No abstract

Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth crisis, resulting in the loss of genomically normal cells prior to the appearance of established, aneuploid cell lines. Mouse embryo cells established and maintained for multiple passages in the absence of serum did not exhibit growth crisis or gross chromosomal aberration. Cells cultured under these conditions were dependent on epidermal growth factor for survival. Proliferation was reversibly inhibited by serum or platelet-free plasma, suggesting that mouse embryo cultures maintained by conventional procedures are under the influence of inhibitory factors.

We have derived serum-free mouse embryo (SFME) cultures in a basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin. These cells are nontumorigenic, lack gross chromosomal aberrations, and exhibit several other unique properties, including dependence on EGF for survival and growth inhibition by serum. We have examined the concentration dependence of the growth stimulatory effects of protein supplements used in the SFME medium formulation and surveyed other supplements that might act as alternative or complementary additions to the culture medium. Insulin could be replaced by insulin-like growth factor I and EGF could be replaced by transforming growth factor alpha in the same concentration range. Transferrin could be replaced by higher concentrations of lactoferrin. Deterioration of cultures in the absence of EGF began within 8 hours of the removal of the growth factor, and could be prevented by the addition of fibroblast growth factor/heparin-binding growth factor. Attachment proteins other than fibronectin were effective on SFME cells, but limited success was obtained when substituting other lipid preparations for HDL. These data introduce a precise system for exploring the unusual characteristics of SFME cells and contribute additional information that may be useful in the extension of these approaches to other cell types and species.

The metazoan parasitic blood f lukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism's complex molluscan-mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro. Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum create widespread disease in tropical developing countries (1). These parasites thwart attempts to produce practical and effective vaccines, and pharmacological approaches are problematic (2). Schistosome eggs are laid by females paired with males and residing in the vasculature of parasitized vertebrate hosts. To be successfully infective, eggs must penetrate the intestinal wall and pass in feces to fresh water. From these released eggs hatch free-swimming miracidia, the first larval form. Successful miracidia encounter and penetrate the appropriate species of snail and develop into sporocysts, the second larval from. In the following weeks, these primary sporocysts asexually generate first-generation and second-generation daughter sporocysts in the snail. From the later-generation sporocysts develop the third larval form, free-swimming cercaria. To be successfully infective in the human or other vertebrate host, cercaria must encounter and penetrate the skin of the host and undergo further changes to form schistosomula. These move through several further developmental stages, ultimately leading to sexual pairing in the veins of the parasitized host and egg production.Approaches to elucidate the mechanisms schistosomes employ to escape host immunity, as well as molecular genetic and pharmacological investigations, have been limited by the lack of an in vitro system for the continuous proliferative culture of the parasite in the absence of the host. Recently, Yoshino and colleagues (3, 4) have reported significant success in limited culture of the intramolluscan stages of S. mansoni and S.japonicum, but these techniques do not allow extended culture of proliferating sporocysts.Here we describe long-term cultures of S. mansoni in vitro and the development from the snail-infective miracidial stage, through generations of mother and daughter sporocysts, t...

Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, display distinctive properties: (i) SFME cells do not lose proliferative potential or show gross chromosomal aberration upon extended culture, (ii) these cells depend on epidermal growth factor for survival; and (ii) SFME cell proliferation is reversibly inhibited by serum. Treatment of SFME cells with serum or transforming growth factor j3 led to the appearance of glial fibrillary acidic protein, a specific marker for astrocytes. The appearance of glial fibrillary acidic protein in cultures was reversed upon removal of transforming growth factor (3 or serum. Cells with properties similar to SFME cells were also isolated from adult mouse brain. These results suggest a role for transforming growth factor (3 in astrocyte differentiation in developing organisms and in response to injury and identify the cell type that has the unusual properties of SFME cells.Serum-free mouse embryo (SFME) cells initiated and maintained multipassage in a rich basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein, and fibronectin display several distinctive properties (1)(2)(3)(4)(5)(6). Mouse embryo cells derived in conventional serum-supplemented medium undergo a period in which proliferative potential is lost or reduced, and then genetically altered cells with indefinite proliferative potential emerge (2,7,8). SFME cells do not exhibit any loss of proliferative potential or gross chromosomal aberration and maintain a relatively stable karyotype when cultured for >10 times the population doublings achieved with karyotypically normal mouse embryo cells in serum-containing medium. Although the period in which reduced growth rate can be detected may be minimal or absent in cultures in serumcontaining medium maintained in a passaging protocol involving high plating densities, the protocols used to derive SFME cells lead to a well-defined period of limited proliferative potential in cultures maintained in serum-containing medium (2, 7) that is not seen when the cells are cultured in serum-free medium. SFME cells also depend on EGF for survival, and cycloheximide or actinomycin D prevents death from EGF deprivation (5), suggesting that cell death requires the synthesis of RNA and protein, a phenomenon of programmed cell death similar to that reported for neuronal cell death in the absence of nerve growth factor (NGF) (9). In addition, proliferation of SFME cells is reversibly inhibited by serum or platelet-free plasma (1, 4, 6), and thus these cells would not be propagated under conventional culture conditions using serum-supplemented medium. As for most mouse embryo cell lines, the precise nature of the cells giving rise to the continuously growing SFME cultures was unknown. The unusual characteristics of SFME cells led us to attempt to determine the cell type represented by these cells.We found that treatment of SFME cells with serum or We a...

No abstract

A heat-sensitive, trypsin-sensitive factor that promoted growth and spreading of cells in serum-free, hormone-supplemented medium was partially purified from human serum. The major portion of the proteins in these preparations migrated upon SDS-polyacrylamide gel electrophoresis with a mobility consistent with molecular weights between 60,000 and 90,000. The spreading activity, which we have termed serum spreading factor, stimulated growth and spreading of a wide variety of cell types. The serum spreading factor was similar to fibronectin in that it showed an affinity for the plastic cell culture substrate but was shown to be distinct from fibronectin by several criteria. This factor may prove useful in studies of cell attachment and spreading and in studies of the relationship of cell shape and cell proliferation.

The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1. Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.