Primary cell cultures of leukosis P-388 as a possible model for the screening of antitumor substances]

A substrain of the ascitic leucosis P-388 was obtained as a result of interchanging passages of P-388 leucosis cells in the primary cultures and abdominal cavity of mice. The tumor cells of the ascitic leucosis P-388 multiplied in the primary suspended culutres as well as in vivo. The substrain lost its hemorrhagic properties. The content of DNA and RNA in the primary cultures of P-388 doubled every 16-18 hours and the number of the cells doubled every 24-26 hours. The cells of the adapted substrain of P-388 grew also in the semiliquid agarized medium forming compact colonies by the 4th-5th day of cultivation. The primary suspended cultures of P-388 were highly sensitive to cytostatics and in particular to vinblastin and kolchamine (alkaloids). In this connection they were recommended for prescreening antitumor compounds.     

Growth kinetics by scanning of granulocytic cell colonies in glass capillaries.

Pure granulocytic colonies were cultivated from mouse bone marrow cells in agar contained in glass capillary tubes using mouse embryo conditioned medium as colony stimulating activity. A random distribution of colonies along the agar gels was achieved under controlled conditions. Only 3 capillaries were needed for a coefficient of variation around 5% provided at least 104 cells were seeded per capillary. The daily growth of single colonies within an agar capillary was followed, using the light scattering properties of the colonies for automatic scanning. The position of the colonies in the capillary greatly affected the scan signal; the consequences of positional changes were studied in detail. Using the mean peak height as growth parameter, the onset of measureable granulocytic colony growth was found between day 2 and 3, the maximum colony size was reached between day 7 and 9, after which the colonies decayed. Other parameters such as colony count and total peak are were determined and their sinificance discussed.     

The in vitro differentiation of density sub-populations of colony-forming cells under the influence of different types of colony-stimulating factor.

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density subpopulations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSFMLCM), post-endotoxin mouse serum (CSFES) and from human urine (CSFHu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macrophage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

The role of intercellular interactions in the activation of T-lymphocyte colony precursors

The influence of the previous cultivation of human peripheral blood mononuclear cells in suspension with mitogen on the consequent PHA-stimulated response in suspension and agar cultures was studied. It has been established that proliferative response of mononuclear cells in suspension with mitogen is not changed, but the intensity of T-cell colony formation in two-layer agar systems in increased after preincubation mainly at the expense of increasing the number of type II colonies and clusters. It is concluded that the free cell-cell contacts are needed to activate T-lymphocyte colony precursors.     

Mononuclear macrophage (M-CFC) colony formation in semisolid media in the absence of exogenous GM-CSF

Human fetal bone marrow (FBM) cells were examined for the ability to form colonies in the absence of exogenous colony-stimulating factor (CSF) in double layer agar, methylcellulose (MC), and in agar-MC (agar underlayer, MC overlayer) culture systems. Without exogenous CSF, macrophage colonies (M-CFC) were formed in a combined culture of agar and MC. Aggregates of 5-40 cells were observed on day 7. Gradually, large compact colonies which survived for 10-12 weeks of cultivation, were formed. They were composed of mononuclear monocytes and multinucleated cells. M-CFC progenitors were nonadherent, but their progeny became adherent during differentiation within the colony. Colony formation was cell-dose-dependent. Depletion of monocytes increased the number of colonies in agar-MC cultures and stimulated the development of some macrophage colonies in MC. Survival of monocyte progenitors was not dependent on CSF. Neither was their proliferation nor partial differentiation in agar-MC cultures. CSF increased M-CFC colony efficiency, however, if it was present when cultures were initiated. Addition of CSF to M-CFC growing for 2-5 weeks in CSF-deprived medium stimulated monocytes proliferation and transformation into macrophages. Epithelioid cells, an increase in the number of giant multinucleated cells, and granulocyte multiplication were also observed. The absolute dependence of macrophage colony formation on CSF described by others might be a result of inadequate culture conditions due to agar rather than an intrinsic physiological requirement.     

Role of mercaptoethanol and endotoxin in stimulating B lymphocyte colony formation in vitro.

Mercaptoethanol is necessary to permit B lymphocyte colony formation in semi-solid agar cultures of cells from normal mouse lymphoid organs. Transfer studies on developing colonies showed that, in part, this was a direct action on B lymphocyte colony cells but evidence was produced that in the presence of mercaptoethanol lymphoid organ cells release a factor promoting colony growth. Endotoxin strongly potentiated B lymphocyte colony formation in vitro by a direct action on colony cells but in the absence of mercaptoethanol did not allow cell survival or proliferation.     

Analysis of colonies developing in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum

An analysis has been made of cell colonies developing in agar cultures from mouse bone marrow cells following stimulation either by neonatal kidney cell feeder layers or AKR lymphoid leukemia serum. Colonies arose by cell proliferation and were mixtures of granulocytic and mononuclear cells. Colonies stimulated by kidney feeder layers reached a mean size of 2000 cells by day 10 of incubation and remained predominantly granulocytic in nature. When bovine serum was substituted for fetal calf serum, cell colonies grew to a smaller size and lost their granulocytic nature, finally becoming almost pure populations of mononuclear cells. Colonies stimulated by AKR leukemic serum reached a mean size of 350 cells by day 10 of incubation. Although these colonies initially were granulocytic in nature, they finally became almost pure populations of mononuclear cells. The colony mononuclear cells actively phagocytosed carbon, and contained metachromatic granules probably derived from ingestion of agar. The mononuclear cells in these colonies may not have been members of the original colony, but may have been incorporated in the colony as it expanded in size, subsequently proliferating in the favourable environment of the colony.     

B lymphocyte colony-forming cells in the SJL/J mouse.

Mercatoethanol-induced B lymphocyte cloning in semi-solid agar has been used to study lymphocyte colony formation by cells from the SJL/J mouse thymus. From the 3rd month of life, the SJL/J mouse thymus. From the 3rd month of life, the SJL thymus develops an increasing frequency of cells forming B lymphocyte colonies in agar. The peak frequency in 6- to 12-month-old mice was one colony per 1000 to 2000 cultured thymus cells. In contrast, 10 to 100 times lower frequencies were found in the thymus of five other inbred mouse strains. The rise in B lymphocyte colony-forming cells correlated well with the age-related rise in Ig-positive cells and approximately 50% of the colony cells reacted with anti-micron-serum indicating the B lymphocyte nature of the colony cells. Colony-forming cells from the thymus showed higher sensitivity than colony-forming spleen cells to cortisol and irradiation. Cell transfer experiments and thymus grafting suggested that the increased frequency of colony-forming cells in the thymus is caused by development of special thymus-seeking B lymphocytes in ageing SJL/J mice. Finally, B lymphocyte colony-forming cells were found to be more frequent in the thymus, spleen, and lymph nodes from healthy aged mice than in lymphoid organs from mice with spontaneous reticulum cell tumors.     

Functional differences between cells from MLR colonies grown in soft agar cultures

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.     

Studies on colony formation in vitro by mouse bone marrow cells. II. Action of colony stimulating factor

An analysis was made of some of the processes involved in the stimulation by colony stimulating factor (CSF) of cluster and colony formation by mouse bone marrow cells in agar cultures in vitro. Colony formation was shown to be related to the concentration and not the total amount of CSF. The concentration of CSF determined the rate of new cluster initiation in cultures and the rate of growth of individual clusters. Colony growth depleted the medium of CSF suggesting that colony cells may utilise CSF during proliferation. Bone marrow cells incubated in agar in the absence of CSF rapidly died or lost their capacity to proliferate and form clusters or colonies. CSF appears (a) to be necessary for survival of cluster-and colony-forming cells or for survival of their proliferative potential, (b) to shorten the lag period before individual cells commence proliferation and (c) to increase the growth rate of individual clusters and colonies.     

Cultivation of frozen mouse bone marrow cells in agar.

The authors attempted to cultivate frozen mouse bone marrow cells in a semisolid medium. They demonstrated that the stem haematopoietic cells of frozen mouse bone marrow were capable of proliferation and of colony formation on agar. The much smaller number of colonies from frozen mouse bone marrow (about 80% fewer) compared with fresh marrow is evidence that part of the stem haematopoietic cell population retains proliferative capacity even after freezing.     

Identification of an epidermal growth factor-related transforming growth factor from rat fetuses

An epidermal growth factor (EGF)-like transforming growth factor was identified in acid/ethanol extracts of 19-day-old rat embryos. This factor had an apparent molecular weight of 55,000 and possessed EGF-competing activity, stimulated DNA synthesis in quiescent Rat-1 fibroblasts, and induced progressive growth of untransformed cells in soft agar. The factor was trypsin sensitive and required intact disulfide bonds for colony stimulating activity. It was determined to be distinct from adult mouse submaxillary gland EGF based on its elution from BioGel P-60 column and its increased colony stimulating activity/unit of EGF-competing activity.     

Formation of eosinophilic-like granulocytic colonies by mouse bone marrow cells in vitro

Formation of granulocytic and macrophage colonies in agar cultures of mouse marrow or spleen cells was stimulated by the addition of medium from pokeweed mitogen-stimulated cultures of mouse spleen cells (PKW-CM). Approximately 5% of the colonies developing were large, dispersed granulocytic colonies (DG-colonies) composed of cells with eosinophilic cytoplasmic granules. The capacity to stimulate DG-colonies was shown by media conditioned by PKW-treated lymphoid and peritoneal cells but not by other cells or organ fragments. Velocity sedimentation studies indicated that cells generating DG-colonies were separable from cells generating regular granulocytic or macrophage colonies. DG-colonies did not survive if transfered to cultures containing other forms of CSF. The active colony stimulating factor in pokeweed mitogen-conditioned medium which stimulates DG-colony formation was antigenically distinct from the factor stimulating granulocytic and macrophage colony formation, was separable electrophoretically from the latter factor and on gel filtration had an apparent molecular weight of 50,000. Although the cells in DG-colonies have not been established to be eosinophils, DG-colonies represent an interesting new system for analysing further aspects of the control of growth and differentiation in hemopoietic populations.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Formation in agar of fibroblast-like colonies by cells from the mouse pleural cavity and other sources

Colonies of elongated fibroblast-like cells (stellate colonies) developed in agar cultures of mouse pleural cavity cells mixed with whole blood. Cultures of pleural cells alone developed only abortive clusters of round cells. The frequency of colony-forming cells in the pleural cavity was highest in neonatal mice (200/10⁵ cells) and fell progressively with aging. Stellate colony-forming cells were not in cell cycle but were radiosensitive. In adult mice, only occasional colony-forming cells were detected in peritoneal cavity, thymic, spleen, lymph node or bone marrow cell populations. Stellate colony formation was not stimulated by the granulopoietic regulator, colony stimulating factor. The active component in whole blood required for stellate colony formation was present in plasma but not serum or washed red or white cells.     

Colony formation in agar by multipotential hemopoietic cells.

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.     

Automated scanning of bone marrow cell colonies growing in agar-containing glass capillaries

An optical scanning system was developed to determine the growth of clusters and colonies of granulocytes and macrophages from mouse bone marrow cells in agar capillary tubes. The system consists of a commercially available photometer with a densitometer attachment, a two-mirror set to receive the light scattered by the cell colonies, a multiple capillary holder and an automatic sample changer. Parameters affecting scanning were examined and optimized: background scatter, instrument adjustments (e.g. signal damping) and threshold settings for clusters and colonies. Combined with the advantageous agar capillary technique, the complete scanning system provides an easy, accurate and sensitive method for rapid quantitation of hemopoietic cell colony formation in vitro.     

Heterogeneity of in vitro colony- and cluster-forming cells in the mouse marrow: segregation by velocity sedimentation.

C57BL bone marrow cells were separated on the basis of their sedimentation velocity at unit gravity and cell fractions cultured in agar using three types of colony stimulating factor (CSF). Colony-forming cells separated as a single peak (s equal 4.4 mm/hr) in cultures stimulated by mouse lung conditioned medium (CSFMLCM) or endotoxin serum (CSFES). Cluster-forming cells were separable into two peaks and the majority were larger than colony-forming cells (s equal 5.7 mm/hr). Partial segregation of colony-forming cells was observed according to the morphological types of colonies generated, large cells tending to generate macrophage colonies and small cells, granulocytic colonies. Large colony-forming cells were more responsive to stimulation by CSF than small cells. Human urine (CSFHU) appeared unable to proliferation of most small colony-forming cells. Colony-forming cells appear to be a highly heterogeneous population with intrinsic differences in responsiveness to CSF and with differing capacities to generate colonies whose cells differentiate to granulocytes of macrophages.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.