- e-book Submicroscopic Cytodifferentiation (Advances in Anatomy, Embryology and Cell Biology)
- Submicroscopic Cytodifferentiation (Advances in Anatomy, Embryology and Cell Biology)
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Number of bids and bid amounts may be slightly out of date. The surface epitheliumis scraped firmly 2 to 3 times with a rubber policeman D. Scraping generatessheets of HOSE and these are rinsed from the scraper and biopsy surface into oneor two culture dishes E. The epithelial sheets attach to the culture dish andHOSE cells grow out from the attached sheets.
Contaminating fibroblasts were removed fromcultures by either scraping the fibroblasts off with a rubber policeman or cuttingthe fibroblastic outgrowths from the coverslips with sterile scissors.
e-book Submicroscopic Cytodifferentiation (Advances in Anatomy, Embryology and Cell Biology)
Both of thesemethods of decontamination were only temporarily successful. Eventuallyfibroblastic contamination returned. Often, fibroblasts overgrew HOSE cultures. Dishes badly contaminated with fibroblasts were discarded. Because of the problemsassociated with HOSE cultures derived from explants: 1 fibroblastic overgrowth; 2 low HOSE cell yield; and 3 laborious technique, attempts were made to improve theculture of surgical specimens.
At the end of the experiment, the cells adherent to thecollagen gels were harvested as described in Materials and Methods section D. LP-9 cells werecentrifuged in Percoll under the following conditions: 1. The density of the cellbands was determined with density marker beads Pharmacia in parallel gradients.
Following centrifugation, each cell band was collected with a Pasteur pipet andplated directly, without washing, into tissue culture dishes lined with glasscoverslips. Percoll Decontamination of Mold Infected CulturesIn the course of one of the Percoll experiments described above, it was noted thatPercoll treatment appeared to separate cells from mold infections. ROSE grow as epithelialmonolayers and produce basement membrane and interstitial extracellular matrixcomponents Auersperg et al. All cultures were maintained in the absence ofany fungicides.
An environmental mold and an environmental yeast were obtained by exposingopened petri dishes of culture medium to the circulating air within a tissue cultureincubator. The infecting mold was identified as belonging to the fungal Cladosporiumspecies and the yeast was identified as Torulopsis candida.
Submicroscopic Cytodifferentiation (Advances in Anatomy, Embryology and Cell Biology)
Both occur commonly inthe environment. Following centrifugation the cells were counted,tested for viability by trypan blue exclusion, and plated directly, without washing,into tissue cuture dishes. To compare the effects of high speed Percoll centrifugationon cell morphology, growth, and extracellular matrix production with the effects ofcentrifugation as used in routine subculturing, uninfected ROSE and HUDF cellswere spun for 5 minutes at bOg in a counter-top clinical centrifuge.
Cultures were maintained for 12 days and themedium was changed as required.
Cells from triplicate wells were harvested atintervals and counted. By thismethod, the biopsy specimen was examined under a dissecting microscope and areasdevoid of anomalies, blood vessels, and papillae were selected for culture. The entirespecimen was rinsed with medium, held surface down over a 35 mm culture dishcontaining two ml of culture medium and the ovarian surface was scraped firmly twoto three times with a white rubber scraper Canlab, Mississauga, ONT attached to aglass rod.
Scraping generated sheets of HOSE cells and these were rinsed from thescraper and the biopsy surface into one or two culture dishes figure 1. Like theexplantation method, the medium used for the transport and the rinsing of thespecimen was collected, centrifuged, and cells plated into a separate 35 mm dish.
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Cultures were left undisturbed for 48 hr and incubated as described in Material andMethods A. In thecourse of this study, Medium became unavailable commercially, so Medium was replaced with Medium Sigma personal communication, Dr. For culture in medium containing 0. The following cell lines were also used in this study: 1. To harvest or subculture cells, cultures were dissociated in 0. All cell counts were made using a hemocytometer and cell viability was determinedby trypan blue dye exclusion. Cultures were routinely examined using a Wild M40inverted photomicroscope or a Leitz Laborlux K inverted photomicroscope andphotographed on Kodak Technical Pan black and white film.
Culture CharacterizationFor histological, immunocytochemical, and immunofluorescent studies cells weregrown to the desired densities on glass coverslips. HOSE cultures between passages were considered early passage cultures, while those between passages were considered late passage HOSE cultures. Immediately prior to use a saturated stock solution of0. The specimens were washed in running tap water for 10 minutes,treated with pararosaniline Schiff reagent for 30 minutes, washed again in runningtap water for 10 minutes.
The coverslips were counterstained briefly withhematoxylin, differentiated in base, rinsed in water, dehydrated in alcohol, clearedin xylol, and mounted. Treatment with human saliva amylase for 60 minutes prior to periodic acidoxidation constituted glycogen digestion so that staining attributable to glycogen wasselectively removed. Control slides consisted of sections of human ovarian tissueobtained from the Dept. Immunofluorescence Staining a. The cultures were fixed for15 minutes in 3. After a 30 minute PBS wash, thecultures were treated with 0.
Primary antisera included sheep anti-type I collagen , rabbitanti-type IV collagen previously absorbed with 0. Antibody controls consisted of specimens stained with normal serum instead ofthe primary antiserum. KeratinAs described previously Auersperg et al. Controlsconsisted of cells stained with PBS instead of primary antibody. Occasionally,cultures of human amniotic cells, which contain both keratin-positive epithelialcells and keratin-negative cells, were used as controls.
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Coverslips were fixedin 3. Controls consisted of cells stained with normal rabbit serum insteadof primary antiserum and positive controls consisted of the rat ovarian surfaceepithelial cell line, ROSE , and HUDF were negative controls. The coverslips were incubated with rabbit anti-PAl-iantiserum provided by Dr. Rheinwald for 30 minutes at roomtemperature, washed in PBS for 15 minutes, incubated with ROTC-conjugated goatanti-rabbit lgG for 30 minutes at room temperature, washed for 30minutes, and mounted in Gelvatol pH 7.
Controls consisted of cells stained withnormal rabbit serum instead of primary antibody and cultures of human mesothelialcells LP-9 served as positive controls. Staining for Mucin and KeratinHOSE cultures were doubly stained for the epithelial markers, mucin and keratinaccording to the method of Wilson et al.
The cultures were then rinsed in 0. The coverslips were develpedfor minutes at room temperature in 0. The reaction was stopped with water and the coverslipswere counterstained in haematoxylin, rinsed in water, blued in 1. Controls consisted of cells stained with PBS instead of primary antibodyas well as cultures of C4-l cells and frozen sections of human cervix and oviductwhich served as controls for keratin and mucin.
The specimens were then critical point dried in aCPD critical-point drying system using liquid carbon dioxide.