Radial histophysiologic gradient culture chamber: Rationale and preparation

Summary Histophysiologic gradient culture methods reconstitute important spatial relationships that occur in nature between a parenchyma and its supporting stroma. At the epithelial-stromal interface, epithelia are firmly attached to the stromal substrate, initiation of renewal takes place, and metabolites are exchanged by a process of diffusion between epithelium and substrate. Other spatial imperatives characteristic of stratified epithelium are high density of cells, gradients of maturation, and continuity of epithelia along the entire course of the stromal-parenchymal interface. In radial gradient culture these relationships of epithelial cells, and supporting substrates are reconstituted. The culture chamber consists of a thin-walled cylinder, 2 to 3 mm in diameter and 3 cm long. The wall is a transparent collagen membrane in whose substance is embedded a reinforcing nylon mesh. To prepare a culture, one end of the cylinder is ligated, 1 or 2 particulate inocula are inserted in the open end of the cylinder, guided toward the ligature, and the open end is ligated. Subsequently, during incubation in a container with medium, the explants attach and proliferate. Proliferation and migration result in the cylinder being completely lined by a complex organoid tissue with structural characteristics of the original tissue. The tissue patterns in radial gradient culture of two human cell lines, RT-4, a bladder cancer, and 87×50, and ovarian cancer, are illustrated.     

Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture

The biology of animal cells is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three-dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three-dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial issues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.     

A method for the isolation and culture of human colonic crypts in collagen gels

Summary Little is known concerning the biological factors that control the proliferation of the stem cells of the colonic mucosa. In part this is due to a lack of systems suitable for studying the proliferation of this mucosa in vitro. We describe a simple technique for the isolation of single viable intact crypts which are free of stroma and which can then be cultured for periods of at least 16 d using a collagen gel culture method. This method of crypt isolation was efficient with the mean yield of viable intact crypts being 1.4 ±1.2×10⁴ ( ± SD) crypts/cm² of mucosa. In culture, mucosal cells only survived for extended periods when the crypts were cultured in collagen gels over a feeder layer of bovine aortic endothelial cells. Cells containing mucus were present in the cultured crypts at all stages of the culture; however we have not been able to demonstrate alkaline phosphatase activity in these crypts. Studies of DNA synthesis after 7 d in culture, using a 18-h pulse label with bromodeoxyuridine (BUdR) has shown that DNA synthesis, as measured by incorporation of BUdR into nuclei, is still occurring in these cultured crypts.     

Isolation and differentation of cloned epithelial cell lines from normal rat mammary glands

Summary Single-cell-cloned cell lines have been established from primary cultures of neonatal rat mammary glands. A representative cuboidal cell line, Rama 704, shows the presence of intermediate filamental proteins keratin and vimentin, and occasional cells express milk fat globule membrane antigens on their apical surfaces. Rama 704 cells grow as a cuboidal pavement in culture and produce hemispherical blisters or domes when confluent. Noteworthy ultrastructural features are the presence of junctional complexes, desmosomes, and apical microvilli typical of epithelia. Cells seeded within floating collagen gels with form a variety of multicellular outgrowths, some of which are ductlike in morphology and are composed of polarized cells surrounding a central lumen. The cuboidal cells produce elongated cells under conditions of high cell density and also when cells float off collagen gels and reattach to the plastic substrate. The former elongated cells have been cloned and three cel lines established: Rama 710, 711, and 712; the latter uncloned elongated cells are termed Rama 704E. The cloned elongated cells show an increase in the amounts of basement membrane proteins deposited, a lack of junctional complexes and microvilli, and an increase in the amount of rough endoplasmic reticulum compared with their parental cells. Rama 704E cells show an enhanced deposition of basement membrane proteins and increased amounts of actin in the cytoplasm over the elongated cell lines and contain microfilaments and pincocytotic vesicles similar to those seen in myoepithelial cells. All the elongated cells and lines fail to form ductlike structures within collagen gels. None of the cell lines form tumors in syngeneic rats although they all produce some tumors in nude mice, which are composed of cords of epithelioid cells and spindle cells in varying proportions. In addition, some of the Rama 704 tumors contain rhabdomyoblastic elements that penetrate the host fat pad. This is the first report of the isolation and characterization of a stable cuboidal cell line from a neonatal rat mammary gland. The Rama 704 cell line shows morphological and biochemical features of mammary epithelial cells and converts at high cell density to elongated cells that have also been cloned.     

Effects of ethanol on rat schwann cell proliferation and myelination in culture

Summary It is possible to treat dissociated embryonic rat dorsal root ganglia in culture to inhibit proliferation of all nonneuronal cells except Schwann cells. Neurons have been shown to produce a mitogenic stimulus for Schwann cells under these conditions. Additionally, myelin-competent neurons induce Schwann cells to elaborate myelin sheaths. Groups of sibling cultures were exposed to various nonlethal concentrations of ethanol (0, 43, 86, or 172 mM) for 4 wk. Culture were assessed weekly by light microscopy in a blind fashion for evidence of Schwann cell proliferation and myelin formation. Ethanol adversely affected both Schwann cell proliferation and myelin formation in culture. No obvious differences in neuronal morphology were observed among the various groups of cultures by light or electron microscopy. These observations suggest that ethanol might interfere with Schwann cell proliferation and myelin formation in culture by one or both of the following means: a) inhibit neuronal production of signals for Schwann cell proliferation and myelination or b) impede Schwann cell responses to neuronal signals. Investigation of these possibilities in culture may provide insight into neuropathologic mechanisms operative in the fetal alcohol syndrome or alcohol-associated peripheral neuropathy in humans. This work was supported by the Department of Veterans Affairs, Washington, D.C.     

Experimental studies on ultralow frequency pulsed gradient magnetic field inducing apoptosis of cancer cell and inhibiting growth of cancer cell

The morphology characteristics of cell apoptosis of the malignant tumour cells in magnetic field-treated mouse was observed for the first time. The apoptotic cancer cell contracted, became rounder and divorced from adjacent cells; the heterochromatin condensed and coagulated together along the inner side of the nuclear membrane; the endoplasmic reticulums(ER) expanded and fused with the cellular membrane; many apoptotic bodies which were packed by the cellular membrane appeared and were devoured by some lymphocytes and plasma. Apoptosis of cancer cells was detected by terminal deoxynucleotidyl transferase mediated in situ nick end labeling(TUNEL). It was found that the number of apoptosis cancer cells of the sample treated by the magnetic field is more than that of the control sample. The growth of malignant tumour in mice was inhibited and the ability of immune cell to dissolve cancer cells was improved by ultralow frequency(ULF) pulsed gradient magnetic field; the nuclei DNA contents decreased, indicating that magnetic field can block DNA replication and inhibit mitosis of cancer cells. It was suggested that magnetic field could inhibit the metabolism of cancer cell, lower its malignancy, and restrain its rapid and heteromorphic growth. Since ULF pulsed gradient magnetic field can induce apoptosis of cancer cells and inhibit the growth of malignant tumour, it could be used as a new method to treat cancer.     

Role of serum and hormones during the growth and development of rat mammary tumor epithelial cells in collagen gel culture

Summary The characteristics of hormone-dependent rat mammary tumors in response to serum and hormones were determined in collagen gel matrix culture. Epithelial cells from 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas were embedded in collagen gel and the effect of estrogen, progesterone, prolactin, insulin, and serum was tested. The total cell number and [³H]thymidine incorporation were used to determine the growth pattern of the cells in culture. It was found that in medium containing 20% porcine serum and supplemented with insulin, estrogen, progesterone and prolactin, both the cell number and [³H]thymidine labeling index increased with time, after an initial lag. Serum seemed to be essential to maintain growth of the tumor cells, because hormones alone, in the absence of serum, were unable to sustain growth of the cells. When estrogen, progesterone, prolactin, and insulin were tested individually in the presence of 20% porcine serum, only estrogen demonstrated a significant stimulatory effect.     

The resistance of epithelia to vascularization: Proteinase and endothelial cell growth inhibitory activities in urinary bladder epithelium

The avascularity of epithelia may be attributed to the presence of an extractable, low-molecular-weight factor. This factor contains potent inhibitors of proteolytic enzymes, as well as a growth inhibitory activity directed against endothelial cells in vitro. It is extracted from the epithelium of bovine urinary bladders by 1 M NaCl. The extract is ultrafiltered through an Amicon XM-50 membrane, then concentrated and dialyzed into a 0.9% NaCl solution, using a UM-2 membrane. This ultrafiltrate, called the UM-2 retentate (UM-2R). contains approximately 6 μg protein/ g tissue. The UM-2R has a low content of uronic acid and is practically devoid of hydroxyproline. SDS-PAGE reveals that the UM-2R consists of six major proteins. The UM-2R contains a Trasylol-like proteinase inhibitor that expresses strong trypsin inhibitory activity. Comparisons between bladder and serum UM-2Rs and electrophoretic mobility assays indicate that this proteinase inhibitory activity is derived from the bladder epithelium and not from the serum. The UM-2R is cytotoxic to cultured endothelial cells. Cultures of other cell types (normal and neoplastic) are not affected. The bladder-derived proteinase and endothelial cell growth inhibitory activities may protect epithelia from vascular invasion.     

Cultivation of the hybridoma cell line MN12 in a homogeneous continuous culture system: Effect of culture age

The stability of the hybridoma cell line MN12 in a long-term homogeneous continuous culture was studied using a panel of analytical methods. These include two flow cytometry methods, for the determination of relative cytoplasmic and membrane IgG content. In addition, the antibody production was determined by an ELISA, and the metabolic state of the cells was determined by means of glucose consumption and lactate production.These results indicate a possible selection of variants of MN12 hybridoma cells with an overall aerobic metabolism, but with a higher glucose consumption rate and a higher lactate production rate. These variants are mainly characterized by a different membrane IgG content and cytoplasmic antibody content. These changes may possibly be affected by the culture age.     

Atmospheric air vs. normal middle ear gas: Effects on in vitro growth and collagen synthesis in normal middle ear fibroblasts

Summary The present study was undertaken to quantitate the effects of atmospheric air and normal middle ear gas on cultured fibroblasts obtained from normal rabbit middle ear mucosa. The cells were exposed to three different gas compositions: 7% O₂:5% CO₂:88% N₂, 21% O₂:5% CO₂:74% N₂, and 75% O₂:5% CO₂:20% N₂. The growth was monitored by measuring the total content of cell protein, the amount of DNA, and the cell division activity. The activity of the synthetic apparatus was determined by the collagen synthesis. For comparison, rabbit skin fibroblasts were grown under identical conditions. The results demonstrated significantly higher replication rate of middle ear fibroblasts at 7% oxygen than at atmospheric air whereas the collagen synthesis was significantly lower at 7%. Furthermore, the responses varied significantly between rabbit middle ear and rabbit skin fibroblasts. Thus the present study substantiates the hypothesis of an influence of atmospheric air on the middle ear mucosa which might be of importance, e.g., in relation to insertion of ventilation tubes or longstanding perforations of the tympanic membrane in otitis media.     

Regulation of growth and differentiation by vitamin a in a cloned fetal lung epithelial cell line cultured on collagen gell in hormone-supplemented medium

Summary Proliferative and differentiative responses to various doses of vitamin A (VA) were studied in the predifferentiated cells of a fetal Syrian hamster pulmonary epithelial line (M3E3/C3), which were cultured on a collagen gel in a hormone-supplemented medium. These predifferentiated cells possessed well-developed endoplasmic reticulum (ER) and Golgi apparatus. At VA doses higher than 8 μg/ml, periodic acid Schiff and slightly alcian blue positive mucuslike granules were produced, which were also detectable electron microscopically. These mucuslike products were rich in sialic acid and resembled quite well those from primary cultures of tracheal epithelial cells of Syrian hamster sucklings when analyzed by column chromatography on various types of gel. At all VA doses studied (2.4, 8, 24 μg/ml), cells grew exponentially with an average population doubling time of around 74 h, whereas in the absence of VA they had a linear growth rate and a population doubling time of 158 h between Days 4 and 11. The uptake of [³H]glucosamine into the whole cell homogenates showed a peak at Day 8, irrespective of VA doses (0 to 24 μg/ml), and at the highest VA dose (24 μg/ml) it exceeded by twofold the control (0 μg/ml) level. At the same time, [¹⁴C]thymidine demonstrated a high peak of uptake on Day 8 at 8 and 24 μg/ml VA. There was virtually no difference between 0 and 2.4 μg/ml VA, with both doses yielding much lower peaks. Based on the results currently presented and previously reported, three successive stages were hypothesized for the mucous differentiation processes in M3E3/C3. The process from the first undifferentiated stage to the second predifferentiated stage with well-developed ER and Golgi apparatus requires both collagen gels and hormones. Differentiationn from the second stage to the third secretory stage with mucous granules is stimulated by VA. These observations indicate that the cell line M3E3/C3 could provide a new system for investigating the mechanisms of mucus differentiation by VA. This study was partly supported by a grant for Humanisierung des Arbeitslebens from Bundesministerium für Forschung und Technologie     

Comparison of isoperoxide patterns in tobacco cell culture and in the intact plant

Analysis by ion-exchange chromatography of the enzymes from cultured tobacco cells and root or leaf tissues of the tobacco plant revealed that the cultured cells contain exclusively cationic peroxidases and the leaf tissues mainly anionic and neutral peroxidases.     

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

Fish cell culture: Characteristics of a cell line from the silver perch,Bairdiella chrysura

Summary A cell line designated SP-1 was established from tissue of the silver perch,Bairdiella chrysura. Cells were fibroblast-like and grew best at 26°C in Leibovitz medium (L-15) containing 15% fetal bovine serum and 0.150m sodium chloride. Passage 1 to passage 9 SP-1 cells contained a chromosome number of 48; at passages 27 and 50 the modal numbers were 51 and 54, respectively. Confirmation of the origin of SP-1 cells was made by the cytotoxic antibody dye-exclusion test. This cell line supported the growth of lymphocystis virus from the silver perch but was not found to replicate various other fish and mammalian viruses. This study was supported in part by fund supplied by the Faculty Research Council of the University of Southern Mississippi and by the National Oceanographic and Atmospheric Administration, Office of Sea Grant No. 04-3-158-58. A portion of these results were presented at the 26th Annual Meeting of the Tissue Culture Association, Motreal, Canada, 1975.     

Infection and development of Nosema bombycis (Microsporida: Protozoa) in a cell line of Antheraea eucalypti

Spores of Nosema bombycis derived from diseased insects were highly purified by Urografin density gradient centrifugation. Antheraea eucalypti cells were inoculated with the purified spores primed with 0.1 n KOH solution to start a continuous propagation of N. bombycis in cell culture. The first increase in the number of infected A. eucalypti cells was observed at 48 hr postinoculation, and it was caused by the secondary infective forms of N. bombycis. The secondary infective forms were produced during the course of sporoblast differentiation. The parasites in cell cultures divided synchronously until 36 hr postinoculation. Mature spores were observed initially 6 days postinoculation at 27°C. The infected cultures were subcultured extensively for more than 1 year with the addition of healthy A. eucalypti cells.     

Induction of apoptosis in normal cultured rat hepatocytes and in Hep3B, a human hepatoma cell line

The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions. Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells. In this study, we compared the effects of several apoptosis-inducing agents – transforming growth factor β₁ (TGF-β₁), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) – on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h. Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining. DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot. Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry. Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA. Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-β₁; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively). OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-β₁, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h. In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis. In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of donor plant genotype and growth environment on anther culture of soft-red winter wheat (Triticum aestivum L.)

The effects of donor plant growth temperature and photoperiod on embryo formation and plant regeneration from cultured anthers in five genotypes of soft-red winter wheat (Triticum aestivum L.) were examined. There were no significant differences between the three environments studied (15°C - 16/8 h light/dark, 20°C - 16/8 h light/dark, and 20°C - 12/12 h light/dark) when frequencies were averaged over genotypes; however, significant genotype and genotype x environment interactions were observed for embryo formation. When averaged over environments, highest embryo and plant production frequencies were exhibited by a line derived from the cross IL 72-2219-1/Amigo. A mean of 8.6 embryos per 100 anthers plated was observed for this genotype grown in the 20°C - 16/8 h light/dark environment. The cultivar Scotty averaged 4.2 plants produced per 100 anthers plated when grown in the 15°C - 16/8 h light/dark environment. The results from this study suggest a potential for increasing embryo and plant production in this material and point toward the need to optimize donor plant growth environmental conditions to maximize response frequencies for specific genotypes of interest.     

Effect of peripheral nerve on the neurite growth from retinal explants in culture

The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined.Cultures of retinal explants,and co-cultures of retinal explants and PN were performed using chick retinal basement memebrane (BM) as substrate.The presence of PN increases the number and length of neurite outgrowth.In addition,a high proportion of neurites situated close to PN tend to grow towards it.Since there was no contact between retinal explants and PN,we suggest that PN might secete diffusible substances to attract the neurites to grow towards it.     

Epidermal growth factor (EGF)-nonresponsive variants of normal rat kidney cell line: response to EGF and transforming growth factor-beta

Anchorage-independent growth in soft agar of normal rat kidney (NRK) fibroblasts depends on both transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) (or TGF-alpha). We have isolated two EGF-nonresponsive cell lines, N-3 and N-9, from chemically mutagenized NRK cells, after selection of mitogen-specific nonproliferative variants in the presence of EGF and colchicine. Saturation binding kinetics with 125I-EGF showed one-half or fewer EGF receptors in N-3 and N-9 than in their parental NRK. Cellular uptake of 2-deoxy-D-glucose was enhanced in all NRK, N-3, and N-9 cell lines by TGF-beta treatment, whereas treatment with EGF significantly enhanced the cellular uptake of the glucose analog in NRK cells, but not in N-3 and N-9 cells. DNA synthesis of NRK during the quiescent state, but not that of N-3 and N-9, was stimulated by EGF. Anchorage-independent growth of N-9 could not be observed even in the presence of both EGF and TGF-beta, whereas that of N-3 was significantly enhanced by TGF-beta alone. EGF stimulated phosphorylation of a membrane protein with molecular size 170 kDa of NRK, but not of N-3, when immunoprecipitates reacting with anti-phosphotyrosine antibody were analyzed. Exposure of NRK cells to EGF increased cellular levels of TGF-beta mRNA, but there appeared little expression of TGF-beta mRNA in N-3 and N-9 cells. Exposure of N-3 cells to EGF or TGF-beta enhanced the secretion of EGF into culture medium, but exposure of NRK or N-9 cells did not. Altered response to EGF of N-3 or N-9 might be related to their aberrant growth behaviors.