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Method Article
Jacob H. Hanna
Weizmann Institute of Science
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2042-9974
License:
This work is licensed under a CC BY 4.0 License. Read Full License
In mammals, morphogenesis and organogenesis take place after the embryo implants into the uterus, which makes it relatively inaccessible for observation and manipulation. While methods for in vitro culture of pre- and peri-implantation mouse embryos are routinely used, approaches for efficient and stable culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. We recently developed highly robust ex utero post-implantation mouse embryo culture platforms, that enable appropriate and faithful development of embryos before gastrulation (E5.5) until the hind limb formation stage (E11). In these protocols, late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings, while extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture protocols. These systems support stable growth of normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis.
Keywords
Mouse embryo, ex utero culture, gastrulation, organogenesis
This is a list of supplementary files associated with this preprint. Click to download.
- Video1.mp4
Movie 1. Embryo dissection. Step by step visualization of material and protocol for dissecting E7.5 embryos for ex utero culture. These guidelines apply also for E5.5 and E6.5 embryos.
- Video2.mp4
Movie 2. Media preparation and pre-heating. Detailed visualization of protocol for preparing ex utero culture media and pre-heating inside the roller culture.
- Video3.mov
Movie 3. Opening the gas regulation module and operating the incubator’s gas flow valve. Part 1. Opening of the gas regulation module to show the internal components and pressure transmitter which controls the gas pressure depending on the Voltage. Part 2. Setting the proper gas flow by turning the gas flow valve and monitoring the rate of bubbles created in the gas outlet tube. Turning the valve counterclockwise results in the absence of gas flow and clockwise turning increases the bubble flow rate.
- Video4.mov
Movie 4. Embryo transfer to a new culture bottle. Transferring of one embryo to a bottle containing freshly prepared pre-heated media.
- Video5.mov
Movie 5. Embryo imaging. Transferring of 3 embryos to a plate for imaging under a stereomicroscope.
- Video6.mp4
Movie 6. Embryo culture in static plates and transfer to the roller culture. Embryos explanted at E5.5/E6.5 are cultured in a CO2 incubator in individual wells of an iBidi plate and subsequently transferred to a bottle for roller culture.
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Posted 30 Mar, 2021
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Associated Publications
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Jacob H. Hanna
Weizmann Institute of Science
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2042-9974
Version History
CURRENT STATUS: Posted
Version 1
Posted 30 Mar, 2021
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
In mammals, morphogenesis and organogenesis take place after the embryo implants into the uterus, which makes it relatively inaccessible for observation and manipulation. While methods for in vitro culture of pre- and peri-implantation mouse embryos are routinely used, approaches for efficient and stable culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. We recently developed highly robust ex utero post-implantation mouse embryo culture platforms, that enable appropriate and faithful development of embryos before gastrulation (E5.5) until the hind limb formation stage (E11). In these protocols, late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings, while extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture protocols. These systems support stable growth of normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis.
This is a list of supplementary files associated with this preprint. Click to download.
- Video1.mp4
Movie 1. Embryo dissection. Step by step visualization of material and protocol for dissecting E7.5 embryos for ex utero culture. These guidelines apply also for E5.5 and E6.5 embryos.
- Video2.mp4
Movie 2. Media preparation and pre-heating. Detailed visualization of protocol for preparing ex utero culture media and pre-heating inside the roller culture.
- Video3.mov
Movie 3. Opening the gas regulation module and operating the incubator’s gas flow valve. Part 1. Opening of the gas regulation module to show the internal components and pressure transmitter which controls the gas pressure depending on the Voltage. Part 2. Setting the proper gas flow by turning the gas flow valve and monitoring the rate of bubbles created in the gas outlet tube. Turning the valve counterclockwise results in the absence of gas flow and clockwise turning increases the bubble flow rate.
- Video4.mov
Movie 4. Embryo transfer to a new culture bottle. Transferring of one embryo to a bottle containing freshly prepared pre-heated media.
- Video5.mov
Movie 5. Embryo imaging. Transferring of 3 embryos to a plate for imaging under a stereomicroscope.
- Video6.mp4
Movie 6. Embryo culture in static plates and transfer to the roller culture. Embryos explanted at E5.5/E6.5 are cultured in a CO2 incubator in individual wells of an iBidi plate and subsequently transferred to a bottle for roller culture.
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