Cells In Diffusion Chambers Research Articles

Normal mononuclear blood cells in diffusion chambers: occurrence of cALLA and TdT.

It is known, that normal peripheral mononuclear cells show remarkable growth and blastic transformation in the diffusion chamber culture system. The rise in cell number is mainly caused by lymphoid cells.

Effects of daily low dose fraction gamma radiation on growth and differentiation of fresh human myeloid leukemia and preleukemic bone marrow cells in diffusion chambers

Effects of daily low dose fraction gamma radiation on growth and differentiation of fresh human myeloid leukemia and preleukemic bone marrow cells in diffusion chambers

Formation of Bone and Cartilage by Marrow Stromal Cells in Diffusion Chambers in Vivo

When freshly isolated rabbit marrow cells were cultured either in vitro or in diffusion chambers in vivo, the hemopoietic cells disappeared and there was a proliferation of the stromal cell population. The colonies formed in vitro were mainly fibroblastic, and this cell type predominated in confluent cultures. Staining for alkaline phosphatase activity and for the Von Kossa reaction was negative in in vitro cultures. However, marrow cell suspensions or fibroblasts harvested from in vitro culture of marrow cells, gave rise to a mixture of bone, cartilage and fibrous tissue in diffusion chambers implanted into the peritoneal cavity. In contrast, only a soft fibrous tissue developed from spleen fibroblasts in diffusion chambers. Differentiation of osteogenic tissue within diffusion chambers fell into two categories: (1) Formation of bone in a fibrous layer surrounding cartilage; (2) intramembranous bone formed directly within fibrous tissue unassociated with cartilage. In both cases alkaline phosphatase activity appeared before the onset of mineralization, and decreased as the first signs of mineral became apparent. The present results suggest that postnatal marrow contains osteogenic precursors with the potential to differentiate via either of the two major paths followed during skeletal development in the embryo. Clonal analysis of the marrow stromal cell population will be required to clarify whether osteo-, chondro-, and fibrogenic cells are the products of one stromal cell line modulated by the microenvironment, or whether there are distinct cell lines for each type.

Generation of suppressor T cells during the primary response of murine spleen cells in diffusion chambers.

A vigorous secondary response to DNP is elicited by dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) in spleen cells from primed mice cultured in Millipore diffusion chambers. However, the same antigen under the same conditions does not elicit a primary response in normal spleen cells. In contract, foreign erythrocytes elicit a primary response and function as an effective carrier for the primary response to a hapten. We found that the failure of haptenated protein to induce a primary response in diffusion chambers can be attributed to the generation of suppressor T cells by the protein. Cultures of T depleted normal spleen cells, supplemented with carrier-specific helper T cells, developed a DNP-specific primary response upon stimulation with DNP-KLH.

Culture of human haemopoietic cells in diffusion chambers

Culture of human haemopoietic cells in diffusion chambers

Sister chromatid exchange induced by promutagens/carcinogens in Chinese hamster cells cultured in diffusion chambers in mice.

SummaryA system for in vivo analysis of sister chromatid exchange (SCE) was described. It involved culturing Chinese hamster V-79 cells in diffusion chambers (DC) in mice. Treatment of the hosts with repeated injections of 5-bromodeoxyuridine permitted the demonstration of differentially stained metaphases in the V-79 target cells after using the fluorescence plus Giemsa technique. Thus, it was possible to determine the number of SCE's under in vivo conditions. Induction of SCE in V-79 cells in vivo were studied following treatment with seven known promutagens/carcinogens: Cyclophosphamide, 1-(pyridyl-3)-3,3-dimethyltriazene, dimethylnitrosamine, diethylnitrosamine, benzo(α)-pyrene, 7,12-dimethylbenz(α)anthracene, 3-methylcholanthrene and three nonmutagens/noncarcinogens: perylene, pyrene, and anthracene. With the exception of DEN, all the promutagens/carcinogens caused a significant increase in the frequency of SCE. DEN was effective at the highest dose studied only after pretreatment with Aroclor 1254, ...

Fetal blood-borne myelo- and thrombopoietic stem cells in diffusion chambers.

The potential of hematopoietic stem cells from cord blood for proliferation and differentiation have been studied by the diffusion chamber technique. In a pilot study, it was shown that blast cell production and myelopoiesis from the original lymphoid cell suspension starts 6 days after implantation. Mature granulocytes and megakaryocytes respectively appear in the chambers from the 16th and 10th days onwards. Myelopoiesis is ten times more active in fetal than in adult blood. There were thrombopoietic stem cells in 7 of 10 cord blood samples but in only one of 10 adult blood specimens. It is concluded that the high hematopoietic activity of the bone marrow at term is reflected by an increase in the number of stem cells circulating in the blood. Myelo- and thrombopoietic stem cells must be sought among the lymphoid cells of the original cord blood cell suspension.

Inhibition of Growth of Normal Murine Granulocytes by Cocultured Acute Leukemic Cells

AbstractCoculture of equal numbers of C1498 murine acute myelogenous leukemia cells and normal mouse bone marrow cells in diffusion chambers (DC) led to complete suppression of normal granulocyte development within a few days. Suppression of granulopoiesis was not observed with irradiation-killed leukemic cells. Direct interaction between the leukemic and normal cells appears to be necessary since there was no inhibition when the two populations were separated by an intervening Millipore filter in double-DC cultures. When the ratio of leukemic to normal cells was reduced from 50/50 to 15/85, proliferation of granulocytic cells remained normal for a longer time but eventually ceased. With both the 15/85 and 50/50 cultures, the ratio of leukemic to normal granulocytic cells rose to about the same level, 6/1-7/1, just before granulopoiesis was suppressed. In both instances macrophage numbers were similar to those observed in control cultures of normal marrow cells alone. When mixtures of leukemic and normal bone marrow cells were assayed by the agar and spleen colony-forming techniques rather than by culture in DC, the formation of colony-forming cells in vitro was unaltered but there was a marked reduction in the formation of normal, differentiated spleen colony-forming cells. This finding suggests that the leukemic cells exert their inhibitory influence at the level of the pluripotential hemopoietic stem cell. The findings with mixed DC cultures of leukemic and normal marrow cells resemble those in the bone marrows of patients with acute myelogenous leukemia and provide an experimental model for elucidating the mechanisms by which leukemia subverts normal hemopoiesis.

Relationship between tumour growth rate and proteic variations in interstitial subcutaneous fluid and serum: Possible thymic control

Some murine tumours stimulate the proliferation of target cells in diffusion chambers implanted subcutaneously near the tumour. The growth of tumours with or without the property to stimulate the proliferation of target cells was studied in relation to the proteic variations in interstitial subcutaneous fluid and serum in normal hosts and thymectomized or thymus-deprived hosts. “Stimulating” tumours were found to exhibit a much faster growth than “non stimulating” tumours, in normal hosts. A significant protein decrease was observed in interstitial fluid of mice bearing “stimulating” tumours as compared to control mice. In contrast, the analysis of interstitial fluid and serum of mice bearing “non stimulating” tumours revealed a significant proteic increase. The study of the growth behaviour of these tumours in T-cell deprived mice revealed a deceleration of the “stimulating” tumours, and a heavy acceleration for the “non stimulating” tumours. No proteic variation as compared to control mice was found in interstitial fluid and serum of tumour bearing T-cell deprived mice. The hypothesis was formulated that some relationship exists between tumour growth rate and proteic variations in interstitial subcutaneous fluid and serum which might be under thymic control.

A modified host-mediated assay using cultured human lymphoid cells in diffusion chambers in mice and cytogenetic effects of cyclophosphamide

A modified host-mediated assay system using cultured human lymphoid cells as target cells is described. The human cells were cultured in diffusion chambers (DC) and implanted into C 3H St mice. Induction of chromosome aberrations in human cells in DC as well as host bone marrow cells was used as an index of mutagenicity of a given compound and its metabolites. The cells from six human lymphoid lines were studied for their growth potential in DC in mice. Cells from two lines, both derived from blood of normal persons, failed to grow. The other four lines proliferated well. The maximum number of cells per DC was reached from 3 to 5 days after implantation and the maximum cell density per DC varied from 4 to 15 times the initial cell concentration. The mitotic index in DC was high on the first and second days and then decreased gradually. There were mitoses in some DC more than 2 weeks after implantation. Cells from DC which had been in hosts for approximately 2 weeks were recultured. Several such cultures proliferated within a week. Cytogenetic effects of cyclophosphamide (CY) in cells from two lines in DC in mice and in host bone marrow were investigated. A high rate of human and mouse cells with chromosome aberrations caused by CY injection was observed.

Formula omitted][formula omitted] evaluation of granulocyte differentiation of diffusion chamber inocula

The semi-soft agar colony assay permits an in vitro analysis of committed myeloid stem cell (CFU-c) proliferation capacities. In this paper this procedure has been used in combination with prior diffusion chamber culturing to determine the effect of host influences upon this committed stem cell population. This “double-seeding” procedure of first culturing bone marrow cells in diffusion chambers and then re-seeding them in agar furnishes data suggesting a relationship between in vivo diffusion chamber transitional lymphocytes and in vitro CFU-c seeding capacities. Diffusion chamber culturing offers a means of monitoring granulopoiesis and selects for enrichment of stem cell numbers. Detection and quantification of diffusion chamber stem cell enrichment is easily assessed by seeding chamber contents into the agar colony assay.

The effect of syngeneic peripheral blood cells on the formation of colonies by normal human bone marrow cells in diffusion chambers in mice.

This paper describes the influence of cells capable of releasing colony stimulating activity (CSA) in vitro on the formation of granulocytic colonies by normal human bone marrow in diffusion chambers in mice. A carbonyl iron method was used to remove phagocytic cells from normal human bone marrow. This treatment prevented spontaneous colony and cluster formation when the cells were cultured in agar in vitro at initial concentrations of 2-5 x 105 cells per ml. However, non-phagocytic bone marrow cells formed granulocytic colonies when inoculated into diffusion chambers at 105 cells per chamber and cultured in 450 R-irradiated or non-irradiated mice. The formation of granulocytic colonies by carbonyl iron treated marrow in diffusion chamber cultures was not consistently enhanced by the admixture of 1.4 x 105 1500 R-irradiated syngeneic light density blood cells (less than 1.077 g/ml) to the chamber inoculum. In contrast, these cells induced cluster or colony formation when added in the same proportion to the marrow cells in agar cultures in vitro. Addition of 1.4 x 105 1500 R-irradiated high density blood cells(greater than 1.077 g/ml) to the inoculum resulted in a slight, non-significant decrease in the number of colonies in diffusion chambers. The stimulating effect of host irradiation on neutrophilic colony formation was independent of the presence of CSA releasing cells in the chamber inoculum.

Fetal hemopoiesis in diffusion chamber cultures. III. The effect of neutropenia

Proliferation and differentiation of granulocytes, macrophages, and both myeloid committed (CFC) and pluripotent (CFU) stem cells in diffusion chamber (DC) cultures of fetal liver were studied in order to evaluate the role of circulating humoral factors in the control of fetal myelopoiesis. When DC with fetal liver cells were implanted into mice rendered neutropenic by pretreatment with cyclophosphamide, more granulocytes and CFC were produced through day 10 as compared to DC implanted into saline pretreated control hosts. A difference in CFU recovery from fetal liver suspensions grown in DC implanted into neutropenic and control hosts was not seen until day 10. Serum CSF concentrations were increased in neutropenic as compared to control host mice 2 and 3 days after implantation of DC. Levels of serum inhibitors of colony growth showed marked variability but, in general, were similar in both groups. These data provide evidence that fetal CFC and fetal myelopoiesis are influenced by a circulating humoral factor present in neutropenic serum. CSF may be the factor, although the data presented in this paper do not establish this with any certainty.

Fetal Hemopoiesis in Diffusion Chamber Cultures. III. The Effect of Neutropenia

Proliferation and differentiation of granulocytes, macrophages, and both myeloid committed (CFC) and pluripotent (CFU) stem cells in diffusion chamber (DC) cultures of fetal liver were studied in order to evaluate the role of circulating humoral factors in the control of fetal myelopoiesis. When DC with fetal liver cells were implanted into mice rendered neutropenic by pretreatment with cyclophosphamide, more granulocytes and CFC were produced through day 10 as compared to DC implanted into saline pretreated control hosts. A difference in CFU recovery from fetal liver suspensions grown in DC implanted into neutropenic and control hosts was not seen until day 10. Serum CSF concentrations were increased in neutropenic as compared to control host mice 2 and 3 days after implantation of DC. Levels of serum inhibitors of colony growth showed marked variability but, in general, were similar in both groups. These data provide evidence that fetal CFC and fetal myelopoiesis are influenced by a circulating humoral factor present in neutropenic serum. CSF may be the factor, although the data presented in this paper do not establish this with any certainty.

Differentiation of small numbers of mouse peritoneal cells into antibody-producing plasma cells in diffusion chambers

Comparatively small numbers, 2.5−10 × 10 4, of primed mouse peritoneal cells cultured in diffusion chambers, can with antigen, be induced to differentiate into plasma cells producing antibody specific to two different priming antigens, horseradish peroxidase and sheep erythrocytes. This number of immunocytes is small enough to permit morphologic analysis of the events in differentiation. The overt response to SRBC was characterized by the appearance between 48 and 72 hr of plasmablasts, many mitotic figures and alterations in the membrane morphology of many lymphocytes. Interactions of plasmablasts and mature plasma cells with macrophages were very prominent and after months of culture plasma cells still clustered specifically above macrophages or mesothelial cells. The response of peritoneal cells to horseradish peroxidase was more subtle than the response to sheep erythrocytes in that differentiation into plasma cells proceeded without major morphologic transitions. The intracellular localization of anti-HRP antibody in the perinuclear space and in patches near or on the plasma membrane of the mature plasma cell suggested discontinuous synthesis of specific antibody. In many mature plasma cells, no anti-HRP antibody could be detected.

Cultivation of leukemic human bone marrow cells in diffusion chambers implanted into normal and irradiated mice

In order to elucidate the question of whether the maturation defect in vivo in acute leukemia is due to environmental or cellular factors, we have cultured human leukemic cells in a nonleukemic milieu, i.e., diffusion chambers implanted into the abdominal cavity of normal and irradiated mice. For each harvest, the cell count was measured and differential counts and the number of peroxidase-positive cells determined. The cell number decreased with time, without significant difference between culture in irradiated (500 rads) and normal mice. The blast cells succeeded only in developing distorted promyelocytes and myelocytes. There was a general pattern of increase in the number of peroxidase-positive cells. The study supports the concept that acute myeloid leukemia (AML) is a disturbance of cellular maturation due to cellular rather than environmental defects.

Cultivation of Leukemic Human Bone Marrow Cells in Diffusion Chambers Implanted Into Normal and Irradiated Mice

In order to elucidate the question of whether the maturation defect in vivo in acute leukemia is due to environmental or cellular factors, we have cultured human leukemic cells in a nonleukemic milieu, i.e., diffusion chambers implanted into the abdominal cavity of normal and irradiated mice. For each harvest, the cell count was measured and differential counts and the number of peroxidase-positive cells determined. The cell number decreased with time, without significant difference between culture in irradiated (500 rads) and normal mice. The blast cells succeeded only in developing distorted promyelocytes and myelocytes. There was a general pattern of increase in the number of peroxidase-positive cells. The study supports the concept that acute myeloid leukemia (AML) is a disturbance of cellular maturation due to cellular rather than environmental defects.

Growth stimulation of tumor cells in diffusion chambers implanted in mice bearing chemically induced tumors(author's transl)

AbstractThe growth of target tumor cells in diffusion chambers implanted subcutaneously into normal mice and into mice bearing chemically‐induced tumor was studied. It was observed that target‐cell multiplication was heavier in mice bearing a tumor less than 20 mm in diameter than in normal mice. In contrast, there was no growth difference between normal mice and mice bearing tumors with a diameter superior to 20 mm. This effect was observed when the target cells were of the same origin as the tumor borne by the mice. When the origin of tumors was different, the multiplication of target cells was stimulated but to a lesser degree. This phenomenon was observed in three out of four tested tumors. If the mice were irradiated with 500 R before tumor grafting and implantation of the diffusion chamber, the stimulation was still fully observable. In contrast, implanting the diffusion chambers intraperitoneally instead of subcutaneously or, alternatively, subcutaneously but far from the tumor, suppressed the stimulation. Also, no stimulation was observed if the grafted tumor was irradiated lethally or if a local inflammation was created by injection of bentonite or Freund's adjuvant. Tumor excision just before implantation of the chamber suppressed the stimulatory effect. Since the diffusion chambers are impermeable to cellular penetration it is concluded that the interstitial fluid of some tumor‐bearing mice contains diffusible factors capable of stimulating the multiplication of tumor target cells, close to the tumor.

Chamber centrifugation: a harvesting technique for estimation of the growth of human haemopoietic cells in diffusion chambers.

A harvesting technique which gives information about the spatial relationships between cells grown in diffusion chambers in mice has been designed. The principle is that cells are centrifuged directly from the chamber clot onto a slide during pronase treatment. The technique has been applied to normal human bone marrow cells grown in diffusion chambers. The slides show that the cells are situated in clusters. A limiting dilution assay of progenitor cells based on the presence of granulopoietic clusters in the chambers demonstrates the use of the method for quantitation of progenitor cells.

Regulation of normal hemopoiesis in the acute leukemia L5222.

Normal hemopoiesis becomes markedly depressed in rats during the development of the rat leukemia L5222. Whether this could be due to the influence of a circulating humoral factor was investigated by comparing the growth of normal bone marrow cells in diffusion chambers implanted into the peritoneal cavity of normal or leukemic hosts. Similar growth in total cell numbers was observed in both groups of hosts, and no significant difference could be detected between them. In an attempt to exclude the possibility that an inhibitor either did not penetrate to the peritoneum or was inactivated by the chamber membrane, normal serum, or serum from a highly leukemic rat was mixed with the bone marrow cell suspension in the chambers. Again, no difference in growth between the 2 groups could be detected. Therefore, the influence of a circulating humoral factor in the depression of normal bone marrow hemopoiesis in this experimental leukemia seems to be ruled out, and the decrease may be attributable to local events in the bone marrow such as short-range factors or cellular interaction between the leukemic and normal hemopoietic cell populations.