Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

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 development^1,2. 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 Science Helsinki Committee (#1566-3).

1. Media preparation

1. Prepare the dissection medium using....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm pore size filter (250 mL)	JetBiofil	FCA-206-250
0.22 µm pore size syringe PVDF filter	Millipore	SLGV033RS
8-well µ-plates glass bottom/ibiTreat	iBidi	80827/80826
Bottle with adaptor cap for gas inlet	Arad Technologies
Bungs (Hole)	B.T.C. Engineering, Cullum Starr Precision Engineering	BTC 06	Used to seal the bottles to the drum
Bungs (Solid)	B.T.C. Engineering, Cullum Starr Precision Engineering	BTC 07	Used to seal the rotating drum
Culture bottles	B.T.C. Engineering, Cullum Starr Precision Engineering	BTC 03/BTC 04	Either Glass Bottles (Small) BTC 03 or Glass Bottles (Large) BTC 04
D(+)-glucose Monohydrate	J.T. Baker
Diamond knife	Fine Science Tools	10100-30/45
Digital Pressure Gauge	Shanghai Benxu Electronics Technology co. Ltd	BX-DPG80
DMEM	GIBCO	11880
Dulbecco's Phosphate Buffered Saline	Biological industries	02-020-1A
Fetal Bovine Serum	Biological industries	04-013-1A
Gas regulation module	Arad Technologies	HannaLab1
Glutamax	GIBCO	35050061	glutamine
Graefe forceps	Fine Science Tools	11052-10
HEPES	GIBCO	15630056
Microsurgical forceps (Dumont #5, #55)	Fine Science Tools	11255-20
Pasteur pipettes (glass)	Hilgenberg	3150102
Pasteur pipettes (plastic)	Alexred	SO P12201
Penicillin/Streptomycin	Biological industries	03-031-1B
Petri Dishes (60 mm and 100 mm)	Falcon	351007/351029
Precision incubator system	B.T.C. Engineering, Cullum Starr Precision Engineering	BTC01	BTC01 model with gas bubbler kit
Pro-coagulant sterile test tubes (5 mL)	Greiner Bio-One	#456005
Rat whole embryo culture serum	ENVIGO Bioproducts	B-4520
Stereoscopic microscope equipped with heating plate	Nikon	SMZ18
Sterile syringes (5, 10 ml) for sera filtration	Pic Solution
Surgical scissors	Fine Science Tools	14094-11