Diffusion Chamber Cultures Research Articles

Proliferation kinetics of Shay chloroleukaemia cells grown in diffusion chambers in vivo.

ABSTRACTThe proliferation kinetics of Shay chloroleukaemia cells was studied by the labelled mitoses technique and some other methods. Detailed estimates of the phase durations of the cell cycle for proliferating cells were obtained using young, exponentially growing diffusion chamber cultures; estimates of kinetic parameters were also obtained for ‘old’ cultures and for local subcutaneous chloroma tumours. The results enabled determination of the growth fraction and the cell loss factor.Cell loss was found to be the dominant factor determining the growth rate of chloroleukaemia cell populations. The mechanism of cell loss was cell death. Some implications of these findings are discussed.

Proliferative properties of malignant cell populations cultured in intraperitoneal diffusion chambers

The proliferative characteristics of four different types of malignant cells cultured in intraperitoneal diffusion chamber (ID-chamber) were investigated, and compared with the corresponding data obtained under conditions of cell culture in vitro and growth in vivo. The cell types were: BICR M1R K cells, a permanent malignant cell culture line originating from a transplantable mammary tumour of the Marshall rat; Fortner A Mel 1 cells, a transplantable malignant melanoma of the golden hamster in the ascitic form; a transplantable ethylnitrosourea-induced leukemia (L 5222) of the BD IX rat; and finally leukemic cells from two human patients with untreated acute myeloblastic leukemia. After an initial decline in the cell number, all cell types investigated proliferated in ID-chambers implanted into BD IX rats, for periods of 8–13 days. The tumour cell populations grew exponentially for 2–5 days, with an actual doubling time corresponding to that obtained during growth in vivo. The human leukemic cells behaved as a “steady-state” system, where the rate of proliferation was apparently compensated by a corresponding rate of cell death. The rate of proliferation in ID-chambers was slower than under cell culture conditions in vitro. Ten min after intravenous injection of hydroxyurea (HU) into the host animals, the HU concentration in the ID-chambers had equilibrated with the blood concentration of HU, when measured on the third day after implantation of the chambers. On the 7th day, the rate of diffusion of the compound between the blood and the chambers was slower, probably due to an inflammatory reaction on the external side of the chambers. The mode of proliferation of the investigated cell populations during diffusion chamber culture may be more representative of the normal growth conditions in vivo than of those in cell culture in vitro.

Inhibition of granulopoiesis by endogenous granulocyte chalone studied with the diffusion chamber technique

This study showed that growth of granulocytes, both normal and leukaemic, is inhibited in a specific manner in diffusion chamber cultures carried by chloroma-bearing host rats (→ elevated granulocyte chalone content in the body). This inhibition could be demonstrated directly by a reduced formation of granulocytes and by measuring the 3 H-thymidine uptake in the cells. Non-granulocytic cell lines (mastocytoma and HeLa cells) were not detectably inhibited when grown in identical conditions. The inhibition of granulocytic cells did not depend on a selective immunological reaction developed against granulocytic cells in the chloroma-bearing host rats.

Differentiation of Leukaemic Cells in Diffusion Chambers

Morphological and cytochemical characteristics of cells obtained from Shay chloroleukaemia tumour growing subcutaneously in the rat and after 0.5–16 d of diffusion chamber culturing were assayed using seven different cytochemical procedures. Further, the cytochemical characteristics of the recent subline of Shay chloroma were compared with those reported in 1962 by other workers.The chloroleukaemia cells did undergo some maturation in diffusion chamber cultures, but full differentiation to polymorphonuclear cells was not seen. The most striking change in the cytochemical pattern of chloroleukaemia cells during the diffusion chamber culturing was a significant increase in alkaline phosphatase activity. There were some cytological differences in the recent chloroma tumour compared with that described 10 years ago.The observations made in the present work show that all properties of cells after a neoplastic transformation are not irreversible: many characteristics of malignant chloroleukaemia cells can alter when these cells are maintained in different host animals or in the diffusion chamber milieu.

The cell specific effect of the granulocyte chalone demonstrated with the diffusion chamber technique.

ABSTRACTThe granulocytic chalone is secreted by mature granulocytes and inhibits 3H‐thymidine incorporation of proliferating granulocytes in vitro. The effect and the cell line specificity of this chalone was assessed with the in vivo diffusion chamber culture technique. Tests were carried out on cultures from normal mouse bone marrow cells and mouse and rat blood leucocytes. The majority of the DNA synthesizing cells in marrow cultures were proliferating granulocytes. Macrophages and immunoblasts proliferated in rat leucocyte cultures, when the chambers had been carried for 5 days in host mice. Repeated chalone or control injections were given i.p. to the host mice during 6–7 hr prior to 3H‐thymidine injection. Isotope uptake of proliferative granulocytes was reduced by the chalone treatment. No such effect was found on the rat immunoblasts and macrophages. The viability of cultured cells was apparently not affected by the chalone treatment.

The Effect of Neutropenia on Myeloid Growth and the Stem Cell in an In Vivo Culture System

Abstract An in vivo diffusion chamber (DC) culture technique was used to evaluate the role of circulating humoral factors in the control of myelopoiesis. Normal mouse bone marrow cell suspensions sealed in DC’s were implanted intraperitoneally into mice or rats rendered neutropenic by pretreatment with cyclophosphamide, and the growth of cells in the DC’s was evaluated at intervals thereafter. When marrow was cultured in hosts with graded neutropenia, a dose-related augmentation of myelopoiesis was demonstrated. The growth of macrophages and of pluripotent stem cells, measured by the spleen colony-forming technique, was also enhanced by culture in neutropenic hosts. These data provide further evidence that the rate of myelopoiesis is influenced by a circulating humoral factor. They further suggest that a humoral or hormonal factor is important in control of the pluripotent stem cell population.

Response of multipotent (CFU) and granulocyte (diffusion chamber assay) progenitor cells and differentiating cells of murine haematopoietic tissues to a perturbation of the steady state.

AbstractRegulation of haematopoiesis was investigated by studying the response of haematopoietic tissues of mice to a perturbation of the steady state by vinblastine (VLB). Progenitor cells were quantified ly limiting dilution analysis of diffusion chamber cultures of haematopoietic cells and by the spleen colony technique. The diffusion chamber technique appears to assay granulocyte progenitor cells and those multipotent progenitor cells that become committed to granulopoiesis during chamber culture. The spleen colony technique probably assays multipotent progenitor cells.Decaying oscillatory responses to VLB were observed for progenitor cells as well as for differentiating cells in bone marrow. The period lengths of the diffusion chamber progenitor cell oscillations might indicate that these were induced by humoral feedback signal(s) from nonproliferative granulocytes. The oscillations of the multipotent progenitor cells of bone marrow were less pronounced and were earlier damped than those of the granulocyte progenitor cells. This may support the hypotesis that multipotent progenitor cells are regulated by more efficient mechanisms, which may depend on short range cell‐cell interactions rather than long range humoral regulators.

Regulation of granulocyte and macrophage formation in diffusion chamber cultures of mouse haematopoietic cells

Bone marrow and blood cells from mice were cultured in peritoneal diffusion chambers in homologous recipients. Granulocytes and macrophages were formed in the chambers. About a quarter of the blood lymphocytes were recovered after the culture period, whilst bone marrow lymphocytes almost disappeared. The number of granulocytes harvested was linearly related to the number of cells implanted, indicating that growth regulatory cell interactions did not take place in the chambers. Regenerating bone marrow produced twice as many granulocytes per progenitor cell as did normal marrow. The cell yield was also increased when irradiated or cyclophosphamide pretreated chamber hosts were employed. Thus properties of the progenitor cells as well as humoral factors from the hosts did influence chamber granulopoiesis. In contrast to the growth results for granulocytes, the formation of macrophages showed a relative decrease when the cell inocula were increased. Pretreatment of the host mice with irradiation or with cyclophosphamide resulted in increased macrophagopoiesis in bone marrow cultures.

Anti-DNP hemolytic plaques by mouse spleen cells in diffusion chambers.

Spleen cells frcan mice immunized with dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) were restimulated with DNP-KLH in vitro, enclosed in cell-impermeable diffusion chambers and implanted in the peritoneal cavity of irradiated recipient mice. At various intervals, the cells were recwered from the chambers and DKP-specific plaque forming cells (PFC) were enumerated. The numbers of nucleated cells in the chambers declined rapidly during the first day after implantstion then increased between days 3 and 6 and remained at between 30 and 40% of the initial number from day 6 to day 10. Doses of DNF-KLH of 5 to 500 ng induced maximal numbers of PFC, while fewer PEC were obtained Kith lower and higher doses. The numbers of PFC/106 cells declined during the first 2 days of culture in diffusion chambers, then increased explosively between days 3 and 4 and reached a peak on day 5 through 8. At peak response, 5% of the nucleated cells were PFC and in one case the proportion of PFC was 12%. Varying the time be...

Responses of cells from different lymph nodes to BSA antigen in diffusion chambers.

Lower titers of anti-BSA antibodies were obtained from diffusion chamber cultures of unprimed mesenteric lymph nodes with antigen than in cultures of thoracic and popliteal lymph nodes. The incidence of cultures with no demonstrable antibodies was highest among the mesenteric lymph node cultures. The serological results were parallelled by the incidence of reactive cellular forms in the chambers.

Formation of granulocytes and macrophages in diffusion chamber cultures of mouse blood leucocytes.

Suspensions of mononuclear cells were prepared from mouse blood by a one stage centrifugation method. Iso‐ and homologous culturing of these cells were then carried out in cell‐impermeable diffusion chambers. These were implanted into the peritoneal cavities of host mice. The culture periods ranged from 1 to 13 days. The proliferation, differentiation and death of the chamber cells were quantitated by total and differential cell counts and by evaluation of 3H‐thymidine incorporation from liquid scintillation counting and radioautography.Proliferating and differentiating granulocytes and macrophages were consistently found in the cultures, whilst lymphocytes from the inoculum were gradually lost during the culture periods.

The Separation and Cultivation of Basal and Differentiating Cells from Hairless Mouse Epidermis

An earlier described method for the separation of basal and differentiating cells from hairless mouse epidermis (1) has been further studied. 1) A more precise quantitative determination of the specificity of the separation procedure has been performed by radioactive labeling of DNA in basal cells. By autoradiography the number of labeled basal cells in each fraction was counted and the purity calculated. The purity of the basal cell suspensions was 87.0 per cent and the differentiating cells 77.8 per cent. As purely morphological judgement is insufficient to distinguish an early differentiating cell from a basal cell, such specificity tests are necessary to ensure a correct separation technique. 2) The viability of the separated cells has been studied by culture in diffusion chambers up to 10 days. The cells appeared intact the first 24 hours, and thereafter a pronounced cell loss took place. The surviving differentiating cells appeared to continue their keratinization ending after 7 days, as expected from their turnover time in vivo. Basal cells either went into differentiation or proliferation, but the latter process was outweighed by the cell loss.

The cellular sources of antibodies in diffusion chamber cultures of alveolar exudates.

Alveolarexsudatzellen von Kaninchen wurden zusammen mit Schaferythrozyten in Diffusionskammern gebracht und diese in neugeborene Kaninchen implantiert. Antikörperbildende Zellen wurden in den Diffusionskammern und im Omentum der Rezipienten festgestellt.

Erythropoiesis in diffusion chambers during hypoxia

Bone marrow suspensions were cultured in diffusion chambers implanted in animals kept for six days under hypoxic conditions. It was shown that under hypoxic stimulation the bone marrow cultures show significant erythropoietic activity based on uptake studies of Fe- 59. This study indicates that in vivo culture in diffusion chambers provides a possible system for analysis of differentiation control factors present under hypoxic stimulation.

The Suppressive Effect of Rauscher Leukemia Virus on the Secondary Antibody Response of Spleen Cells Cultured in Cell-Impermeable Diffusion Chambers

Abstract The effects of Rauscher leukemia virus (RLV) on the immune response to sheep red blood cells (SRBC) were studied in BALB/c mice and in isogeneic spleen cells cultured in cell-impermeable diffusion chambers implanted into irradiated hosts. Primary antibody formation to SRBC was depressed in mice inoculated with RLV. Mice injected with SRBC from 0 to 48 hr after virus inoculation had normal 19 S and depressed 7 S antibody production. Both 7 S and 19 S antibodies were depressed when SRBC were inoculated 3 to 15 days after RLV infection. There was a positive correlation between erythroblastic reaction and immunosuppression in two strains of mice: BALB/c was sensitive and C57BL partially resistant to the erythroblastic and immunosuppressive effects of RLV. Antibody production was not impaired in mice inoculated with RLV mixed with anti-RLV mouse antisera providing evidence for the viral specificity of immunosuppression. Antibody production and plasmacytosis were demonstrated in SRBC-primed spleen cells cultured with SRBC in cell-impermeable diffusion chambers implanted into irradiated isogeneic hosts. RLV inhibited the secondary antibody response to SRBC in intact mice and in spleen cells cultured in diffusion chambers. Increase in blast cells, reduction in plasma cells, and depression of antibody formation were found both in primed spleen cells infected with RLV prior to culture and in primed spleen cells simultaneously exposed to RLV and SRBC in diffusion chamber cultures. This system provides a quantitative approach to the study of mechanisms of oncogenic virus-induced immunosuppression at cellular and subcellular levels.

Stochastic model for the production of antibody-forming cells.

FOR some time it has been thought that the generation of antibody-forming cells (AFC) may involve an interaction between cell types1–3. We present here a stochastic model, based on a two-cell interaction system, for the production of AFC. The assumptions on which the model is based have chiefly come from an interpretation of two types of experimental data, each of which can yield information concerning the number of cells involved. The first is limiting dilution analysis, which is based on the Poisson probability distribution. The second is dose–response analysis, showing the relationship between lymphoid cell dose and the number of AFC generated. These types of experiments involve either the transfer of lymphoid cells to irradiated, compatible recipients or the growth of lymphoid cells in diffusion chamber cultures.

In vitro Antibody Synthesis by Diffusion Chamber Cultures of Spleen Cells

Summary The effect of free antibody on antibody production in the secondary immune response has been studied by use of antigen-stimulated primed spleen cells in diffusion chamber cultures. The cultures were exposed to high concentrations of antibody at various times after the cultures were begun, and antibody production was assayed by determining the amounts of in vitro radioactive amino acid incorporation into specific antibody. Chamber cultures implanted from the start in animals hyperimmunized to the test antigen (sheep red blood cells) failed to exhibit any significant antibody production. When the cultures were grown for 7 to 9 days in normal animals and exposed to free antibody either at the time of in vitro incubation or by transfer into hyperimmune animals for 1 or 2 days prior to the in vitro assessment of antibody production, there was no significant depression of antibody production. This was true even if the cultures were exposed to kiloroentgen doses of x-ray at the beginning of the plateau phase of their immune response to eliminate turnover in the antibody-producing cell population. These results suggest that antibody-mediated regulation of antibody production occurs at the level of the antibody-producing cell population, possibly by preventing antigenic determinants from triggering progenitor cells that participate in the immune response, and not through control of the synthetic or secretory activities of antibody-producing cells.

In vitro Antibody Synthesis by Diffusion Chamber Cultures of Spleen Cells

Summary Techniques for the study of antibody production by primed antigen-stimulated spleen cells in cell-impermeable diffusion chambers have been developed. The chambers were recovered from their host animals and incubated in vitro with radioactive amino acids. Antibody production was measured by determining the amount of radioactivity which would specifically bind to erythrocyte stroma of the type used for immunization. In addition, the effect of kiloroentgen doses of radiation on antibody production by plateau phase cultures was studied in a system in which primed spleen cells in cell-impermeable diffusion chambers were irradiated 6 days after antigen stimulation. One and three days later the chamber cultures were incubated in vitro in the presence of 14C-labeled amino acids. The incorporation of radioactivity into total extracellular proteins, γG-globulin, and specific antibody was measured. Isotope incorporation in cultures which had received 10,000 r of x-radiation was qualitatively and quantitatively similar to that by unirradiated controls—as judged by a variety of criteria. It has been shown previously that irradiation of diffusion chamber cultures of antigen-stimulated spleen cells with kiloroentgen doses of x-radiation in the plateau phase of their response leads to a purification of mature antibody-producing cells and virtually eliminates cell multiplication. It is shown here that the surviving immunocompetent cells are as efficient as unirradiated cells in synthesizing antibodies. Thus, it appears that heavily irradiated cultures of lymphoid cells should be useful for further biochemical studies of antibody synthesis and regulation.

In Vitro and in Vivo Culture of Mouse Peripheral Blood for Chromosome Preparations

SummaryThe in vitro and in vivo culture methods for consistently obtaining mitotic divisions suitable for chromosome analysis from peripheral blood of various mouse strains have been described. In the in vitro culture system pokeweed was used as the mitogenic agent. The DNA-synthesis and mitotic activity commenced much earlier than in the diffusion chamber cultures in which the allogeneic or heterologous environment provided the mitogenic stimulus. In the latter system cells could be maintained for at least 7 days. Mitoses were still frequent at this time.

Pyrogenicity of granulocyte lysosomes.

ARTICLESPyrogenicity of granulocyte lysosomesJC Herion, JK Spitznagel, RI Walker, and HI ZeyaJC Herion, JK Spitznagel, RI Walker, and HI ZeyaPublished Online:01 Sep 1966https://doi.org/10.1152/ajplegacy.1966.211.3.693MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByEnhanced granulocyte function in a case of chronic granulocytic leukemia in a dogVeterinary Immunology and Immunopathology, Vol. 28, No. 2Cationic polyelectrolytes: A new look at their possible roles as opsonins, as stimulators of respiratory burst in leukocytes, in bacteriolysis, and as modulators of immune-complex diseases (A review hypothesis)Inflammation, Vol. 11, No. 4Modulation of locomotor activity of polymorphonuclear cells by cationic substances and cationic lysosomal fractions from human neutrophilsInflammation, Vol. 9, No. 4Mechanisms of feverPharmacology & Therapeutics, Vol. 19, No. 2Isolation and characterization of granulocyte lysosomal proteins and study of their effects on the clotting systemAmerican Journal of Hematology, Vol. 7, No. 3Marrow culture in diffusion chambers in rabbits: III. Effect of endotoxin and leukocyte products on cell productionAmerican Journal of Hematology, Vol. 7, No. 1The influence of natural and synthetic cationic substances on phagocytic activity of human polymorphonuclear cellsExperimental Cell Research, Vol. 117, No. 1Effects of polymorphonuclear leukocytes on endothelial cell growthThrombosis Research, Vol. 12, No. 3Lysosomal EnzymesThe Platelet as an Inflammatory CellLysosomal Hydrolases and Inflammatory MaterialsTHE PLATELET AS AN INFLAMMATORY CELL*16 December 2006 | Annals of the New York Academy of Sciences, Vol. 201, No. 1The Significance of the Lysosome in Toxicology26 September 2008 | CRC Critical Reviews in Toxicology, Vol. 1, No. 3Physiopathology of cellular peptidesNaunyn-Schmiedebergs Archiv f�r Pharmakologie, Vol. 269, No. 2-4Effects of actinomycin on in vivo and in vitro formation of endogenous rabbit pyrogen induced with myxoviruses or stimulated with endotoxinArchiv f�r die gesamte Virusforschung, Vol. 32, No. 2-3The Role of Lysosomes in Immune ResponsesLysosomes and the Reactions of Skin to Ultraviolet Radiation**From the Division of Dermatology Cornell University Medical College, New York, New York 10021.Journal of Investigative Dermatology, Vol. 53, No. 2Cationic Protein-Bearing Granules of Polymorphonuclear Leukocytes: Separation from Enzyme-Rich GranulesScience, Vol. 163, No. 3871Role of Lysosomes in the Pathogenesis of Rheumatoid ArthritisMedical Clinics of North America, Vol. 52, No. 3Induktion von Fieber und Fiebertoleranz durch inkomplette InfluenzavirenZeitschrift für Medizinische Mikrobiologie und Immunologie, Vol. 153, No. 4Arthritis induced by Filipin in rabbitsArthritis & Rheumatism, Vol. 10, No. 4Virusbedingtes FieberKlinische Wochenschrift, Vol. 45, No. 15The extravascular nature of arthus reactions elicited by ferritin. A combined light and electron microscopic analysis of immune states in rabbit ear chambers and mesenteriesZeitschrift f�r Zellforschung und Mikroskopische Anatomie, Vol. 77, No. 4Antimicrobial Specificity of Leukocyte Lysosomal Cationic ProteinsScience, Vol. 154, No. 3752 More from this issue > Volume 211Issue 3September 1966Pages 693-698 Copyright & PermissionsCopyright © 1966 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1966.211.3.693PubMed5927898History Published online 1 September 1966 Published in print 1 September 1966 Metrics