Embryo culture media preparation

A combined in vivolin vitro system for chemical metabolites has been developed that demonstrates the role of metabolism in activating compounds. In this system, rats are treated with a chemical and serum is removed 1 h later and cultured with explanted embryos. This system has been used to demonstrate that metabolites of procarbazine are toxic to explanted embryos, but that procarbazine and a microsomal preparation are not toxic to explanted embryos grown in culture medium (Schmid et al., 1982). 

Normal animal tissues (e.g., skin, kidney, liver) or whole embryos are commonly used to establish primary cell cultures. To prepare tissue cells for a primary culture, the cell-cell and cell-matrix interactions must be broken. To do so, tissue fragments are treated with a combination of a protease (e.g., trypsin or the collagen-hydrolyzing enzyme collagenase or both) and a divalent cation chelator (e.g., EDTA) that depletes the medium of usable Ca or Mg. The released cells are then placed in dishes in a nutrient-rich, serum-supplemented medium, where they can adhere to the surface and one another. The same protease/chelator solution is used to remove adherent cells from a culture dish for biochemical studies or subculturing (transfer to another dish). 

Fig. 10. Effect of ablation of the Cxi neurons on axon growth from the Til neurons in the grasshopper embryo. (A) Operated leg in which the Cxi neurons had been killed at the onset of Til neuron axonogenesis and the embryo allowed to develop further in culture medium. Asterisk indicates debris of Cxi cells. The Til axons possess multiple abnormal branches (arrowheads). (B) Contralateral control limb for (A) The Til axons take a normal pathway to the CNS. (C) Operated limb bud. The Til axons project straight ahead (arrowhead) towards efferent axons from the CNS, rather than turning posteriorly towards the Cxi cell site. (D) Contralateral control limb bud for (C), showing a normal Ti 1 trajectory. Camera lucida drawings from anti-HRP immunohistochemistry preparations. Scale bar = 100 /tm (reproduced with permission from Nature 304, (1983) Macmillan Magazines Limited).

Rat serum This is the basic culture medium for all postimplantation embryo culture, and is used undiluted for embryos cultured with closed yolk sacs (E7-E10 mouse) (6), or diluted to 25% for culture with open yoUc sacs (ElO-Ell mouse) (5). Rat serum is available commercially, at a price, but because of the method of preparation, it is inferior, particularly for the culture of the earlier stages (7,8), to what you can prepare yourself (see Subheading 3.1.3.). 

Fertilization medium (FM) All chemicals should be Sigma-Aldrich tissue culture or embryo-tested grade. Prepare the following concentrated stocks and filto each through a 0.4- jm MiUipore filter into a sterile plastic tube. Store frozen at -20 C. 

Embryos are most successfully cultured in a MM3 medium (Shields and Sang 1977 Currie et al. 1988) at 25 C in a humid chamber. For preparation of the MM3 culturing medium, see Broadie et al. (1992). After filtering (0.22-jxm Millipore filter), MM3 remains stable at 4 C for up to 3 months, although longer storage is not recommended. No striking difference has been detected in quality between MM3 stored at 4°C, -20°C, and -70 C. Fetal calf serum should be added several days (3-7) before use. Some considerations include ... 

Semliki Forest arborvirus was grown in chick embryo tissue culture. The infectious tissue culture liquid was decanted and diluted with medium 199 to give a preparation containing between 10 and 10 mouse IDso virus/ml. 

The preparation of the embryonic cockroach brain neuronal cultures is a complicated procedure which has been described in detail by Beadle and Lees ( ). In brief, the brains were dissected from 23-26 day old embryos of Periplaneta americana and broken up by passage through Pasteur pipettes of decreasing diameter. The cultures were initiated in a medium consisting of 5 parts Schneider s Drosophila medium and 4 parts Eagle s Basal medium, and after 7 days growth were transferred to a... 

Embryos and explants are cultured in dilutions of Normal Amphibian Medium (NAM 14) llOmM NaCl 2mM KCl ImM CalNOji ImM MgSO, 0.1 mAf Na EDTA 2mM sodium phosphate pH 7.5 ImMNaHCOj 50 Xg/mL gentamy-cin. It is convenient to prepare a lOX stock solution of all the components of NAM with the exception of the phosphate buffer, the NaHCOj, and the gentamycin. This lOX NAM salts stock may be autoclaved. We then prepare ... 

Chicken embryo fibroblast cells (CEF) were prepared from the carcasses of 10-day-old embryos and maintained in complete growth medium as described [5]. The cells were used between the third and seventh subcultivation after initiation of the cultures. Cells were labeled in vivo with 125 juCi of [2,8,5 - H]adenosine per ml of culture media (50.5 Ci mmol 1 Ci = 3.7 x 10 Bq New England Nuclear) as described [5]. 

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