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 mainly dependent on serum for proper growth and maintenance of the embryos in the culture^1-9.

Serum has been utilized as one of the major components ranging from 10-100% of the culture media^6-8,10,11.  However, the composition of serum is not well defined and can differ from animal to animal and each time the serum is collected. While laboratory preparation of serum is time consuming and involves stringent procedures, serum procured commercially exhibits considerable variability among different lots and raises experimental costs. Added to these, the serum may contain unknown factors such as growth factors, hormones, or other proteins, which can potentially affect the outcome of certain experiments, especially those that involve the study of signaling molecules important in tissue interactions. Studies have shown that addition of serum to culture can potentially alter the intracellular levels of certain signaling molecules such as cyclic adenosine monophosphate (cAMP) and proteins involved in mitogenic signaling and phosphoinositide 3 (PI 3)-kinase signaling pathways^12-14. Contrary to these, a serum-free culture system provides the advantages of antigen free environment, abstinence from biologically active enzymes that can alter the cellular processes and enables consistency among experiments.

In the present study, we utilized a serum-free culture medium prepared from commercially available stem cell media supplements to culture mid-gestation stage whole embryos in an atmosphere of 95% O₂ / 5% CO₂ in a rolling bottle culture apparatus at 37 °​C^15,16.  Mouse embryos cultured for 16 to 40 hr under these defined conditions exhibited progression in morphological development of overall embryonic body and different structures such as the heart, limbs, brain and eyes indicating appropriate levels of cellular proliferation, migration, differentiation and tissue interactions. Molecular analysis of the embryonic development in the culture for one of the complex organ systems such as the eye revealed the ocular development to be consistent with that observed in ocular tissue in embryos developing in utero (Kalaskar and Lauderdale, in preparation). Thus we show that mouse embryos cultured at mid-gestation stage, exhibit progressive growth and morphological development comparable to that observed in embryos developing in utero.

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 μg/ml) and Amphotericin-B (1.25 μg/ml). The culture medium prepared is similar to that previously described¹⁵ with the following changes: addition of anti-mycotics and using twice the concentration of antibiotics (for details of specific reagents and equipment, see List of Materials).
   Note: Antibiotic concentration as previously used (Moore-Scott, et al.¹⁵) was sufficient for embryo cultures carried up to 24 hr time period. However, when culture was continued beyond 24 hr, we experienced contamination of the culture and this was successfully controlled by doubling the antibiotic concentration.
2. Store the media components at 4 °​C or at -20 °​C as per manufacturer recommendations. KSR and N-2 Supplement should be stored in aliquots at -20 °​C to avoid repeated freezing and thawing. KnockOut DMEM once opened should be utilized within 30 days as per manufacturer’s recommendation. Prior to use, warm the components in a water bath to 37 °​C.
3. Prepare the media under sterile conditions. Disinfect the surface of all the equipment and bottles containing the media components with 70% alcohol spray before placing them in the culture hood.
4. The media components can be mixed either in a sterile beaker or directly in the filter system (Corning). First add the albumin powder to the KnockOut DMEM. Mix thoroughly by gently shaking until the albumin completely dissolves. Then add the N-2 Supplement, KSR and the antibiotics and mix thoroughly using a pipettor. Then filter sterilize the media using a 0.22 µm pore size filter with vacuum suction. Dispense the media into 50 ml conical tubes and store at 4 °​C until used. The media once prepared should be used within 4-5 days for culture.

2. Preparation of Culture Equipment

1. Sterilize the glass culture bottles and rubber corks. Wrap the culture bottles in an aluminum foil and place the rubber bottle corks in a water beaker and sterilize by autoclaving.
2. Sterilize the culture chamber (Precision Incubator Unit) with 70% alcohol spray and warm to 37 °C before starting the culture. The culture chamber is equipped with a rolling disc at the center which holds the culture bottles. Gas flow into the chamber is regulated by a pressure gauge and the flow rate can be adjusted by observing the outflow of air bubbles through water in a glass tube at the end. This rolling bottle culture apparatus is a modified version of the original apparatus introduced by New and Cockroft¹⁷.
3. Connect a gas cylinder containing a gas mixture of 95% O₂ / 5% CO₂ to the culture chamber and adjust the gas flow to “one air bubble per second” which gives a flow rate of 50 cc/min and ensures adequate oxygen supply to the culture embryos.

3. Mouse Embryo Collection and Preparation for Culture

1. To obtain wild type mouse embryos, we utilized mice of C57BL/6J and CD-1 (Charles River Laboratories) genetic background strains.
2. Check the female mice for vaginal plugs every day morning. The noon on the day of finding the vaginal plug should be considered as E0.5 dpc (days post coitus).
3. Collect mouse embryos at E10.5 dpc by euthanizing the pregnant mice following standard protocols. Mice were euthanized using a CO₂ inhalation device as specified by the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2013). To insure death, cervical dislocation was performed after exposure to CO₂.
4. Spray 70% ethanol on the ventral abdominal surface to avoid sticking of abdominal hair to the instruments. Then open the abdominal cavity ventrally using a sharp-blunt operating scissors and a pair of 4-3/4 in microdissecting forceps to locate the uterus. Using a 4 in microdissecting forceps lift the entire uterus and separate it from the body by cutting with a light operating scissors at the uterine body and at the tips of the uterine horns.
5. Quickly rinse the entire uterus in 1x PBS warmed to 37 °C to remove any blood sticking to the uterus and immediately place in DMEM warmed to 37 °C in a Petri dish. Sterilize the instruments by 70% ethanol spray before further use.
   Note: Preheating the solutions including the PBS, DMEM and the culture medium and maintaining them at 37 °​C during the entire procedure is recommended.
6. Under a dissecting microscope, the uterus should be segmentally dissected with a light operating scissors. This results in small openings on either side of the segmented uterus through which a pair of modified (blunted ends) microdissecting tweezers can be gently inserted to widen the opening. This permits the exposure of the placental decidua, which can then be removed by gently tearing with a pair of microdissecting tweezers to expose the parietal yolk sac (PYS) with the Reichert’s membrane. Using one edge of the tweezers gently pierce the Reichert’s membrane along with the PYS and separate from the underlying visceral yolk sac (VYS) layer to expose the embryos with intact VYS. The ectoplacental cone and the trophoectoderm derivatives can be removed either with the placental decidua or with the Reichert’s membrane. Care must be taken to avoid rupture of the VYS as the embryos pop out immediately.
7. The embryos with intact VYS should then be transferred to a Petri dish containing the culture medium warmed to 37 °​C using a plastic transfer pipette.
   Note: Transfer to culture medium immediately after separation from uterus helps for better development of the embryo in culture.
8. A small opening should be made in the yolk sac with a pair of microdissecting tweezers avoiding major blood vessels. A sharp pair of tweezers can be used to make the opening by gently piercing in an area adjacent to the head region. Alternatively two pairs of blunt tweezers can be used to hold the yolk sac and gently tear to make a small opening. Then gently widen the opening by expanding with the tweezers to a size just enough to fit the embryonic head. The amniotic membrane which is closely wrapping the embryo should be gently held away from the body and torn using a pair of tweezers. The embryonic head and later the whole embryo should be gently exteriorized from the yolk sac while maintaining the integrity of the embryonic vasculature with that of the yolk sac¹⁵.
   Note: Any damage to the yolk sac vasculature can potentially affect the development of the embryo in culture. Such embryos with damaged vasculature should not be used for culture and can be used for staging the embryos which can later be discarded.
9. Embryonic staging criteria: Examine the embryos under the dissecting microscope and group by morphological criteria including body and head size, limb and eye morphology and stage by counting the number of somite(s) for at least two embryos from each group.
   Note: Usually embryos obtained from the same litter show differences in development in utero and differ in body size, morphological features and number of somites. However, we found that embryos grouped by our morphological criteria usually exhibited similar somite number (±1). For this reason, it is not required to count somites for each embryo as this would delay the time for starting the culture.
   Note: Do not use the embryos that were used to count the somites for culture. Count the somites after starting the culture for other embryos in order to avoid the time delay in starting the culture. The embryos that were delayed to culture after separation from the uterus usually exhibit poor development.
10. Transfer the embryos immediately to a Petri dish with fresh culture medium warmed to 37 °​C and take it to the culture hood where the embryos should again be transferred to a Petri dish with sterile culture media before putting them into the culture bottles with medium to start the culture.
    Note: The multiple media transfers for the embryos before culture helps in preventing contamination as the different steps of embryo collection and dissection were performed under a dissecting microscope which was not installed in the culture hood.
    Note: When the litter size is large (>12 embryos), process half the embryos and start the culture or put them in a dish with culture medium and place it in a CO₂ incubator at 37 °​C. When embryos were left outside for longer periods (>35-40 min) we observed the heartbeat to slow down and this will affect the later development in the culture.

4. Culturing Mouse Embryos

1. Open the autoclaved culture bottles in the culture hood and properly label them. Transfer 3 ml of culture medium warmed to 37 °​C to each of the bottles and then the mouse embryos should be transferred gently into the culture bottles using sterile plastic transfer pipettes. The culture bottles should then be capped with the rubber bottle corks which help to hold the culture bottles to the rolling disc in the culture apparatus.
   Note: The culture bottles with the media can be prepared and placed on the rolling disc of the culture apparatus before euthanizing the mice. This shortens the time for transfer of embryos into the culture bottles.
   Note: Mouse embryos can be cultured individually or as cocultures with two or three embryos in the same culture bottle.
2. The culture bottles should be aseptically carried to the culture apparatus at 37 °​C and tightly hook them to the holes on the rolling disc with the rubber corks. Turn on the rolling disc to rotate at a constant speed of 35 rotations per minute (rpm), which enables the free floatation of the embryos in the media and also helps in free gas exchange by the embryonic tissue. The gas from the cylinder connected to the culture chamber flows through the rolling disc and the rubber corks into the culture bottles.
3. Culture the embryos for 16-40 hr and regularly check for the gas outflow and culture chamber temperature.
   Note: Mouse embryo manipulation in culture: Let the embryos get adapted to the culture conditions for at least 30 min. Then embryos that perform poorly and showing feeble heartbeat can be discarded and the remaining embryos can be utilized for further manipulation studies. Embryo manipulations such as electroporation^5,9,16, microinjections¹⁸, bead implantation¹⁶, drug treatment and other procedures can be performed at this time. We successfully performed implantation of affi-gel agarose beads treated with certain signaling molecules to study their role in early eye development. The embryos after manipulations can be returned back in a fresh medium and continued in culture.
4. Replace the culture media totally with fresh media at specific intervals after 9-10 hr and 18-19 hr of culture when the culture was continued for 40 hr.
   Note: Culture media replacement may not be required if the culture is stopped by 16-18 hr.
5. Measures of success in culture: Embryonic survival in culture should be determined by visible heartbeat and blood circulation in the body while embryonic growth in culture should be assessed by increase in body size, somite number and morphological development of head, limbs, heart and eyes.
6. In utero developed embryos at E11.0 dpc (~40-41 s) and E12.0 dpc (~49-50 s) should be used as controls for comparing embryos cultured for 16-18 hr and 38-40 hr, respectively.
7. Embryonic development at different time points during culture and for in utero stages can be captured under dissecting microscope. Spot imaging software was utilized to capture the images and later processed using image processing software.

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

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 (10000 IU/ml); Streptomycin (10000 µg/ml; Amphotericin (250µg/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 – 50ml	Corning	430291
Light Operating Scissors	Roboz	RS-6702
Operating Sharp-Blunt Scissors	Roboz	RS-6812
Micro Dissecting Forceps – 4”	Roboz	RS-5211
Micro Dissecting Forceps - Hudson (cWALD) – 4-3/4”	Roboz	RS-5237
Micro Dissecting Tweezers (5/45)	Roboz	RS-5005	Modified - Sharp ends were made blunt
Micro Dissecting Tweezers (5)	Roboz	RS-5060	Modified - Sharp ends were made blunt
Micro Dissecting Tweezers (55)	Roboz	RS-5063	Modified - Sharp ends were made blunt
Instrument Tray	Roboz	RT-1401S
Instrument Tray Lid	Roboz	RT-1401L
Petri Dish-100mm	Fisher Scientific	087571Z
Petri Dish-60mm	Fisher Scientific	0875713A
Petri Dish-35mm	Fisher Scientific	0875711YZ
Filter System ( 0.22um Cellulose Acetate)-150ml	Corning	431153
Filter System ( 0.22um Cellulose Acetate)-250ml	Corning	430756
Filter System ( 0.22um Cellulose Acetate)-500ml	Corning	430758
Pipet-aid Pipetter	Drummond Scientific Co.	D57849
Serological Pipette-10ml	VWR	89130-898
Disposable Serological Pipette-25ml	Corning	4251
Transfer Pipette - 7.7ml	Thermo Scientific	202-20S