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In the last 20 years, one of the main goals in the drug discovery field has been the development of reliable in vitro models. In particular, in 2006 the European Centre for the Validation of Alternative Methods has approved the colony-forming unit granulocytes–macrophages test, which is the first and currently unique test applied to evaluate the myelotoxicity of xenobiotics in vitro. The present work aimed at miniaturizing this in vitro assay by developing and validating a Lab-on-Chip platform consisting of a high number of bioreactor chambers with screening capabilities in a high-throughput regime.

In the last 20 years, one of the main goals in the drug discovery field has been the development of reliable in vitro models. In particular, in 2006 the European Centre for the Validation of Alternative Methods has approved the colony-forming unit granulocytes–macrophages test, which is the first and currently unique test applied to evaluate the myelotoxicity of xenobiotics in vitro. The present work aimed at miniaturizing this in vitro assay by developing and validating a Lab-on-Chip platform consisting of a high number of bioreactor chambers with screening capabilities in a high-throughput regime.

In order to evaluate the potential hematotoxicity of xenobiotics, including candidate anti-cancer drugs, in vitro models of hematopoiesis are used, which involve clonogenic assays on CFU-GM (Colony Forming Unit-Granulocyte-Macrophage). These assays require live and unstained colonies to be counted. Most laboratories still rely on visual scoring, which is time consuming and error prone. As a consequence automated scoring is highly desired. A classification algorithm aimed at emulating the colony recognition and scoring capabilities of a human expert has been developed. A first account will be given herewith. Assays were carried out on CFU-GM progenitors derived from human umbilical cord blood cells and grown in methylcellulose. A three-dimensional (3-D) medium is essential for these assays to simulate the clonogenetic process which takes place in bone marrow. Stacks of images representing slices of a 3-D domain were acquired. Structure and texture information was extracted from each image. Classifier training was based on a 3-D colony model applied to the image stack. The number of scored colonies (assigned class) was required to match the count supplied by the human expert (class of belonging). Successful applications to scoring colonies, which partially overlap and/or are masked by caustics, are described. Whereas the industry's scoring methods all rely on image structure alone and process 2-D data, the classifier described herewith takes texture into account and fuses 3-D data from a whole stack

The pig is widely used as a large animal model for biomedical research and could be an interesting experimental model for studies of the hematopoietic system and its response in physiological and pathological conditions. With the intention of using the pig as a large animal model in hematopoietic research, a clonal assay in methylcellulose was developed and standardized for detection of committed progenitors of the granulocyte-macrophage lineage from adult pig bone marrow and peripheral blood. Progenitor cells were stimulated to proliferate and differentiate in vitro by adding pig leukocyte conditioned medium (LCM) as a source of homologous growth factors. The number of CFU-GM (Colony Forming Unit -Granulocyte-Macrophage) directly depended on the concentration of LCM. The proliferative rate of CFU-GM progenitor cells was determined by the cytosine arabinoside suicide technique. The percentage of bone marrow and peripheral blood CFU-GM cells in S phase of the cell cycle was 34.7% and 22.2%, respectively. The data obtained regarding the number and characteristics of pig bone marrow and peripheral blood CFU-GM confirmed that the organization of the pig CFU-GM progenitor cell compartment is similar and comparable to that in miniature swine, other animal species and humans., Svinja je životinja koja se koristi kao model u različitim biomedicinskim istraživanjima, a mogia bi biti i interesantan model u ispitivanju fiziologije i patolofizioloških promena hematopoetskog sistema. U cilju razvoja eksperimentalnog modela svinje u istraživanju hematopoeze, stanadardizovan je esej za odredivanje i karakterizaciju opredeljenih matičnih ćelija granulocitno-monocitne loze iz kostne srži i krvi odrasle svinje. Stimulacija proliferacije i diferencijacije ovih matičnih ćelija postignuta je dodavanjem medijuma kondicioniranog leukocitima (LCM - Leukocyte conditioned medium) bogatog faktorima rasta. Broj CFU-GM (Colony forming unit- granulocyte-macrophage) je direktno zavisio od koncentracije LCM-a. Procenat CFU-GM ćelija u S fazi ćelijskog cikiusa odredjivan je tehnikom 'suicida' korišćenjem citozin arabinozida (Ara-C) i iznosio je 34.7% za CFU-GM iz kostne srži i 22.2% za CFU-GM iz periferne krvi. Podaci dobijeni za broj i karakteristike CFU-GM iz kostne srži i periferne krvi potvrđuju da je ovaj odeljak matičnih ćelija kod odraslih svinja organizovan na isti način kao i kod minijaturnih svinja, drugih vrsta životinja i ljudi.

The inhibitory effect of human interferon-alpha and interferon-gamma on the colony formation of CFU-GM was studied using peripheral blood and bone marrow cells in patients with chronic myelogenous leukemia (CML) and healthy donors. Both types of interferon inhibited colony growth of CFU-GM in CML and health donors. Interferon-alpha suppressed CFU-GM in some patients with CML more strongly than that in healthy donors. Whereas interferon-gamma suppressed CFU-GM in healthy donors more strongly than that in patients with CML. The CFU-GM in peripheral blood mononuclear cells with CML had significantly lower sensitivity to interferon-gamma than that in bone marrow mononuclear cells with CML. These findings indicate that the inhibitory effect of interferon, at least in part, is mediated by hematopoietic accessory cells in blood and bone marrow. From this study, it was suggested that interferon-alpha is a more active therapeutic agent than interferon-gamma for patients with CML. There were some co-relationship between the clinical effect of interferon-alpha and the sensitivity of CFU-GM in patients with CML. In conclusion, the sensitivity test of CFU-GM is beneficial to screening of therapeutic agents in CML.

type:text, A monoclonal antibody (MoAb) K15 (IgG3) was obtained by fusion between SP2/0, mouse myeloma cell line and spleen cells from BC3Fl mice immunized with K562. a chronic myelocytic leukemia (CML) blastic crisis cell line. MoAb Kl5 precipitated a 21 kilodalton (KD) polypeptide in reduced condition. It stained lymphocytes, monocytes, eosinophiles and small bone marrow cells, but not neutrophiles, platelets, red blood cells (REC) and large bone marrow cells. Depletion of K15+ cells abolished both colony-forming units-granulocyte/monocyte (CFU-GM) and burst-forming units-erythroid (BFU-E). Thus MoAb Kl5 identifies antigens expressed on hematopoietic progenitor cells. The selective retention of this antigen by certain cell lineages may suggest some functional role of this antigen.

type:text, A monoclonal antibody (MoAb) H5 (IgG2b) was obtained by fusion between SP2/0, mouse myeloma cell line and spleen cells from BC3Fl mice immunized with HEL, a erythroleukemia cell line. MoAb H5 precipitated a 26 kilodalton (KD) polypeptide in reduced condition. It reacted with platelets, monocytes, eosinophiles, 13% of lymphocytes, thymocytes and colony-forming units-granulocyte/monocyte (CFU-GM), but not with neutrophiles, red blood cells (RBC) and burst-forming units-erythroid (BFU-E). It also stained T-lymphoid leukemia cell lines but not B cell lines. Thus this antigen was expressed selectively by both myeloid and lymphoid lineages.

A monoclonal antibody (MoAb) K15 (IgG3) was obtained by fusion between SP2/0, mouse myeloma cell line and spleen cells from BC3Fl mice immunized with K562. a chronic myelocytic leukemia (CML) blastic crisis cell line. MoAb Kl5 precipitated a 21 kilodalton (KD) polypeptide in reduced condition. It stained lymphocytes, monocytes, eosinophiles and small bone marrow cells, but not neutrophiles, platelets, red blood cells (REC) and large bone marrow cells. Depletion of K15+ cells abolished both colony-forming units-granulocyte/monocyte (CFU-GM) and burst-forming units-erythroid (BFU-E). Thus MoAb Kl5 identifies antigens expressed on hematopoietic progenitor cells. The selective retention of this antigen by certain cell lineages may suggest some functional role of this antigen.

A monoclonal antibody (MoAb) H5 (IgG2b) was obtained by fusion between SP2/0, mouse myeloma cell line and spleen cells from BC3Fl mice immunized with HEL, a erythroleukemia cell line. MoAb H5 precipitated a 26 kilodalton (KD) polypeptide in reduced condition. It reacted with platelets, monocytes, eosinophiles, 13% of lymphocytes, thymocytes and colony-forming units-granulocyte/monocyte (CFU-GM), but not with neutrophiles, red blood cells (RBC) and burst-forming units-erythroid (BFU-E). It also stained T-lymphoid leukemia cell lines but not B cell lines. Thus this antigen was expressed selectively by both myeloid and lymphoid lineages.