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

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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Long-term exposure to epidermal growth factor results in apoptosis in T51B cells

Biochemistry and Cell Biology ◽

10.1139/o94-057 ◽

1994 ◽

Vol 72 (9-10) ◽

pp. 429-438 ◽

Cited By ~ 4

Author(s):

Caroline J. Brabyn ◽

Douglas J. Franks ◽

Leonard P. Kleine

Keyword(s):

Electron Microscopy ◽

Growth Factor ◽

Epidermal Growth Factor ◽

In Vitro Model ◽

Control Cell ◽

Light And Electron Microscopy ◽

Cell Numbers ◽

Epidermal Growth ◽

Vitro Model

The major objective of this research was the development of an in vitro model for liver homeostasis that would allow the future study of early events in cell proliferation and cell death. The model that was set up involves growing T51B rat liver epithelial cells with a single dose of 1 nM epidermal growth factor (EGF). This results in a period of hyperplasia where the cells reach double the control cell numbers 2 days after EGF addition. This is then followed by a decrease in cell numbers and the cell density returns to around the confluent control level 5 days after EGF addition. The model was investigated to ascertain whether the decrease in cell numbers 3–5 days after EGF addition was due to an increase in apoptosis. The results from light and electron microscopy studies, electrophoresis of T51B cell DNA, and quantification of nuclear fragmentation indicated that the cells do die via an increase in apoptosis. The electron microscopy studies also show that healthy T51B cells can phagocytose apoptotic bodies. This suggests that the model is more physiological than other in vitro models of apoptosis. This work describes the development and characterization an in vitro model of liver homeostasis that closely parallels in vivo systems where animals are given mitogenic stimuli.Key words: apoptosis, epidermal growth factor, T51B liver epithelial cell line, tissue homeostasis.

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The Response of Testis Cells to Low Dose X-Irradiation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099155 ◽

1981 ◽

Vol 39 ◽

pp. 542-543

Author(s):

D. E. Philpott ◽

W. Sapp ◽

C. Williams ◽

Joann Stevenson ◽

S. Black

Keyword(s):

Electron Microscopy ◽

Stem Cell ◽

Light Microscopy ◽

Dose Level ◽

Cross Sections ◽

Low Dose ◽

Light And Electron Microscopy ◽

Cellular Responses ◽

Post Irradiation ◽

X Irradiation

The response of spermatogonial cells to X-irradiation is well documented. It has been shown that there is a radiation resistent stem cell (As) which, after irradiation, replenishes the seminiferous epithelium. Most investigations in this area have dealt with radiation dosages of 100R or more. This study was undertaken to observe cellular responses at doses less than 100R of X-irradiation utilizing a system in which the tissue can be used for light and electron microscopy.Brown B6D2F1 mice aged 16 weeks were exposed to X-irradiation (225KeV; 15mA; filter 0.35 Cu; 50-60 R/min). Four mice were irradiated at each dose level between 1 and 100 rads. Testes were removed 3 days post-irradiation, fixed, and embedded. Sections were cut at 2 microns for light microscopy. After staining, surviving spermatogonia were identified and counted in tubule cross sections. The surviving fraction of spermatogonia compared to control, S/S0, was plotted against dose to give the curve shown in Fig. 1.

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Solving the mechanisms responsible for organelle behavior in vivo by same-cell correlative light and electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120473 ◽

1992 ◽

Vol 50 (1) ◽

pp. 14-15

Author(s):

Conly L. Rieder

Keyword(s):

Electron Microscopy ◽

Quantitative Analysis ◽

Light Microscopy ◽

Living Cell ◽

Light And Electron Microscopy ◽

Time Behavior ◽

Dynamic Interactions ◽

Positive Identification ◽

Cellular Components

The behavior of many cellular components, and their dynamic interactions, can be characterized in the living cell with considerable spatial and temporal resolution by video-enhanced light microscopy (video-LM). Indeed, under the appropriate conditions video-LM can be used to determine the real-time behavior of organelles ≤ 25-nm in diameter (e.g., individual microtubules—see). However, when pushed to its limit the structures and components observed within the cell by video-LM cannot be resolved nor necessarily even identified, only detected. Positive identification and a quantitative analysis often requires the corresponding electron microcopy (EM).

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Faculty Opinions recommendation of Trypanosoma cruzi activates mouse cardiac fibroblasts in vitro leading to fibroblast-myofibroblast transition and increase in expression of extracellular matrix proteins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732607671.793542414 ◽

2018 ◽

Author(s):

Giulio Gabbiani

Keyword(s):

Extracellular Matrix ◽

Trypanosoma Cruzi ◽

Cardiac Fibroblasts ◽

Extracellular Matrix Proteins ◽

Matrix Proteins

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An in vitro model of fibroblast activity and adhesion formation during flexor tendon healing

The Journal Of Hand Surgery ◽

10.1016/0363-5023(94)90181-3 ◽

1994 ◽

Vol 19 (5) ◽

pp. 769-776 ◽

Cited By ~ 12

Author(s):

David L. Packer ◽

George W. Dombi ◽

Ping Yang Yu ◽

Paul Zidel ◽

Walter G. Sullivan

Keyword(s):

In Vitro Model ◽

Flexor Tendon ◽

Adhesion Formation ◽

Tendon Healing ◽

Vitro Model

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Establishment of an in vitro model for analyzing mitochondrial ultrastructure in PRKN-mutated patient iPSC-derived dopaminergic neurons

Molecular Brain ◽

10.1186/s13041-021-00771-0 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Mutsumi Yokota ◽

Soichiro Kakuta ◽

Takahiro Shiga ◽

Kei-ichi Ishikawa ◽

Hideyuki Okano ◽

...

Keyword(s):

Electron Microscopy ◽

Dopaminergic Neurons ◽

Structural Changes ◽

In Vitro Model ◽

Induced Pluripotent Stem Cell ◽

Substantia Nigra Pars Compacta ◽

Ultrastructural Changes ◽

Mitochondrial Morphology ◽

Vitro Model

AbstractMitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson’s disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.

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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 development of isolated ectoderm from axolotl gastrulae

Development ◽

10.1242/dev.80.1.321 ◽

1984 ◽

Vol 80 (1) ◽

pp. 321-330

Author(s):

Jonathan M. W. Slack

Keyword(s):

Electron Microscopy ◽

Electron Microscope ◽

Light Microscopy ◽

Outer Layer ◽

Microscope Examination ◽

Animal Pole ◽

In Vitro Development ◽

Electron Microscope Examination ◽

Vitro Development

The development of ectoderm isolated from the animal pole of axolotl gastrulae is monitored by light microscopy, electron microscopy and analysis of newly synthesized proteins, glycoproteins and glycolipids. When control embryos are undergoing neurulation it is shown that the explants autonomously begin to express epidermal markers and do not express mesodermal markers. However the results suggest that not all the cells become epidermal and electron microscope examination shows that only the outer layer does so, the inner cells remaining undifferentiated.

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