The Culture of Small Aggregates of Amphibian Embryonic Cells in vitro

Abstract

Methods are described for the short term culture of disaggregated amphibian embryonic cells in a medium consisting of about 0·1 per cent protein in Steinberg’s physiological salt solution. In a medium containing whole serum or serum protein cells differentiate according to their presumptive fate in the embryo. The use of a particle-free extract of mouse bone marrow causes ectoderm cells to develop as mesoderm. Gastrula ectoderm cells will differentiate ciliated epidermis providing they are permitted to reaggregate into balls of about thirty cells and upwards. Gastrula mesoderm cells and induced ectoderm cells likewise do not differentiate unless aggregated, but the majority of aggregates fail to differentiate. This seems to be because these cells have exacting requirements for precise concentrations of the protein component in the medium. There is some variation between species in this respect, and requirements vary between ovulations in the case of Rana pipiens and Xenopus laevis. Ciliated epidermal cells differentiate in cohesive balls that detach from the glass. All other cell types differentiate as thin outgrowths of cells attached to the glass by cytoplasmic fringes or pseudopodia. The first visible event in the differentiation of non-epidermal cells is their attachment to the glass by pseudopodia or cytoplasmic fringes, and the rapid expansion of these contributes to the deformation of the cell from the spherical embryonic form to that approximating to the form of the differentiated cell. This rapid change in form and activity is termed germination. It appears to be a necessary preliminary for cytodifferentiation, the rapid utilization of yolk and the build up of large mitochondrial populations. Germination precedes the formation of striated muscle fibres, vacuoles in chordal cells and other indications of cytodifferentiation by a time interval that is characteristic for each cell type within a species. The time and order in which cells differentiate in culture is related to the behaviour of comparable cells in the embryo. It is concluded that, by the neurula stage, the control of the time of germination is vested in the individual cells. It is concluded that the development of neural and mesodermal cells involves two necessary periods of decisive change, one at the end of gastrulation unconnected with any gross morphological changes in the cells, the second, closely following neural closure and involving the in vivo counterpart of germination in culture, by which the potentialities of the cells are mobilized in the interests of cytodifferentiation. Both these important epigenetic events coincide with changes in the adhesive properties of cells.