Method Article

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

DOI:

10.3791/2170

⸱

August 28th, 2010

 , 
1Division of Developmental Neuroscience, United Centers for Advanced Research and Translational Medicine (ART), Tohoku University Graduate School of Medicine, 2The Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST)

In This Article

Summary

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Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Abstract

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Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Protocol

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1. Setting up the WEC system

  1. Joint a 0.22 μm membrane filter to the inlet silicon tube within WEC system a).
  2. Open the valve of a gas cylinder containing O2 (95%) and CO2 (5%). Flow the gas mixture into the drum via the bubbling bottle containing autoclaved water.
  3. Adjust the flow volume of gas mixture to 50 cc.
  4. Insert the out let silicon tube into a water bottle and check out the flow of gas mixture.
  5. Rotate the drum at the speed of 20 rpm/min.

2. Preparation of culture medium

  1. Thaw rat immediately-centrifuged (IC) serum at 37.0°C ....

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Discussion

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There are two critical steps in rodent WEC for the success. First, the dissection procedure should be accurate not to damage embryos, especially blood vessels. Second, the procedure should be as quickly as possible because oxygen and nutrient are no longer supplied via the placenta after isolation from the uterus. This is critical for older embryos. In the case of rat WEC after E12.5, we should transfer dissected embryos into culture bottles within 30 minutes.

We have analyzed migration patte.......

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Disclosures

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No conflicts of interest declared.

Acknowledgements

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We thank Mr. Hajime Ichijo for video-recording and helpful advices for editing the video. We also thank Drs.Yuji Tsunekawa and Kaichi Yoshizaki for kind assistant for video-recording. This work is supported by KAKENHI on Young Scientist B and on Priority Areas- Molecular Brain Science from MEXT of Japan. We acknowledge the support of Global COE Program "Basic and Translational Research Center for Global Brain Science" from MEXT of Japan and The Core Research for Evolutional Science and Technology (CREST) from Japanese Science and Technology Corporation from Japanese Science and Technology Agency (JST).

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
WEC system (10-0310)ToolIkemoto Rika010-0310Another small model is also available.
Silicon plug without a holeToolIkemoto Rika010-032-08
Gas mixture cylinderToolNikko Sanso-Containing 95% oxygen & 5% carbon dioxide. Custom order.
Gas regulatorToolOno SeisakushoWR-11
0.22 μm filter Millex GSToolEMD MilliporeSLGS033SS
Large scissorsToolNapoxB-7H
ForcepsToolNapoxA-3-2
Disposal Petri dish (90 mm x 15 mm) ToolIwaki, Asahi Techno Glass Corp.SH90-15Deep-type dish is the best for dissection.
IsofluraneReagentAbbott LaboratoriesB506For anesthesia
Pentobarbital sodiumReagentMerck & Co.-For anesthesia
Tyrode’s salineReagentAccording as the protocol in Ref. 2. Save at 4°C.
Timed-pregnant Sprague-Dawley ratAnimalCharles River Laboratories-
Culture glass bottleToolIkemoto Rika010-032-05
Disposal culture bottleToolIkemoto Rika010-032-06
Silicon plug with holeToolIkemoto Rika010-032-07
Aluminum foilToolAny Supplier-
Autoclaving bagToolHogyHM-26For culture bottles
Autoclaving bagToolHogyHM-14AFor silicon plugs
0.45 μm filter Millex HAToolEMD MilliporeSLHA033SS
50 ml conical tubeToolBD Biosciences352070
100 ml beakerToolIwaki, Asahi Techno Glass Corp.TE-32
Rat IC serumReagentCharles River Laboratories-Refer to: Mr. Kunihiko Morisaki,
TEL: +81-(0) 45-474-9336; FAX: +81-(0) 45-474-9340
D(+)-GlucoseReagentWako Pure Chemical Industries, Ltd.041-00595
Antibiotic-antimyotic liquidReagentGIBCO, by Life Technologies15240
Ophthalmic straight scissorsToolNapoxMB50-7For cutting the uterine wall
Ophthalmic curved scissorsToolNapoxMB54-2For cutting the yolk sac
Forceps #5 ToolVigorT6715
Forceps #5FToolRegineT6819
Glass pipetteToolMade by hand using Pasteur pipette.
Autoclaving bagToolHogyHM-4For glass pipettes
Binocular microscopeToolLeica MicrosystemsMz7s
LightToolLeica MicrosystemsCLS 150XD
Oil stoneToolUchida Yoko833-2000For sharpening of forceps
Machine oilToolUchida Yoko835-0000For sharpening of forceps

References

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  1. New, D. A. T. Intoroduction. In mammalian postimplantation embryos. In Mammalian Postimplantation Embryos A Practical Approach. Copp, A. J., Cockroft, D. L. , IRL Press. Oxford. 1-14 (1990).
  2. Cockroft, D. L. Dissection and culture of post implantation embryos. In Mammalian Postimplanta....

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Tags

Whole Embryo CultureRotator Bottle SystemRodent Embryo CultureEmbryo DissectionGene TransferElectroporationCell Migration AnalysisGFP RFP LabelingOrgan Slice CultureDevelopmental Biology

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