Human cells in suspension 2

Summary When PHA stimulated human lymphoid cells are allowed to proliferate in vitro and chromosomal slides are prepared from the culture after 48,72, 96,120, and 144h of growth, gradual changes in chromosome morphology can be observed after traditional Giemsa staining of the slides. Keeping culture conditions, colcemid exposure, and fixation procedure constant for all samples, it is found that average chromosome length decreases with increasing culture time. A shift from high frequencies of subbanded chromosomes (sample 48 h and sample 72 h) to high frequencies of unbanded and G banded chromosomes (sample 120 h and sample 144 h) takes place simultaneously with the general compaction of the chromosomes. Examination of trypsin-induced G bands as well as examination of untreated G banded chromosomes from all samples clearly indicate that the basic G band pattern is not altered during proliferation and differentiation, although the progressive compaction of the chromosome observed with increasing culture time results in a phenomenon similar to that observed during mitosis, where the compact late metaphase chromosome after trypsin treatment exhibits fewer but more prominent bands than the prophase/prometaphase chromosomes. Thus the progressive compaction of metaphase chromosomes observed during in vitro aging seems to resemble the condensation processes during the G₂ phase and mitosis.It has been suggested that the chromomeres serve as centers for chromosome condensation during mitosis, probably mediated by a sulphydryl-disulphide transition in chromosomal proteins. The data presented here further suggest that the chromomeres may also serve as centers for chromosomal differentiation, presumable by a mechanism similar to that acting during chromosome condensation in mitosis.     

Diffusion chamber and spleen colony assay of murine haematopoietic stem cells

Mouse bone marrow cells have been cultured in diffusion chambers and their capacity to form spleen colonies in irradiated mice investigated after different culture periods. The number of spleen colony-forming units (CFU) in the chambers decreased during the first day of culture. The number then increased rapidly to a level significantly above the original chamber value on the third to fifth day of culture. By that time large numbers of granulocytes and macrophages had also appeared. Histological examination of spleen colonies showed that prior culturing did not alter the ratio between the different types of colonies. Cultured bone marrow cells which were transferred to new chambers retained granulopoietic capacity. This capacity increased between the first and second day of primary culturing. At this time hydroxyurea injections to chamber hosts revealed that the progenitor cells were proliferating. The results show that the granulopoietic progenitor cells of the chambers are stem cells, and that one progenitor cell type is identical with the CFU.     

小型猪骨髓间充质干细胞的分离和培养

目的建立小型猪骨髓间充质干细胞（mesenchymal stem cells,MSCs）的体外分离和培养方法。方法穿刺小型猪髂后上嵴抽取骨髓,经密度梯度法离心得到骨髓单个核细胞,接种后形成单层贴壁细胞。用形态学方法鉴定培养的MSCs。结果经培养存活的MSCs原代和一代呈纺锤型、多边型或星型。至二代起呈均一纺锤型,似成纤维细胞样,长宽比例约为（2～3）？1。体外培养的原代MSCs8～10d达到融合,传代后仍具有较强的增殖能力。结论小型猪MSCs可在体外长期、稳定培养,其分离、培养体系的建立为基础研究和组织工程技术提供了一个有价值的动物模型。     

Genotoxic action of fungicide Conan 5FL (hexaconazole) on mammalian cells in vivo and in vitro

The genotoxic effects of fungicide Conan 5FL (containing 50 g/L hexaconazole) in mouse bone-marrow cells and human lymphocytes have been evaluated. Three different concentrations of Conan 5FL (17.50, 35.00 and 70.00 microg/mL for human lymphocytes and 17.50, 35.00 and 70.00 mg/kg for mouse bone marrow cells) were studied. Conan 5FL induced significant increases (except 17.50 mg/kg for mouse bone marrow) in the frequency of chromosomal aberrations (CAs) in both test systems. This fungicide caused structural and numerical abnormalities in both mammalian cells. These are sister chromatid union, chromatid and chromosome breaks, fragments, dicentric and ring chromosomes, and polyploidy. Significant increase was found in induction and in minimum-maximum numbers of sister chromatid exchanges (SCEs) at all treatments compared with the negative control. Conan 5FL did not affect the replication index (RI) in human lymphocyte cultures, however, it significantly decreased the mitotic index (MI) in all treatment concentrations in both test systems. Using of Conan 5FL should be reconsidered due to its possible cytotoxic, clastogenic and mutagenic effects.     

Cell replication and aging: in vitro and in vivo studies.

The relationship between human aging and cell replication has been investigated using two complementary approaches: in vitro studies of human fibroblasts derived from young and old volunteer members of the Baltimore Longitudinal Study and in vivo examinations of bone marrow cell populations from young and old mice and rats. Total proliferative capacity measured as either the onset of cell culture senescence or as in vitro life span was significantly diminished in cell cultures derived from old human donors when compared to parallel cultures established from young donors. Acute replicative abilities as measured by percent replicating cells, cell pupulation doubling time, cell number at confluency, and colony size distribution were also significantly decreased in human old cell populations. An in vivo cytogenetic technique for measuring cell replication was developed utilizing the differential staining properties of metaphase chromosomes of cells that have replicated in the presence of bromodeoxyuridine. With this technique, cell cycle times have been derived in vivo as well as in vitro. Preliminary in vivo results in both mice and rats indicate that cell replication is slowed in old animal cell populations. Further research will be directed both in vitro and in vivo at discerning the mechanisms for this impairment of cellular replication with aging.     

Genotoxic action of fungicide Conan 5FL (Hexaconazole) on mammalian cells in vivo and in vitro

The genotoxic effects of fungicide Conan 5FL (containing 50 g/L hexaconazole) in mouse bone-marrow cells and human lymphocytes have been evaluated. Three different concentrations of Conan 5FL (17.50, 35.0, and 70.0 μg/mL for human lymphocytes and 17.50, 35.0, and 70.0 mg/kg for mouse bone marrow cells) were studied. Conan 5FL induced significant increases (except 17.5 mg/kg for mouse bone marrow) in the frequency of chromosomal aberrations (CAs) in both test systems. This fungicide caused structural and numerical abnormalities in both mammalian cells. These are sister chromatid union, chromatid and chromosome breaks, fragments, dicentric and ring chromosomes, and polyploidy. Significant increase was found in induction and in minimum-maximum numbers of sister chromatid exchanges (SCEs) at all treatments compared with the negative control. Conan 5FL did not affect the replication index (RI) in human lymphocyte cultures, however, it significantly decreased the mitotic index (MI) in all treatment concentrations in both test systems. Using of Conan 5FL should be reconsidered due to its possible cytotoxic, clastogenic and mutagenic effects. The text was submitted by the authors in English.     

Effects of human leukocyte conditioned medium on mouse haemopoietic progenitor cells.

Medium conditioned by human peripheral blood leukocytes (HLCM) was studied for its in vitro effects on haemopoietic progenitor cells (CFU-s and CFU-c) present in mouse bone marrow. HLCM has poor colony stimulating activity in semi-solid cultures of mouse bone marrow cells, but invariably increases the number of colonies obtained in the presence of plateau levels of semi-purified colony stimulating factor (CSF). In liquid cultures, HLCM appears to contain a potent initiator of DNA synthesis in CFU-s, an activity which coincides with an increased CFU-s maintenance and causes a three- to four-fold increase in CFU-c number. It is apparent from this study that HLCM, in addition to stimulating colony formation in cultures of human bone marrow cells, has a profound in vitro effect on primitive haemopoietic progenitor cells of the mouse, which cannot be attributed to CSF.     

Bone tissue formation in organ cultures of human bone marrow

Bone formation in adult human bone marrow organ cultures is described. When culturing marrow fragments, thick bone lamina is formed. It has well-mineralized trabecular bone matrix with bone cells incorporated and is lined with osteoblast-like cells. In cultures of marrow deaggregated cell suspensions thin layers of the bone are only formed. Osteoclast-like cells develop in the cultures.     

Induction of DNA damage in mammalian cells by hydrogen peroxide generated by glucose oxidase immobilized in agarose slides

A new model system has been developed to study the influence of reactive oxygen species on isolated mammalian cells in conjunction with the comet assay. The glucose-glucose oxidase system was used as a hydrogen peroxide generating source. The level of DNA damage was assessed in the splenocytes and the cells of bone marrow of mouse and in human leukocytes both in untreated cells and in cells treated with hydrogen peroxide generated by glucose oxidase using the alkaline comet assay in vitro. Various options for the location of the enzyme in the slides have been studied: in the layer with the cells, in the layer above the cells, or in solution on the surface of the slides. The option where glucose oxidase was in the upper layer of 0.5% agarose over the layer of the cells was optimal. It provided separation of the enzyme from the cells and avoided obstruction to the hydrogen peroxide exposure. For the whole blood study, the content of endogenous glucose must be taken into account. This approach can be used to study the level of DNA damage induced in vitro and for the detection of DNA repair, thereby expanding the possibilities of the method, while the experiments are conducted under controlled conditions.     

Cytochemical reactions of human hematopoietic cells in liquid culture.

Growth of human bone marrow in liquid suspension cultures has been used to study normal hematopoietic cell differentiation and abnormalities in blood diseases. A variety of cytochemical stains were applied to human marrow cells cultured in vitro for up to 14 days. AS-D- CHLOROACETATE ESTERASE AND ALPHA-NAPHYHYL BUTYrate esterase were most useful in distinguishing different cell lines in culture. Peroxidase activity disappeared with mononuclear phagocyte morphogenesis and diminished with culture in intermediate and mature granulocytes. Acid phosphatase activity and methyl greed pyronin staining intensity increased with macrophage maturation.     

Chromosomal damage induced by maleic hydrazide in mammalian cells in vitro and in vivo

The induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Ch.Ab.) by the herbicide maleic hydrazide (MH) has been investigated in Chinese hamster ovary (CHO) cells grown in vitro and in bone marrow cells of mice treated in vivo. MH induces SCE and Ch.Ab. in CHO cells without metabolic activation; however, no induction of SCE was found in the in vivo experiments.     

Reverse chromosome painting for the identification of marker chromosomes and complex translocations in leukemia.

BACKGROUND: Chromosome banding techniques and in situ hybridization reveal the majority of chromosomal aberrations. However, difficulties remain in cases of highly contracted chromosomes, poor quality of the metaphases or the presence of markers with the involvement of several chromosomes. Here, it is demonstrated that reverse painting can be applied successfully starting with bone marrow cells from primary acute myelocytic leukemias (AML). METHODS: This was accomplished by culturing the leukemic cells with a cocktail of various growth factors, which yielded sufficient numbers of cells in cycle to harvest chromosomes for sorting. Aberrant chromosomes were flow-sorted and amplified by degenerate oligonucleotide-primed PCR. The resulting products were labeled by nick-translation and hybridized on normal metaphase spreads. RESULTS: Two patients with marker chromosomes in their leukemia cells were analyzed in detail. The hybridization pattern displayed the composition of the aberrant sorted chromosome. Results were compared with conventional cytogenetic analyses that were performed on material obtained from the same aspirate. The reverse-painting technique enabled identification of aberrations that were not detected by conventional cytogenetic analysis. CONCLUSIONS: Primary AML cells can be cultured in vitro, using optimal culture conditions, facilitating the production of high quality flow karyotypes, suitable for sorting of marker chromosomes to produce DOP-PCR derived chromosome painting probes for reverse painting. Valuable additional cytogenetic information can thus be obtained about complex chromosomal rearrangements or structural aberrations that could not be completely resolved by conventional cytogenetic analysis.     

Clastogenicity of carbazole in mouse bone marrow cells in vivo

The protective effect of Nigella sativa seed extract and its main constituents thymoquinone (TQ) was studied on mouse cells infected with schistosomiasis. Bone marrow cells in the in vivo experiments and spleen cells in the in vitro one were used to evaluate the potentially protective effect of these natural compounds on the induction of chromosomal aberrations. Karyotyping of the mice cells illustrated that the main abnormalities were gaps, fragments and deletions especially in chromosomes 2, 6 and some in chromosomes 13 and 14. Both N. sativa extract and TQ were considered as protective agents against the chromosomal aberrations induced as a result of schistosomiasis.     

Generation of human plaque-forming cells in culture: tissue distribution, antigenic and cellular requirements.

A system for the induction of specific, hemolytic plaque-forming cells from normal human lymphocytes in vitro (HcPFC) has been established and cells from various normal lymphoid tissues have been investigated. Normal values for anti-SRBC HcPFC responses in cultures of 107 Ficoll-Hypaque separated lymphocytes range from 2000 (bone marrow) to 7000 (spleen) and 15,000 (tonsillar and peripheral blood lymphocytes). HcPFC responses to ovalbumin were lower by factor of 2 to 4. Anti-SRBC as well as anti-ovalbumin responses required the cooperation of T lymphocytes and IgM-bearing B lymphocytes and the magnitude of the response was antigen dose dependent. Addition of adherent cells as well as of 2-mercaptoethanol enhanced the response. On the basis of the data obtained in experiments examining the role of B and T lymphocytes, a tentative model of cellular interaction has been postulated, suggesting a major role for antigen concentration in the modulation of the response via reactive T lymphocytes.     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

Bimolane induces multiple types of chromosomal aberrations in human lymphocytes in vitro

Bimolane has been commonly used in China for the treatment of psoriasis and various types of cancer. Patients treated with bimolane have been reported to have an increased risk of developing therapy-related leukemias. Although bimolane has been identified as a human leukemia-inducing agent, little is known about its genotoxic effects, and a systematic study of the types of chromosomal alterations induced by this compound has not been performed. In this study, a combination of immunochemical, molecular and conventional cytogenetic techniques has been used to study the chromosomal alterations induced by bimolane in cultured human lymphocytes. Immunochemical staining with the CREST antibody indicated that bimolane induces micronuclei (MN) originating primarily from chromosome breakage. Interestingly fluorescence in situ hybridization (FISH) with differentially labeled chromosomes 1 and 9 centromeric probes indicated that bimolane also caused non-disjunction and polyploidy. Consistent with this, an expedited analysis of Giemsa-stained metaphase chromosomes in bimolane-treated lymphocytes revealed a high frequency of polyploidy/hyperdiploidy as well as dicentric chromosomes, and premature centromeric division (PCD). In addition, bimolane was also found to produce binucleated cells, possibly through an interference with normal functioning of intermediate filaments. As a follow-up to these studies, three different types of commercially available bimolane formulations obtained from different Chinese manufacturers were also evaluated. The effects seen with the formulated bimolane were similar to those seen with the synthesized compound. Our studies indicate that bimolane effectively induces a variety of cellular and chromosomal changes in cultured lymphocytes and that similar alterations occurring in bone marrow stem cells could contribute to the development of the secondary cancers seen in bimolane-treated patients.     

Survival of human bone marrow progenitor cells after freezing: Improved detection in the colony-formation assay

If cryopreserved suspensions of human bone marrow were stimulated by human placental conditioned medium in the same way as fresh unseparated marrows, less than 40% of granulopoietic progenitor cells (CFU_c) was identified. By adding α-thioglycerol (0.6 mM) to the culture medium, the concentration of detectable CFU_c in cryopreserved bone marrow was increased by a factor of 3.4, and the recovery of CFU_c after cryopreservation rose to 90%. The low recovery of CFU_c after freezing in the absence of α-thioglycerol is due to the destruction of accompanying cells. Noncolony-forming cells normally present in the fresh human marrow promote colony formation in cultures stimulated by placental conditioned medium. Their effect can be replaced by α-thioglycerol. It is concluded that, in order to detect all CFU_c independent of the cellular composition of the marrow suspension, this supplement is essential for CFU_c cultures stimulated by conditioned medium.     

Relative biological effectiveness and dose rate effect of tritiated water on chromosomes in human lymphocytes and bone marrow cells

Tritriated water (HTO) is a major toxic effluent from the nuclear power industry, that is released into the environment in large quantities. The low dose radiation effect and dose rate effect of HTO on human lymphocytes and bone marrow cells have not been well studied. The present study was therefore undertaken to investigate the HTO dose-response relationship for chromosomal aberrations in human lymphocytes and bone marrow cells at low in vitro radiation doses ranging from 0.1 to 1 Gy. Lymphocytes (G₀ stage) and bone marrow cells were incubated for 10–150 min with HTO at a dose rate of 2cGy/min (555 MBq/ml). The relative biological effectiveness (RBE) of HTO was calculated with respect to ₆₀Co γ-rays for the induction of dicentric and centric ring chromosomes at low radiation doses. The RBE value for HTO β-rays relative to ⁶⁰Co γ-rays was 2.7 for lymphocytes and 3.1 for chromatid aberrations in bone marrow cells. Lymphocytes were also chronically exposed to HTO for 6.7–80 h at dose rates of 0.5 cGy/min (138.5 MBq/ml) and 0.02 cGy/min (5.6 MBq/ml). There was a 71.5% decrease in the yield of dicentrics and centric rings at the dose rate of 0.02 cGy/min, indicating a clear dose rate effect of HTO. The RBE value for HTO relative to ¹³⁷Cs γ-rays was 2.0 at a dose rate of 0.02 cGy/min, suggesting that low HTO dose rates produce no increase of the RBE values and that the values may be constant between 2 and 3 within these dose rates. These results should prove useful in assessment of the health risk for humans exposed to low levels of HTO.     

Action of granulopoiesis-stimulating cytokines rhG-CSF, rhGM-CSF, and rmGM-CSF on murine haematopoietic progenitor cells for granulocytes and macrophages (GM-CFC)

The aim of this study was to provide new data to the knowledge of mechanisms by which recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) enhance the numbers of colonies growing from hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in the murine bone marrow. The in vitro technique for cultivating GM-CFC from normal bone marrow cells was used. For evaluation of stimulatory actions of the drugs studied, the factors themselves or sera of mice given these factors were added to the cultures. The factors or the sera were present in the cultures either as the only potentially stimulatory agents or acted jointly with a suboptimum concentration of recombinant murine interleukin-3 (rmIL-3). It was found that both rhG-CSF and rmGM-CSF stimulate the proliferation of GM-CFC by a combination of direct mechanisms (direct actions on the target cells) and indirect effects (effects mediated through the induction of other cytokines and/or growth factors in the murine organism). The rhGM-CSF exhibited somewhat weaker in vitro effects in comparison with the other two factors and only indirect effects were noted. Additional in vivo experiments documented that, in spite of differences in mechanisms of action of the individual drugs studied on murine bone marrow cells in vitro, equal in vivo doses of the factors induce quantitatively similar effects on the production of GM-CFC in vivo.     

Induction of chromosomal damage in mammalian cells in vitro and in vivo by sulfapyridine or 5-aminosalicylic acid.

Sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA) are the two primary metabolites of the anti-inflammatory drug salicylazosulfapyridine (SASP). These two metabolites were studied for induction of chromosomal damage in mammalian cells, in vitro and in vivo, in an attempt to understand better the genetic effects produced by SASP in humans and laboratory mice. To this end, SP and 5-ASA were tested for induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Abs) in Chinese hamster ovary (CHO) cells in vitro. In addition, they were tested in vivo for induction of micronuclei (MN) in mouse bone marrow polychromatic erythrocytes (PCE). SP gave positive results in the in vitro SCE test and the in vivo MN test, and negative results in the in vitro Abs test. 5-ASA was negative in all three tests. These results indicate that it is the SP metabolite of SASP that is necessary for the induction of chromosomal damage reported to occur in humans and mice after treatment with SASP.