scholarly journals HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS

1969 ◽  
Vol 43 (3) ◽  
pp. 506-520 ◽  
Author(s):  
M. N. Berry ◽  
D. S. Friend
Keyword(s):  
Gap Junctions ◽  
Rat Liver ◽  
Structural Integrity ◽  
Cell Injury ◽  
High Yield ◽  
Isolated Cells ◽  
Nylon Mesh ◽  
Parenchymal Cells

A new technique employing continuous recirculating perfusion of the rat liver in situ, shaking of the liver in buffer in vitro, and filtration of the tissue through nylon mesh, results in the conversion of about 50% of the liver into intact, isolated parenchymal cells. The perfusion media consist of: (a) calcium-free Hanks' solution containing 0.05% collagenase and 0.10% hyaluronidase, and (b) magnesium and calcium-free Hanks' solution containing 2 mM ethylenediaminetetraacetate. Biochemical and morphologic studies indicate that the isolated cells are viable. They respire in a medium containing calcium ions, synthesize glucose from lactate, are impermeable to inulin, do not stain with trypan blue, and retain their structural integrity. Electron microscopy of biopsies taken during and after perfusion reveals that desmosomes are quickly cleaved. Hemidesmosome-containing areas of the cell membrane invaginate and appear to pinch off and migrate centrally. Tight and gap junctions, however, persist on the intact, isolated cells, retaining small segments of cytoplasm from formerly apposing parenchymal cells. Cells which do not retain tight and gap junctions display swelling of Golgi vacuoles and vacuoles in the peripheral cytoplasm. Cytoplasmic vacuolization in a small percentage of cells and potassium loss are the only indications of cell injury detected. By other parameters measured, the isolated cells are comparable to normal hepatic parenchymal cells in situ in appearance and function.

Isolation and Culture of Single Cell Types from Rat Liver

2016 ◽  
Vol 201 (4) ◽  
pp. 253-267 ◽  
Author(s):  
Qidi Zhang ◽  
Ying Qu ◽  
Zhenghong Li ◽  
Qingqing Zhang ◽  
Mingyi Xu ◽  
...  
Keyword(s):  
Single Cell ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Ethylene Glycol Tetraacetic Acid ◽  
High Yield ◽  
Isolated Cells ◽  
Liver Sinusoidal Endothelial Cells ◽  
Pathology Of The Liver

There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p < 0.05) and improved LSEC purity (93.6 ± 3.6 vs. 82.5 ± 5.6%, p < 0.01). The isolated cells could also be cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future.

scholarly journals THE ENZYMATIC PREPARATION OF ISOLATED INTACT PARENCHYMAL CELLS FROM RAT LIVER

1967 ◽  
Vol 35 (3) ◽  
pp. 675-684 ◽  
Author(s):  
Roger B. Howard ◽  
A. Kent Christensen ◽  
Frederic A. Gibbs ◽  
Leroy A. Pesch
Keyword(s):  
Rat Liver ◽  
Structural Integrity ◽  
Light And Electron Microscopy ◽  
Hepatic Metabolism ◽  
Endogenous Respiration ◽  
Treatment Utilization ◽  
Tissue Preparation ◽  
Enzymatic Preparation ◽  
Isolated Cells ◽  
Parenchymal Cells

Suspensions of isolated parenchymal cells were prepared from rat liver by incubation with collagenase and hyaluronidase followed by mechanical treatment. Utilization of 0.15% collagenase together with 0.15% hyaluronidase yielded adequate numbers of cells for experimental purposes. As shown by light and electron microscopy, approximately 75% of the isolated cells retain their structural integrity. The cell suspensions are capable of maintaining endogenous respiration in the presence of 1% albumin for periods of time up to 8 hr. These cell preparations consist almost entirely of parenchymal cells and offer a unique tissue preparation for the study of hepatic metabolism.

A comprehensive study on the isolation and characterization of the HeLa S3 nuclear matrix

1991 ◽  
Vol 98 (3) ◽  
pp. 281-291
Author(s):  
P. Belgrader ◽  
A.J. Siegel ◽  
R. Berezney
Keyword(s):  
Nuclear Matrix ◽  
Intermediate Filament ◽  
Structural Integrity ◽  
Rnase A ◽  
Matrix Proteins ◽  
Intermediate Filament Proteins ◽  
Isolation And Characterization ◽  
Hela S3

Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones. The importance of intermolecular disulfide bonds, RNA and 37 degrees C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodium tetrathionate and 37 degrees C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37 degrees C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix. The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality.

Uptake and Degradation of Tissue Plasminogen Activator in Rat Liver

1988 ◽  
Vol 59 (03) ◽  
pp. 474-479 ◽  
Author(s):  
Monica Einarsson ◽  
Bård Smedsrød ◽  
Håkan Pertoft
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rat Liver ◽  
Liver Cells ◽  
Terminal Phase ◽  
Tissue Plasminogen ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

SummaryThe mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(α), and the terminal phase, t1/2(β), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells.Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells.Competitivb inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types.These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.

scholarly journals Mouse spleen lymphoblasts generated in vitro. Their replication and differentiation in vitro

1978 ◽  
Vol 147 (2) ◽  
pp. 297-315 ◽  
Author(s):  
RM Steinman ◽  
BG Machtinger ◽  
J Fried ◽  
ZA Cohn
Keyword(s):  
Proliferative Activity ◽  
Plasma Cells ◽  
Calf Serum ◽  
Mouse Spleen ◽  
High Yield ◽  
Short Life Span ◽  
Bovine Plasma ◽  
Flow Microfluorometry

Mouse spleen lymphoblasts induced with lipopolysaccharide and fetal calf serum were obtained in high yield and purity in their first proliferative cell cycle by floatation in dense bovine plasma albumin columns (3). The blasts were maintained in vitro for 3 more days. The cultures were examined in bulk on each day, and in addition, those cells in S phase initially were tagged with [(3)H]thymidine and followed continuously in vitro. Grain count dilution data indicated that most blasts divided but twice over a 2- to 3-day interval in vitro. [(3)H]Thymidine pulse radiolabeling and flow microfluorometry suggested that at least 50-70 percent of the proliferating blasts withdrew from proliferative activity after 2-3 days of culture. Morphologic studies demonstrated that lymphoblasts persisted as such for 1-2 days in vitro and then matured into typical plasma cells. Many of the blastprogeny had small nuclei and considerable basophilic cytoplasm on Giemsa-stained cell smears; abundant rough endoplasmic reticulum by electron microscopy; and readily detectable cytoplasmic Ig by immunocytochemistry. Reversion of blasts to small lymphocytes could not be detected; however, some blasts persisted even after 3 days of culture. The viability of the cultured lymphoblast was followed by initially tagging the cells with [(3)H]thymidine as well as several other techniques. Little cell death was documented during the first day of culture. The number of labeled progeny increased twofold whereas the grain count halved. But 40- 50 percent of the cell-associated label was lost during each of the second and third days, and fewer labeled progeny than predicted by grain count dilution were identified. The culture medium could not be implicated in this loss of lymphoblast progeny, and we suggest that the maturation of the lymphoblast to a short-lived plasma cell was responsible. Therefore mitogen-stimulated B blasts seem to mature into typical plasma cells after just two cycles of cell division. The plasma cells resemble those produced in situ during an immune response in their cytologic features, withdrawal from active proliferative activity, and short life-span.

scholarly journals Expression of Glutamine Synthetase in Macrophages

2000 ◽  
Vol 48 (3) ◽  
pp. 415-421 ◽  
Author(s):  
Johannes Georg Bode ◽  
Thorsten Peters-Regehr ◽  
Ralf Kubitz ◽  
Dieter Häussinger
Keyword(s):  
Glutamine Synthetase ◽  
Rat Liver ◽  
Protein Level ◽  
Constitutive Expression ◽  
Laser Scanning Microscopy ◽  
Mrna Levels ◽  
Liver Parenchymal ◽  
Liver Macrophages ◽  
Parenchymal Cells

We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages.

Histamine at low concentrations aggravates rat liver BRL-3A cell injury induced by hypoxia/reoxygenation through histamine H2 receptor in vitro

2013 ◽  
Vol 27 (1) ◽  
pp. 378-386 ◽  
Author(s):  
Tiansheng Wu ◽  
Xiaoliang Gan ◽  
Shaoli Zhou ◽  
Mian Ge ◽  
Zheng Zhang ◽  
...  
Keyword(s):  
Rat Liver ◽  
Cell Injury ◽  
Histamine H2 Receptor ◽  
H2 Receptor ◽  
Low Concentrations ◽  
Hypoxia Reoxygenation

scholarly journals The Isolation of Human Glioblastoma Cells: An Optimised Protocol

2021 ◽  
Vol 49 (1, 2, 3) ◽  
pp. 4
Author(s):  
Tomaz Velnar ◽  
Uros Maver ◽  
Roman Bosnjak ◽  
Lidija Gradisnik
Keyword(s):  
Cluster Formation ◽  
Culture Conditions ◽  
High Yield ◽  
Isolated Cells ◽  
Isolation Technique ◽  
Standard Culture ◽  
Cranial Tumour ◽  
Human Gbm ◽  
High Level

<p><strong>Objective. </strong>The aim of this study was to establish an optimised protocol for glioblastoma (GBM) cell isolation from brain resection samples, with a high yield and low risk for contamination.</p><p><strong>Methods. </strong>Human GBM cells can be obtained following cranial tumour operations. In sterile conditions, the fragments of viable tissue removed during surgery were collected. The tissue was cut and mechanically coarsely decomposed. The sediment was harvested after centrifugation, the cells were seeded in suspension, and supplemented with a special medium (Advanced DMEM) containing high level nutrients and antibiotics.</p><p><strong>Results. </strong>In an appropriate environment, the isolated cells retained viability and proliferated quickly. Attachments were observed after ten hours, and proliferation after two days. The time to full confluence was about one week. The cells were stable. Under standard culture conditions, cell proliferation and cluster formation were observed. Cell viability was 95%.</p><p><strong>Conclusion. </strong>The protocol described for isolation is easy, quick and affordable, leading to stable GBM cells. The isolation technique provides sufficient quantities of isolated cells that may be used as an important new tool for <em>in vitro </em>research. The availability of this system will permit the study of cell properties, biochemical aspects, and provides the potential of therapeutic candidates for pathological disorders in a well-controlled environment.</p>

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