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.
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.
1. Setting up the WEC system
2. Preparation of culture medium
3. Anesthesia of the rat and isolation of the uterus
4. Removal of embryos from the uterus
5. Dissection of rat embryos
6. Whole embryo culture
7. Notes
8. Representative results
Figure 1 shows procedures of dissection of the rat embryo and cultured rat embryos.
Figure 1. Whole embryo culture of the E12.5 rat embryo. (A) A conceptus dissected from the uterus of the pregnant rat. (B) Removal of the decidua at the placenta side. (C) Removal of Reichert’s membrane. (D) Opening the yolk sac. (E) The rat embryo culturing in the bottle for 6 hr after the beginning of WEC. (F) The rat embryo cultured for 42 hr. d, decidua; R; Reichert’s membrane; p, placenta; ys, yolk sac; e, embryo.
0 hr | 12 hr | 24 hr | 36 hr | 48 hr | |
E12.5 rat embryo | 95% 50 cc | 95% 75 cc (10 hr) |
95% 100 cc | 95% 125 cc (34 hr) | 95% 150 cc |
Table 1. Optimal oxygen condition.
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 patterns of neural crest cells in the wild type and Pax6 mutant rat embryos by labeling small numbers of cells with fluorescent dye DiI or transplanting DiI-labeled cells in rat embryos of the wild type and mutant background 7. A fate map of the anterior neural plate has been drawn in the mouse from labeling neuroepithelial cells with DiI and culturing for 24 hr 8. WEC has also been used for transplantation of cells expressing GFP into the telencephalon of E12.5 rat embryos to examine cell autonomy in migration defects shown in the Pax6 mutant rat 9. Various inhibitors can be applied to the culture medium to examine signaling pathways involved in axis formation and cell cycle during development 10, 11. Furthermore, we can simply introduce expression plasmids and siRNA into developing brains of cultured embryos by electroporation to analyze gene functions 12,13,14. We also investigated behavior of neuroepithelial cells labeled with fluorescent protein in organ culture or slice culture following WEC (see ref. 14 and references therein) 14. Therefore, rodent WEC is very useful not only for analyzing cell lineage but also for elucidating gene functions by loss-of-function and gain-of-function experiments.
The authors have nothing to disclose.
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).
Name | Type | Company | Catalog Number | Comment |
Setting up WEC | ||||
WEC system (10-0310) | Tool | Ikemoto Rika | 010-0310 | Another small model is also available. |
Silicon plug without a hole | Tool | Ikemoto Rika | 010-032-08 | |
Gas mixture cylinder | Tool | Nikko Sanso | – | Containing 95% oxygen & 5% carbon dioxide. Custom order. |
Gas regulator | Tool | Ono Seisakusho | WR-11 | |
0.22 μm filter Millex GS | Tool | Millipore | SLGS033SS | |
Anesthesia and isolation of the uterus | ||||
Large scissors | Tool | Napox | B-7H | |
Forceps | Tool | Napox | A-3-2 | |
Disposal Petri dish (90 mm x 15 mm) |
Tool | Iwaki | SH90-15 | Deep-type dish is the best for dissection. |
Isoflurane | Reagent | Abbott | B506 | For anesthesia |
Pentobarbital sodium | Reagent | Schering-Plough Animal Health | – | For anesthesia |
Tyrode’s saline | Reagent | – | – | According as the protocol in Ref. 2. Save at 4°C. |
Timed-pregnant Sprague-Dawley rat | Animal | Charles Rivers Laboratories Japan | – | |
Preparation of culture medium | ||||
Culture glass bottle | Tool | Ikemoto Rika | 010-032-05 | |
Disposal culture bottle | Tool | Ikemoto Rika | 010-032-06 | |
Silicon plug with hole | Tool | Ikemoto Rika | 010-032-07 | |
Aluminum foil | Tool | Any company | – | |
Autoclaving bag | Tool | Hogy | HM-26 | For culture bottles |
Autoclaving bag | Tool | Hogy | HM-14A | For silicon plugs |
0.45 μm filter Millex HA | Tool | Millipore | SLHA033SS | |
50 ml conical tube | Tool | Becton Dickinson | 352070 | |
100 ml beaker | Tool | Iwaki | TE-32 | |
Rat IC serum | Reagent | Charles Rivers Laboratories Japan | – | Refer to: Mr. Kunihiko Morisaki, TEL: +81-(0) 45-474-9336; FAX: +81-(0) 45-474-9340 |
D(+)-Glucose | Reagent | Wako | 041-00595 | |
Antibiotic-antimyotic liquid | Reagent | Gibco | 15240 | |
Dissection of embryos | ||||
Ophthalmic straight scissors | Tool | Napox | MB50-7 | For cutting the uterine wall |
Ophthalmic curved scissors | Tool | Napox | MB54-2 | For cutting the yolk sac |
Forceps #5 | Tool | Vigor | T6715 | |
Forceps #5F | Tool | Regine | T6819 | |
Glass pipette | Tool | – | – | Made by hand using Pasteur pipette. |
Autoclaving bag | Tool | Hogy | HM-4 | For glass pipettes |
Binocular microscope | Tool | Leica | Mz7s | |
Light | Tool | Leica | CLS 150XD | |
Oil stone | Tool | Uchida Yoko | 833-2000 | For sharpening of forceps |
Machine oil | Tool | Uchida Yoko | 835-0000 | For sharpening of forceps |