Michael Balls

The relationship between cell sizes, respiration rates and survival of amphibian tissues in long-term organ cultures

Abstract 1. 1. Respiration rates of freshly isolated Amphiuma, Diemictylus, Siredon, Trituras, Xenopus and rat tissues, and cultured Amphiuma tissues were measured. 2. 2. The sizes of erythrocytes, erythrocyte nuclei and hepatocyte nuclei of the same species were measured. 3. 3. The success of long-term organ cultures of amphibian tissues may be due to their low respiration rates and large cell sizes.

Amphibian cells in vitro: I. Growth of Xenopus cells in a soft agar medium and on an agar surface

Abstract Cells of nine amphibian permanent cell lines grew in or on agar with plating efficiencies of 10–85%. Xenopus primary strain normal cells or lymphoid tumour cells usually did not grow in the agar medium, but sometimes formed detached, morula-like balls of cells in liquid medium on a hard agar surface

Amphibian cells in vitro: II. Effects of variations in medium osmolarity on a permanent cell line isolated from Xenopus

Abstract The growth of Xenopus cells of a permanent aneuploid cell line was investigated, using media with osmolalities from 90 to 750 mOs/kg. Media from 120 to 320 mOs/kg gave high growth rates, and the optimum growth medium was at 190 mOs/kg. Labelled thymidine was incorporated by the cells over the range 90 to 750 mOs/kg.

Hormonal control of glucose production by Amphiuma means liver in organ culture.

Abstract The effects of glucagon, insulin, and adrenalin on glucose production by Amphiuma means liver organ cultures maintained at 25° were studied. Glucagon stimulated gluconeogenesis from pyruvate and alanine, and adrenalin treatment resulted in a more rapid glycogenolysis than glucagon treatment. Glucagon increased the tissue levels of glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), and fructose-1,6-diphosphatase after 40 hr, while insulin decreased GOT and GPT levels after 72 hr. The presence of pyruvate in the medium significantly reduced nitrogenous excretion by the liver fragments. We conclude that cultured A. means liver responds to glucagon, insulin, and adrenalin in ways that are compatible with the known effects of these hormones in mammals.

Organ culture of Amphiuma means liver: control of glycogen content.

Abstract 1. 1. Amphiuma means liver fragments retained their glycogen for at least 35 days in organ culture. 2. 2. Glycogen levels fell when adrenalin was added, then increased when insulin and/or extra glucose were added to the culture medium.

Organ culture of adult Amphiuma means kidney: Effects of media and urea on ammonia production, glycogen content and cell proliferation

Abstract 1. 1. Amphiuma means kidney fragments were cultured for up to 6 weeks in three different media (Leibovitz L15 (L15), Minimum Essential Medium (MEM), MEM + urea). 2. 2. Ammonia was released into the culture medium for up to 5 weeks; the amount produced was greater in L15 medium, which has a higher amino acid content than MEM. 3. 3. Kidney fragments contained glycogen after 5 weeks in culture in all three media. 4. 4. There was an increase in mitotic incidence in culture, which was significantly greater in one animal in medium with added urea. 5. 5. The relationship between function and proliferation in the kidney is discussed.

The effects of phenformin on glucose, glycogen and lactate metabolism in Amphiuma means liver organ cultures

Abstract 1. Although phenformin has been in use as an oral hypoglycaemic agent for nearly 20 yr, the biochemical basis of its action is still poorly understood. 2. Adult amphibian liver organ cultures were used in studies on the effects of the drug on glucose utilisation, lactate production and glycogen level. 3. Phenformin concentrations in the range 4·8 × 10 −4 M to 4·8 × 10 −6 M caused an increase in glucose utilisation and in lactate production, and led glycogenolysis. 4. We conclude tthat the effect of the drug is to increase glucose utilisation at the expense of increased lactate output. The hypoglycaemic effect probably depends on both the dose phenformin given and the glycogen status of the liver.

Organ Cultures of Normal and Neoplastic Amphibian Tissues

Although the organ culture technique has been widely used in studies on the growth and differentiation of mammalian tissues, there are few records of the use of the technique with the tissues of metamorphosed amphibians. We have recently shown that a variety of organs from recently metamorphosed, immature Xenopus may be maintained in culture for at least 6 days, with mitotic incidences comparable to or even higher than those in vivo (1).

The long-term organ culture of tissues from adult Necturus maculosus, the mud puppy

Fragments of Necturus maculosus liver, spleen and kidney were cultured at 25°C in 50% Minimal Essential Medium (MEM) or 50% Leibovitz L‐15 Medium (L‐15) for up to 49 days. The integrity of tissue structure was evaluated, hepatocyte cell and nuclear volumes were measured, the respiration rates of freshly‐isolated and cultured liver fragments were determined, and the mitotic incidences in cultured liver, spleen and kidney were estimated. The addition of adrenalin caused a reduction in the glycogen content of liver cultures, and the subsequent addition of insulin resulted in a net increase in glycogen synthesis. Glycogen levels fell in fragments cultured in L‐15, but rose in cultures in MEM. Arginase and ornithine transcarbamylase levels fell gradually throughout a 49‐day culture period in L‐15. Evidence presented supports the position that the survival of tissues in vitro is related to cell size and respiration rate. These experiments show that N. maculosus is a suitable donor of tissues for long‐term organ culture studies on the maintenance and control of tissue‐type specific structure and function.

Amphibian organ culture as a basis for investigations on the specific and general effects of drugs.

Abstract 1. Since a variety of tissues from adults of a number of amphibian species survive and function normally in long-term organ culture, it is argued that such cultures have great potential value for pharmacological investigations. 2. Preliminary studies were carried out on the effects of physiological doses of diazoxide, frusemide, propranolol, isoprenaline and phentolamine on glycogen level and on glucose uptake or release in Amphiuma means liver cultures. 3. Since all the results were consistent with the known effects of these drugs in man, it is concluded that amphibian organ culture will be useful to the pharmacologist.