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PubMed1990

A band of F-actin containing podosomes is involved in bone resorption by osteoclasts.

Isolated rabbit osteoclasts cultured on devitalized thin bone slices excavate resorption lacunae that can be visualized with brightfield or phase-contrast microscopy. Superimposition of the brightfield images of such resorption lacunae and the fluorescence images of the corresponding osteoclasts after fixation and staining with rhodamine-conjugated phalloidin revealed that a bright fluorescent band of F-actin-containing podosomes precisely outlined the resorption lacunae in stationary osteoclasts. When the resorption lacunae were being extended laterally, the clearly delineated band of podosomes corresponded to the advancing edge of the resorbing osteoclast and the most recently excavated part of the lacunae. Reshaping and reorganization of the bright bands preceded development of the lateral boundary of the lacunae. Podosomes forming these bands were highly dynamic, changed in size and location, and appeared and disappeared continuously. Their lifespan varied between 2 and 12 min. Similar bands were also seen in vivo in bone-resorbing osteoclasts on the endocranial surface of growing calvariae. Podosomes disappeared in osteoclasts treated with calcitonin, resulting in the disruption of the fluorescent bands. Our results suggest that podosomes are an essential part of the resorption apparatus of osteoclasts.

Actins↗
PubMed1996

Effects of triiodothyronine on morphology, growth behavior, and the actin cytoskeleton in mouse osteoblastic cells (MC3T3-E1).

We investigated the effects of thyroid hormone treatment on morphology, growth behaviour, and cytoskeletal structures of long-term cultured MC3T3-E1 cells. Morphological investigations were carried out on native cells by phase contrast microscopy and on epon-embedded semithin sections. The area covered by the cell and matrix layers (tissue-like area), percent extracellular matrix, average height of tissue-like area, and length and height of single cells were measured histomorphometrically on the cross sections. F-actin was analyzed histochemically and quantitated after fluorochrome-labeled phalloidin staining using confocal microscopy and fluorometry. Significant differences between control and T3-treated cells were found after confluency, but not in subconfluent cultures. Control cells continued to proliferate forming multilayers, and produced increasing amounts of extracellular matrix. In contrast, T3-treated cells stopped to proliferate forming two cell layers at the maximum. These cells were flattened, distinctly enlarged, and polygonal in shape. Histochemical staining for F-actin revealed three different staining patterns, depending on the position of the cell within the multilayer of control cultures. Basal cells contained a large number of thick stress fibers in parallel arrangement. Intermediate cells exhibited only a few thick actin filament bundles located at the outermost periphery. The superficial cells were characterized by a large number of thin, parallel-oriented microfilament bundles extending across the entire cytoplasm. The actin pattern of T3-treated cells resembled that of the basal cell layer of the control cells. The amount of F-actin increased with the prolonged T3 treatment. We conclude from these data that the known specific cellular responses to T3 treatment are accompanied by significant morphological alterations indicating pivotal effects of thyroid hormones on osteoblastic differentiation.

3T3 Cells↗
PubMed2006

A relevant in vitro eukaryotic live-cell system for the evaluation of plasmodial protein localization.

Understanding the functional genomics and proteomics of plasmodia underpins the development of new approaches to antimalarial chemotherapy. Although genome databanks (e.g. PlasmoDB) and biocomputing tools (e.g. PlasMit, PlasmoAP, PATS) are useful in providing a global albeit predictive view of the myriad of about 5000 genes, only 40% are annotated, with few cases of endorsed subcellular localizations of the corresponding proteins in animal models. Progress in plasmodial protein trafficking has been hampered by the lack of a simple yet reliable method for studying subcellular localization of plasmodial proteins. In this study, we have used a combination of fluorescent markers, organelle-specific probes, phase contrast microscopy, and confocal microscopy to locate a selection of signal peptides from 10 plasmodial proteins in CHO-K1 cells. These eukaryotic cells serve as an in vitro living system for studying the cellular destinations of four mitochondrial-targeted TCA cycle proteins (citrate synthase, CS; isocitrate dehydrogenase, ICDH; branched chain alpha-keto-acid dehydrogenase E1alpha subunit, BCKDH; succinate dehydrogenase flavoprotein-subunit, SDH), two nuclear-targeted proteins (histone deacetylase, HDAC; RNA polymerase, RPOL), two apicoplast-targeted proteins (pyruvate kinase 2, PK2; glutamate dehydrogenase, GDH), and two cytoplasmic resident proteins (malate dehydrogenase, MDH; glycerol kinase, GK). The respective localizations of these malarial proteins have complied with the selected molecular targets, viz. mitochondrial, nuclear and cytoplasmic. Interestingly, MDH that is widely known to be resident in eukaryotic mitochondria was found to be cytoplasmic, probably due to the absence of molecular target sequences. Since the localization of plasmodial proteins is central to the authentication of their pathophysiological roles, this experimental system will serve as a useful a priori approach.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2005

Mylar and Teflon-AF as cell culture substrates for studying endothelial cell adhesion.

The textured and opaque nature of Dacron and ePTFE has prevented the use of these fabrics in conventional cell culture techniques normally employed to optimize cell attachment and retention. This lack of optimization has led, in part, to the poor performance of endothelialization strategies for improving vascular graft patency. Here we show that thin, transparent films of Mylar and Teflon-AF are viable in vitro cell culture mimics of Dacron and ePTFE vascular graft materials, particularly for the study of protein mediated endothelial cell (EC) attachment, spreading and adhesion. Glass substrates were used as controls. X-ray photoelectron spectroscopy (XPS) and contact angle analysis showed that Mylar and Teflon-AF have surface chemistries that closely match Dacron and ePTFE. (125)I radiolabeling was used to quantify fibronectin (FN) adsorption, and FN and biotinylated-BSA "dual ligand" co-adsorption onto glass, Mylar and Teflon-AF substrates. Native human umbilical vein endothelial cells (HUVEC) and streptavidin-incubated biotinylated-HUVEC (SA-b-HUVEC) spreading was measured using phase contrast microscopy. Cell retention and adhesion was determined using phase contrast microscopy under laminar flow. All surfaces lacking protein pre-treatment, regardless of surface type, showed the lowest degree of cell spreading and retention. Dual ligand treated Mylar films showed significantly greater SA-b-HUVEC spreading up to 2 h, but were similar to HUVEC on FN treated Mylar at longer times; whereas SA-b-HUVEC spreading on dual ligand treated Teflon-AF was never significantly different from HUVEC on FN treated Teflon-AF at any time point. SA-b-HUVEC retention was significantly greater on dual ligand treated Mylar compared to HUVEC on FN treated Mylar over the entire range of shear stresses tested (3.54-28.3 dynes/cm(2)); whereas SA-b-HUVEC retention to dual ligand and HUVEC retention to FN treated Teflon-AF gave similar results at each shear stress, with only the mid-range of stresses showing significant difference in cell retention to Teflon-AF.

Biocompatible Materials↗
PubMed2006

Morphological and growth alterations in Vero cells transformed by cisplatin.

Cisplatin is an antineoplastic agent used to treat solid tumours, such as ovarian, testicular and bladder tumours. However, studies in vitro and in vivo have shown that cisplatin is mutagenic, genotoxic and tumorigenic in other tissues and organs. In this work, we examined the effect of cisplatin on Vero cells, a fibroblast-like cell line. The morphological characteristics were investigated using phase contrast microscopy, scanning electron microscopy and the actin cytoskeleton was labelled with fluorescein isothiocyanate-phalloidin. Cell proliferation was assessed based on the growth curve. Cultured Vero cells treated with cisplatin showed behavioural and morphological alterations associated with cellular transformation. The transformed cells grew in multilayers and formed cellular aggregates. The proliferation and morphological characteristics of the transformed cells were very different from those of control ones. Since transformed Vero cells showed several characteristics related to neoplastic growth, these cells could be a useful model for studying tumour cells in vitro.

Actins↗
PubMed2005

Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture.

The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used as an alternative basement membrane-containing matrix to support RPE growth and differentiation. Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0.1 mM Ca(++) and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000-9000 cells cm(-2) in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca(++) concentration in the medium was increased to 1.8 mm for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24 hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca(++) switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.

Amnion↗
PubMed2004

Water-mediated lysis of lens epithelial cells attached to lens capsule.

PURPOSE: To investigate the effect of distilled deionized water (DDW) on lens epithelial cells (LECs) attached to the lens capsule. SETTING: Wound Healing Research Laboratory, Center for Vision Research, Westmead Hospital, Sydney, NSW, Australia. METHODS: Anterior capsulotomy specimens taken during routine cataract surgery were divided in half. One half was immersed in DDW and the other half in culture medium (control) for 1 to 5 minutes and photographed at intervals by phase-contrast microscopy. In further experiments, the capsules were exposed to DDW for 1 or 2 minutes and placed in culture for 1 week to determine whether LECs survive treatment and are capable of repopulating the lens capsule. RESULTS: Distilled-deionized water induced marked swelling of the cytoplasm within 60 seconds of treatment. At 120 seconds, there was disruption of the plasma membranes, with few intact cells remaining. In the control capsules, confluent monolayers of LECs covered the entire capsule surface with a halo of LECs growing on the surrounding plastic well. Viable LECs were observed in 1 of 3 capsules treated for 1 minute with DDW. These did not reach confluence or grow off the capsule onto the surrounding well. No viable LECs were seen on capsules exposed to DDW for 2 minutes. CONCLUSIONS: Short exposure of LECs to DDW induced extensive and rapid cell lysis. Distilled-deonized water may be a useful agent for instillation in the capsular bag during sealed-capsule irrigation to prevent posterior capsule opacification.

Cataract Extraction↗
PubMed2005

Hematuria in thin basement membrane nephropathy.

Thin basement membrane nephropathy (TBMN) often is diagnosed clinically when there is persistent dysmorphic or glomerular hematuria, but minimal proteinuria, normal kidney function, and no other obvious cause. This study investigated hematuria in patients with TBMN. A total of 112 patients with biopsy examination-proven TBMN were studied. All had hematuria at the time of presentation, with a mean urinary red blood cell (RBC) count of 256 +/- 250 x 10 3 /mL. Seventy-five (67%) patients attended for review over a median of 48 months (range, 3-120 mo) and provided a total of 485 urine specimens. Twenty-one patients (28%) had no hematuria by phase-contrast microscopy on at least 1 occasion. These corresponded to 32 urine specimens (7% of total). Of the 21 patients, the most recent urinary RBC counts were within the normal range in 11 (52%), but hematuria had recurred in the other 10 (48%). Hematuria is persistent in most patients with TBMN, but occasionally it resolves or is intermittent.

Diagnosis, Differential↗
PubMed2006

Challenges in cryopreservation of clouded leopard (Neofelis nebulosa) spermatozoa.

The clouded leopard (Neofelis nebulosa) is an endangered species that is difficult to breed in captivity. Species management could benefit from the use of artificial insemination (AI) with frozen-thawed spermatozoa, but there have been no detailed studies of sperm cryosensitivity. The purposes of this study were to: (1) re-characterize seminal characteristics in the clouded leopard 20 years after the first descriptive studies Wildt et al., [Wildt DE, Howard JG, Hall LL, Bush M. Reproductive physiology of the clouded leopard. I. Electroejaculates contain high proportions of pleiomorphic spermatozoa throughout the year. Biol Reprod 1986; 34: 937-947]; and (2) conduct a comparative cryopreservation study on the feasibility of sperm from this species surviving a freeze-thawing stress. Ejaculates were collected from five adult males and subjected to standard analysis, followed by a two-step straw freezing protocol that evaluated the impact of thawing, dilution, centrifugation and in vitro culture (through 4 h) on sperm motility and acrosomal integrity. Additionally, we assessed the impact of both a traditional permeating cryoprotectant (glycerol at a final dilution of 4%) and an unconventional nonpermeating trisaccharide; raffinose (R) at a final dilution of 4% or 8%, with or without 4% glycerol on sperm cryosurvival. The clouded leopard produced an extremely poor quality ejaculate; although approximately 70% of fresh sperm were motile, >80% were malformed. Phase contrast microscopy revealed that 40% of all sperm had abnormal acrosomes, but Coomassie blue staining indicated that acrosomal abnormalities existed in almost 70% of spermatozoa. Upon freeze-thawing, sperm motility declined markedly (P < 0.05) by an average of 40%, regardless of diluent used. Interestingly, raffinose was as effective as glycerol in protecting both sperm motility and acrosomal integrity. Although no acrosomal damage was seen immediately after thawing, < 6% morphologically normal intact acrosomes were present by the last measured time point. In conclusion, the clouded leopard is a rare felid that (at least in North American zoos) is producing extraordinarily poor quality ejaculates. There are so many sperm with unexplained deranged acrosomes that it will be particularly challenging to use traditional AI with thawed sperm as an adjunct management tool.

Animals↗
PubMed2000

Functional characterization of purified zinc transporter from renal brush border membrane of rat.

Major zinc binding protein purified from renal brush border membrane (BBM) (R. Kumar, R. Prasad, Biochim. Biophys. Acta 1419 (1999) 23) was reconstituted into liposomes and its functional characteristics were investigated. Physical incorporation of the major zinc binding protein into the proteoliposomes was checked by SDS-PAGE, which showed a single band on silver staining. The structural integrity of the proteoliposomes was assessed by phase contrast microscopy, which revealed the proteoliposomes as globular structures and intact boundaries. Further structural integrity/leakiness of the proteoliposomes was checked by monitoring efflux of Zn(2+) from the pre-loaded proteoliposomes in the presence of either 2 mM Ca(2+) or Cd(2+) or Zn(2+). It was observed that even after 2 h of the initiation of efflux, 85-95% of Zn(2+) was retained in the proteoliposomes, thereby indicating that proteoliposomes were not leaky and maintained structural integrity during the uptake study. Zinc uptake into the proteoliposomes followed Michaelis-Menten kinetics with affinity constant (K(m)) of 1.03 mM and maximal velocity (V(max)) of 1333 nmol/mg protein per min. The uptake process followed first-order kinetics with a rate constant (k) of 1. 09x10(-3) s(-1). The specificity of zinc transport system was determined by studying the interaction of divalent cations viz. Ca(2+) and Cd(2+) with the zinc uptake. It was observed that Cd(2+) competitively inhibited the zinc uptake process with inhibitory concentration (K(i)) of 2.9 mM. Kinetic analysis of inhibitory effect of Cd(2+) on zinc uptake revealed an increase in K(m) to 1.74 mM without influencing V(max). Zn(2+) uptake into the proteoliposomes was found to be temperature sensitive and Arrhenius plot showed a breakpoint at 27 degrees C. The apparent energies of activation (E(a)) were found to be 7.09 and 2.74 kcal/mol below and above the breakpoint, respectively. The initial velocity of Zn(2+) uptake increased with the increase in outwardly directed proton gradient ([H](i) greater than [H](o)). The Zn(2+) uptake was inhibited by DCCD, thereby suggesting the involvement of -COOH groups in the translocation of Zn(2+) across the lipid bilayer. The ratio of acidic to basic amino acids (1.26) strongly indicates that it is an acidic protein. The cysteine content in this protein was insignificant, which further corroborates the possibility that the acidic amino acids might be prominent candidates for binding to zinc. The findings of the present study confirms that 40 kDa major zinc binding glycoprotein purified from renal BBM is a zinc transporter involved in the influx of Zn(2+) into the epithelial cells of the renal tubular system.

Amino Acids↗
PubMed1976

Geometry of normal mammalian platelets by quantitative microscopic studies.

The shape distributions of normal and hardened human and rabbit erythrocytes and platelets were obtained for edge-on orientations of a few hundred freely rotating cells from analyses of microphotographs obtained similarly as by Ponder(1930, Q. J. Exp. Physiol. 20:29) by phase-contrast microscopy at 800 X magnification. Major average diameters (d) and thicknesses (t) were estimated for both normal and hardened cells, and were used to calculate an average geometric axis ratio, rp = t/d, which increases to unity as cells become more spherical. Our fixation procedure did not alter these shape parameters: rp was unchanged for erythrocytes, with d and t values similar to those reported by Ponder (1930); platelets had d X t = 3.6 +/- 0.7 mum X 0.9 +/- 0.3 mum and 3.1 +/- 0.4 mum X 0.6 +/- 0.3 mum, respectively, for human and rabbit cells, with rp = 0.26 and 0.20, respectively. Agreement in rp was found with data obtained by a novel rheo-optical method which allows for a direct statistical averaging for large populations (greater than 100 X 10(3) cells). Histograms and linear correlation studies were made of the above three parameters (d,t,rp), as well as volume (V), total surface area are (S), and sphericity index (S.I.) calculated for both "prolate ellipsoid" and "disc with rounded edges" models. Results indicate very high linear correlations between rp - t, rp - S. I., and d -S, with high correlations for t - V,d -V and S. Data are in agreement with the few reports in the literature determined by other methods, with the best model for platelets appearing to be an oblate spheroid.

Animals↗
PubMed1996

The bundling of actin with polyethylene glycol 8000 in the presence and absence of gelsolin.

Actin filament and bundle formation occur in the cytosol under conditions of very high total macromolecular concentration. In this study we have utilized the inert molecule polyethylene glycol 8000 (PEG) as a means of simulating crowded conditions in vitro. Column-purified Ca-actin was polymerized in the absence and presence of gelsolin (to regulate mean filament lengths between 50 and 5000 mers) and PEG (2-8%) using various concentrations of KCl and/or 2 mM divalent cations. Bundling was characterized by the scattered light intensity and mean diffusion coefficients obtained from dynamic light scattering, as well as by fluorescence and phase-contrast microscopy. The minimum concentration of KCl required for bundling decreases both with increasing concentration of PEG at a fixed mean filament length, and with decreasing filament length at a fixed concentration of PEG. In the absence of divalent cation, bundling is reversible on dilution, as determined by intensity levels, diffusion coefficients, and microscopy. However, with either 2 mM Mg2+ or Ca2+ added, bundling is irreversible under conditions of higher PEG concentrations or longer filaments, indicating that osmotic pressure effects cannot fully explain actin bundling with PEG. Weaker divalent cation-binding sites on actin as well as disulfide bonds appear to be involved in the irreversible bundling.

Actins↗
PubMed2003

The human adult cardiomyocyte phenotype.

AIM: Determination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology. METHODS: Atrial and ventricular cardiomyocytes were obtained from patients undergoing cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured with or without serum. The relative cell attachment promoting efficiency of several reagents was evaluated and compared. Morphological changes during long-term culture were assessed with phase contrast microscopy, morphometric analysis and immunocytochemistry or RT-PCR of sarcomeric markers including alpha-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin. RESULTS: The isolation method produced viable rod-shaped atrial (16.6+/-6.0%, mean+/-S.E.; n=5) and ventricular cells (22.4+/-8.0%, mean+/-S.E.; n=5) in addition to significant numbers of apoptotic and necrotic cells. Cell dedifferentiation was characterized by the loss of sarcomeric structure, condensation and extrusion of sarcomeric proteins. Cells cultured with low serum recovered and assumed a flattened, spread form with two distinct morphologies apparent. Type I cells were large, had extensive sarcolemmal spreading, with stress fibers and nascent myofibrils, whilst type II cells appeared smaller, with more mature myofibril organisation and focal adhesions. CONCLUSION: Characterization of the redifferentiation capabilities of cultured adult cardiac myocytes in culture, provides an important system for comparing cardiomyocytes differentiating from human stem cells and provides the basis for an in vitro transplantation model to study interaction and communication between primary adult and stem cell-derived cardiomyocytes.

Actinin↗
PubMed1987

A predictive model for determining asbestos concentrations for fibers less than five micrometers in length.

The controversy of whether small asbestos fibers are biologically significant has not been resolved. The present standard method for evaluating asbestos fiber concentrations in workroom air excludes fibers less than 5 micron long even though it has been shown that small fiber concentrations dominate in a dust cloud. This research project was conducted to develop a mathematical model whereby one could predict small (less than 5 micron length) asbestos fiber concentration based on the fiber count concentration determined by phase contrast microscope analysis. Dry chrysotile asbestos was aerosolized into a chamber and sampled by membrane filtration. Segments from each filter were analyzed by both the NIOSH technique using phase contrast microscopy (PCM) and scanning electron microscopy (SEM) at 2000 X for fiber concentrations. A linear relationship was found to exist between the natural logarithm of the SEM-determined concentration and the natural logarithm of the PCM-determined concentration (r = 0.852). Using these data, a mathematical model was developed to predict SEM concentrations based on PCM counts. This model may have application in retrospective epidemiological studies for estimating small fiber exposure levels to determine if small fibers play a role in disease production. The greatest utility would be in those retrospective studies where the only exposure information available is based on PCM counts.

Air Pollutants↗
PubMed1985

p21ras. Heterogeneous localization in transformed cells.

The cellular targets for the Kirsten murine sarcoma virus (KiMSV)-transforming protein, p21ras, are unknown. Other studies have indicated that the mature form of p21 is distributed diffusely on the cytoplasmic face of the plasma membrane. However, after fixation without buffer washes, indirect immunofluorescent staining of sparse cultures revealed a particularly well preserved cellular architecture and a strikingly heterogeneous subcellular distribution of p21 in transformed normal rat kidney (NRK) cells but not in their untransformed counterparts. The transformed cells included A KiMSV-transformed NRK line. NRK cells newly transformed with KiMSV. A temperature-sensitive (ts) KiMSV-transformed NRK line. An uninfected, spontaneously transformed NRK line in which p21 was neither phosphorylated nor overproduced. In the tsKNRK line p21 was abundant at both permissive and non-permissive temperatures; however, its distribution was heterogeneous at the permissive temperature only. Observation of this array of cells indicates that the transformation-associated p21 distribution does not require overexpression of the gene, nor phosphorylation of the protein, nor the viral oncogene. Furthermore, it is reversible in the tsKNRK cells, and so appears to be highly correlated with acquisition of a transformed morphology. Accumulations of p21 occurred preferentially in subcellular locations similar to those where ruffles were observed by phase contrast microscopy and lamellar and villous extensions were observed by scanning electron microscopy (SEM). Since enhanced ruffling is a morphological correlate of transformation in a variety of cells, the distribution of p21 observed here may relate to its function as a transforming molecule.

Cell Line↗
PubMed2003

Changes in the refractive index of lens fibre membranes during maturation--impact on lens transparency.

PURPOSE: Local variations in refractive index are the physical cause of light scattering in a material or tissue and also induce phase changes of propagating light waves. The goal of this study was to analyse local differences in refractive index by phase contrast microscopy of sections of human lenses. METHODS: Refractive index was estimated by immersion refractometry. Cryo-sections of quick-frozen human donor lenses were embedded in a graded series of bovine serum albumin solutions, and in immersion oil, ranging in refractive index from 1.34 to 1.52. RESULTS: Fibre membranes in the lens cortex prove to have a refractive index considerably above that of fibre cytoplasm at the same location. Fibre membranes in the lens nucleus have a refractive index approximately the same as that of fibre cytoplasm at the same location. CONCLUSION: In the lens cortex, transparency is obtained by a high spatial order of the lens fibre lattice to compensate for the light scattering caused by differences in refractive index between fibre membranes and cytoplasm. In the lens nucleus, high spatial order is less important, because the minor differences in the refractive index between fibre membranes and fibre cytoplasm lead only to minimal scattering.

Cryopreservation↗
PubMed2003

Isolation of a putative keratocyte activating factor from the corneal stroma.

Fetal bovine serum has commonly been used to expand the population of keratocytes in culture. Tissue extracts, however, have also been used to grow other cell types. We prepared a DMEM/F12 extract of corneal stroma and compared the growth and morphology of collagenase-isolated keratocytes cultured in DMEM/F12, or DMEM/F12 containing either stromal extract or fetal bovine serum. Cell proliferation was measured by 3H-thymidine and BrdU incorporation as well as by DNA quantitation. The extract was fractionated by gel filtration. Cell morphology was assessed by phase-contrast microscopy. Culture in both extract and serum stimulated keratocytes to proliferate, but keratocytes cultured in the extract grew more slowly due to a longer cell cycle and to a lower final density because of greater sensitivity to contact inhibition. Keratocytes cultured in serum became fibroblastic while those cultured in extract retained the dendritic morphology of quiescent keratocytes. The stimulating factors in the corneal extract were more sensitive to heat inactivation and of higher molecular weight than the stimulating factors in serum. These results indicate that the mitogenic activity in extract and serum are different and that the phenotypes resulting from growth in serum and extract are also different. Keratocytes cultured at low cell densities in the corneal extract may mimic keratocyte activation, an initial and crucial event for keratocytes during the corneal wound healing process.

Animals↗
PubMed1985

Pneumocystis carinii: growth variables and estimates in the A549 and WI-38 VA13 human cell lines.

Recent studies indicate that rat Pneumocystis carinii can be propagated in the A549 cell line, an alveolar epithelioid cell line derived from human lung carcinoma. In the present study, growth of P. carinii was compared in the A549 cell line and the WI-38 VA13 subline 2RA, an SV40 transformed derivative of the human fetal fibroblast cell line with epithelioid morphology. Similar P. carinii growth occurred in both cell lines under optimal conditions, but the WI-38 VA13 cell line was usually more sensitive to changes in the culture system. Growth of P. carinii was affected by temperature, environmental gas mixture, motion of the cultures, and source and concentration of serum additives, but not by the presence of antibodies in the medium. A technique was developed for quantitating P. carinii in the lung inoculum which permitted analysis of P. carinii growth during the first 24 hr of culture. Inverted microscope and oil immersion phase-contrast microscopy were very helpful in monitoring the organism's stages of development and viability. Thus, this culture system should be helpful in establishing standard methodology for in vitro work with P. carinii.

Ammonium Chloride↗