Nonadherent Population Research Articles

Different functional and morphological characteristics in a nonadherent subpopulation of human macrophages recovered by bronchoalveolar lavage.

The alveolar macrophages (AMs) constitute a morphologically and functionally heterogenous cell population. The adhesive properties of these cells are important for their role in host defence. To focus on the heterogeneity, a population of nonadherent macrophages were characterized functionally and morphologically. These cells were then compared with the total alveolar macrophage population. Alveolar cells (> 95% alveolar macrophages), were recovered by bronchoalveolar lavage (BAL) from healthy smokers. Nonadherent macrophages were separated by adhesion. The phagocytic capacity and the autofluorescent properties of the cell populations were determined in flow cytofluorometric assays. In addition, electron microscopic evaluation was performed. The alveolar macrophage adhesion to wells coated with albumin increased in a time-dependent manner. After 15 min, median 47%, interquartile range 42-52% (uncoated wells 68%, 67-72%) of the alveolar macrophages were attached; and after 60 min, 56%, 51-58% (uncoated wells 73%, 71-76%) of the alveolar macrophages were attached. The nonadherent alveolar macrophage population had less phagocytic capacity. The cell autofluorescence increased with increasing cell size and cell complexity/granularity in both populations. The nonadherent cells were more autofluorescent, indicating an increased granularity/complexity. These findings were confirmed with electron microscopy. Thus, the nonadherent alveolar macrophages had more cytoplasmic inclusions than the total alveolar macrophage population (volume density median 0.39, interquartile range 0.35-0.46 and 0.31, 0.26-0.34, respectively), but less surface protusions. We conclude that in lavage fluid from human smokers there is an ultrastructurally specific subpopulation of alveolar macrophages, showing less adhesive properties and impaired phagocytic capacity in vitro. These macrophages may be older alveolar cells or, alternatively, airway macrophages. Since only alveolar macrophages from smokers were investigated, we cannot draw any conclusions regarding alveolar cells from nonsmokers. Nevertheless, the heterogeneity of the lavaged cells should be taken into consideration when the functional ability of the alveolar macrophages are evaluated.

P-364 Clinicopathological and immunogenetical study of tiopronin induced hepatic injury

Thymic stromal cell line TS-9 was found to selectively bind a subpopulation of normal murine thymocytes. Selective binding allowed the isolation and phenotypic characterization of the adherent and nonadherent subpopulations of thymocytes. Flow cytometric analysis of fluorescently labeled thymocytes revealed that the adherent and nonadherent populations differ in maturity, with the adherent population enriched in immature thymocytes of the PNAhi, Thy-1hi, CD3−/lo, and CD4+/CD8+ double positive surface phenotype. A quantitative microwell assay was developed to measure the binding of thymocytes to TS-9. Thymocytes labeled with vital DNA stain Hoechst 33342 were allowed to bind to TS-9 in microwells and the intense fluorescence of this label was readily detected with a scanning fluorometer. The binding was trypsin-sensitive and hyaluronidase and PI-PLC resistant. The binding was also temperature dependent and sensitive to cytochalasin B. A panel of monoclonal antibodies to cell surface antigens including CD2, LFA-I/ICAM-I, and Thy-1 was screened in a quantitative binding assay for their ability to inhibit the binding of thymocytes to TS-9. The binding was partially inhibited by the C3C12 monoclonal antibody which recognizes the recently identified and apparently unique gp23, gp45 complex expressed on murine Stromal cells.

PGE 2 induces the transition from non-adherent to adherent bone marrow mesenchymal precursor cells via a cAMP/EP 2-mediated mechanism

When mesenchymal precursor cells from bone marrow are cultured in the presence of dexamethasone, the existence of distinct non-adherent and adherent populations can be demonstrated. The addition of PGE 2, forskolin, or dibutyryl-cAMP can induce a transition from the former to the latter and this may be an important mechanism in the bone anabolic effects of PGE 2. On the other hand, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, and sulprostone, an agonist for the PGE 2 receptor EP 1/EP 3 subtypes, had no effect. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), had a synergistic effect in combination with PGE 2, whereas neomycin, an inhibitor of inositol phosphate activity, had no effect, and LiCl, an inhibitor of inositol triphosphate metabolism, had an inhibitory effect on the PGE 2-induced transition. Consistent with this, the addition of PGE 2 to non-adherent bone marrow cells caused a 100% increase in cAMP synthesis. These results suggest that the induction of the transition from non-adherent to adherent osteoblast precursor is mediated by the EP 2-PGE 2 receptor subtype via an increase in intracellular cAMP synthesis.

IL-10 mediates immunosuppression following primary infection with Toxoplasma gondii in mice.

Suppression of the host immune response by Toxoplasma gondii has been observed in both human and experimental murine infection. In this study, inbred mice were infected with T. gondii. At day 7 post-infection, the lymphoproliferative response to both mitogen and superantigen as well as parasite antigen were found to be significantly depressed. Using a transwell system, it was determined that the reduced proliferative response was due to soluble factor(s) being expressed by splenocytes from the infected mice. Isolation of the splenocytes into an adherent and nonadherent population suggested that both macrophages and T cells were able to produce at least one soluble factor. Tissue culture supernatant derived from the splenocytes of the infected mice contain increased levels of IL-10, whereas measurable IL-2 levels could not be quantitated. At day 7 post-infection, both a biologic assay for IFN-gamma in culture supernatant and the expression of IFN-gamma mRNA in the splenocytes were reduced. Antibody to IL-10 was able to partially neutralize (almost 50%) the in vitro immune downregulation of the tissue culture supernatant. Anti-IL-10 in combination with a nitric oxide (NO) antagonist was able to reverse the inhibitory activity of the culture supernatant by 85%. Since IL-10 is a potent antagonist of IFN-gamma, it may represent a critical cytokine involved in mediating T. gondii induced immunosuppression in the infected host.

Characterization of an inhibitory seric factor from tuberculosis anergic patients that acts on non-adherent PPD reactive cells.

Non-adherent cells from PPD+ tuberculosis patients (TBP PPD+) and from healthy individuals treated with whole tuberculosis anergic immune sera or with its protein A-Sepharose IgG fraction, or with sera fraction separated by PPD-Sepharose chromatography, were submitted to immunofluorescence assays. Anti-human IgG or IgM FITC-conjugate were used to reveal the assays, and results were expressed by a fluorescence percentage or fluorescence index. The presence of IgG over the surface of PPD+ non-adherent cells was detected. High fluorescence percentages were observed only in those PPD+ cells treated with whole anergic serum or with its IgG fraction. Positive fluorescence index values were obtained only in those PPD+ cells treated with anergic serum, meanwhile fluorescence index was always negative when non-bound fractions from PPD-Sepharose were used. Results suggest that non-adherent population are the cell targets for the serum inhibitory factor, which previously has been detected to inhibit antigen response in PPD reactive cells and, point out the specific behavior of this factor, since it was eliminate by PPD-Sepharose chromatography. The IgG nature of the factor was demonstrated by SDS-PAGE and immunoelectrophoresis.

Stimulation of in vitro hematopoiesis by a murine fetal hepatocyte clone through cell-cell contact.

We have previously shown that a fetal liver-derived epithelial cell clone, FHC-4D2, could support hematopoiesis in vitro through its colony-stimulating factor (CSF) activities in a short-term culture. In this study, since FHC-4D2 cells were found capable of maintaining hematopoietic progenitors in the coculture for a long time, we examined how FHC-4D2 could exert hematopoietic supporting activity in a long-term culture by coculturing adult bone marrow (BM) cells or fetal liver (FL) cells on a monolayer of FHC-4D2 cells. This clone could maintain the colony-forming unit of granulocytes and macrophages (CFU-GM) of BM for > or = 12 weeks under the coculture condition, but the fibroblastic cell clone from the fetal liver, FHC-4A3, could not support the survival of CFU-GM, even for 1 week. In addition to BM CFU-GM, the FHC-4D2 clone also supported the survival of FL CFU-GM, burst-forming unit of erythroid cells (BFUe), and colony-forming unit of mixed progenitors (CFU-Mix) for longer than 4 weeks. When BM cells were separated by a membrane filter from the FHC-4D2 cells in the coculture, the comparable number of CFU-GM was maintained at day 3, but virtually no hematopoietic progenitors were detected at the end of the first week. CFU-GM were present in both nonadherent and adherent cells to the FHC-4D2 cells at day 3 of the coculture, but at day 7, the adherent population contained greater number of CFU-GM. CFU-GM derived from the adherent cells formed larger colonies and contained more bipotential CFU-GM than the nonadherent population. When BM cells from mice given 5-fluorouracil were cocultured with FHC-4D2 cells under the limiting dilution condition, interleukin-3 (IL-3)-responsive CFU-GM were induced from immature hematopoietic progenitor cells that were otherwise unresponsive to IL-3. From these data we conclude that the FHC-4D2 clone could generate and maintain IL-3-responsive hematopoietic progenitors via close contact and that, in the fetal liver, the contact between hepatocytes and hematopoietic cells may be critically important in inducing the differentiation of resting, IL-3-unresponsive immature hematopoietic cells into CFU-GM (progenitors responsive to IL-3) and in triggering the self-renewal of CFU-GM.

Frequent harvesting from perfused bone marrow cultures results in increased overall cell and progenitor expansion

The establishment of prolific long-term human bone marrow cultures has led to the development of hematopoietic bioreactor systems. A single batch expansion of bone marrow mononuclear cell populations leads to a 10- to 30-fold increase in total cell number and in the number of colony forming units-granulocyte/macrophage (CFU-GMs), and a four- to tenfold increase in the number of long-term culture initiating cells (LTC-ICs). In principle, unlimited expansion of cells should be attainable from a pool of stem cells if all the necessary requirements leading to stem cell maintenance and division are met. In this article, we take the first step toward the identification of factors that limit single batch expansion of ex vivo bone marrow cells in perfusion-based bioreactor systems. One possible constraint is the size of the growth surface area required. This constraint can be overcome by harvesting half the cell population periodically. We found that harvesting cells every 3 to 4 days, beginning on day 11 of culture, led to an extended growth period. Overall calculated cell expansion exceeded 100-fold and the CFU-GM expansion exceeded 30-fold over a 27-day period. These calculated values are based on growth that could be obtained from the harvested cell population. Growth of the adherent cell layer was stable, whereas the nonadherent cell population diminished with increasing number of passages. These results show that the bioreactor protocols published to date are suboptimal for long-term cultivation, and that further definition and refinement is likely to lead to even greater expansion of hematopoietic cell populations obtained from bone marrow. More importantly, these results show that the LTC-IC measured during the single pass expansion do have further expansion potential that can be realized by frequent harvesting. Finally, the present culture conditions provide a basis for an assay system for the identifications provide a basis for an assay system for the identification of the factors that determine the long-term maintenance and replication of human stem cells ex vivo.

Phenotypic characterization of CD4-/alpha beta TCR+ and gamma delta TCR+ T cells with a transendothelial migratory capacity.

During inflammation, both CD4+ and CD4- T lymphocytes extravasate into perivascular tissues by adhering to and migrating through the vascular endothelium. These studies were undertaken to characterize the phenotype of CD4- T cells that have a capacity to migrate through endothelium. Results show that CD4- T cells exhibit a greater capacity to migrate through endothelial cells (EC) than CD4+ T cells; and that TCR-gamma delta+ T cells exhibited the greatest migratory capacity. The migrating CD8+ T cell population was enriched in CD45RO+/L-selectin-/LFA-1bright/CD29bright/CD 44bright cells. TNF-alpha-activated EC did not support increased CD4- T cell transendothelial migration and changes in the phenotype of the migrating CD8+ T cells. The migrating CD4- T cell population was enriched in VLA-2+ T cells and expressed increased densities of VLA-4, VLA-5, and VLA-6. The migrating TCR-gamma delta+ T cell population contained both CD8dim and CD8- T cells. Moreover, the migrating gamma delta T cell population was not different from the initial or nonadherent population in that it contained CD45RO+, CD45RA+, and L-selectin+ cells. Finally, migrating TCR-gamma delta+ T cells contained cells expressing V delta 2 and V gamma 9 TCR chains, but these were not enriched compared with the initial population. These studies have characterized the CD4- T cells that are capable of transendothelial migration in vitro. The results are consistent with the conclusion that unique subpopulations of CD8+ alpha beta and CD8+ and CD8- gamma delta T cells gain access to inflammatory sites by virtue of their intrinsic ability to migrate across the endothelium.

Recombinant protein fragments from haemorrhagic septicaemia rhabdovirus stimulate trout leukocyte anamnestic responses in vitro

This work shows that viral protein fragments are capable of stimulating fish anamnestic immunological responses in leukocytes from the rainbow trout (Oncorhynchus mykiss, W.). Recombinant protein fragments of glycoprotein and nucleoprotein from the rhabdovirus causing viral haemorrhagic septicaemia of trout (VHSV), were cloned and expressed in Escherichia coli, Yersinia ruckeri (a trout pathogen) and Saccharomyces cerevisiae. The recombinant protein fragments stimulated anamnestic responses in leukocyte cultures derived from the anterior kidney of survivors of VHSV infection but not from uninfected trout. Two types of stimulatory anamnestic responses were detected, (i) a stimulation of lymphoproliferation as measured by thymidine incorporation assays and (ii) an increase in number, spreading and size of cells as determined by fibrin-clot and/or flow cytometry techniques. The evidence presented suggests that both adherent and non-adherent trout cell populations are needed for the immunological response to VHSV in this primitive vertebrate. The possible use of in vitro lymphoproliferation assays as a preliminary screening method for candidate fish vaccines prior to their testing in vivo is discussed.

Constitutive activation of integrin alpha 4 beta 1 defines a unique stage of human thymocyte development.

Our understanding of thymocyte development and of the positive and negative selection events involved in shaping the repertoire of mature T lymphocytes has been greatly facilitated by the use of transgenic and gene knockout animals. Much less is known about the factors that control the homing and population of the thymus by T cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. As the integrins represent a candidate group of cell surface receptors that may regulate thymocyte development, we have analyzed the expression and function of alpha 4 beta 1 and alpha 5 beta 1 on human thymocytes. A major portion of double positive (CD4+ CD8+) human thymocytes express alpha 4 beta 1 in a constitutively active form and adhere to fibronectin and vascular cell adhesion molecule 1. alpha 4 beta 1 expression is similar on adherent and nonadherent populations, thus, activity reflects the receptor state and not simple expression. The adherent cells are immature, expressing high levels of CD4/CD8 and low levels of CD3 and CD69. In contrast, nonadherent cells possess the phenotype of thymocytes after positive selection, expressing intermediate levels of CD4 and/or CD8 and high levels of CD3 and CD69. The adherent population fails to respond to activation with anti-CD3 and fibronectin, whereas nonadherents exhibit an alpha 5 beta 1-dependent proliferation. Differential regulation of alpha 4 beta 1 and alpha 5 beta 1 receptors may provide a mechanism controlling cellular traffic, differentiation, and positive selection of thymocytes.

Infection of human bone marrow stromal cells by hepatitis B virus: implications for viral persistence and the suppression of hematopoiesis.

Suspension cultures of bone marrow cells (BMC) were challenged with hepatitis B virus (HBV) to study interactions between the virus and the nonadherent and adherent BMC populations. Virus-challenged BMC developed an adherent stromal layer that differed in cellular composition from that of mock-infected cultures, showing a threefold increase in cells of the monocyte-macrophage lineage with an accompanying decrease in cells of the granulocytic lineage. Both viral envelope hepatitis B surface and core antigen expression was detected in adherent and nonadherent cell populations up to 10 days after virus challenge, which decreased thereafter. HBV DNA was still detectable in adherent cells 3 weeks after virus challenge, as shown by polymerase chain reaction analysis. These data indicate that HBV can infect not only bone marrow colony-forming cells but also the stromal cell populations involved with the regulation of hematopoiesis in vivo. Such virus-cell interactions could contribute to the immune dysfunction and bone marrow failure occasionally reported for patients with HBV infection as well as acting as an important site for HBV latency and persistence.

Characterization of adherent T cells to human endothelial cells in patients with HTLV-I-associated myelopathy

To clarify the phenomenon of increased adherence of T cells to endothelial cells (EC) in patients with HTLV-I-associated myelopathy (HAM), we determined the surface markers and expression of lymphocyte function antigen-1 (LFA-1) in T cells adherent or nonadherent to EC. The percentage of activated or HLA-DR + T cells and the expression of LFA-1 in the adherent cell population were significantly higher than those in the nonadherent cell population. Moreover, the CD4 to CD8 ratio of the HLA-DR + cells in the EC-adherent T cells was significantly higher than that in the nonadherent cells. Collectively, these results indicate that increased adherence of T cells to EC in HAM patients is based on the increase of activated T cells with high density LFA-1 expression in the peripheral blood. Moreover, CD4 + HLA-DR + cells exhibited more adhesive activity to EC than CD8 + HLA-DR + cells, suggesting that activated CD4 + cells, rather than activated CD8 + cells, may be important as the first trigger for T cell-infiltration to the central nervous system in the immunopathogenesis of HAM.

Human fetal antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells.

Human fetal antibody-dependent cellular cytotoxicity (ADCC) has not been reported previously. Most investigations have failed to document any cytolytic activity among fetal lymphocytes. The purpose of this study was to investigate ADCC activity in the human fetus and identify and characterize the effector cell populations in the fetus. Fetal spleen cells were separated into single-cell suspensions and assayed with 51Cr-labeled herpes simplex 1-infected Chang liver target cells. Significant ADCC activity was detected in 19 of 26 (73%) of freshly assayed fetal spleen cell preparations from fetuses of 17-24 wk gestational age. This activity, however, was significantly less than concurrently run adult peripheral blood mononuclear cells. After plastic adherence the fetal spleen ADCC activity from nonadherent cells was not significantly different from whole spleen preparations. Surprisingly, ADCC activity in nonadherent fetal cells dropped significantly after exposure to latex beads, an effect not seen in nonadherent adult lymphocytes. Thus, either fetal monocyte-derived (macrophages) fetal spleen cells do not efficiently adhere to plastic or a unique nonadherent population of latex-sensitive immunocytes is capable of mediating ADCC activity in the fetus. We suspect the former conclusion to be the more plausible; however, fluorescence-activated cell sorter staining of fetal cells was not sufficient to confirm these suspensions by fluorescence-activated cell sorter analysis.

A comparative study of T and B lymphocytes in rainbow trout (Oncorhynchus mykiss) following their separation by nylon wool adherence and lectin agglutination techniques

A nylon wool separation technique was employed to separate rainbow trout leucocytes into adherent and non-adherent populations. The non-adherent population showed a greater response to concanavalin A (ConA) and a lesser response to lipopolysaccharide (LPS) than did the adherent population in the spleen, kidney and peripheral blood. The great majority (>90%) of thymocytes were in the non-adherent population. The non-adherent population from the spleen, kidney and peripheral blood showed signicantly (P>0.05) higher numbers of acid phosphatase-positive lymphocytes than the adherent population, but there was no significant difference in the pattern of immunochemical staining using a mouse anti-trout IgM monoclonal antibody.

Relationship between cell adherence and proteoglycan synthesis in cultures of human peripheral blood mononuclear cells: effects of concanavalin A.

Human, blood-derived mononuclear cells (MC), stimulated with Concanavalin A (Con A), synthesized a chondroitin sulfate (CS) proteoglycan (PG), which was elaborated largely by T-cells. Following Con A stimulation, the majority of MC adhered to the culture dish by day 2, but as incubation progressed to day 10 the proportion of non-adherent (NAd) MC increased in a fashion which approximately paralleled the accumulation of PG in the medium. Cell kinetic studies suggest that, following Con A stimulation, there was an inverse relationship between the amount of newly synthesized cellular PG and adherence, which appears to be related to a reciprocal effect on PG synthesis of the declining adherent (Ad) cell density with time of culture. In the stimulated cultures, NAd cells contained much more newly synthesized CS/cell than Ad cells up to day 6 of incubation. Cell type analysis, using monoclonal antibodies against specific cell surface markers, suggested that the higher PG synthesis in the NAd population may, at least in part, be due to a greater proportion of T-helper cells.

Salmonella typhimurium induces IFN-gamma production in murine splenocytes. Role of natural killer cells and macrophages.

IFN-gamma is a cytokine known to play an important role in host defense against Salmonella typhimurium. The lymphoid cells required for in vitro production of IFN-gamma after S. typhimurium stimulation of mouse spleen cells was investigated. Spleen cells depleted of cells bearing NK1.1, asialo GM1, Thy 1.2, or CD5 resulted in a significant reduction in IFN-gamma production after stimulation with S. typhimurium. In contrast, Con A-induced IFN-gamma production was only slightly reduced after depletion of NK1.1- or asialo GM1-bearing cells. Spleen cells from SCID mice produced elevated levels of IFN-gamma after stimulation with S. typhimurium. IFN-gamma production by SCID spleen cells was dependent upon asialo GM1+ T cells, suggesting that NK cells were the cells producing IFN-gamma in response to S. typhimurium. Splenic adherent cells were required for optimal IFN-gamma production. However, direct contact between the adherent and nylon wool nonadherent (NWNA) cell populations was not essential. IFN-gamma production was observed when the adherent and NWNA cell populations were physically separated or when supernatant from S. typhimurium-stimulated adherent cells was added to NWNA cells. Optimal IFN-gamma production was dependent on the presence of TNF-alpha, inasmuch as addition of antibody to TNF-alpha to spleen cell or NWNA cell cultures significantly reduced IFN-gamma production. However, addition of rTNF-alpha did not induce IFN-gamma production by NWNA cells. These findings document the existence of a T-independent mechanism for early IFN-gamma production in response to S. typhimurium, and show that TNF-alpha is necessary but not sufficient for the production of IFN-gamma.

Human Herpesvirus-6-Associated Suppression of Growth Factor-Induced Macrophage Maturation in Human Bone Marrow Cultures

AbstractCultures of marrow mononuclear cells were exposed to medium derived from cell cultures infected with several different strains of human herpesvirus-6 (HHV-6) immediately before the addition of either of two growth factors, granulocyte-macrophage colony-stimulating factor and interleukin-3. Exposure to any of the viral preparations suppressed the outgrowth of nonspecific esterase-positive adherent macrophages induced by the factors by more than 90%. The nonadherent cell populations in the infected cultures were numerically similar to those in uninfected control cultures, demonstrating the absence of a nonspecific cytotoxic effect of the viral materials. Infectious virus was not necessary for the macrophage outgrowth suppression. These findings suggest that HHV-6 either encodes or induces a soluble mediator or mediators that can interfere with the responses of bone marrow to growth factors and possibly block the normal differentiation of macrophages from marrow precursors.

Human herpesvirus-6-associated suppression of growth factor-induced macrophage maturation in human bone marrow cultures

Cultures of marrow mononuclear cells were exposed to medium derived from cell cultures infected with several different strains of human herpesvirus-6 (HHV-6) immediately before the addition of either of two growth factors, granulocyte-macrophage colony-stimulating factor and interleukin-3. Exposure to any of the viral preparations suppressed the outgrowth of nonspecific esterase-positive adherent macrophages induced by the factors by more than 90%. The nonadherent cell populations in the infected cultures were numerically similar to those in uninfected control cultures, demonstrating the absence of a nonspecific cytotoxic effect of the viral materials. Infectious virus was not necessary for the macrophage outgrowth suppression. These findings suggest that HHV-6 either encodes or induces a soluble mediator or mediators that can interfere with the responses of bone marrow to growth factors and possibly block the normal differentiation of macrophages from marrow precursors.

Relative Sensitivity of Leukemic (CML) and Normal Progenitor Cells to Treatment with the Photosensitizer Benzoporphyrin Derivative and Light

Normal bone marrow and peripheral blood committed progenitor cells were found to be significantly more resistant to treatment with the photosensitizer benzoporphyrin derivative (BPD) and white fluorescent light (11 J/cm2) than were clonogenic cells from two cell lines (K562 and EM2) derived from nonremission patients with myelogenous leukemias. Bone marrow and peripheral blood committed progenitor cells from normal donors were also found to be more resistant to this treatment than were equivalent cells from patients with chronic myelogenous leukemia (CML). Normal bone marrow mononuclear cells grown in long-term marrow culture (LTMC) following treatment with BPD and light showed that no differences in stem cell productivity existed between control and treated samples. When bone marrow from CML patients was treated in the same manner, the numbers of progenitors detected in the nonadherent population during culture were greatly reduced compared to control material. Two rounds of PCR using nested primers to detect BCR-ABL mRNA showed that while this treatment significantly decreased the numbers of stem cells bearing the Philadelphia chromosome, it did not eliminate them.

Properties of blast colonies obtained from trout head-kidney in fibrin clot cultures

Different tissue dissociation protocols and serum supplements resulted in a variety of primary cultures from rainbow trout spleens. These were classified as either haemopoietic or non-haemopoietic. In haemopoietic cultures, a complex stromal layer developed on the cell culture surface; round cells appeared on the stromal layer; and ultimately some round cells were released into the medium as a non-adherent population. Many non-adherent cells were able to reattach in new culture vessels. Most of these had the characteristic shape and unique motility of dendritic cells. Cultures of these cells were subcultivated 2–4 times but continuous cell lines did not arise. Haemopoietic cultures resulted with collagenase dissociation and maintenance in high foetal bovine serum (FBS) concentrations. Non-haemopoietic cultures contained adherent cells, including macrophages and fibroblasts, but did not develop a non-adherent population. These cultures resulted with 10% FBS or horse serum and after explant outgrowth or dissociation with trypsin. Both culture types should be useful for studying the regulation of haemopoiesis and the functions of specific immune cells in fish.