Peter J. Goldblatt

Trout hepatocyte culture: Isolation and primary culture

SummaryRainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92×106 liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochromep450 activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d of culture. Culture temperature also influenced thep450 activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and the appearance of bundles of intermediate filaments also were observed with increased time in culture.

MOUSE LIVER CELL CULTURE II. Primary Culture

SummaryMouse hepatocytes in primary culture were characterized. Hepatocytes were isolated by the two-step hepatic portal vein perfusion method described previously. An optimal cell attachment of 43% was noted after 2 h incubation in 10% fetal bovine serum. Minimal attachment (lees than 7%) occurred in serumless medium. Serum concentrations above 10% and attachment durations greater than 2 h resulted in no increased attachment of viable cells. Nonviable cells, however progressively attached when both of these parameters were increased. Survival data of the cells in culture resembled those reported for rat hepatocytes in primary culture. A progressive decrease in survival was noted following initial attachment until only approximately 15% of initially plated cells remained viable and attached after 8 d culture. The decrease in survival was accompanied by morphologic changes including flattening and elongation of the cells, some multinucleation, and disruption of monolayer groups.

Strain A/J Mouse Lung Adenoma Growth Patterns Vary When Induced by Different Carcinogens*

The histogenesis of mouse lung adenomas is currently being investigated in several laboratories. Based upon studies of a limited number of carcinogens in different mouse strains, some investigators suggest that all lung adenomas in mice are derived from alveolar type II cells, whereas others suggest a Clara cell origin for a majority of the tumors. This report differs from previous investigations in that 12 different carcinogens were evaluated for the types of lung tumor growth patterns they induced in a single mouse strain (strain A mice). The carcinogens aflatoxin B, (AFB,), benzo(a)pyrene (BP), 1,2-dimethylhydrazine (DMH), 3-meth-ylcholanthrene (MCA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosomethylurea (MNU) induced tumors with a predominately solid/alveolar growth pattern, whereas N-nitrosodiethylamine (NDEA) induced predominantly papillary tumors. Most of the other carcinogens induced a higher proportion of lung tumors with the solid/alveolar growth pattern than with the papillary growth pattern; however, ratios between the 2 growth patterns varied. If, as suggested by others, solid tumors are derived from alveolar type II cells and papillary tumors from Clara cells, then carcinogens may differ with respect to their ability to transform one cell type or the other.

Tumor-localizing and photosensitizing properties of hematoporphyrin derivative in hamster buccal pouch carcinoma

The tumor-localizing and photochemotherapeutic properties of hematoporphyrin derivative (HPD) were examined in 7, 12 dimethylbenzanthracene (DMBA)-induced oral cancers in the Syrian hamster. Oral tumors in hamsters injected with HPD (50 micrograms per gram of body weight) exhibited bright salmon pink fluorescence when exposed to long-wave ultraviolet light 24 hours after intraperitoneal HPD injection. Adjacent tumor-free mucosa did not fluoresce. Similarly, tumors not treated with HPD, normal mucosa treated with HPD, and normal mucosa not treated with HPD did not fluoresce. Tumors in animals that received HPD and photochemotherapy (PCT) were examined for gross and microscopic pathologic changes following the phototreatment. Tumors displayed edema, hemorrhage, and cellular necrosis that progressed with the time of sampling after photochemotherapy. Complete tumor necrosis was evident in the majority of oral tumors 24 hours after HPD PCT.

Hematoporphyrin Derivative Photochemotherapy of Experimental Bladder Tumors

Recent studies have shown that disruption of tumor blood flow is a major consequence of hematoporphyrin derivative photochemotherapy. A series of experiments was undertaken on the transplantable N-(4-(5-nitro-2-furyl)-2-thiazolyl)-formamide induced urothelial tumor in Fischer 344 rats to determine a dose response for both hematoporphyrin derivative and light. Tumor blood flow was used as the biologic criteria of response. Hematoporphyrin derivative doses of 10 micrograms./gm. body weight or above were necessary to cause a significant decrease in tumor blood flow when the tumors were illuminated with 360 joules/cm.2 of noncoherent red light (greater than 590 nm.). With a constant hematoporphyrin derivative dose of 20 micrograms./gm. body weight, significantly lower tumor blood flows were observed with fluences of 240 joules/cm.2 and above. In order to correlate dose response to tumor regression, experiments were done in which tumor dry weights were determined 3 weeks after completion of photochemotherapy (360 joules/cm.2). Hematoporphyrin derivative doses of 10 micrograms./gm. body weight or above were necessary to induce tumor regression. These studies support the hypothesis that disruption of tumor blood flow is a tumoricidal mechanism of hematoporphyrin derivative photochemotherapy.

Glyceraldehyde-3-Phosphate Dehydrogenase and Other Enzymatic Activity in Normal Mouse Lung and in Lung Tumors

The histogenesis of chemically induced mouse lung adenomas is currently being debated. Tumors induced by a variety of chemicals and in a number of different strains exhibit growth patterns having a solid/alveolar appearance, a papillary appearance, or a mixture of both. Ultrastructural observations suggest that solid tumors are derived from the alveolar type II pneumocyte and that papillary tumors arise from the bronchiolar Clara cell. However, recent immunocytochemical investigations have concluded that most mouse lung tumors are derived solely from the alveolar type II cell. Enzyme histochemical methods have previously been utilized to identify Clara cells in pulmonary cell isolates and also to characterize mouse lung tumors. This report demonstrates a difference in glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity in type II pneumocytes and Clara cells. Solid tumors and type II cells appear to have a similar G3PD activity, and this activity is different from that observed in papillary tumors and bronchiolar cells. These findings support morphological evidence that suggests mouse lung tumors are phenotypically different and may arise from at least two different cells of origin.

EFFECT OF FREEZING AND THAWING ON CERTAIN NUCLEAR AND MITOCHONDRIAL ENZYMES OF MOUSE LIVER

1. The effects produced by a rapid freezing and thawing on the activities of some enzymes of nuclear (nicotinamide adenine dinucleotide (NAD)-pyrophosphorylase) and mitochondrial (succinoxidase, succinic dehydrogenase, and glutamic dehydrogenase) origin are presented. These effects are compared with those produced by sonic treatment of fresh tissue. 2. The activity of NAD-pyrophosphorylase was unaffected by the freezing procedures employed, namely dry ice at –79°C, isopentane at –155°C, and propane at –175°C, or by sonic treatment of homogenates of fresh tissue. 3. Succinoxidase activity was decreased to some extent by all the procedures employed. Sonic treatment produced a 47% loss in the enzyme activity, while freezing on dry ice, in isopentane, or in propane produced losses of 26, 29, and 37%, respectively. 4. The activity of succinic dehydrogenase was least affected by sonic treatment or by freezing on dry ice. Quenching in isopentane or in propane, however, produced 11% and 23% loss respectively in the dehydrogenase activity. 5. Free glutamic dehydrogenase activity, which is low in fresh-tissue preparations, was increased to the same extent by all methods employed. The unsedimentable activity depended upon the procedure used, however. Sonic treatment and freezing with propane, isopentane, or dry ice gave 78, 48, 33, and 20%, respectively, of the total enzyme activity. 6. The fact that the increase in unsedimentable glutamic dehydrogenase activity roughly parallels a decrease in succinoxidase activity suggests that a rapid freezing followed by thawing produces alterations in the mitochondrial membranes.

Evaluation of an immunoisolation membrane formed by incorporating a polyvinyl alcohol hydrogel within a microporous filter support.

An immunoisolation membrane formed by incorporating a high water content polyvinyl alcohol (PVA) hydrogel into a microporous polyether sulfone (PES) filter has been investigated in this study. The PVA hydrogel is formed in situ within the filter pores via glutaraldehyde (GA) crosslinking under acidic conditions. The tortuous nature of the microporous filter pores securely anchors the embedded hydrogel to provide excellent structural integrity. The high void fraction of the PES filter support (>80%) and high water content of the PVA hydrogel (>85% water by weight) allow excellent solute transport rates, while an appropriate level of glutaraldehyde crosslinking supplies the required molecular size selectivity. In vitro permeability measurements made with solutes covering a wide range of molecular sizes demonstrate high transport rates for small nutrient molecules with rapidly diminishing permeabilities above a molecular weight of approximately 1,000 Dalton. Implantation experiments show that the membrane properties are not deleteriously affected by prolonged in vivo exposure or common sterilization techniques. Thus, this hybrid hydrogel/filter membrane system offers a promising approach to the immunoisolation of implanted cells.

Correlation Of Tumor Blood Flow To Tumor Regression After Hematoporphyrin Derivative (HPD) Photodynamic Therapy To Transplantable Bladder Tumors

Hematoporphyrin derivative (HPD) photodynamic therapy (PDT) has proved effective in the treatment of selected neoplasms (1). The effectiveness of this form of therapy rests on the retention of the systemically administered HPD in neoplastic tissue and its photoactivation with visible light resulting in ‘photodynamic’ tumor destruction. Although it is generally agreed that singlet oxygen liberated during HPD-photodynamic therapy is responsible for the biologic damage created by PDT, the mechanisms of cell death have not been clearly defined (2). Previous studies in our laboratory have demonstrated a rapid and sustained decrease in tumor blood flow after HPD-photodynamic therapy (3,4). The present study was undertaken to correlate changes in tumor blood flow with tumor regression after HPD-PDT.

Tracer technique to measure in vivo chemical transport rates within an implantable cell transplantation device

An in vivo tracer technique that uses radiolabeled insulin as the tracer molecule has been developed to assess the rate of chemical transport between the cell transplantation chamber of an implantable bioartificial device and the hosts circulatory system. The device considered here employs site-directed neovascularization of a porous matrix to induce capillary growth adjacent to an immunoisolated cell implantation chamber. This device design is being investigated as a vehicle for therapeutic cell transplantation, with the advantages that it allows the cells to perform their therapeutic function without the danger of immune rejection and it avoids damaging contact of blood flow with artificial surfaces. A pharmacokinetic model of the mass transport between the implantation chamber, the vascularized matrix, and the body has been devised to allow proper analysis and understanding of the experimental tracer results. Experiments performed in this study have been principally directed at evaluation of the tracer model parameters, but results also provide a quantitative measure of the progression of capillary growth into a porous matrix. Measured plasma tracer levels demonstrate that chemical transport rates within the implanted device increase with the progression of matrix vascular ingrowth. Agreement between the fitted model curves and the corresponding measured concentrations at different levels of capillary ingrowth demonstrate that the model provides a realistic representation of the actual capillary-mediated transport phenomena occurring within the device.