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Osteochondral differentiation and the emergence of stage-specific osteogenic cell-surface molecules by bone marrow cells in diffusion chambers.

The osteochondral potential and emergence of osteogenic cell-surface molecules by avian marrow cells was evaluated in in vivo diffusion chamber cultures. The chambers were inoculated with unselected marrow cells from young chick tibiae and implanted intraperitoneally into athymic mice. At the light microscopic level, morphologic evidence of de novo bone and cartilage formation, including specific immunostaining by antibody probes, was observed in 14 out of 16 chambers incubated for 20 days or longer. In order to monitor the osteogenic differentiation of the marrow-derived cells, indirect immunofluorescence was performed with monoclonal antibodies against stage-specific cell surface antigens on cells of the embryonic osteogenic lineage. The binding of these and other specific monoclonal antibodies in the developing tissue indicates that the cell surface and extracellular matrix molecules expressed by descendants of marrow-derived mesenchymal progenitor cells are indistinguishable from their in vivo counterparts found in embryonic skeletal structures. Furthermore, the experiments reported here describe the first molecular identification of osteogenic cells by probes which are selective for stage-specific surface antigens on cells of the osteogenic lineage. Importantly, bone formation by these marrow-derived cells appears to occur through a lineage progression which is similar to that observed for embryonic tibial osteoblasts. In summary, these data support the use of diffusion chambers inoculated with avian marrow to study aspects of osteogenic and chondrogenic differentiation.

Animals↗

Tumor cells cultured in vivo in diffusion chambers induce hematological alterations.

We have studied in BALB/c mice the hematological alterations of the host induced by the growth of tumor cells in diffusion chambers (DC) In this model, host-tumor interactions are only mediated by soluble factors. Tumor cells proliferate and grow in DC up to 15 days after implant. Our results show a reversal of the granulocyte-lymphocyte ratio in peripheral blood, with lymphopenia and a relative increase of myeloid progenitors in the bone marrow of mice bearing DC with M3 tumor cells (M3TC).

Adenoma↗

Studies on growth and enzymatic characteristics of human mammary tumours grown in heterologous host.

Human mammary tumours were grown in diffusion chambers in the heterologous host--Charle's Foster rat. The effect of oophorectomy-induced alterations of the hormonal environment of the host in breast tumour cells grown in the diffusion chamber were studied with respect to their growth patterns and enzymatic characteristics. The tumour cells not only survived but actively proliferated as indicated by the increase in cell count and formation of cell sheets as well as by the presence of mitotic figures. Cytochemical studies of certain enzymatic activities, i.e. succinic dehydrogenase, alkaline phosphatase, acid phosphatase and beta-glucuronidase indicate--except for a slight depression of the overall activity--that the distribution patterns are more or less maintained following diffusion chamber culture. The alteration of the hormonal environment by oophorectomy influences the cell growth and the enzymatic activity of the human tumour cells inside the diffusion chambers. The results clearly indicate that D.C. culture technique provides a useful method for assessment of growth and hormonal responsiveness of human tumours.

Acid Phosphatase↗

[Use of technique of culturing cells in diffusion chamber in host-mediate for testing chromosomal effects of gossypol acetate on human lymphocytes].

Human peripheral blood lymphocytes were cultured in diffusion chamber in mice after short-term administration of gossypol acetate. The induced chromosomal aberrations of human target cells indiffusion chamber were used as indices of DNA damage. The results showed that the mean frequency of chromosomal aberration of the human lymphocytes in Gossypol-treated mice was slightly higher than that in the control group, but the difference between them was not statistically significant (p 0.05).

Animals, Laboratory↗

Development of a mechanical testing and loading system for trabecular bone studies for long term culture.

A highly accurate (+/-3%) mechanical loading and measurement system combined with a trabecular bone diffusion culture-loading chamber has been developed, which provides the ability to study trabecular bone (and possibly) cartilage under controlled culture and loading conditions over long periods of time. The loading device has been designed to work in two main modes, either to apply a specific compressive strain to a trabecular bone cylinder or to apply a specific force and measure the resulting deformation. Presently, precisely machined bone cylinders can be loaded at frequencies between 0.1 Hz to 50 Hz and amplitudes over 7,000 microepsilon. The system allows accurate measurement of many mechanical properties of the tissue in real time, including visco-elastic properties. This paper describes the technical components, reproducibility, precision, and the calibration procedures of the loading system. Data on long term culture and mechanical responses to different loading patterns will be published separately.

Animals↗

Temperature-dependent inhibition of murine granulocyte-monocyte precursors.

The response of nucleated bone marrow cells from C3H mice to hyperthermic temperatures of 41.5 to 49.5 degrees for a fixed heating period of 30 min has been determined. The threshold temperatures for cell lysis, loss of trypan blue exclusion, and histological evidence of cell injury were greater than 49.5 degrees, 45.5 degrees and 43.5 degrees, respectively. Growth of mature granulocyte-monocytes from precursors was evaluated in Millipore diffusion chamber culture. There was a biphasic decrease in granulocyte-monocyte growth after exposure to temperatures of 41.5 to 45.5 degrees. The ratio of granulocytes to monocytes in proliferated cultures was not altered after hyperthermia. Pluripotent and committed granuloid stem cells were assayed by the spleen colony and plasma clot diffusion chamber techniques. These also showed a biphasic decrease with increase in temperature, with committed stem cells having a greater thermal sensitivity at lower temperatures.

Animals↗

Growth of the Reh cell line in diffusion chambers. Evidence for differentiation along the T- and B-cell pathway.

Cells of the Reh line, originally derived from an ALL of the 'Non-T, Non-B' type were cultured in diffusion chambers implanted intraperitoneally into perirradiated CBA mice. At different intervals over a period of 20 d changes in surface characteristics were examined by labelling the cells with AcALLG, ATCG as well as with polyvalent AIg. The evaluation was performed by using direct immunofluorescence. In addition, the ability to form rosetts with SRBC, AET-treated sheep erythrocytes, mouse red blood cells and EoxAC was tested. On day 0 of the diffusion chamber culture the cells only carried cALLA, and no rosette formation was observed. In the course of the diffusion chamber culture the cells unequivocally developed T-cell antigen, and in 1 of 2 experiments they further acquired a receptor for forming AET- and E-rosettes. Conversely, in the other experiment a receptor for mouse red blood cells was detected in a considerable portion of the cells. Our data show that the rather undifferentiated Reh line cells in vitro are able to develop features of mature T-cells and attributes of early B-cells during the diffusion chamber culture. In vitro they apparently retain a bivalent potentiality of lymphatic maturation. The diffusion chamber system proves to be a suitable tool for promoting differentiation in these cells.

Animals↗

Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia.

Exposure of cultured cells to changing gaseous environments is used as a model for understanding both the immediate and long-term effects of such exposures on lung cells in vivo. We conducted experiments with polystyrene tissue culture flasks and plates to determine the time course of changes in oxygen concentration occurring under in vitro conditions. Only a few minutes were required for the concentration of oxygen in the environmental chamber to reach equilibrium with that of the flushing gas. However, >3 h were required for the oxygen content in the medium in the tissue culture flasks and plates to achieve equilibrium. The low solubility of oxygen in aqueous solutions and the limited diffusion of oxygen through a boundary layer of gas above the medium are the major barriers to rapid oxygen transport into the culture medium. The delay in achieving the desired PO(2) within the culture medium limits the temporal precision of any assessment of the correlation of cellular events with the concentration of oxygen to which those cells are exposed.

Animals↗

Embryoid bodies cultured in in vivo diffusion chambers show reduced tumorigenicity while retaining expression of F9 antigens.

Embryoid bodies of the mouse teratocarcinoma OTT6050 were dissociated into single cells and cultured in diffusion chambers implanted into the peritoneal cavities of mice. The syngeneic host mice, into which the cells of embryoid bodies cultured in the diffusion chambers had been injected, survived much longer than those which received the original cells of embryoid body. But in the case of the F9 cells, obtained in the same culture conditions, only a slight decrease in tumorigenicity was observed. By contrast, the F9 antigenic expression was observed on both F9 and embryoid body cells cultured in diffusion chambers. Judging from the determination of adult-type antigenic expressions, the differentiation of the cells in chamber was negligible. These results suggest that the tumorigenic activity of the embryoid body cells cultured in vivo in a diffusion chamber is almost suppressed, but that they continue in an undifferentiated state.

Animals↗

Glass ring-wall sealing to prevent leakage from electrode culture chambers: a technical note.

Entellan is a good sealing substance for culture chamber walls and does not interfere with the growth of the cultured DRG cells. If the Entellan becomes brittle the glass ring can easily be loosened by placing the chamber with the sealed ring for 24 hrs in xylene. Repeated rinsing with xylene and subsequently with hydrated steps of ethanol (100%-70%-30%) allows the ring-wall and chamber floor with its electrodes to be reused.

Animals↗

Resistance of lymphokine-activated T lymphocytes to cell-mediated cytotoxicity.

We observed that lymphokine-activated T lymphocytes, obtained in short- and long-term cultures following stimulation with recombinant interleukin-2 (rIL-2), are resistant to cell-mediated cytotoxicity. In particular, lymphokine-activated killer (LAK) cells do not undergo self-lysis or lysis by alloreactive cytotoxic T lymphocytes (CTL), in line with recent reports concerning CTL clones. Similar findings were further confirmed in a lectin-dependent cell cytotoxicity assay. LAK cell lysis resistance was not due to an inability to recognize itself, since inactivated LAK cells used as cold competitors inhibited tumor cell lysis in a dose-dependent manner. In contrast, the addition on Day 0 of irradiated LAK cells or alloreactive CTL, as well as a CTL clone having LAK-like activity to rIL-2-stimulated cultures abrogated or strongly reduced LAK cell generation. Therefore, LAK cell precursors were most likely susceptible to the lytic activity of differentiated cytotoxic cells, as no inhibition was detected when cell to cell contact was prevented by using a diffusible chamber culture system. These findings, on the whole, suggest that the emergence of the lysis-resistant phenotype is most likely the result of a selective process occurring in vitro that leads to the elimination of lysis-susceptible lymphocytes present in culture.

Animals↗

Hematopoiesis-promoting activity of rat liver biliary epithelial cells: involvement of a cell surface molecule, liver-regulating protein.

Stromal cell lines from bone marrow and other blood-forming organs including fetal liver have been found to support hematopoiesis. In this paper, we demonstrate that rat liver biliary epithelial cells (RLEC), most likely originating from primitive bile ductules, are able to support long-term hematopoietic cell growth as well as burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) production. RLEC have previously been shown to express a cell surface molecule named liver-regulating protein (LRP), which is involved in the long-term maintenance of hepatocyte functions in a coculture system. In addition, LRP-like molecules have been found in spleen, thymus, lymph nodes, and peripheral blood cells. In the present study, we found that hematopoietic cells and several stromal cell types from bone marrow were LRP-positive, and immunoprecipitation revealed polypeptides similar to those found in RLEC. We then investigated the biological role of LRP on hematopoiesis using short-term RLEC and bone marrow stromal cell culture systems. Addition of specific anti-LRP antibody to both systems reduced hematopoietic cell proliferation and committed progenitor production, whereas it did not directly affect the clonal proliferation and maturation of these progenitors in methylcellulose assays. Moreover, using diffusible chamber cultures that suppress direct contacts with hematopoietic cells, we observed low cell growth and no effect of monoclonal antibody (mAb) L8 treatment. All these results strongly argue for a cell proximity signal mediated by RLEC and bone marrow stromal cells and for the involvement of LRP-like molecules in this signal in liver and bone marrow hematopoietic function.

Animals↗

Suppression of granulopoiesis in diffusion chambers by syngeneic clonal acute myelogenous leukemia cells or peritoneal exudate macrophages.

To determine the mechanism by which acute myelogenous leukemia (AML) cells suppress normal marrow granulopoiesis, diffusion chambers containing Wistar/Furth (W/Fu) rat marrow cells, peritoneal exudate (PE) macrophages or W/Fu AML clone-3 cells were implanted intraperitoneally into syngeneic irradiated rats. Growth of each population over 21 days in single or double diffusion chambers (in which cell populations were separated by a Nucleopore filter) was compared to that of mixed populations. Double diffusion chamber culture of homologous or heterologous combinations had no detectable effect on growth kinetics of any of the three cell populations compared to single chambers. In contrast, normal granulocyte proliferation was significantly depressed by single chamber co-cultivation with one tenth the number of PE macrophages or AML cells. Mixing PE macrophages with AML cells produced no preferential population suppression. AML cells differentiation was not detected under any set of conditions. These studies demonstrate that physical contact with proliferating normal macrophages as well as AML cells will suppress granulopoiesis in diffusion chambers.

Animals↗

Diffusion chamber colony-forming unit (CFU-d): a primitive stem cell.

Assay of hematopoietic precursor cells in diffusion chambers (DCs) implanted intraperitoneally in experimental animals provides a powerful tool for studying stem cell kinetics in vivo. In this system, the effect of cell migration (which complicates whole animal studies) is eliminated because the membranes utilized in the construction of the chambers are impermeable for cells, while permitting free passage of molecules present in the humoral phase of the host. As judged by light microscopy, conditions in the DC cultures primarily favor macrophage and granulocyte growth. However, the use of in vitro and in vivo subculture to further analyze chamber contents has demonstrated that the system supports proliferation of early hematopoietic progenitors. Additionally, cells capable of rescuing lethally irradiated mice proliferate in DC cultures. Development of the plasma clot DC technique has revealed that most of the growth occurs in colonies which are derived from single cells (CFU-d). Characterization of these cells indicates that they are at least as primitive as other colony-forming cells and, also based on subculture studies, can differentiate along several hematopoietic lineages. In addition to normal CFU-d, both embryonal and leukemic cells can give rise to granulocytes, macrophages, megakaryocytes and erythroid cells in the DC cultures. Evaluation of the effects of humoral factors on hematopoietic cell proliferation and differentiation in the system has led to the identification of both stimulators and inhibitors that may be different from the well-characterized cytokines. Thus, the system seems to be useful for detecting molecules controlling the most primitive stages of hematopoiesis. We believe that the DC culture technique holds enormous potential in the study of stem cell proliferation and differentiation in vivo.

Animals↗

A compatible support system for cell culture in biomedical research.

The lack of a suitable system to culture epithelial cells for a long period under a luminal-antiluminal medium gradient, was the reason to develop a new system. It consists of an interchangeable sheet of permeable support material, which is set in place by two tight fitting holding rings. For special demands the supports can be coated with extracellular matrix proteins improving cellular attachment and terminal differentiation. The handling of the sheets by forceps proceeds easily and quickly, thus fastening the transfer of cultured cells without additional manipulations. The sheets can be transferred to a newly developed microperfusion chamber on which an apical and a basal perfusion over a long culture period parallel to a transepithelial electrophysiological registration becomes possible. The chamber has an extremely low amount of fluid dead space. The separate perfusion of cultured cells under isotonic, hypotonic or hypertonic conditions opens new possibilities. Thus, culture can be performed under most natural conditions e.g., that found within the kidney.

Animals↗

An in vitro model based on cell monolayers grown on the underside of large- pore filters in bicameral chambers for studying thyrocyte-lymphocyte interactions.

In the processes underlying thyroid autoimmunity, thyrocytes probably act as antigen-presenting cells exposing T-cell epitopes to intrathyroid lymphocytes. To study the interactions between lymphocytes and thyrocytes, which are arranged in a tight, polarized monolayer, we developed a new in vitro model based on human thyrocytes grown on the underside of a filter placed in a bicameral chamber. Thyrocytes from Graves' disease glands were plated onto the upper face of a 8-mum-pore polyethylene terephthalate culture insert filter placed in the inverted position and grown for 24 h before the insert was returned to the normal position for a week in the cell culture plate wells. Thyrocytes grown in the presence of thyroid stimulating hormone, forming a homogeneous monolayer on the underside of the filter, reached confluence after 8 days in vitro. The cells developed a transepithelial electrical resistance >1,000 Omega.cm(2), and the ZO-1 tight junction protein showed a junctional pattern of distribution. Thyrocytes showed a polarized pattern of thyroperoxidase and thyroid stimulating hormone receptor expression in the apical and basolateral positions, respectively. They were also found to aberrantly express DR class II human leukocyte antigen and an Fc immunoglobulin receptor (FcgammaRIIB2) in the basolateral and apical positions, respectively. Autologous intrathyroidal T lymphocytes cocultured for 24 h across the filter with the thyrocyte monolayer proliferated and remained in the upper chamber without any leakage occurring through the epithelial barrier, which makes this model particularly suitable for studying the cell-cell interactions involved in antigen processing.

Cell Communication↗