so as to ensure the purity of the oviduct epithelial cell. Centrifuging the collected liquid at 3000r/min for 5min, discarding the supernatant, adding DMEM containing 10% FBS, repeatedly centrifuging for 2 times, discarding the supernatant, and placing the obtained precipitate into a 6-well plate containing 2 ml of DMEM containing 10% FBS for culture; after culturing for 3 days, replacing a fresh culture medium, and digesting for later use when the cells grow over the bottom of the 6-pore plate;
B. feeder layer co-culture cell droplet preparation: digesting oviduct epithelial cells overgrowing with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 104The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO2Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;
or,
(2) cumulus granulosa cell feeder layer droplet preparation:
placing the cultured mature oocyte in a maturation solution, repeatedly blowing and beating cumulus cells except the oocyte by using a liquid transfer gun, completely blowing and picking up the oocyte, keeping the remaining cumulus granular cells in the solution, and adjusting the cell density to 104The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO2Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;
or,
(3) porcine fetal fibroblast feeder layer droplet preparation
A. Preparation of porcine fetal fibroblasts: the specific operation method refers to the method disclosed in ZL201310381274.5 Hubei white pig fibroblast line;
B. feeder layer co-culture cell droplet preparation: digesting pig fetal fibroblast overgrown with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 104The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO2Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;
4) co-culturing single embryo and feeder layer cells:
(1) co-culture of oocyte and feeder layer cell with zona pellucida attachment
Changing a cell culture medium DMEM in the microdroplets into a fresh embryo culture medium NCSU-23 2 hours before the oocytes are placed into the microdroplets for later use;
selecting in-vitro mature oocytes with first polar bodies and uniform cytoplasm obtained in the step 2) to be in an embryo culture medium NCSU-23 balanced for 2 hours in advance, washing for 3 times, and then putting the oocytes into the co-culture feeder layer single-cell microdroplets prepared in the step 3) for co-culture, wherein 1 oocyte is put in each microdroplet; carrying out half-amount liquid change within 48 h;
or,
(2) co-culture of zona pellucida-removed oocytes and feeder cells
Changing a cell culture medium DMEM in the microdroplets into a fresh embryo culture medium NCSU-23 2 hours before the oocytes are placed into the microdroplets for later use;
after in vitro mature oocytes are parthenogenetically activated, selecting in vitro mature oocytes with a first polar body, uniform cytoplasm and complete cell membranes, digesting the in vitro mature oocytes with pronase with the working concentration of 0.25% to remove zona pellucida, washing the zona pellucida-free oocytes in an embryo culture medium NCSU-23 balanced for 2 hours in advance for 3 times, placing the zona pellucida-free oocytes into single embryo culture microdroplets of the co-culture feeder layer prepared in the step 3) for co-culture, placing 1 oocyte in each microdroplet, and performing half-amount liquid change for 48 hours;
5) obtaining the parthenogenetic blastocysts of the pigs in 7 days: and (4) counting the embryo cleavage rate in 36h, observing the development condition of the embryo under a microscope after co-culture for 7d, and counting the blastocyst development rate.
Preferably, the single embryo co-cultured in step 4) of the above method is any one of an in vitro fertilized embryo, a single sperm injection embryo, and a somatic cell nuclear transfer embryo.
The invention has the following advantages:
the method for co-culturing the feeder layer cell microdroplets and the single embryos is used for in-vitro culture of the single embryos of the pigs for the first time. The better development result is obtained, the development process of single embryo culture is consistent with that of the traditional multi-embryo culture, and the parthenogenetic development blastocyst of the pig can be obtained 7 days from the single embryo with the zona pellucida attached or removed. The feeder layer cell microdroplet provided by the invention is simple to prepare, can provide reliable raw materials for researching the development of a single embryo according to the method provided by the invention, and can completely meet the requirements of tracking observation and further research on the development process of the single embryo of the pig. The single embryo culture method provided by the invention can be widely applied to single embryo culture of various species.
Drawings
FIG. 1 is a schematic representation of porcine oviduct epithelial cells in accordance with the present invention;
FIG. 2 is a schematic representation of porcine cumulus granulosa cells of the present invention;
FIG. 3 is a schematic representation of porcine fetal fibroblasts of the present invention;
FIG. 4 is a schematic representation of feeder cells co-cultured single embryo developmental blastocysts according to the invention;
A. the oocyte attached with the zona pellucida develops to a blastocyst; B. the oocyte with the zona pellucida removed develops to a blastocyst;
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way. [ example 1 ] preparation of oocytes matured in vitro in pigs
After the ovaries of the sows are collected in a grain and meat food processing factory in Wuhan City, the ovaries are immediately put into sterilized normal saline with 35 ℃ added with double antibiotics for 2 to 4 hours and then sent back to a laboratory. Ovarian tissue is washed with 75% ethanol for 1min, washed with sterilized and preheated physiological saline for 3 times, and then follicles with a diameter of 3-8mm on the surface of the ovary are aspirated by a sterile syringe equipped with a standard 20-gauge needle (containing a small amount of equilibrated DPBS), and the aspirated solution is injected into a 50mL conical centrifuge tube and placed in a conical centrifuge tube in a 39 ℃ water bath. Cumulus-oocyte complex (COC)S) After precipitation, the eggs were washed 2 times with DPBS (GIBCO) and examined under a stereomicroscope. CoCs that are morphologically normal, cytosolically homogeneous and have at least 3 granulosa cell envelopes were picked up, washed 3 times with DPBS containing 5% Fetal Bovine Serum (FBS), then 3 times with maturation medium, and transferred to five-well cell culture plates containing 500. mu.L of maturation medium and covered with paraffin oil. 100-150 COCs are put in each drop. The liquid is pre-treated with CO2And balancing in the incubator for more than 2 hours. Culturing in vitro at 39 deg.C for 42-44 hr with 5% CO2And saturation humidity.
The in vitro maturation medium was TCM-199(GIBCO) +0.1% polyvinyl alcohol +3.05mM glucose +0.91mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin +10% (V/V) follicular fluid +10IU/mLPMSG (Ningbo hormone products works) +10IU/mL hCG (Ningbo hormone products works).
[ example 2 ] parthenogenetic activation of oocytes matured in vitro in swine
COCs matured in vitro for 42-44h were transferred into DPBS containing 0.1% hyaluronidase for digestion and gently blown with a pipette gun repeatedly to remove cumulus cells, washed 3 times with pre-warmed activation solution, placed in a fusion tank with activation solution, electrically activated with BTX-2001 fusion instrument with activation parameters: 30 μ s, 1.1kv/cm, 1 direct current pulse. The activated oocytes were washed three times with pre-warmed embryo culture solution NCSU-23 and then kept ready for use.
The formula of the activating liquid comprises: 0.25mol/L mannitol +0.5mol/L calcium chloride +0.5mol/L magnesium sulfate +0.5mol/LHEPES +0.01% PVA.
Example 3 Co-culture feeder layer cell droplet preparation
(1) Preparation of microdroplets of oviduct epithelial cell feeder layer
A. Isolation and primary culture of oviduct epithelial cells: putting a sow oviduct after obtaining an oviduct of a sow, adding sterile physiological saline added with double antibodies at 30-37 ℃ into the sterile physiological saline to be brought back to a laboratory within 2h, shearing redundant oviduct membranes by using sterile scissors, shearing the oviduct into small sections with the length of 2cm, washing for 3 times by using sterile physiological saline added with double antibodies at 35 ℃, putting the small sections into a disposable plastic dish with the diameter of 35mm, flushing epithelial cells while observing under a body type microscope, clamping a fracture on one side of the oviduct by using a pair of tweezers in the operation process of holding a watch by the left hand, directly and slowly injecting pancreatin the working concentration of 0.25 percent from the fracture of the oviduct clamped by using a pair of tweezers by using a 1 ml disposable syringe by the right hand to flush the oviduct epithelial cells, collecting pancreatin the dish after repeating for 2-3 times, quickly adding DMEM containing 10 percent of the pancreatin the same volume to stop digestion, and avoiding excessive digestion, the cell is dead, and the time of pancreatin in the oviduct is controlled within 5min so as to ensure the purity of the oviduct epithelial cell. The collected liquid was centrifuged at 3000r/min for 5min, the supernatant was discarded, DMEM containing 10% FBS was added thereto and centrifugation was repeated 2 times to discard the supernatant, and the resulting precipitate was cultured in 6-well plates containing 2 ml of DMEM containing 10% FBS. After 3 days of culture, the fresh culture medium is replaced, and the cells can be digested for later use when growing over the bottom of the 6-well plate.
B. Feeder layer co-culture cell droplet preparation: digesting oviduct epithelial cells overgrowing with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 104One/ml and ensuring that the cells are uniformly present in the cell suspension, using this cell suspension to prepare CO-cultured feeder layer single embryo culture droplets, i.e., 10. mu.L of cell suspension is aspirated by a pipette measuring 10. mu.L, and cell droplets are made on disposable 35mm dishes, each 10. mu.L, covered with paraffin oil, placed at 39 ℃ and 5% CO2Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture.
(2) Cumulus granulosa cell feeder layer droplet preparation: placing the cultured mature oocyte in a maturation solution, repeatedly blowing and beating cumulus cells except the oocyte by using a liquid transfer gun, completely blowing and picking up the oocyte, keeping the remaining cumulus granular cells in the solution, and adjusting the cell density to 104The cells in the cell suspension are uniformly existed, the cell suspension is used for preparing CO-culture feeder layer single embryo culture microdroplets, namely 10 mu L of cell suspension is sucked by a pipette with the range of 10 mu L, cell microdroplets are made on a disposable capsule with the diameter of 35mm, each microdroplet is 10 mu L, the microdroplet is covered with paraffin oil and placed at the temperature of 39 ℃ and 5 percent CO2Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture.
(3) Porcine fetal fibroblast feeder layer droplet preparation
A. Preparation of porcine fetal fibroblasts: the specific operation method refers to the preparation method disclosed in Chinese patent Hubei white pig fibroblast line.
B. Feeder layer co-culture cell droplet preparation: in a common processDigesting pig fetal fibroblast overgrown with 6-well plate with 0.25% pancreatin 48h before culture for 2min, adding DMEM containing 10% FBS to stop digestion, lightly blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 104And ensuring that the cells exist uniformly in the cell suspension, and preparing a co-culture feeder layer single embryo culture microdroplet by using the cell suspension, namely sucking 10 mu L of cell suspension by using a pipette with the range of 10 mu L and making the cell microdroplet on a disposable capsule with the diameter of 35 mm. Each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO2Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture.
Example 4 Co-culture of cells
(1) Co-culture of oocyte and feeder layer cell with zona pellucida attachment
The cell culture medium DMEM in the microdroplets was replaced with fresh embryo culture medium NCSU-23 2h before the oocytes were placed in the microdroplets for future use.
After parthenogenetic activation of the in vitro mature oocytes, the in vitro mature oocytes with the first polar body, uniform cytoplasm and complete cell membranes are selected to be washed for 3 times in an embryo culture medium NCSU-23 balanced for 2 hours in advance and then placed into the single embryo culture microdroplets of the co-culture feeder layer prepared in the embodiment 3 for co-culture, and 1 oocyte is placed in each microdroplet. And half amount of liquid change is carried out for 48 hours. And (5) counting the embryo cleavage rate at 36h, and counting the blastocyst development rate at 7 d.
(2) The zona pellucida-removed oocytes were co-cultured with feeder cells.
The cell culture medium DMEM in the microdroplets was replaced with fresh embryo culture medium NCSU-23 2h before the oocytes were placed in the microdroplets for future use.
After parthenogenetic activation of the in vitro mature oocytes, the in vitro mature oocytes with the first polar body, uniform cytoplasm and complete cell membranes are selected and digested by pronase with the working concentration of 0.25% to remove the zona pellucida, the oocytes without the zona pellucida are washed for 3 times in an embryo culture medium NCSU-23 balanced for 2 hours in advance and then are placed into the co-culture feeder layer single embryo culture microdroplets prepared in the embodiment 3 for co-culture, and each microdroplet is placed with 1 oocyte. And half amount of liquid change is carried out for 48 hours. And (5) counting the embryo cleavage rate at 36h, and counting the blastocyst development rate at 7 d.
Example 5 Single embryo in vitro culture experiment Using conventional droplet culture method
② a experiment prepares micro-droplets on a 35mm disposable dish for single embryo in-vitro culture, and mainly carries out a control experiment, wherein ② a micro-droplets are divided into three groups, namely, 5 mu L/droplet, 10 mu L/droplet, 50 mu L/droplet and 100 mu L/droplet, ② a micro-droplets are divided into three groups, ② a micro-droplets are cultured in vitro with different culture mediums on ② a basis of ② a micro-droplet size groups, ② a micro-droplets are cultured in vitro with a fresh pig embryo culture medium NCSU-23, ② a micro-droplets are cultured with a blastocyst in a five-hole plate, ② a micro-droplets are cultured with a fresh pig embryo culture medium NCSU-23, ② a micro-droplets are cultured with a fresh embryo in vitro culture medium, ② a micro-droplets are cultured with a fresh embryo culture medium NCSU-23, ② a micro-embryo in vitro culture medium is directly cultured with a fresh embryo NCSU-23, ② a micro-embryo in ② a micro-hole plate, ② a micro-hole is cultured with a fresh embryo in ② a micro-embryo culture medium, ② a micro-embryo in ② a micro-hole, ② a micro-hole in ② a micro-hole, ② a micro-hole in.
Each experiment was repeated at least three times, and each control group had no less than 100 embryos.
As a result: the microdroplet size is 10 mu L/drop, the culture effect is better by adding 10% FBS into TCM199, the embryo cleavage rate is 60%, the embryo develops to 10% of 16 cells, no morula and blastocyst exist, and the embryo is mainly blocked at the 4-cell stage. When NCSU-23 is used for culture, the cells develop to 8 cell death, and the cleavage rate is 50%.
Example 6 in vitro culture of Single embryos Using traditional Microdrop method with feeder cells
⑤ the ⑤ experiment ⑤ prepares ⑤ micro ⑤ - ⑤ droplets ⑤ of ⑤ feeder ⑤ layers ⑤ on ⑤ a ⑤ 35 ⑤ mm ⑤ disposable ⑤ dish ⑤ for ⑤ single ⑤ embryo ⑤ in ⑤ - ⑤ vitro ⑤ culture ⑤, ⑤ the ⑤ micro ⑤ - ⑤ droplets ⑤ are ⑤ 10 ⑤ mu ⑤ L ⑤/⑤ droplet ⑤, ⑤ a ⑤ control ⑤ experiment ⑤ is ⑤ mainly ⑤ carried ⑤ out ⑤, ⑤ each ⑤ feeder ⑤ layer ⑤ cell ⑤ is ⑤ replaced ⑤ according ⑤ to ⑤ a ⑤ culture ⑤ ② ⑤ in ⑤ B ⑤, ⑤ A ⑤, ⑤ the ⑤ feeder ⑤ layer ⑤ cells ⑤ comprise ⑤ cumulus ⑤ granular ⑤ cells ⑤, ⑤ porcine ⑤ fetal ⑤ fibroblasts ⑤ and ⑤ oviduct ⑤ epithelial ⑤ cells ⑤, ⑤ B ⑤, ⑤ the ⑤ culture ⑤ ② ⑤ comprises ⑤ the ⑤ following ⑤ components ⑤, ⑤ firstly ⑤, ⑤ a ⑤ fresh ⑤ porcine ⑤ embryo ⑤ culture ⑤ ② ⑤ NCSU ⑤ - ⑤ 23 ⑤ is ⑤ adopted ⑤ for ⑤ single ⑤ embryo ⑤ in ⑤ - ⑤ vitro ⑤ culture ⑤, ⑤ secondly ⑤, ⑤ NCSU ⑤ - ⑤ 23 ⑤ with ⑤ blastocysts ⑤ cultured ⑤ in ⑤ five ⑤ pore ⑤ plates ⑤ is ⑤ directly ⑤ subjected ⑤ to ⑤ single ⑤ embryo ⑤ in ⑤ - ⑤ vitro ⑤ culture ⑤, ⑤ thirdly ⑤, ⑤ NCSU ⑤ - ⑤ 23 ⑤ with ⑤ blastocysts ⑤ cultured ⑤ in ⑤ the ⑤ five ⑤ pore ⑤ plates ⑤ is ⑤ directly ⑤ subjected ⑤ to ⑤ single ⑤ embryo ⑤ in ⑤ - ⑤ vitro ⑤ culture ⑤, ⑤ fourthly ⑤, ⑤ TCSU ⑤ - ⑤ 23 ⑤ with ⑤ blastocysts ⑤ cultured ⑤ in ⑤ 25 ⑤ percent ⑤ and ⑤ TCM ⑤ - ⑤ 23 ⑤ are ⑤ directly ⑤ subjected ⑤ to ⑤ single ⑤ embryo ⑤ in ⑤ - ⑤ vitro ⑤ culture ⑤, ⑤ ④ ⑤, ⑤ 50 ⑤% ⑤ of ⑤ NCSU ⑤ - ⑤ 23 ⑤ with ⑤ blastocysts ⑤ cultured ⑤ in ⑤ 50 ⑤ percent ⑤ and ⑤ fresh ⑤ NCSU ⑤ - ⑤ 23 ⑤ with ⑤ blastocysts ⑤ cultured ⑤ in ⑤ five ⑤ pore ⑤ plates ⑤ are ⑤ directly ⑤ subjected ⑤ to ⑤ single ⑤ embryo ⑤ in ⑤ - ⑤ vitro ⑤ culture ⑤, ⑤ thirdly ⑤, ⑤ fresh ⑤ NCSU ⑤ - ⑤ 23 ⑤ with ⑤ blastocysts ⑤ cultured ⑤ in ⑤ 75 ⑤ percent ⑤ and ⑤ fresh ⑤ NCSU ⑤ - ⑤ 23 ⑤ with ⑤ amino ⑤ acids ⑤ added ⑤, ⑤ 25 ⑤ percent ⑤ and ⑤ fresh ⑤ NCSU ⑤ - ⑤ 23 ⑤ with ⑤ amino ⑤ acids ⑤ added ⑤ in ⑤ - ⑤ FBS ⑤, ⑤ and ⑤ fresh ⑤ amino ⑤ acids ⑤ added ⑤ in ⑤ the ⑤ five ⑤ pore ⑤ plates ⑤ are ⑤ added ⑤, ⑤ and ⑤ NCSU ⑤ - ⑤ 5 ⑤, ⑤ 199 ⑤, ⑤ 10 ⑤, ⑤ 5 ⑤, ⑤ 199 ⑤, ⑤ 5 ⑤, ⑤ 3 ⑤, ⑤ 5 ⑤. ⑤
Each experiment was repeated at least three times, and each control group had no less than 100 embryos.
As a result: after the feeder layer cells are added, the in vitro culture effect of the single embryo is better than that of the cells without the feeder layer cells. Compared with the traditional method of TCM-199, the method has the advantages that fresh pig embryo culture medium NCSU-23 and feeder layer cells are better in effect than the fresh pig embryo culture medium NCSU-23 and feeder layer cells added with 10% FBS, and the blastocyst rate of in-vitro culture of the oviduct epithelial cells as single-layer single embryos is higher and reaches 8.3%. Both pig fetal fibroblasts and cumulus cells as feeder layers show blastocysts, but are lower than oviduct epithelial cells.
Example 7 control of Single embryo feeder droplet Co-culture and conventional Multi-embryo Co-culture development Processes
Performing group culture on oocytes subjected to in vitro maturation activation: A. co-culturing in feeder layer cell microdroplets, each microdroplet containing an oocyte; B. placing into five-hole plate according to conventional method, covering with paraffin oil, culturing, and culturing with 100-150 pieces/500 μ L culture medium; C. culturing by conventional micro method, 10-15 pieces/μ L culture medium.
TABLE 1 control of development Processes of Single embryo feeder layer droplet Co-culture and conventional Multi-embryo Co-culture
As shown in Table 1, the development of single embryo cultures was consistent with the development of the traditionally used multiple embryo cultures.
Example 8 blastocyst development Rate statistics
Obtaining the parthenogenetic blastocyst of the pig in 7 days. After the co-culture for 7d, the embryo development condition is observed under a microscope of 400X, and the blastocyst development rate is counted. See fig. 4.
Through determination and control tests, the in-vitro culture method of the porcine parthenogenetic activated embryo single embryo in the embodiment can obtain the embryo which normally develops to a blastocyst.
In summary, the feeder layer cell droplets and the target embryo are co-cultured to obtain a single embryo with normal development, the feeder layer cell droplets provided by the invention are simple to prepare, reliable raw materials can be provided for researching the development of the single embryo according to the method provided by the invention, and the single embryo culture method provided by the invention can be widely applied to the single embryo culture of various species.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (2)
1. A pig single embryo in vitro culture method is characterized by comprising the following steps:
1) preparing mature oocytes in vitro of pigs:
collecting ovaries of sows; washing ovarian tissues with 75% alcohol for 1min, then cleaning with sterilized and preheated normal saline for 3 times, then using a sterile syringe with a standard No. 20 needle, wherein a small amount of balanced DPBS is arranged in the sterile syringe, extracting ovarian follicles with the diameter of 3-8mm on the surface of the ovary, injecting the extracted solution into a 50mL pointed-bottom centrifuge tube, and placing the centrifuge tube in a pointed-bottom centrifuge tube in a 39 ℃ water bath kettle; waiting cumulusOocyte Complexes (COC)S) After precipitation, eggs were examined under a stereomicroscope after washing 2 times with DPBS; picking up COCs which are normal in shape and uniform in cytoplasm and are wrapped by at least 3 layers of granular cells, washing the detected COCs for 3 times by using DPBS (double DPBS) containing 5% fetal calf serum, then washing the COCs for 3 times by using in-vitro maturation culture solution, and transferring the COCs into a five-hole cell culture plate which contains 500 mu L of in-vitro maturation culture solution and is covered with paraffin oil; placing 100-150 COCs per drop; the liquid is pre-treated with CO2Balancing in an incubator for more than 2 hours; culturing in vitro at 39 deg.C for 42-44 hr with 5% CO2Saturated humidity;
the in vitro maturation culture solution is: TCM-199+0.1% polyvinyl alcohol +3.05mM glucose +0.91
mM sodium pyruvate +0.57mM L-cysteine +100IU/mL penicillin + 100. mu.g/mL streptomycin +10% (V/V) follicular fluid +10IU/mL PMSG +10IU/mL hCG;
2) parthenogenetic activation of porcine in vitro mature oocytes:
COCs matured in vitro for 42-44h were transferred into DPBS containing 0.1% hyaluronidase for digestion and gently blown with a pipette gun repeatedly to remove cumulus cells, washed 3 times with pre-warmed activation solution, placed in a fusion tank with activation solution, electrically activated with BTX-2001 fusion instrument with activation parameters: 1 direct current pulse of 30 mus and 1.1 kv/cm; the activated oocyte is washed for three times by embryo culture solution NCSU-23 preheated in advance for standby;
the formula of the activating liquid comprises: 0.25mol/L mannitol +0.5mol/L calcium chloride +0.5mol/L magnesium sulfate +0.5mol/L HEPES +0.01% (V/V) PVA;
3) co-culture feeder layer cell droplet preparation:
(1) preparation of microdroplets of oviduct epithelial cell feeder layer
A. Isolation and primary culture of oviduct epithelial cells: putting a sow oviduct after obtaining an oviduct of a sow, adding sterile physiological saline added with double antibodies at 30-37 ℃ into the sterile physiological saline to be brought back to a laboratory within 2h, shearing redundant oviduct membranes by using sterile scissors, shearing the oviduct into small sections with the length of 2cm, washing for 3 times by using sterile physiological saline added with double antibodies at 35 ℃, putting the small sections into a disposable plastic dish with the diameter of 35mm, flushing epithelial cells while observing under a stereoscopic microscope, clamping a fracture on one side of the oviduct by using forceps of a clock and a watch in the operation process, sucking pancreatin with the working concentration of 0.25 percent by using a disposable syringe with the volume of 1 ml in the right hand, directly and slowly injecting from the fracture of the oviduct clamped by the forceps to flush the oviduct epithelial cells, collecting pancreatin the dish after repeating for 2-3 times, quickly adding DMEM containing 10 percent of FBS with the same volume to stop digestion, and avoiding excessive digestion, cell death, controlling the time of pancreatin in the oviduct within 5min so as to ensure the purity of the oviduct epithelial cells; centrifuging the collected liquid at 3000r/min for 5min, discarding the supernatant, adding DMEM containing 10% FBS, repeatedly centrifuging for 2 times, discarding the supernatant, and placing the obtained precipitate into a 6-well plate containing 2 ml of DMEM containing 10% FBS for culture; after culturing for 3 days, replacing a fresh culture medium, and digesting for later use when the cells grow over the bottom of the 6-pore plate;
B. feeder layer co-culture cell droplet preparation: digesting oviduct epithelial cells overgrowing with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 104The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO2Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;
or,
(2) cumulus granulosa cell feeder layer droplet preparation:
placing the cultured mature oocyte in a maturation solution, repeatedly blowing and beating cumulus cells except the oocyte by using a liquid transfer gun, completely blowing and picking up the oocyte, keeping the remaining cumulus granular cells in the solution, and adjusting the cell density to 104The cells in the cell suspension are uniformly existed, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, 10 mu L of cell suspension is sucked by a pipette with the range of 10 mu L, and the cell microdroplet is made on a disposable capsule with the diameter of 35mm(ii) a Each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO2Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;
or,
(3) porcine fetal fibroblast feeder layer droplet preparation
A. Preparation of porcine fetal fibroblasts: the specific operation method refers to the method disclosed in ZL201310381274.5 Hubei white pig fibroblast line;
B. feeder layer co-culture cell droplet preparation: digesting pig fetal fibroblast overgrown with 6-well plate with 0.25% pancreatin 48h before co-culture for 2min, adding DMEM containing 10% FBS to stop digestion, gently blowing into single cell suspension with 1 ml pipette, and adjusting cell density to 104The number/ml of the cells in the cell suspension is ensured to exist uniformly, the cell suspension is used for preparing the co-culture feeder layer single embryo culture microdroplet, namely, a pipettor with the measuring range of 10 mu L is used for sucking 10 mu L of cell suspension, and the cell microdroplet is made on a disposable capsule with the diameter of 35 mm; each droplet size 10. mu.L of overlay paraffin oil, placed at 39 ℃ in 5% CO2Culturing in an incubator with saturated humidity, and culturing for 48h for cell co-culture;
4) co-culturing single embryo and feeder layer cells:
(1) co-culture of oocyte and feeder layer cell with zona pellucida attachment
Changing a cell culture medium DMEM in the microdroplets into a fresh embryo culture medium NCSU-23 2 hours before the oocytes are placed into the microdroplets for later use;
selecting in-vitro mature oocytes with first polar bodies and uniform cytoplasm obtained in the step 2) to be in an embryo culture medium NCSU-23 balanced for 2 hours in advance, washing for 3 times, and then putting the oocytes into the co-culture feeder layer single-cell microdroplets prepared in the step 3) for co-culture, wherein 1 oocyte is put in each microdroplet; carrying out half-amount liquid change within 48 h;
or,
(2) co-culture of zona pellucida-removed oocytes and feeder cells
Changing a cell culture medium DMEM in the microdroplets into a fresh embryo culture medium NCSU-23 2 hours before the oocytes are placed into the microdroplets for later use;
after in vitro mature oocytes are parthenogenetically activated, selecting in vitro mature oocytes with a first polar body, uniform cytoplasm and complete cell membranes, digesting the in vitro mature oocytes with pronase with the working concentration of 0.25% to remove zona pellucida, washing the zona pellucida-free oocytes in an embryo culture medium NCSU-23 balanced for 2 hours in advance for 3 times, placing the zona pellucida-free oocytes into single embryo culture microdroplets of the co-culture feeder layer prepared in the step 3) for co-culture, placing 1 oocyte in each microdroplet, and performing half-amount liquid change for 48 hours;
5) obtaining the parthenogenetic blastocysts of the pigs in 7 days: and (4) counting the embryo cleavage rate in 36h, observing the development condition of the embryo under a microscope after co-culture for 7d, and counting the blastocyst development rate.
2. The method for in vitro culturing porcine single embryos of claim 1, wherein the single embryos co-cultured in step 4) are any one of in vitro fertilized embryos, single sperm injection embryos and somatic cell nuclear transfer embryos.
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| CN111548986A (en) * | 2020-05-29 | 2020-08-18 | 安徽农业大学 | Simple and efficient method for preparing embryo culture liquid drops |
| CN114107180A (en) * | 2021-10-21 | 2022-03-01 | 湖北省农业科学院畜牧兽医研究所 | Method for cloning cells without transparent belt body and polymerizing embryos in 1-cell stage and culturing embryos in vitro |
| CN114107180B (en) * | 2021-10-21 | 2024-03-08 | 湖北省农业科学院畜牧兽医研究所 | Zona pellucida-free somatic clone 1-cell stage embryo polymerization and in vitro culture method |
| CN114208770A (en) * | 2021-12-17 | 2022-03-22 | 江苏集萃药康生物科技股份有限公司 | Method for obtaining sterile mice |
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