Name: ex utero culture of mouse embryos from pregastrulation to advanced organogenesis
Uploaded: 2021-10-19T15:00:00
Description: Watch this Scientific Journal Video about Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis at JoVE.com

This work was funded by Pascal and Ilana Mantoux; European Research Council (ERC-CoG-2016 726497-Cellnaivety); Flight Attendant Medical Research Council (FAMRI); Israel Cancer Research Fund (ICRF) professorship, BSF, Helen and Martin Kimmel Institute for Stem Cell Research, Helen and Martin Kimmel Award for Innovative Investigation; Israel Science Foundation (ISF), Minerva, the Sherman Institute for Medicinal Chemistry, Nella and Leon Benoziyo Center for Neurological Diseases, David and Fela Shapell Family Center for Genetic Disorders Research, Kekst Family Institute for Medical Genetics, Dr. Beth Rom-Rymer Stem Cell Research Fund, Edmond de Rothschild Foundations, Zantker Charitable Foundation, Estate of Zvia Zeroni.

All animal experiments were performed according to the Animal Protection Guidelines of Weizmann Institute of Science and approved by relevant Weizmann Institute IACUC (#01390120-1, 01330120-2, 33520117-2). Healthy pregnant women were asked to give their informed consent to collect blood from their umbilical cord, as approved by the Rambam Medical Center Helsinki Committee (#RMB-0452-15). Healthy adults were asked for their informed consent to have blood collected according to the guidelines of the Weizmann Institute of S...

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The culture protocol presented herein can sustain proper and continuous mouse embryo development ex utero for up to six days, from E5.5 to E11. Previously, embryos at these developmental stages could develop normally in culture only for short periods (up to 48 h)15. The coupling of the gas regulation module to the roller culture incubator for precise control of oxygen concentration and hyperbaric gas pressure is critical for the proper mouse embryo culture described herein. Increasing the...

Intrauterine development of the mammalian embryo has limited the study of the early stages of postimplantation development1,2. The inaccessibility of the developing embryo hampers the understanding of key developmental processes occurring after the embryo implants into the uterus, such as the establishment of the animal body plan, specification of the germ layers, or the formation of tissues and organs. Moreover, the very small size of the early postimplanted embryo makes it difficult to observe by intravital imaging in utero before&#160;E103. The inability to observe and manipulate living embryos at these stages has restricted the study of early postimplantation embryogenesis to snapshots during development.
Protocols for in vitro culture of preimplantation mammalian embryos are well established, reliable, and regularly utilized4. Nevertheless, attempts to establish ex utero culture systems capable of supporting proper mammalian postimplantation embryo growth had limited success5. A variety of culture techniques have been proposed for over a century, mainly by culturing the embryos in conventional static plates6,7,8 or rotating bottles (roller cultures)5,9,10. These platforms proved helpful in expanding the knowledge on mammalian development after implantation11,12, despite being highly inefficient for normal embryo survival and limited to short periods. The embryos began to display developmental retardation and morphological anomalies as early as 24-48 h after culture initiation.
This study provides a detailed description for setting up the ex utero embryo culture system that allows continuous development from pregastrulation to advanced organogenesis stages over up to six days of postimplantation development13. This paper describes the improved roller culture protocol that supports the growth of E7.5 embryos (neural plate and headfold-stage) until the hind limb formation stage (~E11) and the extended culture from E5.5/E6.5 by combining culture on static plates and roller culture platforms.

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			<td>0.22 &#181;m pore size filter (250 mL)</td>
			<td>JetBiofil</td>
			<td>FCA-206-250</td>
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			<td>0.22 &#181;m pore size syringe PVDF filter</td>
			<td>Millipore</td>
			<td>SLGV033RS</td>
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			<td>8-well &#181;-plates glass bottom/ibiTreat</td>
			<td>iBidi</td>
			<td>80827/80826</td>
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			<td>Bottle with adaptor cap for gas inlet</td>
			<td>Arad Technologies</td>
			<td></td>
			<td></td>
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			<td>Bungs (Hole)</td>
			<td>B.T.C. Engineering, Cullum Starr Precision Engineering</td>
			<td>BTC 06</td>
			<td>Used to seal the bottles to the drum</td>
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			<td>Bungs (Solid)</td>
			<td>B.T.C. Engineering, Cullum Starr Precision Engineering</td>
			<td>BTC 07</td>
			<td>Used to seal the rotating drum</td>
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			<td>Culture bottles</td>
			<td>B.T.C. Engineering, Cullum Starr Precision Engineering</td>
			<td>BTC 03/BTC 04</td>
			<td>Either Glass Bottles (Small) BTC 03 or Glass Bottles (Large) BTC 04</td>
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			<td>D(+)-glucose Monohydrate</td>
			<td>J.T. Baker</td>
			<td></td>
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			<td>Diamond knife</td>
			<td>Fine Science Tools</td>
			<td>10100-30/45</td>
			<td></td>
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			<td>Digital Pressure Gauge</td>
			<td>Shanghai Benxu Electronics Technology co. Ltd</td>
			<td>BX-DPG80</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11880</td>
			<td></td>
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			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Biological industries</td>
			<td>02-020-1A</td>
			<td></td>
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			<td>Fetal Bovine Serum</td>
			<td>Biological industries</td>
			<td>04-013-1A</td>
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			<td>Gas regulation module</td>
			<td>Arad Technologies</td>
			<td>HannaLab1</td>
			<td></td>
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			<td>Glutamax</td>
			<td>GIBCO</td>
			<td>35050061</td>
			<td>glutamine</td>
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			<td>Graefe forceps</td>
			<td>Fine Science Tools</td>
			<td>11052-10</td>
			<td></td>
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			<td>HEPES</td>
			<td>GIBCO</td>
			<td>15630056</td>
			<td></td>
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			<td>Microsurgical forceps (Dumont #5, #55)</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
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			<td>Pasteur pipettes (glass)</td>
			<td>Hilgenberg</td>
			<td>3150102</td>
			<td></td>
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			<td>Pasteur pipettes (plastic)</td>
			<td>Alexred</td>
			<td>SO P12201</td>
			<td></td>
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			<td>Penicillin/Streptomycin</td>
			<td>Biological industries</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
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			<td>Petri Dishes (60 mm and 100 mm)</td>
			<td>Falcon</td>
			<td>351007/351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision incubator system</td>
			<td>B.T.C. Engineering, Cullum Starr Precision Engineering</td>
			<td>BTC01</td>
			<td>BTC01 model with gas bubbler kit</td>
		</tr>
		<tr>
			<td>Pro-coagulant sterile test tubes (5 mL)</td>
			<td>Greiner Bio-One</td>
			<td>#456005</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat whole embryo culture serum</td>
			<td>ENVIGO Bioproducts</td>
			<td>B-4520</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic microscope equipped with heating plate</td>
			<td>Nikon</td>
			<td>SMZ18</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringes (5, 10 ml) for sera filtration</td>
			<td>Pic Solution</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td></td>
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ex utero culture of mouse embryos from pregastrulation to advanced organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

The roller culture conditions described for E7.5 embryos (late-gastrulation stage) support constant and normal embryo growth with an average efficiency close to 75% after 4 culture days (Figure 2 and Table 1). The efficiency of embryo development may vary across diverse mouse genetic backgrounds but is consistently robust (Figure 2C). Supplementation with HBS instead of HCS yields an efficiency of ~68% after 4 days of ex utero culture, ...

Watch this Scientific Journal Video about Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis at JoVE.com

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

This method will be fundamental for understanding gastrulation along a formation in mammals. By allowing experimentation in living embryos during embryonic stages that have long been hidden inside the maternal uterus. It allows, continues efficient and appropriate growth of mouse embryos outside the uterus for an extended period of up to 6 days.Quick and adequate embryo dissection and pregastrulation of the gas concentration and pressure are the most critical steps for successful embryo growth. To begin culturing 7.5 day old or more advanced mouse embryos, turn on the rotator and the heating unit of the roller culture incubator system for at least 1 hour before the embryo dissections. Also, add autoclave water to the gas inlet bottle and the outlet test tube.Next, turn on the gas regulator module by pressing the main switch, then use the respective controllers to set the oxygen and carbon dioxide values to 5%Open the gas regulator and move the voltage switch in the pressure transmitter to set the gas pressure to approximately 6.5 to 7 pounds per square inch. Confirm the gas pressure value using a digital pressure gauge. Monitor the gas flow by checking the rate of bubbles created inside the outlet water filled test tube allocated inside the precision incubator.Set the proper bubble rate by closing or opening the gas flow valve on the water bottle's lid. Ensure that the gas flow allows the formation of bubbles at a rate of 2 to 4 bubbles per second or set the bubble flow at the first point where bubbling comes out to the water filled outlet tube. Next, fill the culture bottles with 2 milliliters of culture medium.Use the hollowed silicon bungs to seal the bottles and plug them into the hollowed rotating drum for pre-equilibration for 1 hour. Keep the empty spaces in the rotating drum sealed using the solid bungs. Next, dissect the embryos from a euthanized pregnant female mouse.Clean the abdomen with 70%ethanol and cut the skin and abdominal wall using scissors. Locate one end of the uterus and cut at the intersection between the ovary and the uterus. Continue cutting along the uterus until the other end and transfer to a 100 millimeter Petri dish filled with DPBS.Quickly wash the conceptuses in DPBS and cut in pairs to facilitate embryo handling. Move all the pairs of conceptuses to pre-equilibrated dissection medium in a 60 millimeter Petri dish, then cut into the individual conceptuses. Next, using a pair of gross forceps, remove the uterine wall of the conceptuses by tearing the uterine tissue, then using fine, microsurgical forceps, cut the tip of the pear-shaped decidua.Insert the forceps next to the embryo parallel to its long axis and open the forceps to split the decidua into halves. Finally, using fine forceps, grasp the the embryo from the decidua and peel the parietal yolk sack off the embryo, leaving the intact ectoplacental cone attached to the egg cylinder. Select the embryos in the neural plate, or early head fold stage, that show no damage in the epiblast.Transfer 5 to 6 selected embryos per bottle to the pre-equilibrated glass culture bottles and place the bottles in the rotating culture system at 37 degrees Celsius, in an atmosphere of 5%oxygen and 5%carbon dioxide. To culture pregastrulation and early gastrulation embryos and static plates, add 250 microliters of freshly prepared ex-utero embryo culture medium to each well of an 8 well plate. Place the plates inside a carbon dioxide incubator at 37 degree Celsius for pre-equilibration.After dissecting the embryos out of the uterus as demonstrated previously, transfer the individual embryos into each well of the 8 well plate using a micro pipette and place the plate inside the incubator with 5%carbon dioxide at 37 degrees Celsius. Image the embryos under a stereo microscope and select for culture only those with a well-formed amniotic cavity with no evident damage and without the Reichert's membrane. The roller culture conditions described in this protocol for 7.5 day old embryos support constant and normal embryo growth with an average efficiency close to 75%after 4 culture days.The efficiency of embryo development varies across diverse mouse genetic backgrounds but is consistently robust. Supplementation with HBS instead of HCS yields an efficiency of approximately 68%after 4 days of culture. The development of 6.5 embryos and static plates is correctly recapitulated with an efficiency of more than 90%using ex-utero embryo culture medium with both HCS and HBS.Cultures starting from pregastrulating embryos show a 46%efficiency of proper development to the early somite stage and nearly 17%of the embryos complete proper development after 6 days of culture. The morphogenesis and tissue development proceed properly until 42 somites. Representative developmental defects observed in embryos for cultures initiated from embryonic day 7.5, 6.5, and 5.5 are shown here.Embryos with minor damage to the epiblast or embryos retaining the Reichert's membrane should be discarded. Early embryos will not grow properly or display severe developmental delays while attachment of the embryonic epiblast to the surface of the plate will cause the failure of further embryo development. The main abnormalities observed in defective embryos are the development of the posterior region outside the yolk sack or defects in the growth of the neural folds.In cultures developed from 5.5 day old embryos are frequently observed developmental defect is the presence of a small, underdeveloped epiblast. Using this method, researchers are able to perform a variety of physical or chemical manipulations in developing post-implanted embryos. For instance, manipulation, cellular transplantation, or lineage tracing.This method not only paves the way for studying mouse post-implantation developing in great detail, but also for implementing similar culture methods in embryos from other species, such as human.

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