The effect of a synthetic tripeptide nervous tissue cultured in vitro]

Explants from chick embryo PNS (ganglion trigeminale) and from CNS of embryonal rats (hippocampus) and dissociated cells from chick embryo cerebral hemispheres were cultivated in maximow chambers in the presence of various concentrations of placental serum and of a chemically synthesized tripeptide Gly-His-Lys. 1. The presence of tripeptide in the nutrient medium with a low concentration of serum did not compensate the outgrowth of nerve fibers, that take place in the growth medium. 2. In the presence of tripeptide in the nutrient medium with low concentration of serum the index of growth area increased significantly. 3. Within the first days in cell cultures 0,01 microgram tripeptide pro ml medium stimulated the outgrowth of neuronal processes. 4. The experiments indicated, that the tripeptide did not replace the serum. The possible role of tripeptide as a system in controlling neuron-glial ratio in vitro is discussed.     

RESPIRATION BY CULTIVATED ASTROCYTES AND NEURONS FROM THE CEREBRAL HEMISPHERES

-Rates of oxygen uptake were measured in chick and/or rat astrocytes and neuronal cells cultivated for 2–4 weeks in Falcon flasks or Rose chambers. All the preparations were found to have respiratory rates between 0.4 and 0.8 × 10^?5μl/h O₂ per cell. Based upon measurements of cell diameters these values were recalculated to about 570 μmol/g wet wt. for the neuronal cells and 130 μmol/g wet wt. for the glial cells. The results are compared with previous data of oxygen uptake by neurons and glial cells separated by other procedures.     

Effect of substance P on nerve fiber regeneration in tissue culture

Explants of the ganglion trigeminale from chick embryos (PNS) and of the hippocampus from fetal rats (CNS) were cultivated in maximow chambers with growth medium or maintanance medium. Varied concentrations of substance P (SP . 3 CH3COOH . 4 H2O) were added. 1. The effect of substance P (SP) is related to concentration. In the presence of 10(-7)M SP in the growth medium and of 10(-4)M SP in the maintanance medium the cultivation of PNS cultures indicates positive results. These doses are suitable. 2. Within the first 24 hours in vitro SP stimulates the index of area in PNS cultures. The index of characterizes the relation of the outgrowth zone to the explant. In CNS cultures a significant difference of this effect was not observed. 3. The index of growth of nerve fibers may compare the test cultures with the control cultures. SP significantly increases the index of fiber growth in PNS cultures. A stimulation of CNS cultures was observed, significance was not found. 4. From the beginning of the cultivation with SP up to 48 hours in vitro the growth of nerve fibers significantly increases in the treated cultures in comparison with the control cultures. After this time the growth of nerve fibers decreased and a morphological conformity of test cultures and controls was observed. 5. The role of SP is discussed in specific activity on PNS tissue in vitro. The reactive neurons may be from the medio dorsal group of cells of the sensible ganglion.     

Autoradiographic study of RNA synthesis in isolated cells in culture from chick embryo spinal ganglia

Summary Dissociated cells from 9, 12 and 15 day-old chick embryo spinal ganglia were cultivated in presence of total embryo-extract, brain embryo extract, or total embryo extract supplemented with purified nerve growth factor (NGF). The cells were maintained during 4 days in Maximow assembly and during 1 month in Rose chamber. Neurons showed growth of nerve fibres. The non-neural cells evolved to spindle cells, Schwann cells, or fibroblasts.Ribonucleic acid (RNA) synthesis was followed with tritiated uridine by autoradiography. Some nerve cells showed tritiated uridine incorporation. The highest incorporations for short-term cultures were at 15 hours in presence of NGF, at 48 hours in presence of total or brain extract, and for long-term cultures at 8 days. These periods corresponded to the highest growing activity of the nerve fibres. After 4 days all the non-neural cells incorporated tritiated uridine.The tritiated uridine was first incorporated into the RNA of the nucleus and, afterwards was found also in the cytoplasm. The presence of brain extract or of NGF stimulates the incorporation of labelled uridine into RNA. No labelling was found in the nerve fibres, even after 4 hours incubation.Chargée de Recherche au C.N.R.S.This communication is a part of the Doctorat és-Sciences thesis, presented by Mrs. J. Treska-Ciesielski.With the technical assistance of Mrs. M. F. Knoetgen and A. Bieth.     

Cultivation and Differentiation of Dissociated Cells of Chick Embryo Encephalon

Dissociated cells from cerebral hemispheres of chick embryo at stages 17–18, 12–13 and 9–10, were cultivated for seven days. The cells were cultivated either completely covered with the nutrient medium in an atmosphere containing 5 percent CO₂ or they were covered by only a thin film of nutrient medium in contact with air.
For the embryos at stages 17–18 or 12–13, under both culture conditions neurons differentiated after 3 or 4 days in culture, while for the embryos at stage 9–10, no neuronal differentiation occurred under either condition. The cells remained morphologically undifferentiated and formed aggregates of about 50 cells. Some fibroblasts were found to grow on the collagen matrix.
It is concluded that dissociated cells from embryos at stage 9–10 are incapable of auto differentiation under the present culture conditions. The possible causes of this inability to differentiate are discussed.     

Regulation of glucose transport in chick fibroblasts: bicarbonate, lactate and ascorbic acid.

The rate at which chick embryo fibroblasts in primary or secondary culture transport glucose or 3-O-methyl glucose is strongly influenced by the presence of bicarbonate ion in the culture medium. Cells growing or maintained on glucose at physiologic concentration (5.5 mM) have an 8 to 10 fold higher rate of glucose uptake than their counterparts cultivated without bicarbonate. These cells also produce more lactate as a consequence of their more rapid intake of glucose. The hydrogen acceptors, methylene blue and dehydroascorbate added to the culture medium reduce the cell capacity to transport glucose and 3-O-methyl glucose to levels obtaining in the bicarbonate-free medium. There is a concomitant reduction in glucose utilized by cells during 24 hours and further reduction in lactate formed per molecule of glucose metabolized.     

Effect of Solcoseryl on nerve tissue under in vitro conditions]

Explants from trigeminal ganglia and skin of chick embryos and hippocampus from fetal rats were cultivated in Maximow assembly in the presence of Solcoseryl (Solco AG, Basel), a blood extract of calf. Solcoseryl in vitro did not influence the regeneration of nerve fibers from CNS explants. A stimulatory effect of Solcoseryl in vitro by 1% concentration on the outgrowth of new processes in explants of PNS was demonstrated. It is discussed: under optimal concentration Solcoseryl may be important for the influence of the composition of the medium in which explants of the nerve system and skin are cultivated.     

Cultivation in vitro of the testis cord of the newt, Triturus pyrrhogaster

Testis cords of Triturus pyrrhogaster were cultivated in vitro on (a) medium with chick embryo extract and calf serum, (b) medium with newt gonad extract, (c) Trowell 's medium T8 and (d) liquid synthetic medium 199. Of the four media utilized, medium 199 gave the best result for long-term maintenance of the normal histological structures of the testis cords. Addition of insulin (5 μ/ml) to medium 199 resulted in a remarkable improvement for the maintenance of the testis cord and the migration of columnar cells of the peritoneal epithelium into the primordial germinal tissue occurred as in the intact testis of this animal. Trowell 's medium T8 was proved inadequate. Medium with chick embryo extract and calf serum retained most of the germ cells healthy but caused gradual decrease in height of the columnar cells. Testis cords cultivated on the same medium in combination with Xenopus testis maintained normal histological structure for 18 days, whereas, those kept in contact with Xenopus ovary showed involution within the same period. Newt testis extract brought about transformation of somatic elements of the germinal tissue into fibroblastlike cells which was followed by the disintegration of germ cells. Ovary extract did not cause selective destruction on the somatic or germinal elements.     

Long-term cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain (D) of Rous sarcoma virus.

Attempts have been made to keep in vitro, for extended periods of time, cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D. Roller cultures of transformed chick cells kept in serum-deficient medium can be maintained without subcultivation for up to 6 months. The confluent cultures continuously release viruses and viable tumor cells into the medium. The released cells can be plated and have characteristics of growth and morphology which are relatively stable with time until the culture degenerates. Cells released at later stages of the culture produced substantially more viruses than those released earlier, suggesting that cell selection or differentiation occurs during long-term cultivation in low serum concentration. Long-term cultures of untransformed chick embryo fibroblasts can also be maintained in the same way. The release of viable cells by these confluent cultures, however, is negligible.     

Cultivation and growth of dissociated neurons from chick embryo cerebral cortex in the presence of different substrates

Summary Dissociated cells from 7-day old chick embryo cerebral hemispheres were cultivated for one month in Rose chambers. Four different culture conditions were employed in the composition of the matrix on which the cells were cultivated: collagen alone, collagen plus embryonic extract, collagen plus plasma and collagen plus plasma and embryonic extract.Within the first 48 hours of cultivation the cells formed processes under all four culture conditions. In the presence of plasma the dissociated cells remained well isolated; in the other culture conditions many cells reassociated into clumps.After 2–3 weeks in cultures on collagen or collagen plus embryonic extract many polygonal cells developed and formed a layer upon which typical neurons and oligodendrocyte-like cells were observed. After 3 weeks the polygonal cells began to transform into astrocyte-like cells. In the presence of plasma the cell bodies of the neuroblasts remained small and round. The processes developed generally consisted of one long and many short thick fibres; all processes had a bulbous appearance. In 3–4-weeks old cultures the cells which remained viable, were morphologically unchanged.The differences in the morphological aspects of the cells cultivated on plasma and those cultivated on collagen alone or with embryonic extract are discussed.This work was supported in part by the Délégation Générale à la Recherche Scientifique et Technique and the Mind Science Foundation. Thanks to Prof. Dr. Z. Lodin, Czechoslovak Academy of Sciences, for his continuing help. We are grateful to Mrs. M. F. Knoetgen for technical assistance.     

Brain histogenesis in vitro: Reconstruction of brain tissue from dissociated cells

Summary Comparative studies of the aggregative behavior of cells dissociated from different areas of embryonic chick and mouse brains show that each of the regionally differentiated lobes (cerebrum, optic tectum, and cerebellum), and the stem areas (diencephalon and medulla), form characteristic aggregates distinctive in size and shape. Bispecific co-aggregates are produced by commingling dissociated mouse cerebrum cells with chick cells from various brain regions, or from non-nervous tissues; the size of these co-aggregates and the extent of internal sorting out of cell types is closely related to the degree of homology between the interacting cell populations, e.g. co-aggregates of the closely homologous mouse and chick cerebral cell types contain homogeneous tissue fabrics of intermingled mouse and chick cells. Cell surface constituents involved in selective recognition and association of nerve cells were sought and cell-free supernatant preparations were obtained from short-term monolayer cultures of embryonic cerebrum cells (of either mouse or chick origin) which caused a striking, specific enhancement of aggregation of homologous cerebrum cells. These materials had no such effect on heterologous tissues tested: optic tectum, cerebellum, medulla, neural retina, liver, kidney or limb bud. These findings are discussed in relation to control mechanisms governing normal brain histogenesis and to the specificity of neural associations. This work was supported by United States Public Health Service research grant HD-01253 to Aron Moscona and by the Louis Block Fund of the University of Chicago.     

An improved organ culture method for adult mammalian lung

Summary An improved method for maintaining adult rat lung in submerged organ culture is described in which the alveoli were inflated with agar and 200-μm-thick hand-cut sections were mounted in Rose chambers. The conventional single-compartmented Rose culture chamber was modified by adding a second chamber separated from the first by a gaspermeable membrane. One compartment functioned as an air reservoir and the other housed the explants submerged in nutrient medium. Visking dialysis membrane used underneath the explants prevented cell outgrowth and facilitated the exchange of nutrients and waste products at the glass-tissue interface. Because of the excellent optical properties of the Rose chamber and the thinness of the explants, individual cell types can be identified in the living tissue. The explants were studied with time-lapse cinematography, light microscopy, histology, and with erythrosine B for dye exclusion. With this modified system the functional life span of the explants was increased from 1 week to 1 month. This study was supported by NHLBI Grant No. HL15098-05.     

An analysis of the fate of the chick wing bud apical ectodermal ridge in culture

Stages 20 and 25 chick apical ectodermal ridge have been cultured in nutrient medium containing fetal bovine serum and the tissues have been examined for dying cells at 0, 6, 12, 18, and 24 hr. By 12 hr, an average of 43% of the cells were dying. By 24 hr, stage 20 ridge had lost its integrity and stage 25 ridge contained an average of 50% dying cells. These results are in agreement with the observations of R. L. Searls and E. Zwilling (1964, Dev. Biol. 9, 38-55) on isolated stage 20 ridge. In subsequent experiments, ridge ectoderm was cultured in serum-containing medium to which insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenium (5 ng/ml) or insulin (5 micrograms/ml) had been added. Under these conditions the ectoderms remained viable even after 24 hr in vitro.     

The circumfusion system for multipurpose culture chambers. I. Introduction to the mechanics, techniques, and basic results of a 12-chamber (in vitro) closed circulatory system

A self-contained mechanical system for circulating nutrient fluid through 12 tissue culture chambers is described in detail. This system utilizes nonperforated cellophane membranes in the chambers which separate the circulating nutrient from the tissue culture environments. The nutrient, therefore, is dialyzed through the cellophane of each chamber; some cell products are retained in the microenvironment between the closely apposed cellophane and cover slip, whereas the other cell products move from chamber to chamber in the circulating nutrient. The resultant environmental conditions directed by the circumfusion systems are highly favorable for maintaining the differentiation of chick embryo tissues over protracted periods; a number of micrographs are shown.     

THE GOLGI COMPLEX IN LIVING OSTEOBLASTS

Embryo chick leg bones were cultivated under sheets of dialysis cellophane in multipurpose culture chambers. The osteoblasts emigrating in this environment contained phase gray juxtanuclear bodies from which phase white droplets emanated. These were concluded to be the Golgi complex and are described with respect to their living morphology, microscopically observed functional activity, vital staining, and cytochemical reactions.     

Formation of multinucleated giant cells from circulating blood cells cultivated in diffusion chambers.

Autologous circulating rabbit blood cells have been cultivated in Millipore diffusion chambers implanted intraperitoneally for 13 days. During this period multinucleated giant cells were formed within the diffusion chambers, confirming a hematogenous origin of these cells. The diffusion chamber technique might be helpful for the investigations of factors initiating the formation of multinucleated giant cells.     

Multinucleation of Skeletal Muscle in vitro

Healthy, mature, spontaneously contracting muscle was cultivated from explants of 13-day chick embryos for periods up to 4 months in the multipurpose chamber (Rose, 1954) using cellophane-strip technique (Rose et al., 1958) with silicone gaskets, Eagle's medium including 10 per cent horse serum reinforced with 300 mg-per cent of glucose, and the teased type of explant. This method provided optically ideal conditions for the study of muscle fibers with oil immersion, phase contrast time-lapse cinematography at 1 frame per minute without apparent damage for periods as long as 10 days. In no case was mitosis, amitosis, or nuclear "budding" observed in the course of muscle development. Multinuclear muscle fibers have been shown with cine technique to result from both myoblast fusion and polar extension of preformed (explanted) muscle tissue. Myoblast fusion was the only demonstrable way of giving rise to multinucleation. Nuclear membrane "wrinkling" was shown to be merely a temporary distortion that occurred during nuclear migration and rotation. It is suggested that this phenomenon may be responsible for numerous reports of amitosis in the genesis of muscle fibers. The histological development of new straps resulted from an orderly sequence of events. Included in these were polar extension, nuclear migration, rotation, and fixation. Following these events there was increased mitochondrial activity, myofibril formation, and cross-banding. Spontaneous contractions were seen throughout the entire course of differentiation in vitro but became more regular and stronger in the later stages.     

Interaction of influenza virus with chicken fibroblasts: A proton magnetic resonance study

It has been shown that high resolution ¹H NMR spectroscopy can be used to study the replication of influenza virus in chicken embryo fibroblasts. Marked changes in the NMR spectrum were observed during the course of viral infection and these depended on the presence of culture medium. Only minor changes were observed when cells were maintained in phosphate buffered saline. These data distinguish influenza from other RNA enveloped viruses which have been reported to cause major spectral changes in the absence of nutrient medium.     

Neurotrophic effect of nerve extract on development of tetrodotoxin-sensitive spike potential in skeletal muscle cells in culture.

The effect of the presence of nerve extracts on the development of tetrodotoxin (TTX)-sensitive sodium channels in cultures of dissociated embryonic chick skeletal muscle cells was examined by measuring the maximum rate of rise of TTX-sensitive spike potential. The addition of the nerve extract prepared from brain or spinal cord of chick embryos to the culture medium caused an increase in the channel density. Extracts of non-neural tissues, i.e., lung, kidney, and muscle, were ineffective. Liver extract, however, produced an effect similar to the nerve extracts. These results suggest that the TTX-sensitive sodium channels in the muscle cell membrane are regulated by a diffusible chemical substance independently of innervation, and that this substance resides in neural tissues, and perhaps also in liver.     

A simple method for culturing chick embryo liver and kidney parenchymal cells for microscopic studies

Differentiated parenchymal cells were cultured from chick embryo livers or kidneys. Tissues were trypsinized, fragmented under a dissecting microscope, suspended in culture medium and separated into microscopic cell clumps with a syringe and wide bore needle. These cell clumps were grown in culture chambers and after light microscopic study were fixed and sectioned for electron microscopy. Both liver and kidney parenchymal cells appeared as clusters of epithelial cells containing round nuclei surrounded by numerous large mitochondria. Electron microscopy revealed well-differentiated cytoplasm which, in the liver cells, contained glycogen rosettes closely associated with smooth endoplasmic reticulum.