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Biomedical subjects

B Nicolaissen

Publications and source records attributed to B Nicolaissen.

At least 19 recordsLinked to original sources
PubMed1996

Isolation and in vitro propagation of human choroidal fibroblasts. Morphology and characterization of the cultures.

Subretinal ingrowth of choroidal fibroblasts is of importance in several clinical settings such as age-related macular degeneration or after traumas such as laser treatment and infection. In the present investigation we describe a method for isolation and propagation of human choroidal fibroblasts in vitro. Outgrowth from the outseeded choroid was studied by means of phase contrast microscopy, scanning electron microscopy and by immunohistochemical methods. The secondary cultures were found to contain almost only fibroblasts (more than 95% of the cells), some contaminating retinal pigment cells were also found. Choroidal fibroblasts contribute to the fibrovascular subretinal scarring process, and our cell culturing system seems to be a suitable tool for studying factors regulating the behavior of these cells in vitro.

Aged↗
PubMed1995

In vitro studies of conjunctival cells from eyes with and without pseudoexfoliation.

The aim of the study was to examine for possible differences between cell cultures derived from eye with and without pseudoexfoliation. In both populations, scanning electron microscopy showed flattened epithelial cells and also spindleshaped fibroblast-like cells. The presence of these cell types was further confirmed by immunohistochemical demonstration of cytokeratin and vimentin in the cultured cells. The cells maintained in vitro showed a linear increase in uptake of leucine during a 12-h period. Within this period, the leucine recovered in the TCA precipitable fraction was considerably higher than the nonbound fraction. In cultures maintained in medium with and without L-ascorbic acid, the presence of L-ascorbic acid significantly increase the uptake of leucine into TCA precipitable material, and to a similar extent in cultures from the two populations. In conclusion, cells derived from eyes with and without pseudoexfoliation material and maintained in vitro showed similar morphology, presence of intermediate filaments, as well as uptake of leucine under various culture conditions.

Ascorbic Acid↗
PubMed1992

[Surgery of strabismus performed during topical anesthesia using adjustable sutures].

Retrobulbar, peribulbar and general anaesthesia for strabismus operations all share the disadvantage that they do not permit preoperative evaluation of the effect of the operation. To overcome this problem, we now operate selected cases in topical anaesthesia, using adjustable sutures. In addition to using adjustable sutures in recession, we have developed a method of adjustable resection. During a six month period, 70% of our grown-up patients were operated by this method. We find it a useful supplement to traditional strabismus surgery, also in reoperations and in operations on paretic muscles.

Anesthesia, Local↗
PubMed1992

Topical anesthesia and adjustable sutures in strabismus surgery.

Our experience with the use of topical anesthesia and adjustable sutures in a one-stage operation for treatment of strabismus is discussed, and selected cases are presented. In addition to using the method in recession, we have also developed a method for using adjustable sutures in resection of muscles. We have also found the method useful in reoperations and in operations on paretic muscles.

Administration, Topical↗
PubMed1992

In vitro ingrowth of choroidal cells on Bruch's membrane.

Full thickness eyewall explants were made from human eyes, the retina and retinal pigment epithelium were removed, and the explants were maintained in vitro in Ham's F-10 medium with 20% foetal bovine serum. Cultured explants were examined by scanning and transmission electronmicroscopy after varying lengths of time. When in vitro, choroidal cells migrated across the cut edge of the explant onto the denuded Bruch's membrane where they subsequently showed production of extracellular matrix. In the human eye, ingrowth of choroidal cells on Bruch's membrane is found in macular degenerations, inflammatory chorioretinal disorders and subsequent to trauma. The in vitro model system presented in our study could facilitate evaluation of the dynamics underlying such ingrowth and also of factors affecting the course of this process.

Autoradiography↗
PubMed1992

Amino acid incorporation in cell cultures from eyes with pseudo-exfoliation material.

Conjunctival tissue was removed from eyes with and without pseudo-exfoliation material during cataract surgery. Cells derived from these samples were maintained in vitro. Confluent cultures were examined by transmission electron microscopy, and non-confluent as well as confluent cultures were examined for their ability to incorporate 3H-leucine and 3H-proline into TCA-precipitated material. Cells derived from both types of samples showed deposition of extracellular matrix, and a similar incorporation of the two amino acids. It is concluded that in vitro studies allow examination of basic metabolic patterns under varying conditions, and also a search for possible differences in such functions between tissues with and without production of pseudo-exfoliation material.

Anterior Eye Segment↗
PubMed1991

Outgrowth of cells from human conjunctival explants onto cornea in vitro.

The epithelium was removed from human corneas, and samples of conjunctival tissue were cultured as explants on the denuded corneal surface for 1 and 2 weeks. Cells migrating from the conjunctival explants onto the corneal surface produced a multilayer where cells on the surface generally showed a flattened appearance. The apical membrane of these cells demonstrated villi as well as microplicae. Surface projections were also detected on cells in the deeper layers of the epithelium. Neighbouring cells were connected by junctional complexes. After 2 weeks, however, a lack of intercellular junctions in some areas resulted in the formation of intraepithelial cystoid spaces. Basal cells were connected to the underlying basement membrane by hemidesmosomes. Although transdifferentiation of the cells into a corneal epithelium was not observed within the 2 weeks, the present system provides a tool for studies on factors affecting reepithelialization of corneal epithelial defects by conjunctival cells.

Adolescent↗
PubMed1991

Morphology and proliferation of human corneal epithelium in organ culture.

Organ cultures of central and peripheral human corneal epithelium were maintained for 1 and 2 weeks in Hams F10 medium supplemented with 20% foetal bovine serum, and in medium conditioned by conjunctival epithelial cells. Light microscopy and scanning electron microscopy showed no obvious differences in morphology and survival between central and peripheral explants, or between explants cultured in conditioned and non-conditioned medium. Live cells and areas with confluent growth were seen in all types of explants. The results demonstrate that differences between growth of central and peripheral corneal epithelium in vitro do not necessarily reflect different proliferative potential in vivo. The previously reported lack of survival of central corneal epithelium in culture may reflect an insufficient in vitro system.

Aged↗
PubMed1990

Culture of iris tissue from human eyes with and without pseudoexfoliation.

Samples of iris tissue were removed from human eyes with and without pseudoexfoliation syndrome and maintained in vitro for several weeks. The samples were cultured on tissue culture plastic as well as on biological basement membranes. The morphology of the cultures was examined by phase contrast-, scanning electron-, and transmission electron microscopy. Out-growth of cells with similar shapes was observed from normal iris as well as from pathological tissue on both type of substrate. In sections the outgrowing cells formed multilayered structures. In these areas the individual cells were separated by spaces containing extracellular material. The PE positive specimens revealed typical PE aggregates both within the native tissue and between the outgrown cells. Production of viable cell cultures from human eyes with pseudoexfoliation may facilitate the search for morphological and biochemical abnormalities in this syndrome.

Anterior Eye Segment↗
PubMed1989

DNA synthesis in human retinal pigment epithelium in organ culture.

Retinal pigment epithelium from human eyes was maintained in organ culture, and DNA synthesis was visualized by autoradiography of cultures exposed to labelled thymidine. In Ham's F10 medium, labelling of cells was observed after 7 as well as after 14 days in vitro. At both stages the labelled cells were characteristically located adjacent to areas of epithelial damage. Cells remote from such areas did not incorporate thymidine, This pattern was observed in epithelia removed from the posterior pole as well as from the periphery of the fundus. Several types of routine media and media with human as well as foetal bovine serum (FBS) were found to support DNA synthesis in the cultured epithelium, labelled cells were not observed in epithelium maintained in Ham's F10 without serum, or with low serum concentration. Our study demonstrates, that DNA synthesis does take place in organ cultured human retinal pigment epithelium when the appropriate culture conditions are used. Our findings indicate that induction of DNA synthesis in the cells is not promoted by an indiscriminate effect of the medium but related to the presence of epithelial damage.

Culture Media↗
PubMed1989

Behavior of human RPE cultured on Bruch's membrane and on necrotic debris.

Degeneration and necrosis of the retinal pigment epithelium (RPE) is found in several conditions associated with reactive alteration of the cell layer, and is probably involved in the formation of drusen and age-related macular degeneration. In the current study we describe a novel system for culturing human RPE. This system permits evaluation of the effect of necrotic material between the epithelium and Bruch's membrane, of drusen, and of other alterations in Bruch's membrane on the behavior of the RPE. In this system, dissociated RPE cells were seeded onto isolated Bruch's membrane. Cell behavior was assessed in areas with and without drusen, and also in areas covered with necrotic cells and fragmented debris. On both exposed Bruch's membrane and on the membrane covered with debris, the cells developed a polygonal shape, with a varying density of apical microvilli. The cultures were monolayered with some overlapping. In areas containing fragmented debris and dead cells, seeded RPE cells were found to adhere to the apical surface of necrotic cells, but were also seen to intrude between the necrotic debris and underlying Bruch's membrane, appearing to clear the membrane of such debris. Our study demonstrates the stability of epithelial histology of human RPE under not previously tested conditions. Further, it shows that the present system permits exposure of these cells to several in vivo physiological and pathological changes in a controlled in vitro environment.

Cells, Cultured↗
PubMed1988

Isolation and culture of basal cells of the human corneal epithelium.

The present study introduces a method that permits the isolation of a pure population of viable basal cells of the human corneal epithelium. We demonstrate that this population can be maintained in culture with a maintained epithelial phenotype, DNA-synthesis, migratory and mitotic activity. The isolation procedure permits evaluation of the adhesion between cells in the epithelium and of the surface morphology and histological organisation of the basal cells. This organization is demonstrated to be far more complex than previously recognized. The culture system permits evaluation of the in vitro behaviour of a basal cell population without contaminating superficial cells or stromal cells.

Cell Adhesion↗
PubMed1988

The morphology of human cells from the choroid and retinal pigment epithelium grown in vitro on isolated Bruch's membrane.

Cells from samples of human choroidal tissue were collected and seeded onto isolated Bruch's membrane in vitro, and the morphology of the developing cultures was examined by light-, scanning electron- and transmission electron-microscopy. In early cultures, the cells showed a flattened, spread out appearance with short surface villi and a low degree of cellular overlapping. After 48 h, a shift towards a more elongated cell form was observed, and one week old cultures were composed of predominantly elongated cells with a pronounced degree of overlapping. In contrast, human retinal pigment epithelial cells seeded onto sheets of Bruch's membrane maintained a spread out epithelial morphology with a varying degree of overlapping throughout the culture period. The present system permits comparative studies on the behavior of choroidal and pigment epithelial cells under controlled conditions. It may serve as an in vitro model for disorders characterized by growth of these cells on Bruch's membrane in vivo.

Cell Membrane↗
PubMed1988

The human choriocapillaris in organ culture.

4 x 4 mm pieces of human eye walls consisting of the sclera, the choroid and the pigment epithelium were kept in organ culture for 1 month and studied with light and transmission electron microscopy comparing the findings with normal tissue. After 1 month the choriocapillaris still had open vessel lumina lined with flattened endothelial cells containing organelles and vesicles on both inner and outer cell membranes. The endothelial cells lost their fenestrations and polarity in regard to the location of cell nuclei. Otherwise the ultrastructure was similar to that of the choriocapillaris in vitro. This study has shown that choriocapillaris can survive in organ culture for 1 month, and it permits further studies on the behaviour of the choroid under controlled conditions, and on the long-term effects of different types of trauma to the choroid in vitro.

Adult↗
PubMed1988

The effect of a pulsed neodymium-YAG laser on chorioretinal monolayered cell cultures.

The effect of a pulsed YAG laser on monolayers of chorioretinal cells maintained in vitro on plastic surface was examined. At the energy levels used, phase contrast microscopic visible lesions were observed at all focus distances up to 2 mm. Scanning electron microscopy demonstrated curling of detached sheets of cells, displacement of single cells, clustering of cells, and severe cell membrane damage at the edge of the lesions. The present system permits evaluation of the effect of different laser characteristics and application modes on the extent and morphology of damage in monolayers of human cells under easily controlled conditions.

Cells, Cultured↗
PubMed1988

Argon laser lesions in the human RPE in vitro.

Argon laser lesions were produced in human retinas in eyes to be enucleated because of choroidal melanomas. Lesions were subsequently isolated and maintained in vitro for one month and evaluated by light, transmission and scanning electron microscopy. In the cultured lesions ingrowing cells with a well recognizable epithelial morphology were observed to cover Bruch's membrane in the photocoagulated area, and to grow as sheets on sensory retinal tissue and on detached necrotic pigment epithelium. This growth was predominantly monolayered. In areas, however, overlapping, detachment of cells from the epithelium into the photocoagulated debris, and formation of tubulovescicular structures were observed. The present study demonstrates that lesions with a known potential for reactive changes in the retinal pigment epithelium in vivo can be isolated and cultured with a maintained potential for some of the typical reactive patterns. This opens for investigations on their genesis and on agents modifying these patterns under controlled in vitro conditions.

Adult↗
PubMed1987

Scanning electron microscopy of human drusen.

Drusen are small, yellowish deposits that form under the retinal pigment epithelium (RPE) with senescence or under certain pathological conditions. The present study examined these structures under the scanning electron microscope. Tissue came from four eyes of 66- and 75-year-old donors who demonstrated widespread drusen of the posterior fundus noted on postmortem examination. Specimens were prepared by either detaching the RPE from Bruch's membrane, or by cryofracturing the tissue for cross-sectional views. Drusen appeared to be composed of irregularly-shaped globular masses and of distinct spherical entities. These particles varied greatly in size, and were situated between the RPE's basement membrane and the outer collagenous zone of Bruch's membrane. Surface views showed drusen components to be embedded in the collagenous zone of Bruch's membrane. Pits corresponding to the sizes of the globular and spherical masses imply that some particles were lost during tissue processing. Fractured cross sections of the irregularly-shaped globular masses revealed a homogeneous, granular matrix with no distinct ultrastructural features, while some of the fractured spherical components demonstrated an internal core. Transmission electron microscopic analysis on the same specimens that were subjected to SEM corroborated these observations. Analytical x-ray microanalysis (Kevex, Foster City, CA) in the SEM revealed major peaks for calcium and phosphorous in the crystalline spherical components, and primarily potassium and chloride in the globular structures.

Aged↗