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A method was developed for cloning the Lewis lung carcinoma cells (LLC) in vivo in diffusion chambers. This method is analogous to the system introduced by Gordon for cloning hemopoietic precursor cells. No significant differences were observed in the efficiency of the cloning of the tumour cells within the range of 1 to 4 weeks after transplantation (7.7 +/- 0.7% for 240 cultures).
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Factors influencing granulopoiesis have been evaluated using diffusion chambers implanted in the peritoneal cavity of rabbits. An increase in granulopoiesis in chambers implanted in hosts made neutropenic by nitrogen mustard occurs in mice made neutropenic by x-ray or drug. The intraperitoneal injection of leukocytes inhibited the growth of cells in chambers implanted in rabbits. Removal of mature granulocytes from marrow prior to chamber inoculation produced a marked in-increase in cell growth, especially of granulocytes. Mature granulocytes clearly inhibited cell replication and this inhibition involved both myeloid and erythroid elements, although the data suggest a greater effect on myelopoiesis. In contrast to the mouse, erythropoiesis in chambers in rabbits remained prominent for over 1 wk.
Many experimental arguments make some cellular elements which are present in the hemopoietic bone cavities responsible for the bone marrow microenvironment. Diaphyseal fragments of previously curetted rat femurs were cultivated in vivo in diffusion chambers. Histological examination of the chamber membranes showed the presence of fibroblast-like cells. Their osseous origin was demonstrated by the study of the sexual chromatin. A morphological similarity seems to occur between these cells obtained in vivo and those described in in vitro cultures of hempoietic bone or bone marrow.
A flow diffusion chamber designed for studying cells and tissues in culture is described. The chamber contains a plate with a great number of isolated holes, which enables one to perform the cultivation of cells at different distances from the porous membrane separating the cells from the perfused medium. An individual porous membrane can be placed above each hole. Evidence for the selective permeability of domestic membranes under the conditions of cell culture in chamber is presented. The chamber makes possible a simultaneous cultivation of a great number of various cultures with different conditions of mass exchange with common perfused medium, which contributes to intensification of studies.
Methodical approaches for obtaining the viable tumour cells from solid human tumours are developed. Combination of the short-term enzymatic treatment of the tumour tissue pieces and their gradual rubbing through the metal sieve with the decreasing pore sizes permitted obtaining a large number of isolated tumour cells with the high percentage of viability.
Studies were done of cell production by marrow in diffusion chambers implanted in the peritoneal cavity of rabbits subjected to various stimuli to hematopoiesis. In chambers in neutropenic hosts and in hosts injected with endotoxin, animals presumed to have an increased stimulus to granulopoiesis, there was increased production of granulocytes but there was also increased production of red cells. Although red cell production was decreased in chambers in polycythemic hosts, granulocyte production was not different from that in controls. Stimulation of erythropoiesis by erythropoietin injections or by exposure to hypoxia increased red cell production by marrow in the implanted diffusion chambers without diminishing granulopoiesis. Only in chambers in hosts made anemic by bleeding was there an increase in red cell production accompanied by a decrease in granulocyte production. In these anemic hosts induction of neutropenia led to an increase in granulopoiesis without any depression of erythropoiesis.
With cultivation of explants of different human malignant tumors in diffusion chambers at the same study terms, differences in the ratio of epithelial-like and fibroblast-like cell growth were noticed with marked degeneration changes in one of these cel types, and in the ability to form spheroids from epithelial-like cells. These data are discussed in connection with the optimal time of obtaining the cell material ready for a further cultivation of cell suspensions, with the aim to prepare autovaccines for infection of patients with malignant tumors.
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Data on induction of sister chromatid exchange (SCE), 8-azaguanine and ouabain-resistant mutants in V79 cells in diffusion chambers in mice treated with three chemicals were compared to determine the relationship between SCE and mutation. For 1-(pyridyl-3)-3,3-dimethyltriazene, a positive, linear correlation was demonstrated between induced SCE and mutation at both loci. For cyclophosphamide and ifosfamide, it was observed that SCE analysis was a more sensitive biological endpoint than induction of mutation.
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Inhibition of normal mouse hemopoietic stem cells by leukemic cells (C1498) was observed with use of in vitro agar and in vivo diffusion-chamber cultures. The C1498 cells were unresponsive to colony-stimulating activity, and, above a critical threshold, they inhibited normal granulocyte progenitors in agar culture. C1498 cells added to normal marrow in diffusion chambers progressively reduced granulocyte progenitors. The larger, more rapidly growing, C1498 cells showed the most inhibitory effect. Transmembrane culture of C1498 cells adjacent to normal marrow in double diffusion chambers for five to 14 days led to reduction of recovery of granulocyte progenitors (72 +/- 7 per cent of control) and pluripotent stem cells (45 +/- 7 per cent of control) from the normal marrow chambers. These results indicate that leukemic mouse cells inhibit normal mouse-marrow stem cells by releasing a diffusible substance, and this inhibition occurs primarily at the level of the pluripotent stem cell.
When rabbit bone marrow cells are cultured in a diffusion chamber implanted into the peritoneal cavity of an athymic mouse, the stromal cells proliferate, differentiate, and produce tissues that have the morphological features of loose fibrous tissue, woven or chondroid bone, and cartilage. The collagens synthesized during the development of the tissues from 7 to 28 days after implantation were identified using specific antibodies to rabbit types I, II, III, and V and rat type IX collagens, while the glycosaminoglycans were characterized histochemically using the dye, Alcian blue. Fibrous tissue forms in the first week and it contains types I, III, and V collagens and hyaluronan. Bone and cartilage develop within the fibrous tissue from about 12 days onwards. The bone matrix contains types I and V collagens, and chondroitin and keratan sulphates. The cartilaginous matrix contains types II and IX collagens, and chondroitin and keratan sulphates. Small amounts of type III collagen are found in the bone, and types I, III, and V collagens in the cartilage. These are thought to be the remnants of the fibrous matrix and decrease as the matrices mature. It is concluded that the tissue in diffusion chambers, formed by a small number of early precursor cells present in the soft tissues of the endosteum and marrow of young rabbits, contains extracellular matrix macromolecules similar to those found in bone and cartilage.
The response of granulocyte progenitors (CFU-D) from patients with chronic myeloid leukaemia (CML), neutrophilic reaction (NR) and healthy subjects to macrophage-derived stimulatory and inhibitory factors was investigated in diffusion chamber culture. CFU-D from CML and NR demonstrated a normal reactivity to macrophage stimulation but were hyporesponsive to indomethacin-sensitive inhibition. It is also shown that the spleens of patients with Hodgkin's disease contain locally activated macrophages with higher production of indomethacin-sensitive growth inhibiting factor for autologous CFU-D clonal proliferation.
We assessed the effect of phlebotomy on the proliferative activity of less (BFU-E) and more (CFU-E) mature bone marrow-derived erythroid progenitors from patients with polycythemia vera (PV) and polycythemia symptomatic (PS) in vivo in diffusion chamber culture. The cloning efficiency of erythroid progenitors under the effect of normal and increased erythropoietin (Epo) concentrations in PS was comparable with controls, whereas in PV the BFU-E and CFU-E-derived colony formation was significantly higher (p less than 0.01). In PV, the erythroid progenitors formed markedly more colonies in cultures stimulated with higher Epo concentration (p less than 0.01). The results of this study indicate that phlebotomy both in PV and PS does not affect the reactivity of erythroid progenitors to various Epo concentrations.
The humoral effect of resting and phorbol myristate acetate (PMA) preincubated normal monocytes on granulocytic progenitor growth in double diffusion chamber culture (CFU-dG) was assayed. The activation of monocytes by PMA was confirmed in NBT reduction test (increased formazan content) and by enhanced superoxide anions production compared with resting cells. The activatory and inhibitory effect of monocytes on granulocytic colony formation was cell-concentration dependent. The maximal CFU-dG growth promoting effects was seen at 5 X 10(4) of resting monocytes, whereas PMA-treated cells exerted their optimal stimulatory activity at lower cell number (2.5 X 10(4)). Further increase of monocyte concentration resulted in diminished CFU-dG proliferation. It is shown that the decline of colony count at high monocyte number was caused by indomethacin-dependent inhibitors.