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/pubmed?term=((Whole-embryo+culture+and+the+study+of+mammalian+embryos+during+organogenesis%5BTitle%5D)+AND+%22Biological+Reviews+of+the+Cambridge+Philosophical+Society%22%5BJournal%5D)">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>
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<li>White, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-lived+binding+of+Sox2+to+DNA+predicts+cell+fate+in+the+four-cell+mouse+embryo%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postimplantation+mouse+development%3A+whole+embryo+culture+and+micro-manipulation%5BTitle%5D)+AND+%22International+Journal+of+Developmental+Biology%22%5BJournal%5D)">Postimplantation mouse development: whole embryo culture and micro-manipulation.</a> International Journal of Developmental Biology. 42 (7), 895-902 (1998).</li>
<li>Nicholas, J. S., Rudnick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+rat+embryos+in+tissue+culture%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">The development of rat embryos in tissue culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 20 (12), 656-658 (1934).</li>
<li>New, D. A., Stein, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cultivation+of+mouse+embryos+in+vitro%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cultivation of mouse embryos in vitro.</a> Nature. 199, 297-299 (1963).</li>
<li>Rivera-Pérez, J. A., Jones, V., Tam, P. P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+of+whole+mouse+embryos+at+early+postimplantation+to+organogenesis+stages%3A+developmental+staging+and+methods%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">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. T., Terry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+of+explanted+rat+embryos+in+rotating+tubes%5BTitle%5D)+AND+%22Journal+of+Reproduction+and+Fertility%22%5BJournal%5D)">Culture of explanted rat embryos in rotating tubes.</a> Journal of Reproduction and Fertility. 35 (1), 135-138 (1973).</li>
<li>Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparative+and+historical+review+of+culture+methods+for+vertebrates%5BTitle%5D)+AND+%22International+Journal+of+Developmental+Biology%22%5BJournal%5D)">A comparative and historical review of culture methods for vertebrates.</a> International Journal of Developmental Biology. 41 (12), 127-137 (1997).</li>
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<li>Takahashi, M., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transferring+genes+into+cultured+mammalian+embryos+by+electroporation%5BTitle%5D)+AND+%22Development%2C+Growth+and+Differentiation%22%5BJournal%5D)">Transferring genes into cultured mammalian embryos by electroporation.</a> Development, Growth and Differentiation. 50 (6), 485-497 (2008).</li>
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</ol>

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><tbody><tr><td>0.22 &amp;micro;m pore size filter (250 mL)</td><td>JetBiofil</td><td>FCA-206-250</td><td></td></tr><tr><td>0.22 &amp;micro;m pore size syringe PVDF filter</td><td>Millipore</td><td>SLGV033RS</td><td></td></tr><tr><td>8-well &amp;micro;-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&#039;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

6.7K 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

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 ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

Neuroscience

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Biology

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Immunology and Infection

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Isolation and Genotyping of Gastrulating Mouse Embryo for Single-Cell Sequencing

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Summary

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

Neural Tube Closure in Mouse Whole Embryo Culture

Dissection and Culture of Mouse Embryonic Kidney

Isolation and Derivation of Mouse Embryonic Germinal Cells

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development