THE ENZYMATIC PREPARATION OF ISOLATED INTACT PARENCHYMAL CELLS FROM RAT LIVER

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.

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HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS

The Journal of Cell Biology ◽

10.1083/jcb.43.3.506 ◽

1969 ◽

Vol 43 (3) ◽

pp. 506-520 ◽

Cited By ~ 3272

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.

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Isolation and separation of highly enriched fractions of viable mouse gastric parietal cells by velocity sedimentation.

The Journal of Cell Biology ◽

10.1083/jcb.65.2.428 ◽

1975 ◽

Vol 65 (2) ◽

pp. 428-438 ◽

Cited By ~ 36

Author(s):

L J Romrell ◽

M R Coppe ◽

D R Munro ◽

S Ito

Keyword(s):

Structural Integrity ◽

Light And Electron Microscopy ◽

Cell Types ◽

Parietal Cells ◽

Velocity Sedimentation ◽

Isolated Cells ◽

Adult Mice ◽

Tissue Dissociation ◽

Cell Fractions ◽

Gastric Parietal Cells

Methods of tissue dissociation and cell separation have been modified to obtain highly enriched fractions of mouse gastric parietal cells. Suspension of gastric mucosal cells are prepared by pronase digestion of the glandular portion of the stomach from adult mice. By utilizing the velocity sedimentation technique to separate cells of different sizes it is possible to recovery parietal cells, which are larger than the other cell types, in fractions with purity of 75-95%. The homogeneity of cell fractions has been assessed by light and electron microscopy. The ability of the isolated cells to exclude the dye trypan blue, to incorporate labeled substrate, to consume oxygen, and to retain their structural integrity indicates that they are viable and still capable of functional activity.

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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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