Diffusion Chambers In Mice Research Articles

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.

Proliferation of diffusion chamber colony-forming units (CFUD) in cultures of normal human bone marrow in diffusion chambers in mice

Normal human bone marrow contains cells capable of forming colonies of hemopoietic cells in fibrin clots in diffusion chambers implanted intraperitoneally (i.p.) into irradiated mice. The present paper describes the proliferation of such colony-forming units (CFUD) in cultures in vivo. Cells harvested from diffusion chambers after 1–14 days of culture in 450-R irradiated mice contained CFUD, which formed neutrophilic, eosinophilic, or megakaryocytic colonies when tested by secondary culture in fibrin clot chambers. When bone marrow was precultured in irradiated mice at a concentration of 10(6) cells per chamber, an initial fall in the number of neutrophilic CFUD was observed. This decrease was followed by an increase to a maximum at day 2, and then a secondary decrease. The number of neutrophilic CFUD recovered after 2 days of preculture in irradiated mice varied between 60% and 250% of the number present before preculture. Preculture in nonirradiated mice resulted in a significantly lower recovery of neutrophilic CFUD. In vitro treatment of bone marrow cells with hydroxyurea (OHU) after 2 days of preculture in irradiated mice resulted in a 68% +/- 5% reduction in the number of neutrophilic CFUD. In contrast, OHU had no similar effect on precultures from nonirradiated mice. Both the recovery and sensitivity to OHU of eosinophilic CFUD were independent of host irradiation. Similarly, no effect of host irradiation on the recovery or the 3H-thymidine (3HTdR) labeling index of morphologically recognizable granulocytic cells was observed at day 2. The data suggest an effect of humoral host factor(s) on the proliferation of early precursor cells, which are or become committed to differentiate into the neutrophilic pathway in diffusion chambers.

The Survival of Gonococci and Meningococci in Subcutaneous Diffusion Chambers in Mice

Studies are reported on the survival of gonococci and meningococci in Millipore diffusion chambers implanted subcutaneously in normal mice and in pre-immunised mice. The chambers allowed the passage of nutrients and humoral factors but excluded host cells. After an initial fall in the viable count--attributed to lysis by non-specific serum factors--there was evidence of multiplication of Neisseria gonorrhoeae; the subsequent development of specific antibody led to the disappearance of gonococci 16 days after the chambers were implanted. N. meningitidis behaved differently in the implanted chambers. Meningococci did not appear to be lysed by non-specific humoral factors and so the viable count showed no initial fall. The meningococci survived for 49 days, at which time the entire chamber was rejected.

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.

Human granulocytopoietic colonies in diffusion chambers in mice: growth of colonies and the effect of host irradiation.

Normal human non-separated bone marrow cells were cultured in fibrin clots in diffusion chambers implanted intraperitoneally in mice, and harvested at different intervals by a previously described chamber centrifugation technique. This method demonstrates the presence of cell aggregates in the diffusion chambers. When the chambers are implanted in irradiated mice (450 R) and retransplantated into newly irradiated mice every seventh day, a continous increase in number of cells per granulocytopoietic aggregate is observed from day 8 to day 21. This is compatible with the view that the aggregate is observed from day 8 to day 21. This is compatible with the view that the aggregates are colonies. The term 'colony forming unit diffusion chamber' (CFUD) is suggested to denote the ancestor(s) of the colonies. However, formal proof that one colony is derived from one cell is lacking. Preirradiation of mice with 450 R significantly increases the number of neutrophilic granulocytopoietic colonies at day 14, provided the chambers are retransplantated to newly irradiated mice at day 7, indicating that the neutrophilic colony forming unit or its progeny is involved as at least one of the targets of the stimulating effect of host irradiation. In contrast, no effect of host irradiation on the numbers of eosinophilic colonies was observed.

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.

Trypanosoma cruzi: Morphogenesis in diffusion chambers in the mouse peritoneal cavity and attempted stimulation of host immunity

Sequential morphologic changes and antigen producing capacity of Trypanosoma cruzi in peritoneally implanted diffusion chambers were studied. Diffusion chambers were equipped with two Nuclepore filters (0.20 μm pore size) sandwiched between three Lucite rings. Epimastigotes or trypomastigotes and amastigotes were placed in diffusion chambers and surgically implanted into the peritoneal cavity of mice, or placed in in vitro cell culture, or in various types of culture media and incubated at 26 or 37 C. Epimastigotes maintained in diffusion chambers in mice changed into trypomastigotes as evidenced by the presence of numerous transitional stages and the concomitant decrease in the percentage of the former and increase in the percentage of the latter in chambers removed and examined at 16, 24, 36, 48, 72, and 84 hr after implantation. The maximum of 68% trypomastigotes was noted in chambers examined at 84 hr. Amastigotes subsequently appeared, apparently arising from trypomastigotes and reached the highest percentage (49%) obtained at 132 hr. The total number of parasites in chambers decreased slightly during the first 36 hr (20%). Little change in the total number of parasites was noted during the interval of 36–108 hr. A subsequent decrease in numbers of parasites was noted until by 280 hr after implantation, chambers contained less than 2% of the original number of organisms present in the chambers. No similar transformation of epimastigotes was noted in diffusion chambers maintained in cell culture at 37 C or in a cell culture growth medium or LIT medium at 37 or 26 C. No detectable morphological change was noted when trypomastigotes and amastigotes were implanted in diffusion chambers in the peritoneal cavity of mice. The total number of these parasites decreased notably (82%) after 24 hr. Mice receiving diffusion chambers containing epimastigotes implanted at two different intervals (21 days apart), developed only marginal protective immunity when challenged with virulent T. cruzi three weeks after the second implant of chambers, and no protection was afforded those mice implanted with chambers containing trypomastigotes and amastigotes. Sera collected from mice 6 wk after the second implantation of diffusion chambers containing parasites were observed to have antibody titers to T. cruzi as demonstrated by the fluorescent antibody technique and direct agglutination procedure.

Culture of Psoriatic and Uninvolved Human Skin in Diffusion Chambers in Mice

Pieces of psoriatic and uninvolved skin were cultured for as long as 8 days in vivo in diffusion chambers implanted intraperitoneally in adult mice. On the 2nd day, the greatest part of the psoriatic epidermis degenerated and separated from the remaining regenerating basal cells, while the uninvolved skin either survived intact or only the uppermost cell layers degenerated. In autoradiograms made from the cultures, labeling with 3 H-TdR began on the 2nd day in the epidermal cells either at the cut edges or through the whole explant of both psoriatic and uninvolved skin: maximal labeling indices were reached in 2- to 5-day-old cultures. The labeling indices in both the psoriatic and uninvolved epidermis were at about the same level (max ∼ 14%). Thus, in this study there was no obvious difference in the proliferation of epidermal cells derived from the psoriatic and uninvolved skin. These results are similar to the findings obtained with cultured psoriatic and normal skin in vitro.

Human skeletal muscle cultured in diffusion chambers in mice

Human skeletal muscle cultured in diffusion chambers in mice

The functional activity of thyroid isografts within diffusion chambers in mice

Radioiodine was injected into mice each of which had carried 3 types of grafts for 7 to 28 days. Each carried a vascularized graft of thyroid tissue, an unvascularized graft and a graft in a diffusion chamber. All the grafts showed apparently normal biosynthesis of the various iodinated amino acids. The grafts in the diffusion chambers, however, contained much less radioiodine at 5 hours after injection than the host thyroid tissues. Grafts that had been in the diffusion chambers for 7 days resembled the unvascularized grafts in 131I content but at 28 days some resembled the vascularized grafts. The 131I content of the grafts in the diffusion chambers is probably most dependent on the nearness and richness of the host vascular supply.

Studies of heterografts in diffusion chambers in mice.

Journal Article Studies of Heterografts in Diffusion Chambers in Mice Get access Glenn H. Algire, Glenn H. Algire Search for other works by this author on: Oxford Academic PubMed Google Scholar Mary L. Borders, Mary L. Borders Search for other works by this author on: Oxford Academic PubMed Google Scholar Virginia J. Evans Virginia J. Evans Search for other works by this author on: Oxford Academic PubMed Google Scholar JNCI: Journal of the National Cancer Institute, Volume 20, Issue 6, June 1958, Pages 1187–1201, https://doi.org/10.1093/jnci/20.6.1187 Published: 01 June 1958 Article history Received: 10 December 1957 Published: 01 June 1958