Björn Sandström

STUDIES ON CELLS FROM LIVER TISSUE CULTIVATED IN VITRO. I. INFLUENCE OF THE CULTURE METHOD ON CELL MORPHOLOGY AND GROWTH PATTERN.

Abstract The origin of the cells in liver tissue cultures was investigated. It is shown that the majority of cells originate from the sinusoid endothelium, which has the ability to adapt itself to a predominantly anaerobic metabolism. The parenchymal cells lack this ability and grow in vitro only under special conditions.

Variations among cultured cells in glutathione peroxidase activity in response to selenite supplementation

The aim of this study was to devise conditions for manipulation of the activity of selenium-dependent glutathione peroxidase in cell lines by means of variation in culture medium contents of selenite and fetal calf serum. Nine different cell lines were studied. A low glutathione peroxidase activity was, in most cases, obtained by the use of a medium with a low (2%) serum content. Selenite induced in most of the cell lines an increase in glutathione peroxidase activity, with a plateau ranging from 10 nM to 300-1000 nM. Growth-retarding effects of selenite became apparent at 300-2000 nM, showing a large cell line variation. Supplementation with 50-100 nM selenite for 1 week should generally be suitable for maximal glutathione peroxidase induction. The selenium contents of serum batches were highly variable, pointing to the importance of using only one well-defined, preferably low-selenium, batch. The glutathione peroxidase activities varied considerably between cell lines and the selenite-induced increases ranged from negligible to more than 10-fold. The availability of cell lines with such variable responses should be valuable for experiments aimed at evaluating the importance of glutathione peroxidase and selenium compounds independently of glutathione peroxidase for the protection against oxidative insult.

Combined toxicity to HeLa cells of 30 drug pairs, studied by a two-dimensional microtitre method

Abstract The complete pattern of the cytotoxic interaction of two drugs may be obtained in a single test, by incubation of the drugs, diluted at a right angle to each other in the same area of a microtitration plate, with HeLa cells, grown at a density of 5.10 4 cells/ml in Parkers medium 199 plus 5% calf serum at 37°C for 7 days, and the recording of cyto-inhibition in all cups of the plate by microscopy after 24 h, supplemented by the study of pH-change of the medium after 7 days. Thirty drug combinations were tested by this method; most developed an additive pattern, while minor groups developed patterns of no interference, potentiative antagonism or supra-additive synergism.

Ultrastructure of the developing chicken liver before hatching

SummaryThe ultrastructure of the developing chicken liver has been reexamined on specimens fixed by perfusion with glutaraldehyde. This fixation technique gives a preservation of ultrastructural detail superior to that of earlier investigations. Among others the following observations may be pointed out:1.Bile canaliculi with well-developed microvilli and adjacent tight junctions are present already at the 4-day-old stage and then remain essentially unchanged during development.2.A subendothelial space of Disse is not present until about 16 days of incubation.3.The Golgi apparatus does not assume its adult appearance until about 8 days of incubation.4.Glycogen is first observed in the 6-day-old specimens and then continuously increases throughout development. Glycogen particles often accumulate in membranelimited bodies reminding of the glycogen-filled lysosomes found in a certain type of glycogenosis (Pompes disease).5.The mitochondria increase in size and number during development with a conspicuous change from rounded towards more rodshaped and elongated forms.

Non-freezing light- and electron microscopic enzyme histochemistry by means of polyetylene glycol embedding

SummaryA technique is described, by means of which the undesirable effects by freezing and thawing necessary in cryostat histochemistry might be avoided using embedding in polyetylene glycol, which does not affect enzyme activity nor ultrastructural appearance of cellular detail. The technique permits sectioning with an ordinary sliding microtome.

Improved use of the metabolic inhibition test to screen combined drug toxicity to HeLa cells — preliminary study of 61 drug pairs

Abstract A modification of the Metabolic Inhibition Test was used to test the toxicity to HeLa cells of mixtures of two drugs, at equally toxic concentrations, as well as toxicity of the separate drugs in the combination. By a comparison of the toxicity of the combination to the toxicity of both separate drugs, a preliminary determination of the precise type of cytotoxic interaction could be made for 45 out of the 61 combinations, tested as a pilot study of the method. The majority of these 45 combinations interacted synergistically (77%). Used to screen supra-additive interaction, the method revealed 9 such interactions out of 57 combinations.

On the specificity of histochemically demonstrable bile canalicular phosphatase activities

SummaryBile canalicular phosphatase activity in frog, chicken, rat and cat has been studied with respect to substrate specificity, pH optimum and effect of various stimulators and inhibitors.It is concluded that three different bile canalicular phosphatase activities may be demonstrated histochemically: 1. A strong non-specific nucleoside diphosphatase able to split ATP and most diphosphates. Its relevance is, however, uncertain and requires confirmation by biochemical studies. 2. A non-specific alkaline phosphatase, 3. 5′-nucleotidase.Evidence is presented that the bile oanalicular staining reflects real enzyme activity and not artificial non-enzymatic hydrolysis.

A comparison between histochemically demonstrable Golgi-associated and bile canalicular TDP-ase activity in liver

SummaryHistochemically demonstrable Golgi-associated TDP-ase activity in liver cells from cat, chicken, rat and frog has been investigated.This activity is highly substrate-specific, insensitive to aldehyde fixation, ethanol, acetate, lead and most enzyme inhibitors. It is stimulated by divalent manganese, calcium, magnesium and cobalt and optimum pH is at pH 6 to 7.The characteristics are identical for all four species but significant differences exist at a comparison with bile canalicular activity, Golgi-associated activity in other cells and biochemical findings.

Electron microscopy of organic and cultivated chicken embryonic liver.

SummaryThe ultrastructure of embryonic chicken liver cultivated in vitro according to a technique elaborated by one of the present authors, as well as of organic chicken liver of corresponding age have been studied. With regard to growth pattern and cellular structure, the cultivated cells are in the main identical with the organic liver cells except for the endoplasmic reticulum, which is relatively poorly developed, and the mitochondria, which tend to be more spherical in the cultivated cells.

The functional significance of bile canalicular nucleosidephosphatase activity

SummaryThe histochemically demonstrable bile canalicular nucleosidephosphatase activity of the developing chicken and rat liver has been studied and correlated to the structural and functional maturation.It is concluded that the factor responsible for this activity is not bound to the bile canaliculi as anatomical structures per se. Neither does it play an active role in bile secretion but appears to be a plasma ingredient excreted in the bile.It is suggested that the transitional localization of this activity to different parts of the functional liver unit reflects the morphological route of bile secretion.