A Simple Mammalian Whole Embryo Culture System to Study High-Temperature Effects on Development

Abstract

Whole embryo culture is a unique system for developing rodent embryos outside the uterus. This system enables the observation and manipulation of mammalian embryos during specific periods of embryonic development. Established techniques utilize a rotating bottle system that continuously supplies oxygen. However, commercial devices are currently unavailable, making the new implementation of this system virtually impossible. Furthermore, the conventional device had limitations in temperature settings, making embryonic development studies impossible under conditions deviating from the optimal temperature for mammalian embryos. In this study, we developed a simple rotating culture system that can be installed in a commercially available incubator. Embryo growth and physiological status in this devise are equivalent to those in embryos cultured in the conventional system. Using this system, we clarified the harmful effects of hyperthermia during the early stages of mouse embryonic development. This device is fully suitable for whole embryo culture in mammals and provides a foundation for verifying embryonic development states in rodents under various temperature conditions.

I. Introduction

Whole embryo culture system (WEC) is a unique technique to proceed embryogenesis of rodents (mouse and rat) outside of the maternal uterus. The principle of the WEC has been established around 1960 [1], and since then several modifications have applied to improve the culture condition [2–6]. Currently, the WEC system utilizes 1) the rotator that can be inserted with culture bottles, 2) continuous supply of certain amount of oxygen gas, and 3) a incubating system to maintain physiological temperature (around 37°C) [7]. The WEC can be applicable for limited stages of rodent embryogenesis; embryonic day (E) 6.5–12.5 in mice, and E8.5–14.5 in rats. During these period, growth and morphogenesis of cultured embryos are almost perfectly mimicked to the development in uterus [8]. Accordingly, the system has been used in a variety of investigations, such as the effects of teratogens in organogenesis [9, 10], cell transplantation [11], cell lineage tracing [9, 12], and genetic manipulation combining with exogenous gene transfection methods [13, 14]. In previous most cases of experiments, researchers have used a commercially available device for WEC system (Ikemoto Rika Co., Japan), which consists of an incubator equipped with a roller bottle system. However, commercial devices are currently unavailable, making the new implementation of this system virtually impossible. Furthermore, the conventional device had limitations in temperature settings, making embryonic development studies impossible under conditions deviating from the optimal temperature for mammalian embryos.

To circumvent these challenges, we developed a new device for the mammalian WEC. We referred the previous rotating system but simplified the size of apparatus, enabling to be installed in a commercially available incubator. We examined the growth and physiological conditions of culture embryos, and probed that the embryogenesis in our devise are comparable to those in embryos cultured in the conventional system. Using this system, we clarified the harmful effects of hyperthermia during the early stages of mouse embryonic development. This device is fully suitable for whole-embryo culture in mammals and provides a foundation for verifying embryonic development states in rodents under various temperature conditions.

II. Materials and Methods

Animals

Pregnant female mice (CD-1 background) that were originally obtained from Japan SLC were maintained in a 12 hr dark/light cycle at Kyoto Institute of Technology. Noon of the day the vaginal plug was identified was designated as E0.5. All animal experiments were approved by the experimental animal committee of Kyoto Institute of Technology (#100211) and were performed in accordance with the relevant guidelines.

Whole embryo culture

The WEC was performed according to a previous report [7]. Briefly, E10.5 mouse embryos with the yolk sac and placenta were isolated, and dissected in Hanks’ Balanced Salt Solution (HBSS). The embryo was exposed from the yolk sac by cutting the amnion. Isolated embryos were transferred to the glass bottle (IKEMOTO RIKA, Co, Japan) filled with 100% Fetal bovine Serum (FBS, Corning, 35-079-CV) supplemented with D-glucose (2 mg/mL) and antibiotics (penicillin/streptomycin, FUJIFILM Wako, #168-23191, 1/100). The condition of oxygen gas supply was according to a previous report [7]. In both normothermic (37.0°C) and a hyperthermic (39.5°C) conditions, embryos were cultured under continuous gas supply with an atmosphere of 60% O₂, 35% N₂, and 5% CO₂ (0.05 MPa, 150 cc) for 19 hr, followed by incubation under 95% O₂ and 5% CO₂ (0.05 MPa, 100 cc) for an additional 5 hr. During the culture period, the bottles were rotated at 20 rpm. The WEC system was placed in an incubator (MIR-154PJ, PHCbi). The internal temperature of the incubator are stable within a range of ± 0.5°C for 24 hr, consistent with the initial performance tests. Rat immediately centrifuged (IC) serum was purchased from the Jackson Laboratory, Japan. Prior to WEC at the hyperthermic condition, the embryos were pre-incubated at 37.0°C for 1 hr to acclimate them to the culture medium.

Analysis of serum components

The serum components were measured using i-STAT 1 Analyzer and i-STAT CHEM8+ Cartridge (Abbott). The concentration of potassium was measured using LAQUAtwin-K-11 (HORIBA).

Immunostaining

The embryos were fixed with 4% paraformaldehyde (PFA) dissolved in PBS at 4°C for overnight. After washing off the PFA with excess amount of PBS, the embryos were cryoprotected with 20% sucrose solution and embedded in Tissue-Tek (Sakura, Japan). Frozen sections (20 μm in thickness) were obtained with a cryostat (CM1850, Leica, Germany) and incubated with primary antibodies including anti-PH3 (mouse monoclonal, Merck Millipore,#05-806, 1/1000), anti-βIII-tubulin (mouse monoclonal, Merck Millipore, AB9354, 1/500), anti-Sox2 (rabbit polyclonal, Abcam, #ab97959, 1/1000), anti-cleaved Caspase 3 (rabbit polyclonal, Cell signaling technology, #9661, 1/1000), anti-Neurofilament [2H3, Developmental Studies Hybridoma Bank (deposited by Jessell, T. M. and Dodd, J.), 1/100) antibodies. After washing off the primary antibodies, the sections were incubated with secondary antibodies including Alexa-Fluor 488 and 555 conjugated anti-rabbit and mouse antibodies (Invitrogen, #A11001, #A11008, #A21428, #A21422, 1/1000). Nuclear staining was performed with Hoechst 33342 (Thermo Fisher Science, #H1399, 1/1000).

Whole mount immunostaining

For immunostaining, the embryos were cleared using CUBIC (FUJIFILM Wako, #290-80801). The fixed embryos were washed three times in PBS for 2 hr each at 4°C and incubated in 50% CUBIC solution 1 at room temperature for overnight. The embryos were subsequently transferred to 100% CUBIC solution 1 and incubated at room temperature for 2 days. After washing off, embryos were subjected to a blocking step with 10% FBS at room temperature for 2 hr and, followed by incubation with a Neurofilament (2H3, Developmental Studies Hybridoma Bank, 1/100) at 4°C for overnight. After washing off the primary antibodies, embryos were incubated with Alexa Fluor 555–conjugated anti-mouse secondary antibody (1:1000) and Hoechst 33342 (1:1000) at 4°C for overnight. Following immunostaining, embryos were washed and incubated in 50% CUBIC solution 2 at room temperature for overnight, followed by incubation in 100% CUBIC solution 2 at 37°C for 2 hr. All procedures were performed with shaking at 30 rpm, except for the 50% CUBIC solution 2 treatment. Finally, embryos were mounted in mounting solution (solution 1:solution 2 = 2:8). As a negative control, embryos incubated with the secondary antibody alone were used.

In situ hybridization

In situ hybridization was performed according to previous reports [15]. cDNA sequences of mouse ephrin-B2 was inserted into pBluescript SKII vector [16]. After the amplification of the ephrin-B2 fragment containing T7 and T3 promoter region with M13 primer, sense and antisense RNA probes were made by using T7 or T3 RNA polymerase (ROCHE). T3 or T7 polymerase were used for making digoxygenin (DIG)-labeled sense and antisense RNA probes. The sections were treated with proteinase K (10 min, room temperature, QIAGEN) and hybridized with riboprobe in hybridization buffer (50% formamide, 20 × SSC, 10% SDS) at 68°C overnight. Section slides were washed with Phosphate-Buffered Saline + 0.1% tween 20 solution (PBT) and diethylpyrocarbonate (DEPC)-treated deionized water. The sections were washed 3 times with 5 × SSC and 2 × SSC (50% formamide, 20 × SSC, 10% SDS) and incubated with anti-DIG-AP Fab fragments (Merck) at 4°C overnight. All the sections were visualized by NBT (Nitroblue Tetrazolium, Merck)/BCIP (5-Bromo-4-chloro-3-indolyl-phosphate, Merck) reaction.

Image processing and statistical analysis

Sample images were captured by using the microscope, (TF-B, Kennis; SZX7, Olympus; ECLIPSE Ni, Nikon; M165 FC, LEICA or FLUOVIEW FV4000, Olympus) equipped with a cooled CCD camera (DEGITAL SIGHT DS-Fi2, Nikon, DsFi1c, Nikon or DS-F/3, Nikon). Movies were captured by iPhoneSE (2nd generation, Apple) and processed with Adobe Premiere Pro 2025. All captured images were processed with Image J (1.53k) and Photoshop (v2025, Adobe), CellSens FV (ver3.1.1.67). For statistical analysis, the results of three independent experiments were compared. Comparisons between experimental groups were performed using Microsoft Excel (v16.98, Microsoft) and Prism 9 (v10.5.0, GraphPad9. Statistical significance was determined using unpaired Welch’s t-test.

III. Results and Discussion

Based on the previous WEC system, we developed a new culture device in collaboration with a manufacturing company (BEX Co., Ltd.). The system consists of: (1) a main rotor box equipped with rotating wheels, (2) an O₂ gas modulator connected via tubing, and (3) a console display for system control (Fig. 1A, B). Glass bottles containing serum and embryos can be attached to the wheel, with up to 12 bottles accommodated simultaneously. O₂ gas is continuously supplied to the cavity of each glass bottle through the rotor box and vented via a gas outlet. The minimum space required for installation of the apparatus is approximately 60 × 30 × 25 cm (length × width × height), allowing the system to be placed inside a medium-sized commercial incubator.

Fig. 1.

A concise whole embryo culture system for early stages of mouse embryos. (A, B) The platform of a concise whole embryo culture (WEC) system. a: a commercially available incubator, b: a control monitor, c: plastic tube for gas flow, d: a glass bottle filled with sterilized water, e: a rotator box, f: a rotator, g: exhaust hole. (C) An embryo cultured for 24 hr from E10.5. Regular contractions of atrium and ventricle were confirmed (white and black arrowheads in C1–C4). (D, E) Heart rate (E) and serum glucose level before and 24 hr after the culture. (F, G) Bright-field images of E10.5 embryos (F, F1, F2) and cultured for 24 hr (G, G1, G2). (H–J) Quantification of somite number (H), dorsal length (I), and head length (J). (K, L) Immunostaining of embryos at E10.5 (K) and 24 hr WEC (L) with anti-Sox2 and -βIII-tubulin antibodies. Tel: telencephalon, Mes: mesencephalon, FL: forelimb, HL: hindlimb. Bars = 1 mm.

100% rat IC serum [17] has been considered the most suitable medium for WEC [18]. However, due to issues of cost, labor, and ethical considerations in animal research, recent studies have tested alternative media to replace rat IC serum, such as FBS or serum-free media supplemented with additives [19]. In this study, we used 100% commercially available FBS as the culture medium and examined the condition of embryos cultured in our WEC system. Mouse E10.5 embryos were isolated and cultured in our WEC device at 20 rpm (rotations per min) under 60% followed by 95% O₂ at 37.0°C. After 24 hr, we evaluated cardiac activity and blood circulation in the cultured embryos. Out of 20 embryos cultured, 17 embryos exhibited heartbeats with alternating contraction of the atrium and ventricle (Fig. 1C–C4, D, Supplementary Movie S1, Table 1). To evaluate biochemical changes in the culture medium during WEC, we measured serum electrolytes and glucose after the culture. Compared to the serum prior to the culture, glucose levels decreased significantly after 24 hr of WEC, indicating that glucose was consumed by embryonic growth and metabolisms (Fig. 1E). The levels of various electrolytes, urea nitrogen, and creatinine were not changed after 24 hr of WEC (Table 2), suggesting that the embryonic conditions were not significantly altered in the culture. We also assessed the growth and morphological development of the embryos (Fig. 1F–J). After 24 hr of culture, embryo size increased, and the development of the cranium and limb buds had progressed compared to the E10.5 embryos (Fig. 1F–G2). Consistently, the number of somite and head length were significantly increased in embryos cultured for 24 hr (Fig. 1H, J). We also confirmed Sox2- and β III-tubulin-positive neural progenitors and neurons in the embryos cultured for 24 hr, corroborating that neural development is not abrogated in the WEC (Fig. 1K, L). After the WEC, we noticed some embryos showed the reduced heartrates and abnormal vesicle formation (bulla) at the head region (Table 1, Supplementary Movie S2). This might be due to species-inconsistency of the culture medium, or insufficient development of the placenta. Alternatively, higher potassium level in FBS might affect embryonic conditions (Table 2), although majority of embryos showed normal growth without gross morphological abnormalities.

Table 1. .

Embryonic conditions during WEC at 37.0°C and 39.5°C for 24 hr

Temperature	Number of embryos	Embryos with heart beats after WEC	Embryos without heart beats after WEC	Embryos with vesicle after WEC
37.0°C	20	17(5a)/20	3/20	6/20
39.5°C	16	3/16	13/16	1/16

^aThe embryos with decreased blood flow.

Table 2. .

Serum electrolytes and glucose levels in WEC at 37.0°C (FBS)

Sample	Na+ (mmol/L)	K+ (mmol/L)	Cl− (mmol/L)	iCa2+ (mmol/L)	TCO2 (mmol/L)	Glucose (mg/dL)	BUN (mg/dL)	Crea (mg/dL)
Control1	135	12.79	107	0.38	NA	256	19	2.2
Control1	137	12.79	109	0.49	16	257	20	2.1
Control1	137	12.79	110	0.49	16	266	21	2.2
WEC1	142	10.23	117	0.47	10	105	13	1.4
WEC2	137	11.76	113	0.49	10	157	19	1.9
WEC3	137	11.76	112	0.48	10	161	19	2.0
p-value	0.2961	0.0944	0.0521	0.5440	NA	0.0182	0.2693	0.1602

Several studies have reported that maternal heat stress significantly affects early stages of mammalian embryogenesis [20–22]. Hyperthermia significantly increased embryonic mortality, as well as various altered organogenesis, such as neural tube defects, growth retardation, and abnormal neurogenesis, although most studies investigated the impacts of higher temperature for short period (2–3 hr). To examine the effects of higher temperature on embryogenesis for long period, we incubated E10.5 mouse embryos in a hyperthermic condition (39.5°C) for 24 hr, and assessed physiological and morphological alterations after the culture. Serum glucose level was significantly decreased after the culture, suggesting glucose consumption occurred in a hyperthermic condition, as in the case of a normothermic condition (Table 3 and Fig. 2D). Compared to the culture at 37.0°C, a long-term hyperthermia increased the proportion of embryos with abnormal development, such as reduced heart beats and blood flows, and severe growth retardation (Fig. 2A–C, D, F, Table 1 and Supplementary Movie S3). However, we could not find significant difference in the number of phosphorylated histone H3 (PH3)-positive cells in the dorsal part of the telencephalon between normothermic and hyperthermic conditions (Fig. 2G–I). To examine the heat stress affects neuronal differentiation, we performed immunostaining with anti-neurofilament antibody, and identified altered peripheral nerve development in the embryos cultured at 39.5°C (Fig. 2J–N). In particular, abnormal segregation of the dorsal root ganglia (DRG) was evident in the embryos cultured in a higher temperature (Fig. 2M, N). In these embryos, the distributions of DRG were irregular, and some cases adjacent DRG were fused (Fig. 2O, P). We could not find significant difference in active caspase-3-positive cells in DRG of the embryos with hyperthermia, suggesting that abnormal DRG formation is not due to increased cell death (Fig. 2Q–S). Altered DRG morphology observed in this study resembled the phenotypes of Dll1 (a ligand for Notch receptor)-deficient mice [23]. Segmental neural crest migration and ephrin-B expression were altered in the Dll mutant. Concomitantly, we identified abnormal condensation of ephrin-B2-positive cells in the caudal part of sclerotome in embryos cultured at 39.5°C (Fig. 2T, V). Immunostaining with anti-neurofilament antibody revealed irregular intervals of DRGs at the hyperthermic culture condition; these regions corresponded to the locations where clusters of ephrin-B2-positive cells were observed (Fig. 2T–W). A recent study revealed that hyperthermia affects neural crest development via the activation of heat-activated transient receptor potential ion channel 1 (TRPV1) [24]. We have previously shown that temperature-dependent controls of Notch signaling in developing chicken embryos [25]. Thus, it is possible that hyperthermia at early stages of mouse embryos affect neural crest development via altered expression of Notch or its downstream molecules, such as Eph receptors and ephrins.

Table 3. .

Serum electrolytes and glucose levels in WEC at 39.5°C (FBS)

Sample	Na+ (mmol/L)	K+ (mmol/L)	Cl− (mmol/L)	iCa2+ (mmol/L)	TCO2 (mmol/L)	Glucose (mg/dL)	BUN (mg/dL)	Crea (mg/dL)
Control1	139	10.23	111	0.26	32	272	19	2.2
Control1	137	10.23	109	0.26	29	269	19	2.2
Control1	141	10.23	113	0.26	28	278	20	2.2
WEC1	136	10.74	113	0.48	8	163	17	1.8
WEC2	135	11.25	112	0.47	7	173	20	1.9
WEC3	136	10.23	116	0.57	5	130	15	1.7
p-value	0.0921	0.0399	0.1850	0.0162	0.0002	0.0097	0.3009	0.0202

Fig. 2.

The effect of hyperthermia on mouse embryogenesis during WEC. (A–C) Bright-field images of embryos cultured at 39.5°C for 24 hr in the WEC system. (A, A1) An embryo showing normal growth. (B, B1) An embryo showing severe growth retardation. (C, C1) An embryo showing a bulla (vesicle formation) in the head. Note that the bulla are also formed in the embryo cultured at 37.0°C. (D–F) Quantifications of serum glucose level (D), dorsal length (E), and head length (F) in embryos cultured at 37.0°C and 39.5°C. (G, H) Comparison of phosphorylated histone H3 (PH3)-positive mitotic sells in the dorsal telencephalon cultured at 37.0°C (G) and 39.5°C (H). (I) Quantification of PH3-positive cells in the dorsal telencephalon at 37.0°C and 39.5°C. (J–N) Expression of neurofilaments (NF) in embryos cultured at 37.0°C (K, M) and 39.5°C (L, N) for 24 hr. J represents a negative control by staining with secondary antibody alone. M and N are higher magnifications at the trunk of the embryos. (O–R) Distributions of NF (O, P) and Caspase3 (Csp3)-positive cells at the DRG of embryos cultured at 37.0°C (O, Q) and 39.5°C (P, R). (S) Quantification of Caspase3-positive cells in DRG at 37.0°C and 39.5°C. (T–W) Expression of ephrin-B2 and NF in embryos cultured at 37.0°C (T, U) and 39.5° (V, W). Bars = 1 mm (A, B, C, J–L); 500 μm (A1, B1, C1); 50 μm (G, H, O–R); 200 μm (V).

Recent studies have highlighted long-term culture system for post-implantation mouse and primate embryos [26–28]. Furthermore, methods for assembling embryonic models derived from embryonic stem [24] or induced pluripotent stem (iPS) cells have been reported [29], which enable to accelerate the technology of organoid- or assembloid-based stem cell therapies. In contrast, accumulating evidence imply species-differences between rodent and human embryogenesis, such as brain expansion [30, 31] and tail reduction [32–34], although in utero manipulation of early developmental stages has been technically challenging. Thus, the demands for a concise culture system for ex utero mammalian embryos is rapidly increasing, as such systems can be applied not only to classical teratology, but also to a wide range of research areas aimed for the investigating the early stages of mammalian development.

Limitations of the study

Although we succeeded in advancing embryonic development in the new culture system, abnormalities such as decreased heart rate, developmental delay, and vesicle formation were observed in some embryos at 37.0°C. We also confirmed that the potassium concentration in FBS is higher than physiological levels. In contrast, the potassium concentration in rat IC serum is significantly lower than that in FBS (K⁺ in rat IC serum: 3.41 ± 0.3 mmol/L, n = 3 samples), which supports the general view that rat IC serum is suitable for mammalian embryo culture. The development of a new culture medium that addresses issues such as cost and ethics will greatly contribute to the advancement of fetal development and physiological conditions through mammalian WEC using this system.

IV. Conflicts of Interest

The authors declare no conflicts of interests.

V. Acknowledgments

We thank Ms. Misato Kawami, Mariko Yazaki, and Akiko Watanabe for technical assistance, and all members of Biomedical and Developmental Biology lab, KIT, for constructive discussions. This work is supported by JSPS KAKENHI (26K01919), Takeda Science Foundation, Kyoto Health Science Research Integration Center, KIT cutting edge research (to T.N.).

Supplementary Material

Supplementary Movie S1

Supplementary Movie S2

Supplementary Movie S3