In vitrocultivation ofTrypanosoma congolensebloodstream forms in the absence of feeder cell layers

SUMMARYBloodstream forms ofTrypanosoma congolense(2 clones: ILNat3·1 and IL3000, and 4 stocks: IL2079, IL2466, IL3266 and CP-81) were continuously cultivatedin vitroat 34–36 °C in the absence of feeder cell layers, using HMI-93 medium which was modified from Iscove's modified Dulbecco's MEM (Flow Laboratories, Irvine, Scotland). The modification was done by supplementing the medium with 0·05 mM bathocuproine sulphonate, 1·5 mM L-cysteine, 0·5 mM hypoxanthine, 0·12 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 0·16 mM thymidine, 20% (v/v) heat-inactivated young goat serum and 5% (v/v) Serum Plus™ (Hazleton Biologics, Lenaxa, KS, USA). Trypanosomes obtained from two different sources were used to initiate primary cultures: (1) metacyclic forms which were producedin vitroat 27 °C, and (2) bloodstream forms obtained from Balb/c mice which had been infected with the bloodstream forms transformedin vitrofrom the metacyclic forms. Metacyclic forms placed in 25 cm2T-type (T-25) flasks rapidly attached to the bottom of the flasks and transformed to bloodstream forms during the initial 24 h and continued to proliferate. The bloodstream forms isolated from the infected mouse blood by means of diethylaminoethyl cellulose (DE52) column chromatography also continued to proliferate in the flasks. Cultures were maintained by replacing the medium every 24 h. Every 4–5 days, the attached bloodstream forms were resuspended in fresh medium by gentle pipetting and then were subcultured. The method was further simplified by initiating primary cultures directly with 10 μl of the tail blood of infected mice in 24-well culture plates and then by subcultivating either in wells or in T-25 flasks. The shortest population doubling time, 9 h, was achieved by seeding subcultures with 106bloodstream forms/ml. The bloodstream forms propagated in this system were morphologically similar to those seen in infected mouse blood, they were covered with a surface coat as examined by electron microscopy and they were infective to mice.

Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture

The functional gametes of all vertebrates first arise in the early embryo as a migratory population of cells, the primordial germ cells (PGCs). These migrate to, and colonise, the genital ridges (GR) during the early organogenesis period, giving rise to the complete differentiating gonad. PGCs first become visible by alkaline phosphatase staining in the root of the developing allantois at 8.5 days post coitum (dpc). At 9.5 dpc they are found in the wall of the hind-gut and, during the following three days, they migrate along the hind-gut mesentery to the dorsal body wall, and then to the genital ridges. By 12.5 dpc, the great majority of PGCs have colonised the genital ridges. During this period the number of PGCs increases from less than 100 to approximately 4000. In a previous paper (Donovan et al. 1986), we showed that 10.5 dpc PGCs can be explanted from the hind-gut mesentery, and will spread and migrate on feeder cell layers. We showed also that the intrinsic ability of PGCs to spread and migrate changes as they colonise the genital ridges. In this paper, we examine extrinsic factors that control PGC behaviour in vitro. Using PGCs taken from 8.5 dpc embryos, at the beginning of their migratory phase, we show that culture medium conditioned by 10.5 dpc genital ridges causes an increase in the number of PGCs in these cultures. We also show that PGCs migrate towards 10.5 dpc genital ridges in preference to other explanted organs. These experiments show that genital ridges exert long-range effects on the migrating population of PGCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth of mammalian cells on substrates coated with cellular microexudates. I. Effect on cell growth at low population densities.

Mammalian and avian cells cultured on glass or plastic substrates produce microexudates of cellular macromolecules which remain bound to the substrate when the cells are detached. The gross macromolecular composition of microexudates from a range of diploid, heteroploid, and virus-transformed cells was determined with cells labeled with radioisotopes. Significant differences in the amounts of cellular glycoproteins, proteins, and RNA present in microexudates were found between different cell types and between cells of the same type at different stages of growth. Inoculation of cells onto substrates "coated" with microexudates altered their growth behavior. Microexudates from exponentially growing subconfluent homotypic and heterotypic cell populations enhanced the growth of mouse and chick embryo cells seeded at very low densities, but similar microexudates had no effect on the proliferation of cells seeded at higher densities. The enhanced growth of low-density cell populations seeded on microexudates was compared with the growth enhancement produced by feeder cell layers and conditioned medium.