Differences in growth of neurons from normal and regenerated teleost spinal cord in vitro

Summary Explants and dissociated cells from normal adult spinal cord and regenerating cord of the teleostApteronotus albifrons were grown in vitro for periods of 8 to 12 wk. During this time the neurons showed extensive neurite outgrowth. Neurite outgrowth from tissue explants and dissociated cells of regenerated spinal cord starts sooner and is more profuse than that from normal (unregenerated) cord. Neurite outgrowth is maximized by using adhesive substrata and a high density of explants or dissociated cells. Inasmuch asApteronotus does regenerate its spinal cord naturally after injury, whereas mammals do not, this culture system will be useful to study factors that control (permit) regeneration of spinal neurons in this adult vertebrate.     

Protective effect of hypothermia during ischemia in neural cell cultures

Hypothermia offers protection from the effects of ischemia in small animals. We have recently shown that similar to small animals, hypothermia may also be protective in an astrocytic model of simulated ischemia in cell culture. This study was designed to look at the protective effects of hypothermia in cultures of cerebellar granular (glutamatergic) and cortical (GABAergic) neurons. We used LDH release into the medium as an indicator for neuron damage. Experiments were all done in sister cultures, in groups of six cultures at two temperatures (37 and 32 degrees Celsius). The duration of ischemia was three hours in cerebellar granular neuronal cell cultures and six hours in cortical neurons. LDH release was measured immediately after the insult. Hypothermia protected both granular and cortical neurons. In granular cells, LDH release was 62+/–18 at 32 degrees and 212+/–15 at 37 degrees (p=0.02). Cortical neurons showed LDH release of 15+/–2 at 32 degrees and 32+/–2 at 37 degrees (p=0.005). Our study suggests that similar to astrocytes, the protective effects of hypothermia are evident in neuronal cell cultures from the cerebellum and the cerebral cortex. Cell culture systems should prove useful techniques in understanding mechanisms of hypothermic protection during simulated ischemia in neurons from different sites.     

Induction of high polyploidy in Phaseolus cell cultures by the protein kinase inhibitor,K-252a

Summary Somatic polyploidy of species-specific and tissue-specific degrees occurs in almost all plant species studied so far, but nearly nothing is known about the control mechanisms switching the mitotic cycle to an endoreduplication cycle. In order to search for a possible role of the cdc2 kinase, cell suspension cultures of the Runner bean, Phaseolus coccineus (Leguminosae) were treated with K-252a, an inhibitor of protein kinase activity. The treatment resulted in continuous cell cycles without mitosis, and hence induced polyploidy levels up to 2048C. It is, therefore, suggested that phosphorylation of a protein kinase, probably of the cell cycle-important p34^cdc2 type, is involved in the control of endoreduplication.     

DNA fingerprinting of red clover (Trifolium pratense L.) with Jeffrey's probes: detection of somaclonal variation and other applications

Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6 detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F₁ plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within (0.417≤V≤0.548) cultivars.     

Trypanosoma Manulis N. Sp. From the Russian Pallas Cat Felis Manul

ABSTRACT. The morphology of Trypanosoma manulis n. sp. is described from living and stained specimens obtained from the blood of a Pallas cat, Felis manul , from Kazakhstan. the cat was also infected with a Hepatozoon sp. and feline immunodeficiency virus. the morphology of the trypanosome most closely resembles that of Trypanosoma mpapuense Reichenow and Trypanosoma heybergi Rodhain found in bats. Trypanosoma manulis does not grow well in conventional media, but co-culture with African green monkey kidney cells in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum at approximately 27° C resulted in luxuriant growth of trypanosomes. Under these growth conditions, epimastigotes adhered to the surface of the culture flask and to African green monkey kidney cells, as well as forming large rosettes. At 37° C, although growth was poor, transformation of the epimastigotes into the bloodstream forms occurred. This represents the first report of a trypanosome of the subgenus Megatrypanum in a felid.     

Immunomagnetic separation as a final purification step of liver endothelial cells

Summary We describe a fast and reproducible method that can be used as a final step in obtaining pure populations of liver endothelial cells. This method employs endothelial cell specific lectin covalently bound to magnetic polystyrene beads (Dynabeads). Evonymus europaeus agglutinin (EEA)-coated Dynabeads were used to purify monkey liver endothelium from Percoll gradient separated nonparenchymal cells. EEA-coated beads were also successfully used to purify monkey aortic endothelial cells. The endothelial cells grew to confluence as a cobblestonelike monolayer, expressed Factor VIII related antigen, and incorporated acetylated-low density lipoprotein. The magnetic beads seemed not to modify the normal properties of the isolated endothelium, thus facilitating their use in experimental studies. This immunomagnetic separation technique may be applicable for purification of endothelial cells from a wide variety of tissue sources.     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension     

In vitro cultivation methods for coccidian parasite research

The subclass Coccidia comprises a large group of protozoan parasites, including important pathogens of humans and animals such as Toxoplasma gondii, Neospora caninum, Eimeria spp., and Cystoisospora spp. Their life cycle includes a switch from asexual to sexual stages and is often restricted to a single host species. Current research on coccidian parasites focuses on cell biology and the underlying mechanisms of protein expression and trafficking in different life stages, host cell invasion and host-parasite interactions. Furthermore, novel anticoccidial drug targets are evaluated. Given the variety of research questions and the requirement to reduce and replace animal experimentation, in vitro cultivation of Coccidia needs to be further developed and refined to meet these requirements. For these purposes, established culture systems are constantly improved. In addition, new in vitro culture systems lately gained considerable importance in research on Coccidia. Well established and optimized in vitro cultures of monolayer cells can support the viability and development of parasite stages and even allow completion of the life cycle in vitro, as shown for Cystoisospora suis and Eimeria tenella. Furthermore, new three-dimensional cell culture models are used for propagation of Cryptosporidium spp. (close relatives of the coccidians), and the infection of three-dimensional organoids with T. gondii also gained popularity as the interaction between the parasite and host tissue can be studied in more detail. The latest advances in three-dimensional culture systems are organ-on-a-chip models, that to date have only been tested for T. gondii but promise to accelerate research in other coccidians. Lastly, the completion of the life cycle of C. suis and Cryptosporidium parvum was reported to continue in a host cell-free environment following the first occurrence of asexual stages. Such axenic cultures are becoming increasingly available and open new avenues for research on parasite life cycle stages and novel intervention strategies.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.