Defined Tissue Culture Medium Research Articles

Susceptibility of Candida Species to Photodynamic Effects of Photofrin

The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clinically approved photosensitizing agent Photofrin was examined. Internalization of Photofrin by Candida was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concentration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin uptake was poor in C. albicans blastoconidia grown in nutrient broth. However, conversion of blastoconidia to filamentous forms by incubation in defined tissue culture medium resulted in substantial Photofrin uptake. Under conditions where Photofrin was effectively taken up by Candida, irradiated organisms were damaged in a drug dose- and light-dependent manner. Uptake of Photofrin was not inhibited by azide, indicating that the mechanism of uptake was not dependent on energy provided via electron transport. Fungal damage induced by Photofrin-mediated photodynamic therapy (PDT) was determined by evaluation of metabolic activity after irradiation. A strain of C. glabrata took up Photofrin poorly and was resistant to killing after irradiation. In contrast, two different strains of C. albicans displayed comparable levels of sensitivity to PDT. Furthermore, a reference strain of C. krusei that is relatively resistant to fluconazole compared to C. albicans was equally sensitive to C. albicans at Photofrin concentrations of >/=3 microg/ml. The results indicate that photodynamic therapy may be a useful adjunct or alternative to current anti-Candida therapeutic modalities, particularly for superficial infections on surfaces amenable to illumination.

A hypothesis for the development of a defined tissue culture medium of higher plants and micropropagation of hazelnuts

We have hypothesized that the composition of minerals and organic substances in proportions similar to those found in the seed composition could provide an optimum tissue culture medium for micropropagation of higher plants. This tissue culture medium development method avoids factorial treatments, thus requires less labor, fewer explants and reduces the size of experiments necessary for development of a defined tissue culture medium. Applying this approach, we first developed a new tissue culture medium (NM) for hybrid hazelnuts. Growth of explants cultured on NM was compared to that of explants cultured on woody plant medium (WPM) [Proc. Int. Plant Prop. Soc. 30 (1980) 421], NN [Science 163 (1969) 85], DKW [HortScience 19 (1984) 507] and MS [Physiol. Plant. 15 (1962) 473] media. Mean shoot lengths observed on NM were up to threefold higher than that on any other media, and the potential multiplication rates observed on NM (up to 107%) and WPM media (up to 85%) were higher than those on other media. When the composition of NM was further improved (Nas and Read medium (NRM)) in accordance with our hypothesis, shoot lengths (up to threefold) and potential multiplication rates (up to 93%) were further enhanced. A tissue culture medium and a micropropagation protocol are presented that theoretically could produce, from a single-bud explant cultured for 1 year involving nine subcultures, 2.3–8.7 million shoots, even if 20% of shoot loss occurs at each subculture. Results suggest that using mineral content of kernels as the culture medium could allow micropropagation of a recalcitrant species on a commercial scale.

Candida albicans ALS1: domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif

Transfer of budding Candida albicans yeast cells from the rich, complex medium YEPD to the defined tissue culture medium RPMI 1640 (RPMI) at 37 degrees C and 5% CO2 causes rapid onset of hyphal induction. Among the genes induced under these conditions are hyphal-specific genes as well as genes expressed in response to changes in temperature, CO2 and specific media components. A cDNA library constructed from cells incubated for 20 min in RPMI was differentially screened with yeast (YEPD)- and hyphal (RPMI)-specific probes resulting in identification of a gene expressed in response to culture conditions but not regulated by the yeast-hyphal transition. The deduced gene product displays significant identity to Saccharomyces cerevisiae alpha-agglutinin, encoded by AG alpha 1, an adhesion glycoprotein that mediates mating of haploid cells. The presence of this gene in C. albicans is curious since the organism has not been observed to undergo meiosis. We designate the C. albicans gene ALS1 (for agglutinin-like sequence). While the N- and C-termini of the predicted 1260-amino-acid ALS1 protein resemble those of the 650-amino-acid AG alpha 1, ALS1 contains a central domain of tandem repeats consisting of a highly conserved 36-amino-acid sequence not present in AG alpha 1. These repeats are also present on the nucleotide level as a highly conserved 108 bp motif. Southern and Northern blot analyses indicate a family of C. albicans genes that contain the tandem repeat motif; at least one gene in addition to ALS1 is expressed under conditions similar to those for ALS1 expression. Genomic Southern blots from several C. albicans isolates indicate that the number of copies of the tandem repeat element in ALS1 differs across strains and, in some cases, between ALS1 alleles in the same strain, suggesting a strain-dependent variability in ALS1 protein size. Potential roles for the ALS1 protein are discussed.

Hormonal control of proliferation in the Ishikawa endometrial adenocarcinoma cell line

Steroid hormone regulation of proliferation of the Ishikawa human endometrial adenocarcinoma cell line was investigated in defined tissue culture medium. Oestradiol increased cell number following treatment for greater than 8 days; 4-OH tamoxifen, used alone, induced growth in a similar manner to oestradiol and was not antagonistic when used in combination with oestradiol. Progesterone decreased cell number 4 days after treatment but thereafter the effect was lost; the effect of progesterone was abolished in the presence of Phenol Red, consistent with the oestrogenic properties of this indicator. Oestradiol together with progesterone for greater than 8 days resulted in maximal growth and was preceded by an apparent increase in synthesis of a protein of molecular weight 36 kDa pI 8.

Estrogen-dependent DNA synthesis in cultures of Xenopus liver parenchymal cells.

We have recently shown that extensive proliferation of liver parenchymal cells takes place in adult male Xenopus frogs in response to estradiol-17 beta, which also induces synthesis and secretion of vitellogenin, the precursor of yolk proteins. We demonstrate here that liver parenchymal cells from adult male animals can be maintained for several weeks in a defined tissue culture medium containing added insulin, dexamethasone, and triiodothyronine. Under these conditions the cells do not divide, but can synthesize DNA. Maximum DNA synthesis occurs in cells that have achieved monolayer morphology under low plating densities. Estradiol-17 beta causes a dose-dependent increase in the number of cells synthesizing DNA, as well as inducing synthesis of vitellogenin. Estrogen-dependent, but not background, DNA synthesis is inhibited by the antiestrogen tamoxifen. These results imply that estradiol-17 beta acts directly on liver cells to initiate DNA replication, probably by interaction with a receptor protein and induction of specific gene transcription.

Characterization of human pulmonary endocrine cells maintained in vitro.

Endocrine cells located in the epithelium of human fetal airways contain the amine, 5-hydroxytryptamine (5HT), and the peptide, bombesin (BOM), but difficulties in studying these cells experimentally have slowed progress in understanding their functional roles. This investigation describes an in vitro method to maintain pulmonary neuroendocrine cells (PNEC) in organ culture. Bronchial trees from human fetal lungs were dissected free of adherent blood vessels and lung tissue. Explants of the airways were placed in culture dishes containing defined tissue culture medium for five days. Using indirect immunofluorescence, 5HT- and BOM-like immunoreactive cells were observed both in nonincubated airways and in explants maintained for five days in organ culture. The number of 5HT-immunoreactive cells/0.1 mm2 of airway epithelium was not significantly different in the two groups, although there was a significant reduction in 5HT content measured by HPLC after the five-day culture period. The diameter of dense core vesicles and the number of dense core vesicles/micron2 of endocrine cell cytoplasm in cultures were not significantly different from non-incubated controls. Treatment of the explants with the 5HT-synthesis inhibitor p-chlorophenylalanine resulted in a significant reduction both in the number of 5HT-containing cells/0.1 mm2 of airway epithelium and in the 5HT content. These results demonstrate that both 5HT and BOM content in endocrine cells of explants from human fetal airways can be well maintained in organ culture for at least 5 days and that they are responsive to pharmacologic inhibition of 5HT synthesis.

Regulation of tadpole spinal nerve fiber growth by the regenerating limb blastema in tissue culture.

Explants of frog tadpole spinal cord cocultured with blastemas of regenerating hindlimbs provide in vitro evidence that the mesenchymelike cells of the blastema may serve to elicit nerve fiber growth and sprouting into the limb stump. The present study indicates that (1) blastemal mesenchyme results in enhanced, directed nerve fiber growth and its extended survival in defined tissue culture medium; (2) the amount of nerve growth decreases in the presence of blastemas from limbs incapable of complete regeneration; and (3) the quantity of nerve fiber outgrowth decreases as the developmental stage of the spinal cord increases. It is hypothesized that one of the requisites for limb regeneration is the production of a distal population of dedifferentiated cells capable of eliciting nerve fiber outgrowth from competent neural tissue in an amount that is sufficient to promote nerve-dependent regeneration.

Development of an in vitro technique for cytological investigations of slices of Fasciola hepatica : Evaluation by morphological criteria

The development of a new in vitro system for investigating the cytology of Fasciola hepatica is described. The technique was designed to allow test substances, such as drugs and radioactive precursors, to gain direct access to internal cells and organs not normally in contact with the external environment. It involves incubation of 1 mm-thick transverse slices of flukes under controlled physico-chemical conditions resembling those pertaining to sheep's bile ducts. To decide whether a gluco-saline medium or a tissue culture medium was more suitable for maintaining the slices in vitro, experiments were carried out using 4 different media for up to 20 h. Subsequent electron microscope examination of the slices showed that M199, a completely defined tissue culture medium, gave the best ultrastructural preservation. In this medium, slices remained in good condition for about 10 h.

Specific protein synthesis in cellular differentiation: II. The program of protein synthetic changes during chorion formation by silkmoth follicles, and its implementation in organ culture

During their differentiation, the follicular epithelial cells of the silkmoth, Antheraea polyphemus, produce the extracellular proteinaceous eggshell or chorion. Choriogenesis entails continuous changes in cell-specific protein synthesis; the various chorion proteins are synthesized with distinct kinetics. On the basis of protein synthetic profiles, 17 stages of choriogenesis are defined. The average duration of the stages is 3.0 hr, and thus choriogenesis lasts a total of approximately 51 hr. This program of protein synthetic changes is autonomous; i.e., it is implemented with normal kinetics by follicles cultured in isolation in a defined tissue culture medium.

Schistosoma mansoni: Defined system for stepwise transformation of cercaria to schistosomule in vitro

Defined conditions are described for the in vitro production of large numbers of tail-free viable schistosomules. These consist of (1) the centrifugation of cold cercarial suspension and the incubation of the packed cercariae in a minimal volume of medium at 30 C for 40 min to effect tail loss and glandular secretion; (2) the isolation of the bodies by resuspension and sedimentation and (3) the induction of surface changes by incubating the bodies in inactivated serum or a defined tissue culture medium for a further 40 min interval at 37 C with mild agitation. The resultant schistosomules are characterized by the depletion of their penetration gland contents, loss of tail, fluoride and water sensitivities, complement insensitivity, negative “Cercarien-hüllen Reaktion,” and loss of the surface coat as demonstrated by periodic acid-Schiff (PAS) staining and electron microscope observations.

A Comparison of Wound Repair in Cultured Lenses with that Occurring in the Lenses of Living Animals

Wound repair has been studied in needle injured lenses. A comparison has been made between injuries that were allowed to heal in the animal and others made on tissue maintained under in vitro conditions in a completely defined tissue culture medium. Observations were performed by phase and differential interference microscopy on living material. Fixed and stained whole-mounts were also evaluated. The general features of the response in both types of system are similar. The reaction consists of migration followed by increased proliferation. However, in the in situ tissue this reaction is much more consistent and pronounced. The possibilities offered by the in vitro system are discussed.

Studies on collagenase from rheumatoid synovium in tissue culture.

Fragments of synovium from patients with rheumatoid arthritis survive in defined tissue culture medium in the absence of added serum and, after 3-4 days, release into the medium enzyme capable of degrading undenatured collagen. Maximal activity is observed at pH 7-9 but the enzyme is inactive at pH 5. At temperatures of 20 degrees and 27 degrees C, collagen molecules in solution are cleaved into 3/4 and 1/4 length fragments with minimal loss of negative optical rotation, but with loss in specific viscosity of approximately 60%. Above 30 degrees C the fragments begin to denature and denaturation is complete at 37 degrees C. If the enzyme is not inhibited at this stage the large fragments are broken down further to polypeptides of low molecular weight. Reconstituted collagen fibrils and native fibers at 37 degrees C are cleaved to the low molecular weight fragments, although the fibrils are resistant to breakdown at lower temperatures (20 degrees -27 degrees C). It is proposed that the production of such an enzyme by inflamed and proliferating rheumatoid synovium may be responsible for some of the destruction of collagenous structures that accompanies rheumatoid arthritis.

The Influence of NCTC 109, Serum, and Swine Kidney Cell Cultures on the Morphogenesis of Stephanurus dentatus to Fourth Stage, In vitro

Stephanurus dentatus was grown to advanced parasitic stages in a new static in vitro system, free of bacteria and fungi. Criteria for evaluation were based on anatomical characters of the head, stoma, excretory system. and intestine. Descriptions of these characters and observations on morphogenesis not previously reported from in vivo specimens are given in detail. Development to late fourth stage was obtained in cultures consisting of primary monolayers of swine kidney cells overlaid with the following medium (KW-1S): yeast extract (BBL), 112.5 mg; Bacto-peptone (Difco), 140.65 mg; dextrose (BBL), 140.65 mg; swine serum, 50 ml; NCTC 109, 50 ml; and antibiotics to give a final concentration of 1,000 units penicillin G potassium, 1 mg dihydrostreptomycin, and 10 tLg Amphotericin B per ml of medium. When bovine serum was substituted (KW-1) development halted at mid fourth stage. In addition, with both fluid media, the following were observed: only larvae in fourth stage fed on the kidney cell cultures; a definite feeding pattern occurred; larvae fed on rolled cell layers (presumably dead) as well as living cells; digestion and egestion took place, and there was a correlation between feeding behavior and anatomical development of the stoma, excretory system, and intestine. In either medium, without the cell culture, only early fourth stage was attained and yields of this phase of development were reduced to one-fortieth. Media KW-1 and KW-1S evolved from attempts to culture S. dentatus in modifications of Pitts' and Ball's (1953) medium, or in NCTC 109 (McQuilkin et al., 1957) used alone or supplemented with calf serum. The prototype cell-free media, one of which was also supplemented with nonliving tissue, had the following effects on development: (1) the most advanced stage attained was early fourth stage; (2) larvae advanced to late parasitic third stage in media supplemented with either NCTC 109 or serum; (3) development proceeded to early fourth stage when both NCTC 109 and serum were present; (4) a more rapid rate of development to third molt and early fourth stage ensued when NCTC 109 and serum were present at concentrations of 50%, each; and (5) although fourth-stage larvae ingested nonliving tissues when available, development was not enhanced. In swine, Stephanurus dentatus Diesing, 1839 undergoes its entire parasitic development to the fifth stage (young adult) in the liver and to the sexually mature adult in the kidney and perirenal fat (Schwartz and Price, 1929, 1931, 1932; Clunies Ross and Kauzal, 1932). In unpublished observations, one of us (FGT) has noted that young and sexually mature adults invariably have host tissue in their intestine. We assumed, therefore, that host tissues are probably utilized in vivo; and since S. dentatus has been cultured only to early fourth stage in cell-free media (Douvres and Tromba, 1962), we conjectured that host tissue may also be necessary for advanced parasitic development in vitro. Accordingly, attempts were made to cultivate advanced stages of S. dentatus in the following three static systems: (1) eight cell-free media not previously used for S. dentatus; (2) two media (KW and KW-1S) selected from system 1 and Received for publication 8 April 1966. supplemented with nonliving tissues; and (3) two media (KW-1 and KW-1S) selected from system 1 and used as an overlay for primary swine kidney cell cultures. Media KW, KW-1, and KW-1S (Table I) are new formulations that evolved from our attempts to culture S. dentatus in modifications of Pitts' and Ball's (1953) medium or in the chemically defined tissue culture medium NCTC 109 (McQuilkin et al., 1957). The latter was used alone or supplemented with calf serum. Pitts' and Ball's medium, in addition to serum, includes yeast extract, peptone, glucose, and a balanced salt solution (Fenwick, 1939). These investigators were unable to obtain development or growth of Ascaris suum larvae that were hatched from eggs in this medium. Weinstein and Jones (1959), Sawyer and Weinstein (1961, 1962, and 1963), and Weinstein et al. (1963) reported on their attempts to cultivate larval stages of several nematode parasites of vertebrates in NCTC