Publisher Summary This chapter discusses preparation of isolated rat liver cells The early mechanical and chemical methods for liver-cell preparation were relatively successful in converting liver tissue to a suspension of isolated cells The successful preparation of intact liver cells by perfusion with collagenase is technically quite difficult The major method for preparation of nonparenchymal liver cells is based on the selective sensitivity of parenchymal cells toward proteases By incubation of rat liver minces with pronase, most of the liver parenchyma is digested, while nonparenchymal cells remain intact and can be recovered from the incubate Similar results have been reported with trypsin digestion of collagenase-dispersed liver minces, but pronase appears to be more effective The most common procedure is to perfuse the liver briefly with pronase before it is minced and incubated with the enzyme Such direct pronase methods have been used by several investigators with yields of nonparenchymal liver cells reported to be in the range 2–15 × 10 6 cells/gm liver

Preparation of isolated rat liver 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.

https://rupress.org/jcb/article-pdf/43/3/506/1069297/506.pdf

HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS: A Biochemical and Fine Structural Study

50 years ago Peter Mitchell proposed the chemiosmotic hypothesis for which he was awarded the Nobel Prize for Chemistry in 1978. His comprehensive review on chemiosmotic coupling known as the first "Grey Book", has been reprinted here with permission, to offer an electronic record and easy access to this important contribution to the biochemical literature. This remarkable account of Peter Mitchell's ideas originally published in 1966 is a landmark and must-read publication for any scientist in the field of bioenergetics. As far as was possible, the wording and format of the original publication have been retained. Some changes were required for consistency with BBA formats though these do not affect scientific meaning. A scanned version of the original publication is also provided as a downloadable file in Supplementary Information and can be found online at doi:10.1016/j.bbabio.2011.09.018. See also Editorial in this issue by Peter R. Rich. Original title: CHEMIOSMOTIC COUPLING IN OXIDATIVE AND PHOTOSYNTHETIC PHOSPHORYLATION, by Peter Mitchell, Glynn Research Laboratories, Bodmin, Cornwall, England.

Chemiosmotic coupling in oxidative and photosynthetic phosphorylation

https://www.amjmed.com/article/0002-9343(91)90279-7/pdf

Reactive oxygen species in living systems: source, biochemistry, and role in human disease.

Glutathione and its role in cellular functions.

1

During the standard procedure for the preparation of rat hepatocytes, about half of the cellular GSH (reduced glutathione) is lost.




2

This loss is prevented by the addition of 0.1 mM EGTA (but not EDTA) to the perfusion medium.




3

On incubation with and without EGTA, isolated hepatocytes prepared in the presence of EGTA lose GSH. This loss is prevented by near-physiological concentrations of methionine or homocysteine, but not of cysteine.




4

Cysteine, at concentrations above 0.2 mM, causes a loss of GSH. Cysteine also caused a loss of adenine nucleotides and of biosynthetic capacity.




5

Serine together with methionine or homocysteine increases GSH above the value in cells from starved rats in vivo.




6

It is suggested that the deleterious effects of cysteine are caused by hydrogen peroxide formation during the intracellular oxidation of cysteine and/or the formation of mixed disulphides.




7

The beneficial effects of methionine or homocysteine in maintaining GSH may be due to a gradual release of cysteine via the cystathionine pathway, needed for the resynthesis of GSH.

Regulation of the Hepatic Concentration of Reduced Glutathione

https://iubmb.onlinelibrary.wiley.com/doi/pdf/10.1080/15216540500078772

R. Hems

Papers

Regulation of the Hepatic Concentration of Reduced Glutathione

How I became a biochemist.