Correlative light and electron microscopy of dissociated immature rat testicular cells undergoing morphogenesis in vitro

Immature rat testicular cells undergo morphogenesis in primary culture (Davis, 1978). Depending upon the number of dissociated testicular cells added to the culture dish, spherical or tubular aggregates were formed. Spherical aggregates resulted from movement of cells into centers of aggregation and the detachment of these cells from the substratum; on the other hand, tubular aggregates resulted from detachment and retraction of the cell monolayer at certain points along its outer edge. In this investigation, the different methods of formation of aggregates by immature rat testicular cells in primary culture were examined with the scanning electron microscope (SEM). The cell types involved in such morphogenesis and their associations within completely formed structures were examined by transmission electron microscopy (TEM). In addition, the rates of formation of aggregates were established by time-lapse cinemicroscopy. During formation of spherical aggregates, the rate of recruitment of cells into centers of aggregation (0·04± 0·006 μm/min; x±S.E.M., n = 78) was much slower than the rate of cell detachment during formation of tubular aggregates (11·7 ± 1·8 μm/sec; x±S.E.M., n = 110). Although specific roles for each cell type in formation of aggregates have not been determined, the associations of cells within the two types of reformed aggregates appeared to be similar. Myofibroblast cells were located in outer cell layers and Sertoli cells were observed to underlie the layers of myofibroblasts in both types of aggregates. Germinal cells, however, were found on the outer surface of spherical aggregates, but in tubular aggregates they were located on the inner surface. Since spherical and tubular aggregates are formed by different methods, this observation suggests that rearrangement of cells within the aggregates takes place and contributes to the internal morphology of newly-formed aggregates.

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Morphogenesis by dissociated immature rat testicular cells in primary culture

Development ◽

10.1242/dev.44.1.297 ◽

1978 ◽

Vol 44 (1) ◽

pp. 297-302

Author(s):

John C. Davis

Keyword(s):

Primary Culture ◽

Single Layer ◽

Culture Dish ◽

Rat Serum ◽

Immature Rat ◽

Plating Density ◽

Testicular Cells ◽

Initial Culture ◽

Dissociated Cells ◽

Medium Nctc

Dissociated cells from immature (15-day) rat testes maintained in primary culture were shown to undergo morphogenesis. Dissociated cells (1−4 × 106) were cultured in 2 ml medium NCTC-135 containing 10% rat serum at 32·5 °C under an air:CO2 atmosphere (95%:5%). During the initial culture period (1–3 days) the dissociated cells attached to the culture dish and formed a monolayer. After 4–5 days of culture, aggregates of cells formed at discrete foci within the monolayer; these structures averaged 50µtm in diameter and numbered 3−4 × 103/dish. Within 5–6 days of culture some of these aggregates (2−3 × 102) released their contact with the substratum. Histological examination of released aggregates indicated a more regular, tissue-like organization with increasing time in culture. Aggregates at earlier time periods (4–5 days) exhibited concentric rings of cell while by day 6 some aggregates exhibited a vesicular appearance with a single layer of columnar epithelium surrounding a fluid-filled lumen. The type of structure formed varied with plating density; at densities less than 2 × 106/dish the structures were spheroid while at greater densities tubes were formed. Timelapse observations indicated that spheres formed by movements of cells into an aggregating center while tubes formed by a rolling from the edge of the monolayer. The cells comprising the vesicle epithelium appeared to be predominantly one cell type as determined by light microscopy. These results therefore indicate that dissociated testicular cells have the ability to reform tissue-like associations in culture.

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Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

Veterinary and Comparative Orthopaedics and Traumatology ◽

10.1055/s-0038-1632561 ◽

1997 ◽

Vol 10 (01) ◽

pp. 6-11 ◽

Cited By ~ 3

Author(s):

R. F. Rosenbusch ◽

L. C. Booth ◽

L. A. Dahlgren

Keyword(s):

Electron Microscopy ◽

Extracellular Matrix ◽

Light Microscopy ◽

Light And Electron Microscopy ◽

Tendon Healing ◽

Matrix Proteins ◽

Isolated Cells ◽

Superficial Digital Flexor Tendon ◽

Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

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A Method of Maintaining Parenchymal Cells from Adult Rat Liver In Vitro

Journal of Cell Science ◽

10.1242/jcs.11.1.249 ◽

1972 ◽

Vol 11 (1) ◽

pp. 249-260

Author(s):

J. ALWEN ◽

JENNIFER J. GALLHAI-ATCHARD

Keyword(s):

Electron Microscopy ◽

Endothelial Cells ◽

Protein Synthesis ◽

Light And Electron Microscopy ◽

Rat Hepatocytes ◽

Adult Rat ◽

Adult Rat Hepatocytes ◽

Rna And Protein Synthesis ◽

Parenchymal Cells

A method for preparing suspensions of adult rat hepatocytes suitable for maintenance in vitro is described. Cultures were established from the cell suspensions by the squash technique. Cells were examined by light and electron microscopy; histochemically for glycogen, bile, lipid and glucose-6-phosphatase; and by autoradiography for DNA, RNA and protein synthesis. Hepatocytes could be maintained in vitro for at least 3 days and began to aggregate after 1 day. Uridine and leucine were incorporated, but not thymidine. Cultures consisted mainly of hepatocytes, though reticulo-endothelial cells were sometimes present.

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Cytasters induced within unfertilized sea-urchin eggs

Journal of Cell Science ◽

10.1242/jcs.61.1.175 ◽

1983 ◽

Vol 61 (1) ◽

pp. 175-189

Author(s):

R. Kuriyama ◽

G.G. Borisy

Keyword(s):

Electron Microscopy ◽

Sea Urchin ◽

Sea Water ◽

Light And Electron Microscopy ◽

Sea Urchin Eggs ◽

Microtubule Organizing Centres ◽

Treatment Step

Conditions that induce the formation of asters in unfertilized sea-urchin eggs have been investigated. Monasters were formed by treatment of eggs with acidic or basic sea-water, or procaine- or thymol-containing sea-water. A second treatment step, incubation with D2O-containing, ethanol-containing or hypertonic sea-water induced multiple cytasters. The number and size of cytasters varied according to the concentration of agents and duration of the first and second treatments, and also upon the species of eggs and the season in which the eggs were obtained. Generally, a longer second treatment or a higher concentration of the second medium resulted in a higher number of cytasters per egg. Asters were isolated and then examined by light and electron microscopy. Isolated monasters apparently lacked centrioles, whereas cytasters obtained from eggs undergoing the two-step treatment contained one or more centrioles. Up to eight centrioles were seen in a single aster; the centrioles appeared to have been produced during the second incubation. Centrospheres prepared from isolated asters retained the capacity to nucleate the formation of microtubules in vitro as assayed by light and electron microscopy. Many microtubules radiated from the centre of isolated asters, whether they contained centrioles or not. This observation is consistent with many other reports that microtubule-organizing centres need not contain centrioles.

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In vitro study of chorionic and ectoplacental trophoblast differentiation in the mouse

Development ◽

10.1242/dev.34.3.633 ◽

1975 ◽

Vol 34 (3) ◽

pp. 633-644

Author(s):

Danièle Hernandez-Verdun ◽

Chantal Legrand

Keyword(s):

Electron Microscopy ◽

Morphological Study ◽

Light And Electron Microscopy ◽

In Vitro Study ◽

Capillary Network ◽

Vitro Study ◽

Trophoblast Cells ◽

Trophoblast Differentiation ◽

Somite Stage

Mouse chorioallantoic pre-placental structures alone or in association with the embryo were explanted during the 9th day of gestation (7-somite stage) and cultured in a static medium for 24 to 48 h. From the subsequent morphological study of trophoblast differentiation, using both light and electron microscopy, we draw the following conclusions. 1. The allantoic mesoderm cells migrate inside the trophoblastic population but they do not differentiate a capillary network and trophoblast cells phagocytose the existing foetal erythrocytes. 2. In the absence of allantoic mesoderm, chorionic trophoblast cells remain undifferentiated. 3. The development of the chorionic trophoblast is modified in that chorionic trophoblast cells fail to establish close junctions with ectoplacental trophoblast, and some chorionic cells initiate the formation of multinucleated syncytia. The genesis of these syncytia is discussed.

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THE INDUCTION OF MELANIZATION IN GOLDFISH SCALES WITH ACTH, IN VITRO

The Journal of Cell Biology ◽

10.1083/jcb.18.1.181 ◽

1963 ◽

Vol 18 (1) ◽

pp. 181-194 ◽

Cited By ~ 29

Author(s):

Alden V. Loud ◽

Yutaka Mishima

Keyword(s):

Electron Microscopy ◽

Tissue Culture ◽

Silver Nitrate ◽

Light And Electron Microscopy ◽

Subcellular Structure ◽

Acth Stimulation ◽

Cytoplasmic Bodies ◽

Silver Nitrate Staining ◽

Stimulation Of

The induction of melanization in xanthic goldfish scales with ACTH in vitro has been studied by light and electron microscopy utilizing ammoniated silver nitrate staining of premelanin and melanin. The melanized cells (melanophores and melanocytes) and the yellow pigmented cells (lipophores and the newly described lipocytes) were found to possess many similarities at the levels of cellular and subcellular structure. The latter cells contain characteristic cytoplasmic bodies which react positively to the premelanin stain. Changes accompanying ACTH stimulation of goldfish scales in tissue culture suggest that these bodies in the lipocytes and lipophores can become melanized. Electron micrographs illustrate the intermediate staining of newly formed melanin granules in an induced melanocyte and the appearance of a transitional melanolipophore. It is postulated that ACTH can promote the association of the enzyme tyrosinase with the preformed structure of unmelanized granules.

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Effect of basolateral acidification on the frog oxynticopeptic cell

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1990.259.4.g564 ◽

1990 ◽

Vol 259 (4) ◽

pp. G564-G570 ◽

Cited By ~ 1

Author(s):

S. Arvidsson ◽

K. Carter ◽

A. Yanaka ◽

S. Ito ◽

W. Silen

Keyword(s):

Electron Microscopy ◽

Gastric Mucosa ◽

Light And Electron Microscopy ◽

Structure And Function ◽

Intracellular Acidification ◽

Intracellular Acidosis ◽

Normal Morphology ◽

And Function ◽

Oxynticopeptic Cells

The effects of intracellular acidosis induced by acidification of the basolateral (nutrient) perfusate on the structure and function of the oxynticopeptic cell were studied in in vitro frog gastric mucosa. Changing the pH of the unbuffered nutrient perfusate (UNB) from 7.2 to 3.5 acidified the oxynticopeptic cell with no change in potential difference (PD) or resistance (R). Intracellular pH (pHi), PD, and R were 7.05 +/- 0.01, 16 +/- 1 mV, 165 +/- 7 omega.cm2 before and 6.44 +/- 0.01, 16 +/- 2 mV, 170 +/- 9 omega.cm2 after nutrient acidification. Acid secretion (H+) increased from 0.86 +/- 0.07 to 1.88 +/- 0.18 mu eq.cm-2.h-1. Addition of forskolin to tissues perfused with nutrient pH (pHn) 3.5 decreased PD to 2 +/- 2 mV and further increased H+ to 3.07 +/- 0.19 mu eq.cm-2.h-1. By light and electron microscopy oxynticopeptic cells perfused with UNB, pHn 3.5, appeared normal. Oxynticopeptic cells in tissues pretreated with omeprazole and then exposed to UNB, pHn 3.5, had extensive morphological damage. On increasing the pH of the nutrient perfusate from 3.5 to 7.2 there was prompt recovery of pHi in untreated and forskolin-stimulated mucosae (pHi 6.87 +/- 0.06 and 6.85 +/- 0.04) but no recovery of pHi in tissues pretreated with omeprazole or cimetidine (pHi 6.26 +/- 0.04 and 6.44 +/- 0.06, n = 6, 30 min after reexposure to UNB, pHn 7.2). We conclude that in a secreting mucosa intracellular acidification of the oxynticopeptic cell to pHi 6.4 is associated with normal morphology, PD, R, and increased H+, and that intracellular acidosis is not de facto deleterious.

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Maintenance of differentiated sheep thyroid cells in primary culture for three months

Acta Endocrinologica ◽

10.1530/acta.0.1070054 ◽

1984 ◽

Vol 107 (1) ◽

pp. 54-59 ◽

Cited By ~ 5

Author(s):

Carmel Mothersill ◽

C. Seymour ◽

J. F. Malone

Keyword(s):

Electron Microscopy ◽

Sudden Death ◽

Primary Culture ◽

Light And Electron Microscopy ◽

Functional Differentiation ◽

Entire Period ◽

Iodine Uptake ◽

Long Term Effects ◽

Thyroid Cells

Abstract. A method is described which permits culture of primary thyroid cells without subculture for at least 100 days. Cultures are maintained without medium changes for the entire period, and concentrated glucose is added to replenish energy supplies at carefully defined intervals. The cells retain morphological and functional differentiation shown by light and electron microscopy, PAS positive histochemistry, iodine uptake and T4 production for at least 100 days. After this time fairly sudden death of the cultures occurs. Possible mechanisms for the effect are postulated. The technique should make it possible to study long-term effects of drugs/radiation on differentiated cultures without the need for continuous subculture.

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