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There is a need for commercially manufactured equipment for use in post-implantation embryo culture techniques. A simple, inexpensive modification to an existing device is described that enables such an apparatus to be used for the roller-tube technique.
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Mammalian embryo culture systems can be used to study dominant lethal, recessive lethal, and visible mutations that are expressed between fertilization and early stages of organogenesis. The incidence of dominant lethal mutations has been determined for cultured mouse embryos by morphological observations during pre-implantation and early post-implantation growth in vitro, and the mechanisms of dominant lethal mutations have been studied cytogenetically. Recessive lethal mutations have been studied with cultured embryos, although they can be initially detected only with appropriate breeding protocols. Visible mutations that are due to deletions or single-site base alterations in the DNA can be detected in embryos by isoelectric focusing and gel-electrophoresis techniques.--The principal advantage of cultured embryos for detecting mutations and studying the mechanism of action of particular mutagens is that embryos are accessible for analysis before death with a minimum of indirect maternal effects. The primary disadvantages, which may be alleviated with improved culture conditions, are that only a limited amount of tissue is available, attrition and retardation occur, and offspring are not recovered for further breeding studies.
Cell cultures with a different multiplication potential in vitro, depending upon the strain source used, were obtained from mouse embryos belonging to the CVA, C57 Black A2G and Swiss strains. Only the Swiss 12 culture underwent spontaneous transformation and was carried through more than 50 passages in vitro. The Swiss-12 substrate proved not to be contaminated either by viruses or micoplasma. It is less sensitive than other elective cell substrates to infection with attenuated polioviruses, cytopathogenic Coxsackie A9 virus and vaccinia virus, but its sensitivity to infection with Herpes simplex type 1 virus is similar to that of human embryo fibroblasts. After a high number of passages the Swiss-12 substrate permits, in comparison to other cell substrates (human heteroploid Hep-2 line, human embryo fibroblasts), a highly efficient qualitative differentiation between the growth media and calf serum.
In this review an in vitro system is considered to be any developing tissue, organ, or organism other than mammalian embryo in situ. Before the various test systems that have been used or proposed are discussed, attention is given to the questions of whether all chemicals in the environment are in need of biological testing and what criteria should be used in making this judgment. Consideration is also given to selection of the appropriate level of rigorousness to be used in tests of different categories of chemicals. To aid in this, the characteristics of an optimal test are assembled and used as a standard for estimating the potential usefulness of the various in vitro systems. The systems discussed include bacteria and other unicellular organisms, somatic cells in culture, tissue culture, organ culture, intact invertebrate embryos (e.g., drosophila, sea urchins, sand dollars), intact lower vertebrate embryos (e.g., frogs, other amphibians, fish), cultured mammalian embryos, and incubating chick embryos. None of these are regarded as sufficiently validated in terms of comparisons with known teratogenic responses in pregnant mammals to warrant adoption as a reliable test at this time. Intact embryos of drosophila, sea urchins, amphibians, and fish are regarded as promising, but much research is needed to ascertain their predictive validity for mammals. The incubating chick embryo, however, possesses more of the essential features of the optimal system than any of the others. A tentative proposal using the chick is outlined, but it will require considerable further comparison with currently used procedures in pregnant mammals before its reliability can be fully evaluated.
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Organelle translocation in a number of cell types in tissue culture as seen by high-resolution Zeiss-Nomarski differential interference contrast optics was filmed and analyzed by computer. Principal cell types studied included primary chick spinal cord, chick dorsal root ganglion, ratbrain, and various clones of continuous cell lines. Organelle translocations in all cell types studied exhibited frequent, large changes in velocity during any one translocation. The appearance of particles as seen with Nomarski optics was correlated with their fine structures in one dorsal root ganglion neurite by fixing the cell as it was being filmed and obtaining electron micrographs of the region filmed. This revealed the identity of several organelles as well as the presence of abundant neurotubules but no neurofilaments. Primary cell cultures exhibited more high-velocity organelle movements than continuous cell lines. The net progress of an organelle in a given direction was greater in primary neuronal cells than in fibroblasts or continuous cell lines. These findings are correlated with the literature on organelle translocation and axoplasmic transport.
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Addition of 1 mg/ml streptozotocin to serum in which 10-day rat embryos are cultured reduces their growth and viability. There is therefore a risk that administration of this drug to pregnant animals to induce diabetes could also have direct, deleterious effects on the embryos.
Rat embryos at the head-fold stage were cultured in rotating bottles for 2 days in rat serum extensively dialysed against glucose-free BSS, and to which various energy sources and nutrients were added. Optimum growth and differentiation, virtually indentical with that obtained in while serum and to that seen during the corresponding period in vivo, were obtained with the addition of glucose and certain vitamins: pantothenic acid and riboflavin had a general beneficial effect on development, i-inositol suppressed neural tube defects, and folic acid significantly improved growth of the embryos.
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A method and device for obtaining embryo brain slices are described. Results of cultivation of slices of rhombencephalon and mesencephalon are presented obtained in accord with a proposed technique of the "swimming float" (Olenev, 1979). With this cultivation, original topographical relations between brain nuclei and fibrillar tracts remain unchanged that enables us to compare these cultures with histological sections of brain to identify strictly the structures being cultivated. Cultured neurons of nuclei of XII and VIII pairs of cranial nerves have been first described in addition to various divisions of the reticular formation commissura cochlearis dorsalis. Compared to cells examined in the in vivo conditions, neurons of the nucleus cochlearis laminaris, grown in tissue culture conditions, do not make plate-forming structures; dendrites of the neurons of III layer of tectum opticum do not develop. This may be explained by the absence in vitro of any stimulating influence from the acustic ganglion fibres and ganglion cells of the retina on these morphogenetic processes.
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Tick-borne encephalitis virus (TEV), a Flavivirus, and Lipovník virus (LV), a member of the Kemerovo group and complex and possible member of the Rioviridae, were used to infect chick embryo cell (CEC) cultures. LV reproduction was inhibited by preinfection with TEV, mainly in aged cultures. In young CEC cultures, TEV production was stimulated slightly and interferon was sometimes depressed by this superinfection; on the contrary, in aged cultures the superinfecting LV stimulated interferon also when infective LV was not produced because of interference. Double staining by the fluorescent antibody (FA) technique localized both viral antigens often in the same cells. Interference between TEV and LV (and vice versa) was observed in cerebrally infected adult mice. The pathogenicity of LV, when injected first, was increased by TEV superinfection. Examination by the FA technique confirmed interference between TEV and LV also in suckling mouse brains and demonstrated both viral antigens in the same brains when TEV and LV were injected simultaneously.
A human embryo in transition between a morula and blastocyst after culture in vitro was reintroduced into the mother's uterus via the cervix. The resulting pregnancy was closely monitored and was found to be located in the oviduct. The ectopic embryo was removed at 13 weeks gestation.