Kinetics of colony formation by BFU-E grown under different culture conditions in vitro

Colony formation by erythroid burst-forming units (BFU-E) involves a variable number of cell divisions before individual 'subcolonies' begin to appear. Consequently the numbers of subcolonies vary amongst individual bursts. If this observation is interpreted as a reflection of a stochastic process, the number of subcolonies in each individual burst represents the number of divisions by the BFU-E prior to commitment to terminal differentiation. This provides a means for quantitating the probability of erythroid differentiation (pD) and the probability of renewal (1 − pD). In order to determine whether these kinetics of burst formation can be influenced by exogenous factors we used three commercially available media designed for the growth of BFU-E. We found that subcolony numbers per burst ranged from one to 64 and that the cumulative distributions of subcolonies per burst followed a logarithmic curve ( r   > 0.90). Differences were observed in the distribution of subcolonies per burst when BFU-E were grown in different media (  P  = 0.03; Kruskall-Wallis test). The probability of immediate terminal differentiation (i.e. committment to form a subcolony) was 0.25 for two of the media and 0.7 for the third. The corresponding renewal probabilities were 0.75 and 0.3. These data indicate that the proliferation kinetics of BFU-E are susceptible to regulation by exogenous factors.     

Karyotypic variations in human meningioma cell cultures under different in vitro conditions

Summary Examined were how changes of the culture medium, cultivating procedure and cultivating time will influence numerical representation of different karyotypes in a total of 27 cell cultures of meningiomas with two or more cytogenetically distinguishable cell lines.It could be shown that in medium I (80% Mc Coy 5A, 20% fetal calf serum) more cell lines with a higher degree of hypodipoidy occurred than in medium II (50% TC 199, 40% bovine amniotic fluid, 10% calf serum). The number of cells with normal karyotype was higher in cultures which were grown from trypsinized biopsy meterial in stationary flasks when compared to particle cultures in roller tubes.Cells with a normal karyotype also increased after 1–6 subcultures. By demonstration of SV 40 tumor antigen it could be shown in two cultures that these normal mitoses derived from tumoral and not from stromal tissue.This investigation was supported by the Deutsche Forschungsgemeinschaft (Za 32/16)Parts of this study are included in the thesis (M. D.) of B. L.     

Characterization of steroid hormone sensitivity in human breast cancers maintained ex vivo under organotypical culture conditions

 Purpose: The methodology we propose combines the immunohistochemical determination of the oestrogen and progesterone receptors (ER and PgR) with the characterization of the oestradiol- and progesterone-induced influence on cell proliferation in breast cancers in order to characterize their steroid hormone sensitivity at both the “static” and “dynamic” level. Methods: ER and PgR have been immunohistochemically quantified by means of computer-assisted microscopy. Cell proliferation has been determined by means of tritiated thymidine autoradiography in tumour samples maintained in vitro as organotypic cultures. A series of 14 patients was investigated. Results: Of the 14 breast cancers under study, one with an unequivocally “very ER-rich”/“very PgR-rich” immunohistochemical phenotype totally failed to exhibit any modification in its cell proliferation level after both oestradiol and progesterone stimulation. Two cases definitively associated with an “ER-poor”/“PgR-poor” immunohistochemical phenotype nevertheless responded noticeably to the dynamic stimulation of their cell proliferation by oestradiol and progesterone. While our series of cases covers 14 patients only, it suffices to demonstrate the limits of ER and PgR determination in characterizing steroid hormone sensitivity in breast cancer. Discussion: The present work therefore presents an in vitro approach to test growth regulation of human breast cancer by steroid hormones. The clinical value of the present approach should be further determined by showing that steroid hormone-induced modifications in cell proliferation level are actually associated with clinical response. Received: 11 June 1999 / Accepted: 30 November 1999     

Isolation and development in cell culture of myocardial cells of the adult rat

Ventricular myocardial cells isolated, by simultaneous enzymatic and mechanical treatment, from adult rats, were established in long term primary culture. The development of individual cells in vitro was studied using a microscope equipped with a programmable numerically controlled stage and an automatic photomicrographic system. The system was used to repeatedly locate and photograph cells in culture. The cells' characteristic sequence of morphological development is described. Measurements of nuclear number, cell sizc, occurrence of spontaneous contractility, in vitro survivability, and mitotic activity are reported and compared to other similar measurements in vivo and in vitro. Results of a preliminary study of ultrastructure of the cultured cardiomyocytes are also presented.     

Some properties of Bomirski Ab amelanotic melanoma cells,which underwent spontaneous melanization in primary cell culture

Summary Four types of the Bomirski Ab amelanotic melanoma primary cell culture, differing in the presence of calf serum in the medium and in the cell number used for starting the culture, were employed in the study. In all types of cell culture, rapid melanization occurred in the cytoplasm of the cultured cells. Calf serum in the culture medium stimulated both melanization and proliferation of the Ab melanoma cells. The process of melanin synthesis occurred during the logarithmic phase of growth and was over when the cells reached the plateau phase. Heavily melanized cells changed their adhesive properties, lost the ability to divide in vitro, and showed decreasing tumorigenicity down to complete absence, though they retained some parameters of viability. The rate of melanin synthesis was lower in the cells cultured at high cell density than in those at low cell density. Highly melanized cells that did not divide in vitro but were still tumorigenic in vivo caused the growth of tumors those morphology was typical for the amelanotic melanoma, melanin being absent. In conclusion, it may be stated that the present findings suggest the persistence of a highly anaplastic and malignant phenotype of Bomirski amelanotic melanoma, being a result of the regulatory action of the host, while the change in the phenotype in vitro does not rule out autoregulatory influences of the tumor itself on its differentiation level and malignancy.     

TG、VLDL和OX-VLDL对L-02和HLF细胞增殖及合成细胞外基质的影响

目的探讨脂质对肝细胞和成纤维细胞生物学活性的影响。方法以ＭＴＴ法、透明质酸（ＨＡ）放免测定法及３Ｈ－脯氨酸掺入法，观察４种不同浓度的甘油三酯（ＴＧ）、极低密度脂蛋白（ＶＬＤＬ）和氧化极低密度脂蛋白（ＯＸ－ＶＬＤＬ）对无血清培养正常成人肝细胞（Ｌ－０２细胞）和人胚肺成纤维细胞（ＨＬＦ细胞）增殖及合成ＨＡ和胶原蛋白的影响。结果一定浓度的ＴＧ、ＶＬＤＬ和ＯＸ－ＶＬＤＬ可影响ＨＬＦ和（或）Ｌ－０２细胞的增殖，促进其胶原蛋白和ＨＡ的合成，其中以ＯＸ－ＶＬＤＬ的作用最为显著。结论肝内脂质，特别是ＯＸ－ＶＬＤＬ过多，可通过促进肝细胞和成纤维细胞合成细胞外基质诱导肝纤维化形成。     

Adherence and invasion of mouse-adapted H pylori in different epithelial cell lines

AIM： To assess the adhesion and invasion abilities of different mouse adapted H py/or/strains in different cell lines in vitro and investigate their effects on the virulence factors cagA and vacA. METHODS： The adherence and invasion abilities of different N pylori strains in different epithelial cell lines were examined by the gentamycin protection assay. The null mutants of cagA and vacA were processed by direct PCR mutation method. The morphologic changes of different cell lines after N pylori attachment were examined by microscopy.
RESULTS： The densities of adherence to and invasion into cells in vitro were different from those in the mouse infection experiments. 88-3887 strain could invade and adhere to cells stronger than SS1 and X47. All tested strains had better adhering and invasive abilities in SCG-7901 cell. CagA and vacA minus mutants had the same invasion and adherent abilities as their wild types. In all strains and cell lines tested, only AGS cell had the significant hummingbird phenotype after inoculation with the 88-3887 wild-type.
CONCLUSION： Both the host cells and the bacteria play important parts in the invasion and adhesion abilities of Hpylori. CagA and VacA are not related to the ability of invasion and adhesion of Hpylori in different cell lines in vitro.     

实时荧光RT-PCR与细胞培养法在流感病毒检测中的应用

目的探讨实时荧光逆转录聚合酶链式反应(rRT-PCR)在流感病毒检测中的应用,并比较与常规细胞培养方法的差异,以确定两种检测方法各自更适合的应用领域。方法收集烟台市两所国家级哨点医院就诊的流感样病例(ILI)咽拭子标本,应用实时荧光RT-PCR检测流感病毒核酸,阳性标本用狗肾传代细胞(MDCK)培养、分离流感病毒,比较两种检测方法的灵敏度及相关性差异。结果在617份标本中,实时荧光RT-PCR检测阳性152份,阳性率为24.64%;MDCK细胞培养法分离流感病毒79株,阳性率为12.80%。两者的阳性率差异有统计学意义(χ2=28.38,P<0.01)。结论实时荧光RT-PCR和细胞培养方法检测流感病毒具有一致的特异性。实时荧光RT-PCR可作为一种快速有效的流感病毒核酸检测法,适用于公共卫生应急疫情的实验室快速诊断;细胞培养法作为流感病原学监测的基础,用于核实暴发疫情的实验室诊断,对病毒进行抗原性和基因特性分析。     

Comparison of human lung cancer/SCID mouse tumor xenografts and cell culture growth with patient clinical outcomes

Leukemic cell growth in SCID mice has been reported as a predictor of disease relapse. However, there is a paucity of literature regarding xenograft growth and clinical outcomes in non-small cell lung cancer (NSCLC). Seventy-nine specimens from patients with NSCLC were either subcutaneously implanted into SCID mice and/or placed in tissue culture. Retrospective chart review was correlated with stage, histology, necrosis, disease-free interval, and survival. Tumor xenografts were successfully established with 17 of 37 (46%) tumor biopsy tissues. Thirteen of 59 (22%) specimens grew in cell culture. Patients whose tumors grew in SCID mice had no difference in survival compared to those with no growth (n=20, p=0.42). Median survival was 36 months in 13 patients whose tumors grew in cell culture compared to 39 months in 46 patients without growth. Eight of 12 (67%) patients with metastasis showed SCID/human xenograft growth, whereas nine of 25 (36%) without metastases did so (p=0.08). Growth of tumor cells in vitro occurred in 11 of 31 (35%) adenocarcinomas, one of 25 (4%) squamous cell carcinomas, and one of three (33%) large cell carcinomas (p=0.02). Well or moderately differentiated tumors grew in cell culture in only two of 22 (9%), whereas poorly or undifferentiated tumors grew in 11 of 32 (34%) cases (p=0.03). We conclude that neither the ability of a tumor to engraft and grow in SCID mice nor its ability to grow in vitro in cell culture is a reliable predictor of disease outcome or survival in patients with NSCLC. The ability to propagate tumors in vitro appears to be more dependent upon the histological type of tumor and its degree of differentiation.     

不同程度、频率、时限的间歇低氧细胞模型

目的阐述建立不同间歇低氧程度、频率、以及低氧时限细胞模型的过程,并论述这一模型的科学性。方法由计算机程序控制单片机及电磁阀,继而控制预混气源系统气流的通/断,在细胞培养舱中模拟不同程度、频率、以及时限的间歇低氧/再氧合暴露条件。结果培养舱气体相的暴露水平就是预设的暴露条件,而在一般间歇低氧/再氧合循环时,ECV 304细胞实际PO2和PCO2暴露条件为(76.28±1.2930)mmHg~(54.94±1.0502)mmHg和(38.26±1.4943)mmHg~(88.64±1.5027)mmHg。结论本模型基本上可以模拟出阻塞性睡眠呼吸暂停的间歇低氧以及其不同间歇低氧程度、频率、时限的细胞暴露水平,在一定程度上可以满足实验的需要。     

Magnetic Resonance Imaging Assessment of Excess Iron in Thalassemia,Sickle Cell Disease and Other Iron Overload Diseases

Patients with transfusion-dependent anemia develop cardiac and endocrine toxicity from iron overload. Classically, serum ferritin and liver biopsy have been used to monitor patient response to chelation therapy. Recently, magnetic resonance imaging (MRI) has proven effective in detecting and quantifying iron in the heart and liver. Tissue iron is paramagnetic and increases the MRI relaxation rates R2 and R2* in a quantifiable manner. This review outlines the principles and validation of non invasive iron estimation by MRI, as well as discussing some of the technical considerations necessary for accurate measurements. Specifically, the use of R2 or R2* methods, choice of echo times, appropriate model for data fitting, the use of a pixel-wise or region-based measurement, and the choice of field strength are discussed.     

Efficacy and safety of granulocyte,monocyte/macrophage adsorptive in pediatric ulcerative colitis

AIM: To investigate efficacy and safety for granulocyte, monocyte apheresis in a population of pediatric patients with ulcerative colitis.METHODS: The ADAPT study was a prospective, openlabel, multicenter study in pediatric patients with moderate, active ulcerative colitis with pediatric ulcerative colitis activity index(PUCAI) of 35-64. Patients received one weekly apheresis with Adacolumn~ granulocyte, monocyte/macrophage adsorptive(GMA) apheresis over 5 consecutive weeks, optionally followed by up to 3 additional apheresis treatments over 3 consecutive weeks. The primary endpoint was the change in mean PUCAI between baseline and week 12; the secondary endpoint was improvement in PUCAI categorized as(Significant Improvement, PUCAI decrease of ≥ 35), Moderate Improvement(PUCAI decrease of 20 35), Small Improvement(PUCAI decrease of 10 20) or No change(PUCAI decrease of 10). RESULTS: Twenty-five patients(mean age 13.5 years; mean weight 47.7 kg) were enrolled. In the intention-to-treat set(ITT), the mean value for PUCAI improvement was 22.3 [95%CI: 12.9-31.6; n = 21]. In the per-protocol(PP) set, the mean improvement was 36.3 [95%CI: 31.4-41.1; n = 8]. Significant Improvement was recorded for 9 out of 20 patients(45%); 5 out of 20 patients(25%) had Moderate Improvement and one patient(5%) had No Change in PUCAI score at week 12. In the PP set, six out of eight patients(75%) showed Significant Improvement; and in two out of eight patients(25%) Moderate Improvement was recorded. The endoscopic activity index(EAI) decreased by 3 points on average. Seven(7) out of 21(33%) patients in ITT and 4 out of 8(50%) patients in PP have used steroids during the clinical investigation. The mean steroid dosage for these patients in the ITT set decreased from a mean 12.4 mg to 10 mg daily on average from Baseline to week 12.CONCLUSION: Adacolumn~; GMA apheresis treatment was effective in pediatric patients with moderate active Ulcerative Colitis. No new safety signals were reported. The present data contribute to considering GMA apheresis as a therapeutic option in pediatric patients having failed first line therapy.     

Effect of culture conditions on PLP and MAG gene expression in rat glioma C6 cells

The effects of culture conditions on the expression of myelin-specific genes, i.e. proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) in rat glioma C6 cells was studied. Early passage (40–46) cells had higher steady-state level of PLP- and MAG-specific mRNA than late (100) passage cells when grown in defined (serum-free) medium. The PLP gene expression was increased whereas the MAG gene expression was reduced in the presence of 10% fetal calf serum in either passage. The level of both PLP-and MAG-specific messages was also directly related to the cell density indicating cell contact-induced stimulation of the gene expression. Furthermore, the cells apparently secrete factors into the medium, which upregulate the gene expression in autocrine fashion. The results also indicate a dissimilarity of regulatory mechanisms involved in the expression of the PLP and MAG genes.     

Morphometric analysis of intestinal mucins under different dietary conditions and gut flora in rats

Elucidation of the mechanisms that alter the biosynthesis, turnover, and degradation of intestinal mucins is relevant to the understanding of both the normal gut ecosystem and various intestinal diseases. In this study image analysis was used to quantify the effects of diet and microbial flora on the mucin composition of goblet and deep crypt cells, the number and volume density of mucin-containing cells, and the staining density of their stored mucins in the small and large intestine of germ-free and conventionally maintained rats fed two different diets. One was a coarsely ground commercial rodent diet containing crude fiber of cereal origin and the other a purified diet composed of finely powdered ingredients, including cellulose as a source of fiber. The changes in mucin production were also analyzed in germ-free rats colonized with a human flora. Feeding a commercial diet reduced the volume density of cells containing neutral and sulfomucins in the jejunum of conventional rats and the staining density of neutral and acidic mucins in the germ-free rats. Both rat and human floras reduced the number of cells containing acidic and sulfomucins and the staining density of neutral mucins in the small intestine of animals fed on a purified diet. However, inoculation of human flora increased the staining density of stored neutral and sulfated mucins in the cells of the large intestine. The results demonstrate that the dietary changes are influential in modifying the amount and proportion of mucins in the small intestine and the microbial flora in the large intestine.This work was partly sponsored by the EEC-FLAIR Concerted Action Programme No 9.     

Methods in cardiomyocyte isolation, culture, and gene transfer

Since techniques for cardiomyocyte isolation were first developed 35 years ago, experiments on single myocytes have yielded great insight into their cellular and sub-cellular physiology. These studies have employed a broad range of techniques including electrophysiology, calcium imaging, cell mechanics, immunohistochemistry and protein biochemistry. More recently, techniques for cardiomyocyte culture have gained additional importance with the advent of gene transfer technology. While such studies require a high quality cardiomyocyte population, successful cell isolation and maintenance during culture remain challenging. In this review, we describe methods for the isolation of adult and neonatal ventricular myocytes from rat and mouse heart. This discussion outlines general principles for the beginner, but also provides detailed specific protocols and advice for common caveats. We additionally review methods for short-term myocyte culture, with particular attention given to the importance of substrate and media selection, and describe time-dependent alterations in myocyte physiology that should be anticipated. Gene transfer techniques for neonatal and adult cardiomyocytes are also reviewed, including methods for transfection (liposome, electroporation) and viral-based gene delivery.     

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum

Purpose Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines.Methods The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS).Results Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1.Conclusions Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines, but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation, cytokine release); and (iii) whether coculture experiments are included.     

Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macrophage.

Glucose-6-phosphate dehydrogenase (G-6-PD) isoenzymes types of granulocytes were determined in eight women with chronic myelocytic leukemia (CML). The patients were heterozygous at the X-linked G-6-PD locus for the common gene, GdB, and a variant, such as GdA, so that both B and A enzyme types were found in skin cells. In contrast to these normal cells, only one G-6-PD type was found in CML granulocytes. The fact that such single-enzyme phenotypes are found in CML granulocytes, but not in nonleukemic granulocytes, provides strong evidence that the disease has a clonal origin. Single-enzyme phenotypes were also found in erythrocytes, platelets and cultured blood macrophages indicating that these cells have a common stem cell which is the site of the abnormality in CML.In the one studied patient, no evidence was found for involvement of cultured marrow fibroblasts. Clonal origin of CML virtually excludes cell recruitment as a sole pathogenetic mechanism. Either the leukemia arises as a consequence of a rare initial event in a single cell, or a series of events occurs in a clone such that it evolves into CML, or both.     

Iron Mobilization From Transferrin And Non-Transferrin-Bound-Iron by Deferiprone. Implications in the Treatment of Thalassemia,Anemia of Chronic Disease,Cancer and Other Conditions

Iron mobilization from transferrin is one of the most important screening methods for the selection of chelators intended for clinical use in the treatment of iron overload in thalassemia and other conditions. In vitro and in vivo screening of approved and experimental chelating drugs has shown that only the α-ketohydroxypyridines deferiprone (L1) and 1-allyl-2 methyl-3-hydroxypyrid-4-one (L1NAll), are effective in the mobilization of iron from transferrin. Iron mobilization from transferrin and non-transferrin-bound-iron (NTBI) can be used to optimize existing chelation therapy protocols for the treatment of iron loaded patients. New chelation strategies involving L1 and its combination with deferoxamine (DFO) and other chelators can be used to increase iron excretion and reduce or prevent excess iron deposition in the heart and other vital organs of iron loaded patients by comparison to monotherapies. Deferiprone and its combinations may also have potential applications in the treatment of cancer, the anemia of chronic disease and other conditions.     

OLFM4GW112/hGC-1、GRIM19及PIN1基因在12种人肿瘤细胞株中的表达分析

目的检测OLFM4^GW112/hGC-1、GRIM19及PIN1基因在12种人肿瘤细胞株中的表达,并探讨与肿瘤细胞凋亡与周期的关系。方法利用RT-PCR半定量的方法检测GW112、GRIM19与PIN1三种基因在人12种肿瘤细胞株中转录水平。结果RT-PCR结果显示：12种人肿瘤细胞株中GW112、GRIM19与PIN1三种基因均有表达,未出现转录缺失。其中胃癌细胞SGC-7901与脑胶质瘤细胞CHG-5中的GW112表达明显强于GRIM19,而在其余十种细胞株中GW112表达却明显低于GRIM19。实验组细胞中,PIN1基因只在SGC-7901株与CHG-5株中表达较弱,其余表达均较强。结论GW112、GRIM19与PIN1在这些肿瘤细胞中的表达状况很可能与它们的周期调控和细胞凋亡有关,对三种基因的表达研究可进一步对分析三种基因的功能提供依据。     

Clonal growth and self-renewal of human myeloid leukemia cell lines (BRM and DD) in serum-free semi-solid culture

Summary Primary and secondary colony formation of two new human myeloid leukemia cell lines (BRM and DD) were studied in serum-free semisolid cultures. The results indicate that bovine serum albumin and transferrin were essential for clonal growth in chemically defined medium. Insulin contributed only moderately beneficial effects. Initial cell density was also a major modulator of plating efficiency. Positive cooperation between the leukemia cells was shown by using autologous conditioned media. This is the first serum-free culture method that allows self-renewal of human myeloid leukemia cell lines in terms of secondary colony formation in methylcellulose cultures.