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

Summary

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 exhibit morphological development comparable to embryos developing in utero.

Abstract

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 °​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.

Introduction

In vitro culture methods utilizing whole embryos are well suited to study signaling mechanisms involved in embryonic organogenesis that are otherwise difficult to access in utero. Whole embryos provide the tissue integrity and support appropriate for the tissue interactions that are crucial for timely occurrence of signaling mechanisms essential for different cellular processes during organogenesis. While whole embryo cultures provide a platform for a plethora of applications such as transplantation studies, genetic and tissue manipulations, bead implantation studies, toxicological studies, etc., currently utilized embryo culture systems are....

Protocol

Mouse embryo culture:

All experimental procedures were conducted in strict accordance with National Institutes of Health guidelines following protocol # A2010 07-119, which was reviewed and approved by the University of Georgia Institutional Animal Care and Use Committee, which maintains continued regulatory oversight.

1. Preparation of Culture Media

1. Prepare culture medium using commercially available stem cell media and supplements in the following amounts: KnockOut DMEM, KnockOut Serum Replacement (KSR) (10%), N-2 Supplement (1x), Albumin, from Bovine Serum (2%), Penicillin (50 IU/ml), Streptomycin (50....

Results

Development of mouse embryos ex utero depends on multiple factors starting from the time the uterus is isolated from the body to the time the embryos are cultured. As depicted in Figure 1, the procedure involves a series of steps including, separation of the gravid uterus from the body (Figure 1A), isolation of the embryos with intact yolk sac (Figure 1B), exteriorization of the embryos from the yolk sac (Figure 1C) and culturing the embryos in .......

Discussion

Mid-gestation stage mouse embryos were cultured in a serum-free culture media in an atmosphere of 95% O₂ / 5% CO₂ in a rolling bottle culture apparatus at 37 °​C. Embryo development ex utero was critically dependent on multiple factors at each step during the procedure from the time the uterus is isolated from the euthanized mice to the completion of the culture (Figure 1). The most important factor that influenced the development was the time taken to start the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KnockOut DMEM	Invitrogen	10829-018
KnockOut Serum Replacement	Invitrogen	10828-028
N-2 Supplement	Invitrogen	17502-048	Stock: 100x
Albumin, from Bovine Serum	Sigma	A9418-50G	Stock: 100%
Antibiotic - Antimycotic Solution	Cellgro	30-004-Cl	Stock: Penicillin (10,000 IU/ml); Streptomycin (10,000 µg/ml; Amphotericin (250 µg/ml)
DMEM	Cellgro	15-013-CV
Precision Incubator Unit	B.T.C. Engineering Milton Cambridge England	Id.No. 840-374
Glass Bottles for Rotating Unit	B.T.C. Engineering
Silicone Rubber Cork	B.T.C. Engineering
95% O2/5% CO2 Cylinder	AirGas Inc.
Stemi SV11 Apo Dissecting Microscope	Zeiss
Stemi SV6 Microscope	Zeiss
CO2 Water Jacketed Incubator	Forma Scientific	Model: 3110
Culture Hood	Nuaire Biological Safety Cabinets Class II TypeA2	Model: Nu-425-600
Water Bath	Fisher Scientific	IsoTemp205
Weigh Balance	Mettler Toledo	AG285
Centrifuge Tube, 50 ml	Corning	430291
Light Operating Scissors	Roboz	RS-6702
Operating Sharp-Blunt Scissors	Roboz	RS-6812
Microdissecting Forceps, 4 in	Roboz	RS-5211
Microdissecting Forceps - Hudson (cWALD), 4-3/4 in	Roboz	RS-5237
Microdissecting Tweezers (5/45)	Roboz	RS-5005	Modified - Sharp ends were made blunt
Microdissecting Tweezers (5)	Roboz	RS-5060	Modified - Sharp ends were made blunt
Microdissecting Tweezers (55)	Roboz	RS-5063	Modified - Sharp ends were made blunt
Instrument Tray	Roboz	RT-1401S
Instrument Tray Lid	Roboz	RT-1401L
Petri Dish-100 mm	Fisher Scientific	087571Z
Petri Dish-60 mm	Fisher Scientific	0875713A
Petri Dish-35 mm	Fisher Scientific	0875711YZ
Filter System ( 0.22 µm Cellulose Acetate)-150 ml	Corning	431153
Filter System (0.22 µm Cellulose Acetate)-250 ml	Corning	430756
Filter System (0.22 µm Cellulose Acetate)-500 ml	Corning	430758
Pipet-aid Pipettor	Drummond Scientific Co.	D57849
Serological Pipette-10 ml	VWR	89130-898
Disposable Serological Pipette-25 ml	Corning	4251
Transfer Pipette - 7.7 ml	Thermo Scientific	202-20S