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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 E103. The inability to observe and manip....

Protocol

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

Representative Results

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, .......

Discussion

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.......

Acknowledgements

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, Za....

Materials

NameCompanyCatalog NumberComments
0.22 µm pore size filter (250 mL)JetBiofilFCA-206-250
0.22 µm pore size syringe PVDF filterMilliporeSLGV033RS
8-well µ-plates glass bottom/ibiTreatiBidi80827/80826
Bottle with adaptor cap for gas inletArad Technologies
Bungs (Hole)B.T.C. Engineering, Cullum Starr Precision EngineeringBTC 06Used to seal the bottles to the drum
Bungs (Solid)B.T.C. Engineering, Cullum Starr Precision EngineeringBTC 07Used to seal the rotating drum
Culture bottlesB.T.C. Engineering, Cullum Starr Precision EngineeringBTC 03/BTC 04Either Glass Bottles (Small) BTC 03 or Glass Bottles (Large) BTC 04
D(+)-glucose MonohydrateJ.T. Baker
Diamond knifeFine Science Tools10100-30/45
Digital Pressure GaugeShanghai Benxu Electronics Technology co. LtdBX-DPG80
DMEMGIBCO11880
Dulbecco's Phosphate Buffered SalineBiological industries02-020-1A
Fetal Bovine SerumBiological industries04-013-1A
Gas regulation moduleArad TechnologiesHannaLab1
GlutamaxGIBCO35050061glutamine
Graefe forcepsFine Science Tools11052-10
HEPESGIBCO15630056
Microsurgical forceps (Dumont #5, #55)Fine Science Tools11255-20
Pasteur pipettes (glass)Hilgenberg3150102
Pasteur pipettes (plastic)AlexredSO P12201
Penicillin/StreptomycinBiological industries03-031-1B
Petri Dishes (60 mm and 100 mm)Falcon351007/351029
Precision incubator systemB.T.C. Engineering, Cullum Starr Precision EngineeringBTC01BTC01 model with gas bubbler kit
Pro-coagulant sterile test tubes (5 mL)Greiner Bio-One#456005
Rat whole embryo culture serumENVIGO BioproductsB-4520
Stereoscopic microscope equipped with heating plateNikonSMZ18
Sterile syringes (5, 10 ml) for sera filtrationPic Solution
Surgical scissorsFine Science Tools14094-11

References

  1. New, D. A. Whole-embryo culture and the study of mammalian embryos during organogenesis. Biological Reviews of the Cambridge Philosophical Society. 53 (1), 81-122 (1978).
  2. Tam, P. P., Behringer, R. R. Mouse gastrulatio....

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