Organotypic slice culture of E18 rat brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice.     

Effects of dexamethasone on expression and maintenance of cartilage in serum-containing cultures of calvaria cells

Summary The effects of dexamethasone on the ability of cells enzymatically isolated from 21-day fetal rat calvaria to produce cartilage in vitro has been investigated. Primary cultures of single-cell suspensions of rat calvaria were grown for up to 28 days in vitro in -minimal essential medium containing 15% fetal bovine serum, 50 /ml ascorbic acid, 10 mM Na -glycerophosphate and dexamethasone at concentrations of 1 M to 1 nM. Two types of nodules were present in dexamethasone-containing cultures. One has been characterized previously as bone (Bellows et al. 1986). The second morphologically resembled hyaline cartilage, possessed a strong Alcian blue-positive matrix and contained type-II, but not type-I, collagen. Both bone and cartilaginous nodules were spatially distinct and developed in isolation from each other. Cartilaginous nodules were found in the highest number at a dexamethasone concentration of 100 nM. Time-course experiments revealed that while the number of bone nodules increased continuously at least to day 28, the number of cartilaginous nodules remained constant after cultures had reached confluency. When cells were isolated separately from frontal and parietal bones and suturai regions, the greatest number of cartilaginous nodules developed from parietal bones. Since 21-day fetal rat calvaria contains 2 distinct patches of cartilage at the periphery of the parietal bones, it seems likely that this cartilaginous tissue is the origin of the cartilage cells. The results demonstrate that cultures of rat calvaria cells contain chondrocytes and possibly chondroprogenitor cells that are distinct from osteoprogenitors. Results support previous data that 100 nM dexamethasone permits the expression of and maintains the phenotype of chondrocytes in serum-containing cultures in vitro.     

Estimation of the most probable number with a programable pocket calculator.

The most-probable-number method has many potential applications, particularly if many tubes per dilution and many dilution levels are used. Increasing the number of cultures is possible with modern automatic and semiautomatic equipment. However, available tables are not sufficiently detailed to handle data from a large number of culture tubes used in an assay. This paper provides a computer program capable of handling the necessary arithmetic and written for a hand-held, advanced programable calculator.     

Protective potential of hematopoietic cells of the embryonic liver during long-term culture

The influence of the duration of the organ cultivation on the protective potential of mouse fetal liver hemopoietic cells was investigated. The protective potential was evaluated according to a 3-week viability of the lethally irradiated (mean LD88/21) mice following syngeneic transplantation. During 20 days of cultivation the protective potential (calculated per number of injected cells) remained at the initial level. With more prolonged cultivation (24-62 days) the protective potential was retained only in part of cultures and the mean effect was reduced. No parallelism has been revealed between the totipotent hemopoietic precursors capable of replacing the hemopoietic cells of an irradiated recipient and the CFUs in long-cultivated fetal liver cultures.     

A Procedure for the Measurement of Hormone Receptors Using a Cell Harvesting Machine

Abstract

A procedure has been developed that will rapidly measure the binding of hormones and growth factors to suspensions of intact cells. Radiolabeled ligand is bound to cells in 96-well cluster plates, and the cells are then collected and washed on glass fiber filters using a cell harvester. Up to twelve wells can be processed simultaneously in less than a minute. This rapid washing and harvesting method is convenient to use with a large number of samples, and the amount of bound ligand is saturable and proportional to cell number. This procedure should be applicable to suspension cultures, loosely adhering monolayer cultures, and other cellular preparations that will bind to porous filters. Using this procedure, we have shown that HeLa S₃ cells grown in suspension cultures saturably bind to epidermal growth factor (EGF). The quantity of EGF bound to HeLa S₃ cells can be increased by adding glucocorticoids to the culture medium.     

Ploidy-dependent genomic stability in the tissue cultures of ornamental Phlox drummondii Hook

Comparisons of the chromosome numbers, 2C nuclear DNA amounts and karyomorphology were made in explant cultures of diploid (2n = 2x = 14) and autotetraploid (2n = 4x = 28) Phlox drummondii. In 6–36 week old calli derived from diploid internodal segment explants, and in cells of root tips regenerated from such callus, marked differences were observed in chromosome number. The chromosome numbers ranged from 2n = 14 to 2n = 100 and DNA amounts from 8.20 to 63.20 pg in the diploid derived callus, while the extent of variation was much reduced in the regenerated roots. In contrast, the autotetraploid cultures were characterised by the maintenance of the same chromosome number and DNA amounts as the mother plant. Modified chromosome structures were not apparent in any of the cultures. The possible reasons for the chromosomal instability at the diploid level and stability at the tetraploid level are discussed.     

Quantification of cell nuclei isolated from hepatocytes by cell lysis with nonionic detergent in citric acid

A method was developed for determining the number of nuclei of hepatocytes cultured on collagen gel using a nonionic detergent, Nikkol BO-10TX. The cells were recovered in a test tube after solubilizing the gel by incubating it with the detergent in 0.1 M citric acid and then centrifuging the mixture. Nuclei were isolated from the cells with the same detergent solution and collected by centrifugation. The numbers of nuclei in cultures, scored with a hemocytometer or an electronic particle counter, were proportional to the lactate dehydrogenase activities of the cells. This method was also applicable for scoring the number of nuclei of hepatocytes cultured on collagen-coated plastic.     

Action of retinol on viable cell recovery and clonogenic potential of HTC cells after in vitro hyperthermia]

The aim of this study was to investigate if the association of both hyperthermic and Retinol treatment of HTC hepatoma cells could be useful in antitumor therapy. Treatment with 5 microM Retinol was carried out before or after hyperthermia (42 degrees C or 44 degrees C, one hour; in the latter case it was performed in cells already thermo-selected. We took into consideration two parameters, i.e. the number of the collected vital cells (evaluated by the trypan blue-exclusion test) and the clonal efficiency of these cells (calculated as number of colonies obtained from 250 cells cultured for 5 days). Thermal treatment alone caused a decrease of the number of the collected vital cells and of their clonal efficiency only in the cell cultures incubated at 44 degrees C. Instead the control thermo-selected cells, both at 42 degrees C and at 44 degrees C, showed both decreased clonal efficiency and yield of the vital cells. Compared with the control cultures treated with 0.1% Ethanol, used as vitamin A solvent, only cell cultures treated with Retinol before hyperthermia showed a decreased number of collected viable cells, nevertheless their clonal efficiency was unchanged.     

[3H]5-Hydroxytryptamine Binding to Brain Astroglial Cells: Differences Between Intact and Homogenized Preparations and Mature and Immature Cultures

[3H]5-hydroxytryptamine ([3H]serotonin) binds with high affinity (KD 2-12 nM) to a finite number of sites on brain astroglial cells. The number of binding sites in the C6 glioma line is decreased significantly (Bmax = 315 versus 30 fmol/mg) by homogenization. In intact primary cultures, derived from newborn rat brain, the number of binding sites is far greater in cultures of immature astrocytes than in cultures treated with dibutyryl cyclic AMP (Bmax = 1,520 versus 580 fmol/mg). A role for these receptors in development is suggested.     

Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells

These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly, while exfoliation of cells into the medium as well as formation of cornified envelopes were relatively low. The growth response to pyruvate was concentration-dependent in these cell cultures. In the absence of pyruvate, the extent of terminal differentiation to keratinization gradually increased. This was characterized by a cessation of growth after one week, and an increase in exfoliation until all cells had sloughed from the dish. Accompanying these changes was a marked increase in the formation of cornified envelopes. Cells undergoing DNA synthesis were present throughout 2 weeks of culture in pyruvate-deprived medium, even as the total number of cells was diminishing. Several compounds, including other 2-oxocarboxylic acids, were ineffective growth substitutes for pyruvate. These results indicate that the requirement for pyruvate is quite stringent in these cultures and that one way pyruvate promotes the growth of tracheal epithelial cells is by inhibiting terminal differentiation.     

Somatic embryogenesis and plant regeneration in avocado (Persea americana Mill.): influence of embryogenic culture type

Embryogenic cell lines of Persea americana were classified as SE- or PEM-type based on their morphology in maintenance medium. PEM-type cultures consisted of proembryonic masses (PEMs) with occasional development of proembryos and early-stage somatic embryos, while SE-type cultures consisted of somatic embryos from globular to cotyledonary stages with only a low frequency of proembryos and PEMs. Moreover, histological analysis revealed signs of cellular organisation, with a higher proportion of small cells in peripheral regions of the callus mass in the SE-type, but not in PEM-type cell lines. The effects of time in suspension, inoculum density and maintenance medium on the ability of avocado cell lines to produce mature somatic embryos were evaluated. Morphological differences observed during proliferation were correlated to the subsequent capacity of cultures to develop mature somatic embryos. The period of time in suspension and inoculum density were critical factors that influenced the ability of cultures to undergo maturation. Optimal conditions differed between SE-type cultures and PEM-type cultures. In SE-type cultures, the highest number of somatic embryos was observed in cultures grown in suspension for 9?days and 0.4?g inoculum; in PEM-type cultures a slight increase in mature somatic embryos production could be observed with 4?g inoculum size and 14?days in suspension. The presence of agar in the maintenance medium of SE-type cultures was essential for the maturation of somatic embryos.     

Determination of cell number in monolayer cultures

Determining the cytostatic or cytotoxic effects of various conditions on monolayer cells requires techniques that are rapid, reproducible, and able to monitor these effects as a function of time. Methods currently used to monitor cytostasis or cytotoxicity are either static or indirect; that is, they are designed to test effects of various treatments either at single time points or on associated cellular processes, such as membrane integrity. Because of these limitations in extant techniques, we undertook this study to improve methods for the rapid determination of cell number in monolayer cultures. We have arrived at conditions of staining cell nuclei with crystal violet under fixed regimens which allow rapid and reproducible quantification of cell number in cultures grown in 24-well miniwells. Quantification is possible by solubilizing the adsorbed dye into a solution of Triton X-100 and determining optical density (O.D.) using spectrophotometry. The present communication documents that O.D. is linearly related to cell number with a sensitivity of ca. 500 cells and that the technique is applicable to study agents which affect cell proliferation.     

Growth of bone marrow cells of normal and Rauscher virus infected mice in suspension cultures stimulated by CSA.

Bone marrow cells of normal and Rauscher virus (RLV) infected CBA/J mice were cultured under stimulation by postendotoxin serum. Cellular morphology and the number of granulocytic committed stem cells from day 1-5 after the onset of the cultures were studied with different amounts of postendotoxin serum added. There was a good correlation between total cellularity, the number of immature granulocytopoietic cells and the number of colony forming unit cells (CFUc) in suspension with the amount of postendotoxin serum. Postendotoxin serum delayed the appearance of macrophages in the cultures for 1-2 days. Cells from RLV infected animals showed a rather normal differentiation of the morphological recognisable cells 5 and 21 days after infection, but the CFUc survival in vitro 3 and 5 weeks after infection was reduced.     

Characterization of a γ-glutamyl transpeptidase positive subpopulation of endothelial cells in a spontaneous tube-forming clone of rat cerebral resistance-vessel endothelium

A spontaneous tube-forming clone of rat cerebral resistance-vessel endothelium was characterized in long-term serial culture. In this study, a clone, RV-150 ECT, of cerebral resistance vessel endothelial cells in long-term culture has been shown to have a subpopulation of γ-GTP positive cells that are present in all cultures regardless of confluency status or tube-forming stages. In pre-confluent and confluent cultures, the γ-GTP positive cells are few in number, stain weakly, and are randomly distributed in the monolayers. In monolayer post-confluent cultures, γ-GTP positive cells increase in number, stain strongly, and begin to show signs of non-random distributions. In early post-confluent cultures that have become a mixture of monolayer and multilayer cells, there is a further increase in γ-GTP positive cells which begin to form distinct groupings. In mid post-confluent fultures, the multilayered areas of the culture have begun clustering to form clear multicellular aggregates. The γ-GTP positive cells at this stage are reduced in number and are predominately associated with the cell clusters. In late postconfluent cultures, the multicellular clusters develop clear cell cords between/among the clusters. At this stage the γ-GTP positive cells are associated exclusively with cell clustters. With cord development, the γ-GTP positive cells are associated with bothe clusters and cords, and are reduced in number apparently because of selective degeneration of these cells. The results of this study demonstrate that a phenotypically distinct subpopulation of endothelial cells exhibits characteristic features of the blood-brain barrier, namely γ-GTP. The ability of these cells to express this property in long-term serial culture suggests that this may represent a useful in vitro model to study the growth and differentiation of bloodbrain barrier vessels. © 1993 Wiley-Liss, Inc.     

Selectable recombinant herpesvirus saimiri is capable of persisting in a human T-cell line.

Strategies were developed to insert the neo resistance marker into the junction between L DNA and the right terminal repetitive H DNA of herpesvirus saimiri. Recombinant viruses were selectable in permissive epithelioid cultures. The human T-cell line Jurkat could be infected persistently with the Neor virus; the cells contained episomal viral DNA in high copy number. This selectable vector should be generally applicable for gene expression in human T cells.     

Analysis of correlation between some parameters depending on cell proliferation and life span of "stationary phase aging" cultures and maximum life span of mammals

Earlier we developed a "stationary phase aging" model and introduced a definition of life span of "stationary phase aging" cell cultures. In this model the cells grow after seeding in flasks without subcultivation and medium change. They reach cell saturation density, stop dividing, gradually degrade ("stationary phase aging") and perish. By the term "culture life span" we designate the time from cell seeding until culture death. We designate the culture as dead when the number of living cells is less than 10 per cent of their number at saturation density of cell culture. The life span of transformed Chinese hamster cells was found to be proportional to the duration of their growth from cell seeding up to saturation density, as well as to the number of cell culture doublings and to be inversely proportional to the velocity of cell culture doubling for the same growth period. Maximum life span of mammals is known to be proportional to pregnancy duration and to the age at puberty. We found that maximal life span of mammals was proportional to the number of cell population doublings and inversely proportional to the velocity of cell population doubling during embryonal period or for the time from zygote to growth termination. The dependences for cell cultures and for mammals are analogous to each other.     

Osteoprogenitor viability in cell populations isolated from rat femora is not affected by 24 h storage at 4 degrees C

This study was initiated to determine whether partially dissected bones of rats could be refrigerated for 24 h in saline without losing viability of progenitor cells, specifically osteoprogenitors. This is directly applicable to studies involving bone tissue requiring overnight shipment, for example, studies involving space flown animals, grafting experiments, or transplantation. We evaluated cell populations isolated from the proximal femur of 6-week-old male Fisher 344 rats. Explants from the left femur were prepared and placed into culture immediately following dissection, while the right femur was cleaned, fragmented, and stored in saline at 4 degrees C for 24 h, after which explant cultures were initiated. After 11 days of explant culture, cells were collected from outgrowths, counted, and plated to initiate experiments. Plated cells were grown for either 15 or 21 days. To determine if storage affected the total number of colony forming progenitors, alkaline phosphatase positive colonies, or the number of osteoprogenitors, were counted. There was no significant difference in any of the types of colony forming units examined between cell populations derived from freshly prepared samples or those stored for 24 h, indicating that storage at 4 degrees C of bone tissue for 24 h in saline does not affect the osteogenic potential or the number of osteoprogenitors of the cell populations isolated.     

Transdifferentiation of putative neuronal cells of neural retina into lens: A demonstration by chick-quail chimeric cultures

Summary To elucidate the cell-type origin of lens cells, which differentiate in stationary cultures of neural retina, chimeric cultures between chick and quail cells were made to recombine xenoplastically the different cell fractions separated from 8- to 9-day cultures of 3.5-day-old embryonic neural retinal cells by means of centrifugation in Percoll. Extensive lentoidogenesis occurred in the recombination of the N2-fraction (consisting mostly of small round cells) with the E-fraction (containing a number of flattened epithelial cells). Taking advantage of the difference in electrophoretic mobility of chick and quail -crystallin, it was shown that this lens-specific protein, synthesized in the chimeric cultures, was mostly of the species-specificity of N2. Microscopic observations of histological sections of cell sheets of quail N2- and chick E-fraction chimeric cultures revealed that most cells with -crystallin, as identified by means of immunohistological detection, are provided with a nuclear marker characteristic of quail. By determining the level of activity of choline acetyltransferase and by examining the stainability with a fluorescent dye (Merocyanine-540), it was suggested that cells in the N2-fraction are primitive neuroblast-like cells. Thus, we can conclude that putative neuronal cells in early cultures of avian embryonic neural retina can transdifferentiate into lens cells.     

Correlations in the utilization of carbon sources in the range of griseus species of the genus Streptomyces

The author proposes a new scheme for arranging the data about the assimilation of carbon sources in tables; this is illustrated with the Griseus species belonging to the Streptomyces genus and with the information about them presented by Bergey [2]. In the scheme, carbon sources in the tables are arranged in the order of their diminishing availability for streptomycetes, and the lists of species within the morphological groups are constructed according to the property of a carbon source not being assimilated. Such an arrangement of data in the tables is very clear and convenient to use; it allows one to make optimal schemes for determining the assimilation of carbon sources by actinomycetes, and makes it easy to divide any large number of cultures into groups according to a similar assimilation of carbon sources. The scheme has been used to analyse the data available in science and to establish correlations in the assimilation of carbon sources by species of the Griseus series belonging to the Streptomyces genus.     

Receptor development and hormone actions. Effect of insulin and epinephrine on the glycogen content of newborn rat liver cultures

In the 9-day cultures of liver cells isolated from newborn rats epinephrine was found to increase the number of glycogen containing cells, and caused an elevation of the glycogen content of individual liver cells. Insulin treatment had a weak effect on the number of glycogen containing cells and its effect on intracellular glycogen content was negligible. The experiments indicate that the epinephrine receptor develops in the fetal period and is reactive in the newborn animal, while the insulin receptor is not. It was remarkable that in this period of life and under in vitro conditions the effect on cellular glycogen deposition of epinephrine and insulin is similar.