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 Science Helsinki Committee (#1566-3).
1. Media preparation
<ol>
	<li>Prepare the dissection medium using 500 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) without phenol red and L-glutamine, supplemented with 10% fetal bovine serum, 5 mL of penicillin/streptomycin, and filter-sterilize using a 0.22 &#181;m filter. 
	NOTE: Keep the dissection medium at 4 &#176;C for up to 1 month.</li>
	<li>Prepare ex utero embryo culture medium (EUCM) freshly every culture day inside a biological hood using 25% of embryonic medium plus 50% rat serum and 25% human umbilical cord blood serum (HCS) or human adult blood serum (HBS).</li>
	<li>To prepare embryonic medium, add 5 mL of glutamine, 5 mL of HEPES, and 5 mL of penicillin/streptomycin in 500 mL of DMEM without phenol red and L-glutamine. Prepare 10-15 mL aliquots and store them at 4 &#176;C for up to two months. 
	NOTE: Double the antibiotic concentration if experiencing any contamination.</li>
	<li>Heat-inactivate the rat serum at 56 &#176;C for 30-45 min and filter through a 0.22 &#181;m polyvinylidene difluoride filter. Use the serum for culture on the same day of inactivation. Thaw HCS or HBS and use it immediately for experiments. 
	&#8203;NOTE: Commercial rat serum provides consistent results (a minimum of 4 lots were tested without evident variation). Alternatively, high-quality rat serum can be obtained in-house as described previously8,14. If HCS is not available, replace it with in-house-collected HBS. Rat serum, HCS, and HBS can be refrozen once and kept at -20 &#176;C for use on a different day. Thaw and combine the refrozen serum with a larger volume of freshly thawed serum and do not refreeze it.</li>
</ol>
2. Collection of human umbilical cord blood serum and human adult blood serum
<ol>
	<li>To isolate HCS, collect umbilical cord blood from healthy pregnant women on the day of the scheduled caesarian delivery.
	<ol>
		<li>Double-clamp the umbilical cord 5-7 cm from the umbilicus and transect between the clamps immediately after delivery of the infant.</li>
		<li>Manually draw blood using a large-bore 14 G needle and a 50 mL syringe directly from the umbilical vein while the placenta remains in situ to avoid any traces of hemolysis. 
		NOTE: Collect blood after the infant is removed from the field of surgery, and umbilical blood has been drawn for clinical tests.</li>
	</ol>
	</li>
	<li>Dispense the collected blood to 5 mL procoagulant sterile test tubes and immediately transfer them to 4 &#176;C for 15 min to allow complete coagulation. Centrifuge the coagulated test tubes at 2,500 &#215; g for 10 min at 4 &#176;C.</li>
	<li>Discard tubes showing signs of hemolysis (pink-red serum). Collect the serum (yellow-color) using a 5 mL pipette and filter it through a 0.22 &#181;m filter. Heat-inactivate the serum immediately in a 56 &#176;C water bath for 45 min.</li>
	<li>Prepare 1-1.5 mL aliquots of the inactivated serum and store them at -80 &#176;C for up to six months. If needed, ship at -70 &#176;C using dry ice.</li>
	<li>For adult human blood serum collection, draw blood from healthy adults and immediately isolate serum following the same protocol described for umbilical cord blood serum. Store the HBS as heat-inactivated and filtered aliquots at -80 &#176;C for up to six months. 
	&#8203;NOTE: Human umbilical cord serum and adult human serum prepared freshly in-house gave superior results to commercially available serum. Collect HCS from healthy women, over the age of 18 and under 40 years scheduled for cesarian section delivery. Exclude women who gave vaginal birth and women with any chronic illness or active medical conditions, including gestational diabetes or hypertension.</li>
</ol>
3. Ex utero roller culture of embryos from E7.5 to E11
<ol>
	<li>Setting up the roller culture incubator and gas regulator
	<ol>
		<li>Culture E7.5 or more advanced embryos using the roller culture incubator system. Turn on the rotator and the heating unit at 37 &#176;C for at least 1 h before the embryo dissections. Add autoclaved water to the gas inlet bottle and the outlet test tube. 
		NOTE: Place a thermometer adjacent to the rotating drum and frequently check the stability of the temperature inside the incubator. Open the incubator as quickly as possible as prolonged opening time will increase the temperature and affect embryo development. When necessary, add more autoclaved water to the gas inlet bottle and the outlet test tube to keep them to a minimum of half capacity during culture. Protect the embryos inside the incubator from light by covering the incubator with a cloth.</li>
		<li>Turn on the gas regulator module by pressing the main switch and subsequently turning on the Oxygen/Nitrogen and CO2 controllers (Figure 1A). Set the oxygen and CO2 values to 5% using the respective controllers. Open the gas regulator and set the gas pressure to ~6.5-7 pounds per square inch (psi) by moving the voltage switch in the pressure transmitter (Figure 1B). Confirm the gas pressure value using a digital pressure gauge.</li>
		<li>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/opening the gas flow valve on the lid of the water bottle. Ensure that the gas flow allows the formation of bubbles at a rate of 2-4 bubbles per second or set the bubble flow at the first point where bubbling comes out to the water-filled outlet tube.</li>
		<li>Fill the culture bottles with 2 mL of culture medium and plug them into the hollowed rotating drum for preequilibration for 1 h. Use the hollowed silicon bungs to seal the bottles to the drum. Keep sealed the empty spaces in the rotating drum using the solid bungs. 
		NOTE: The absence of bubbles in the outlet tube (no gas coming through the system) or an exceptionally high gas flow may affect embryo development. In the case of a lack of bubble flow to the outlet test tube, check that all silicon bungs are properly positioned in the rotating drum and sealing the system, and check that the water bottle is closed properly and all the tubing is connected correctly. A lack of bubbling coming into the water bottle may indicate a malfunction in the gas regulator.</li>
	</ol>
	</li>
	<li>Dissection of mouse embryos from pregnant mice
	<ol>
		<li>Preequilibrate the dissection medium inside an incubator at 37 &#176;C and 5% CO2 for a minimum of 1 h. Leave the lid slightly open to allow gas exchange.</li>
		<li>Sacrifice the pregnant female by cervical dislocation, clean the abdomen of the female with 70% ethanol, and cut the skin and abdominal wall with scissors. Locate one end of the uterus and cut at the intersection between the ovary and the uterus. Next, cut along the uterus until the other end and transfer it to a 100 mm Petri dish filled with room temperature Dulbecco&#39;s phosphate-buffered saline (DPBS). 
		NOTE: Use of non-hormone-primed, naturally mated females is recommended.</li>
		<li>Quickly wash the conceptuses in DPBS and cut in pairs to facilitate embryo handling. Move all the pairs of conceptuses to preequilibrated dissection medium in a 60 mm Petri dish and cut into individual conceptuses.</li>
		<li>Remove the uterine wall of the conceptuses by tearing the uterine tissue using a pair of gross forceps. Use fine microsurgical forceps to 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.</li>
		<li>Finally, leave the intact ectoplacental cone attached to the egg cylinder by grasping the embryo from the decidua and peeling the parietal yolk sac off the embryo using fine forceps. 
		NOTE: To avoid affecting the embryo, perform dissections on a microscope equipped with a heating plate at 37 &#176;C within a maximum of 30-40 min. Dissect one litter of embryos at a time.</li>
		<li>Immediately after dissection, transfer the embryos to a new plate filled with equilibrated dissection medium using a glass Pasteur pipette to prevent the embryos from sticking to the tissue debris.</li>
		<li>Select those embryos in the neural plate/early head fold stage showing no damage in the epiblast and transfer 5-6 embryos per bottle to the preequilibrated glass culture bottles. 
		NOTE: To prepare the glass Pasteur pipette, cut the opening of the pipette to an adequate size to fit the embryos using a glass cutter, and keep it in ethanol to avoid contamination. Wash the glass pipette with PBS before transferring embryos. When transferring the embryos to the culture bottle, make sure to carry over as little volume of the dissection medium as possible to avoid diluting the EUCM.</li>
		<li>Place the bottles in the rotating culture system at 37 &#176;C, in an atmosphere of 5% O2 and 5% CO2.</li>
		<li>Each day of culture, remove the bottles one by one from the incubator to evaluate embryo development under a stereomicroscope. Make sure to close the empty hole in the drum with a solid bung when removing a bottle. Cut the tip of a sterile plastic Pasteur pipette to fit the embryo size and move the embryos to a Petri dish to facilitate observation. Make sure that the embryos are always covered by medium. 
		NOTE: Minimize the time for which the embryos are outside the incubator to avoid detrimental effects in the embryos due to a decrease in body temperature. Handle the embryos carefully to avoid rupture of the yolk sac blood vessels, affecting embryo survival.</li>
		<li>At culture day 1 (equivalent to E8.5), transfer groups of 3 embryos to a new bottle with 2 mL of fresh and preequilibrated EUCM containing extra 3 mg/mL of D-glucose and a gas mixture of 13% O2, 5% CO2.</li>
		<li>After 48 h (equivalent to E9.5), transfer groups of two embryos to a new bottle with fresh preheated EUCM plus 3.5 mg/mL of glucose in a gas atmosphere of 18% O2 and 5% CO2.</li>
		<li>At 72 h of culture (equivalent to E10.5), move each embryo to an individual bottle containing 1.5-2 mL of fresh medium supplemented with 4 mg/mL of glucose and a gas supply of 21% O2 and 5% CO2. 
		NOTE: Embryos reach their maximum growth about midnight of culture day 4.</li>
		<li>Use fine forceps to remove the yolk sac and amnion, and detach the umbilical cord for proper observation of embryo morphology. Turn off the gas regulation module and precision incubator. 
		&#8203;NOTE: Preequilibrate the culture medium for 1 hour by incubation inside a glass bottle in the roller culture with an adequate gas atmosphere according to the embryo stage. Clean the culture bottles and all incubator glassware after every use by performing three washes with running distilled water followed by an overnight wash submerged in 70% ethanol. Rewash three times with running distilled water, let the bottles dry overnight, and sterilize by autoclave. Likewise, autoclave the silicon plugs regularly. Thoroughly clean all dissection tools with 70% ethanol and dry-sterilize them.</li>
	</ol>
	</li>
</ol>
4. Extended embryo culture from E5.5/E6.5 to E11
<ol>
	<li>Culture pregastrulation (E5.5) and early gastrulation (E6.5) embryos until the early somite stage (E8.5) in static plates.</li>
	<li>Preequilibrate the dissection medium for 1 h in an incubator with 5% CO2 at 37 &#176;C. 
	NOTE: To allow gas exchange, do not close the lid of the tube completely.</li>
	<li>Prepare the culture plates by adding 250 &#181;L of freshly prepared EUCM to each well. Place the plates inside a CO2 incubator at 37 &#176;C for preequilibration. 
	NOTE: Although 8-well plates are well-suited for embryo growth, culture can be done in any other plate by adjusting the volume of the medium according to the size of the plate.</li>
	<li>Dissect egg cylinder embryos out of the uterus following the technique described for E7.5. Remove the parietal yolk sac of the embryo and leave the intact ectoplacental cone attached to the egg cylinder.</li>
	<li>Transfer individual embryos into each well of the 8-well plate using a micropipette and place the plate inside the incubator with 5% CO2 at 37 &#176;C.</li>
	<li>Image the embryos under a stereomicroscope and select for culture only those with a well-formed amniotic cavity, with no evident damage and without the Reichert&#39;s membrane. 
	NOTE: Use 20 &#181;L pipette tips to transfer E5.5 embryos and 200 &#181;L tips to transfer E6.5 embryos. In the case of cultures starting at E6.5, HCS can be replaced by freshly collected HBS.</li>
	<li>Remove half of the medium and add 250 &#181;L of freshly prepared prewarmed EUCM after 24 h of culture, ensuring that the embryos are always immersed in the culture medium. In the case of cultures starting at E5.5, after two days of culture (equivalent to E7.5), remove 200 &#181;L and add 250 &#181;L of new EUCM. 
	NOTE: During static culture, embryos might attach to the plate (mainly when using plastic plates), which will severely compromise embryo development. Attachment of the embryo to the plate is more frequent on the first day of culturing E5.5 embryos. To prevent attachment, carefully push the embryos away from the plate surface using fine, sterile forceps. Verify that only the ectoplacental cone remains attached to the surface of the plate.</li>
	<li>Transfer the embryos into the roller culture at the early somite stage (4-7 somites; after three days of culture for embryos explanted at E5.5 and two days for cultures started at E6.5), following the same indications described previously for E8.5 stage embryos. 
	NOTE: Transferring the embryos to the roller culture at somite stages different than indicated above results in failure of further development. In contrast with E7.5 ex utero cultures, it is possible to maintain the embryos in a constant atmosphere of 21% oxygen and 5% CO2, providing a slightly higher efficiency of embryo development than atmospheres with dynamic oxygen concentration.</li>
</ol>

<ol>
	<li>New, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-embryo+culture+and+the+study+of+mammalian+embryos+during+organogenesis.">Whole-embryo culture and the study of mammalian embryos during organogenesis.</a> Biological Reviews of the Cambridge Philosophical Society. 53 (1), 81-122 (1978).</li><li>Tam, P. P., Behringer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+gastrulation:+the+formation+of+a+mammalian+body+plan.">Mouse gastrulation: the formation of a mammalian body plan.</a> Mechanisms of Development. 68 (1-2), 3-25 (1997).</li><li>Huang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+mouse+embryos.">Intravital imaging of mouse embryos.</a> Science. 368 (6487), 181-186 (2020).</li><li>White, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+binding+of+Sox2+to+DNA+predicts+cell+fate+in+the+four-cell+mouse+embryo.">Long-lived binding of Sox2 to DNA predicts cell fate in the four-cell mouse embryo.</a> Cell. 165 (1), 75-87 (2016).</li><li>Tam, P. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+mouse+embryos+in+vitro.">Cultivation of mouse embryos in vitro.</a> Nature. 199, 297-299 (1963).</li><li>Rivera-P&#233;rez, J. A., Jones, V., Tam, P. P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+whole+mouse+embryos+at+early+postimplantation+to+organogenesis+stages:+developmental+staging+and+methods.">Culture of whole mouse embryos at early postimplantation to organogenesis stages: developmental staging and methods.</a> Methods in Enzymology. 476, 185-203 (2010).</li><li>New, D. A. T., Coppola, P. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a+second+neural+axis+by+the+mouse+node.">Induction of a second neural axis by the mouse node.</a> Development. 120 (3), 613-620 (1994).</li><li>Aguilera-Castrejon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+utero+mouse+embryogenesis+from+pre-gastrulation+to+late+organogenesis.">Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.</a> Nature. 593 (7857), 119-124 (2021).</li><li>Takahashi, M., Makino, S., Kikkawa, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+serum+suitable+for+mammalian+whole+embryo+culture.">Preparation of rat serum suitable for mammalian whole embryo culture.</a> Journal of Visualized Experiments: JoVE. 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J.H.H is an advisor to Biological Industries Ltd and has submitted a patent application covering the roller and static culture conditions described herein (filed by J.H.H. and the Weizmann Institute of Science). Other authors declare no competing interests.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m pore size filter (250 mL)</td>
			<td>JetBiofil</td>
			<td>FCA-206-250</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m pore size syringe PVDF filter</td>
			<td>Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well &#181;-plates glass bottom/ibiTreat</td>
			<td>iBidi</td>
			<td>80827/80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle with adaptor cap for gas inlet</td>
			<td>Arad Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>D(+)-glucose Monohydrate</td>
			<td>J.T. Baker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond knife</td>
			<td>Fine Science Tools</td>
			<td>10100-30/45</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Biological industries</td>
			<td>02-020-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biological industries</td>
			<td>04-013-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas regulation module</td>
			<td>Arad Technologies</td>
			<td>HannaLab1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>GIBCO</td>
			<td>35050061</td>
			<td>glutamine</td>
		</tr>
		<tr>
			<td>Graefe forceps</td>
			<td>Fine Science Tools</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>GIBCO</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical forceps (Dumont #5, #55)</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes (glass)</td>
			<td>Hilgenberg</td>
			<td>3150102</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes (plastic)</td>
			<td>Alexred</td>
			<td>SO P12201</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biological industries</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
	</tbody>
</table>

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. ...

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Research

JoVE Journal

Developmental Biology

6.4K Views.  Weizmann Institute of Science. 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.

Video: Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...