The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Takahashi, Masanori; Osumi, Noriko

doi:10.3791/2170

Cited by 14 publications

(14 citation statements)

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“…Rat serum was prepared by immediate centrifugation of rat blood (IC serum) as described for ex vivo culture of mouse post-implantation embryos (Takahashi and Osumi, 2010). In our experiments, freshly isolated IC serum from overnight-fasted rats or commercially available rat IC serum improved survival of cultured embryos.…”

Section: Development Of In Vitro Chimera Assaymentioning

confidence: 99%

Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells

et al. 2015

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Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra-and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/ process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs.

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Section: Development Of In Vitro Chimera Assaymentioning

confidence: 99%

Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells

et al. 2015

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“…1 embryos dissected at either 7.5 or 8.5 dpc and cultured in CRSD had overt signs of abnormal development, whereas those cultured in CRSFN appeared to develop to the equivalent in utero stage. Embryos that are grown in culture for 24 hours from the late primitive streak stage (7.5 dpc) are expected to develop ~ 6 somites and to exhibit morphological landmarks of cranial neural development (Downs and Davies 1993;Kaufman and Bard 1999;Rivera-Perez et al, 2010;Takahashi and Osumi 2010;Van Maele-Fabry et al, 1993). The majority of embryos cultured in CRSD did not develop somites and their allantois often protruded through the yolk sac.…”

Section: Resultsmentioning

confidence: 99%

“…The development of murine WEC protocols therefore focused on achieving reasonable culture success using rat serum (since one rat typically yields sufficient sera to culture twenty embryos (Hunter et al, 1988)). A number of studies have shown that rat serum supports ex utero growth of post-implantation stage mouse embryos for at least 24 hours, providing that blood is withdrawn from the animal gently, immediately centrifuged, the fibrin clot carefully removed, and the sera heat-inactivated (Cockroft 1991;Harris 2012;New 1978;New et al, 1976;Piliszek et al, 2011;Rivera-Perez et al, 2010;Steele and New 1974;Takahashi and Osumi 2010;Tam and Snow 1980;Van Maele-Fabry et al, 1991). The exacting requirements for the production of mouse embryo-culture-grade rat serum means commercial preparations are generally not able to support the ex utero growth of mouse embryos.…”

Section: Introductionmentioning

confidence: 99%

“…The system that enables murine WEC was developed in the 1970's (Ellis- Hutchings and Carney 2010;New 1978;New et al, 1973;) and refined over the next two decades such that by the 1990's a standard protocol for WEC was established (Fujinaga 2000;Gray and Ross 2011;Harris 2012;Piliszek et al, 2011;Rivera-Perez et al, 2010;Takahashi and Osumi 2010). The method involves immersion of embryos in nutrient rich media, the supply of appropriate gas mixtures and rotation of embryos in the media at 37ºC.…”

Section: Introductionmentioning

confidence: 99%

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Successful whole embryo culture with commercially available reagents

Glanville-Jones¹,

Woo²,

Arkell³

2013

Int. J. Dev. Biol.

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Since its development in the 1970's, whole embryo culture (WEC) has provided an important method of growing and observing murine embryos ex utero. During WEC, embryos are immersed in a combination of rat serum and cell culture media, and supplied with heat and appropriate mixtures of CO 2 and oxygen that mimic growth conditions in utero. One significant factor limiting the widespread use of WEC is the perception that commercially produced rat serum is inadequate to support normal rates of embryonic growth and development. Conversely, production of serum 'in-house' is technically demanding, time-consuming and expensive. The current study aimed to identify a WEC medium comprising commercially manufactured rat serum that would produce cultured embryos of comparable standard to those grown in utero. A mixed culture medium, composed of 50% commercial rat serum and 50% F12 Ham's cell culture medium with an N-2 neuronal cell growth supplement, was shown to support both a rate of growth, and the development of a range of features comparable to that which normally occur in vivo. Furthermore, the F12 (N-2) supplemented rat serum displayed a very low propensity to induce morphological abnormalities during the culture period. The study establishes a novel method of successful WEC using readily available commercial reagents and should enable the broader use of WEC.

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“…In WEC, mouse and rat embryos can grow ex vivo, (i.e., outside of the uterus). Although the WEC technique was often used in teratology by adding various chemical compounds into the culture medium, this technique has also been used in various developmental biology studies to examine unique developmental mechanisms in mammals [2][3][4] . For example, WEC is combined with other techniques, such as cell labeling, in wild-type and mutant embryos by using fluorescent dye 5 , cell transplantation 6 , and gene introduction via lipofection 7 and electroporation [8][9][10][11][12][13] .…”

Section: Introductionmentioning

confidence: 99%

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Takahashi

Makino

Kikkawa

et al. 2014

JoVE

Self Cite

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Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation. Video LinkThe video component of this article can be found at

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The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Cited by 14 publications

References 10 publications

Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells

Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells

Successful whole embryo culture with commercially available reagents

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

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