Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

人结肠癌CW-2干细胞获取方法研究

目的运用3种不同的方法分离纯化人结肠癌CW-2干细胞,并对其分离纯化效率进行比较,探讨获得癌干细胞的有效方法。方法采用单纯无血清悬浮培养、无血清悬浮培养联合化疗药物、流式细胞分选技术分别富集人结肠癌细胞株CW-2干细胞;然后运用流式细胞术、NOD—SCID小鼠致瘤实验和Transwell侵袭实验分析比较3种方法的富集效率。结果无血清悬浮培养细胞．无血清悬浮培养联合化疗药物处理细胞和流式细胞仪分选技术分选后细胞中具有结肠癌干细胞特性的CD44＋EPCAM_细胞分别为（59．39±4．55）％、（74.36±6．78）％、（86．43±8．43）％;3群细胞的成瘤能力和侵袭能力都存在显著统计学差异（P值〈0．05）：流式细胞分选技术分选后细胞〉无血清悬浮培养联合化疗药物处理细胞〉单纯无血清悬浮培养细胞。结论流式细胞分选技术富集癌干细胞的能力强于单纯无血清悬浮培养和无血清悬浮培养联合化疗药物,无血清悬浮培养联合化疗药物又强于单纯无血清悬浮培养。     

Demonstration of neuronal activities in primary cultures from fetal mouse hypothalamus maintained in a serum-free medium

Fetal neuronal cells dissociated from mouse hypothalamus are able to grow in a serum-free medium for two to four weeks. Several neuronal activities have been measured during the in vitro cell development. Whereas glutamic acid decarboxylase (EC 4.1.1.15) activity disappears, those of thyroliberin (TRH) and tyrosine hydroxylase (EC. 1.14.16.2) remain stable, and acetylcholine transferase one (EC 2.3.1.6) rises earlier than in a serum supplemented medium. These results show that primary cultures grown in serum-free medium offer a promising model for studying neuronal activity differentiation.     

Calcifying bacteria and carbonic anhydrase]

Carbonic anhydrase activity was measured on bacteria known for their calcifying power. The results obtained allow us to conclude that carbonic anhydrase is not present in their enzymatic equipment. Nevertheless, the addition of carbonic anhydrase in culture media increases the growth of the cultures and their calcifying power. This result is due to the direct action of the enzyme on the pH of the culture medium. The results obtained could be applied to the mechanism of dental plaque calcification.     

Nicotinamide desamidase in the culture medium of neuroblastoma cells]

Important amounts of nicotinic acid appear in the growth medium of neuroblastoma cell cultures. It is shown that an enzyme released by the cells into the growth medium deamidates the nicotinamide supplied by the growth medium to nicotinic acid.     

Role of serum components in the binding and phagocytosis of oxidatively damaged erythrocytes by autologous mouse macrophages

To investigate the role of autologous serum components in the recognition of damaged cells by macrophages, we examined the binding and phagocytosis of damage oxidatively damaged red blood cells with Cu2+ and ascorbate (oxRBCs) by autologous resident mouse peritoneal macrophages. The binding of oxRBCs by macrophages was independent of the presence of serum. However, phagocytosis by macrophages increased with serum concentration, and macrophages showed little ingestion of oxRBCs in a serum-free medium. Macrophages neither bound nor appreciably ingested native RBCs (before oxidation) in either the absence or presence of autologous serum. Mouse macrophages ingested significantly more native as well as oxRBCs in the presence of heat-inactivated fetal calf serum than in the presence of heat-inactivated mouse serum. Pretreated oxRBCs with normal serum were rarely ingested by macrophages in a serum-free medium. Phagocytosis of oxRBCs was significantly inhibited by depletion of IgG or calcium from serum, by heat inactivation of complement, or by antiserum against mouse C3. These results demonstrate that serum components such as IgG, C3, and calcium are involved in phagocytosis of oxRBCs by autologous macrophages.     

Effet de divers bois sur certains micro-organismesin vitro

Summary Mycobacterium tuberculosis was brought into contact with stoved beech-tree wood in physiological saline solution. After a contact of 6, 12, and 24 h, respectively, between the beech-wood and the suspension of microorganisms in saline, this latter was sowed on Loewenstein culture medium. The suspensions which were in contact with the beech-wood for 6 h, gave cultures not different from the controls without wood. With the 12 h ones, the growth of mycobacterium tuberculosis was greatly delayed, whereas with the 24 h ones the cultures remained sterile.     

Use of aggregating cell cultures for toxicological studies

Summary Relatively simple techniques are now available which allow the preparation of large quantities of highly reproducible aggregate cultures from fetal rat brain or liver cells, and to grow them in a chemically defined medium. Since these cultures exhibit extensive histotypic cellular reorganization and maturation, they offer unique possibilities for developmental studies. Therefore, the purpose of the present study was to investigate the usefulness of these cultures in developmental toxicology. Aggregating brain cell cultures were exposed at different developmental stages to model drugs (i.e., antimitotic, neurotoxic, and teratogenic agents) and assayed for their responsiveness by measuring a set of biochemical parameters (i.e., total protein and DNA content, cell type-specific enzyme activities) which permit a monitoring of cellular growth and maturation. It was found that each test compound elicited a distinct, dose-dependent response pattern, which may ultimately serve to screen and classify toxic drugs by using mechanistic criteria. In addition, it could be shown that aggregating liver cell cultures are capable of toxic drug activation, and that they can be used in co-culture with brain cell aggregates, providing a potential model for complementary toxicological and metabolic studies.     

Use of aggregating cell cultures for toxicological studies

Relatively simple techniques are now available which allow the preparation of large quantities of highly reproducible aggregate cultures from fetal rat brain or liver cells, and to grow them in a chemically defined medium. Since these cultures exhibit extensive histotypic cellular reorganization and maturation, they offer unique possibilities for developmental studies. Therefore, the purpose of the present study was to investigate the usefulness of these cultures in developmental toxicology. Aggregating brain cell cultures were exposed at different developmental stages to model drugs (i.e., antimitotic, neurotoxic, and teratogenic agents) and assayed for their responsiveness by measuring a set of biochemical parameters (i.e., total protein and DNA content, cell type-specific enzyme activities) which permit a monitoring of cellular growth and maturation. It was found that each test compound elicited a distinct, dose-dependent response pattern, which may ultimately serve to screen and classify toxic drugs by using mechanistic criteria. In addition, it could be shown that aggregating liver cell cultures are capable of toxic drug activation, and that they can be used in co-culture with brain cell aggregates, providing a potential model for complementary toxicological and metabolic studies.     

Effects of TSH and cyclic AMP on the human thyroid cells cultured in a chemically defined medium

Summary In a serum-free, chemically defined medium human thyroid cells elongated remarkably and resembled fibroblastic cells. They retained the cyclic AMP response to TSH and the supplement of medium with TSH or dibutyryl cyclic AMP permitted the preservation of epithelial nature by the cells. Cyclic AMP of the cells of epithelial nature was higher than those of fibroblastic appearance.     

Effects of TSH and cyclic AMP on the human thyroid cells cultured in a chemically defined medium.

In a serum-free, chemically defined medium human thyroid cells elongated remarkably and resembled fibroblastic cells. They retained the cyclic AMP response to TSH and the supplement of medium with TSH or dibutyryl cyclic AMP permitted the preservation of epithelial nature by the cells. Cyclic AMP of the cells of epithelial nature was higher than those of fibroblastic appearance.     

结肠腺癌中结肠癌干细胞的分离、鉴定

目的从人结肠腺癌组织中分离、鉴定结肠癌干细胞,并初步观察其生物学特性。方法利用新鲜结肠腺癌组织,无血清悬浮成球培养,流式细胞检测ESA、CD44表达情况,体外观察其诱导分化及CK20、Muc表达情况,Balb／C小鼠移植观察其成瘤情况。结果从人原代结肠腺癌中分离、纯化EpCAM^high CD44^＋结肠癌干细胞,结肠癌原代细胞中EpCAM^highCD44^＋细胞比例为1．7％～38％（平均5．4％）。单克隆形成实验证实结肠癌组织中存在肿瘤干细胞。其比例为（2．07±0．11）％,分离获得的EpCAM^highCD44^＋细胞能在无血清培养基中“成球”,在血清诱导下能贴壁分化;将EpCAM^highCD44^＋细胞移植在Balb／C裸鼠体内,表现出很强的致瘤性,移植瘤中EpCAM^highCD44^＋细胞比例为3．6％～43．2％（平均15．2％）,所有的移植瘤经组织学测定,均形成腺管样结构,表达结肠特异性分化标志物CK-20、中性上皮粘蛋白（neutral epithelial mucins,Muc）。结论人结肠腺癌组织中存在EpCAM^highCD44^＋细胞群,具有和普通干细胞相类似的无限增殖、自我更新和分化能力。     

The mesenchymoangioblast,mesodermal precursor for mesenchymal and endothelial cells

Mesenchymoangioblast (MB) is the earliest precursor for endothelial and mesenchymal cells originating from APLNR⁺PDGFRα⁺KDR⁺ mesoderm in human pluripotent stem cell cultures. MBs are identified based on their capacity to form FGF2-dependent compact spheroid colonies in a serum-free semisolid medium. MBs colonies are composed of PDGFRβ⁺CD271⁺EMCN⁺DLK1⁺CD73^? primitive mesenchymal cells which are generated through endothelial/angioblastic intermediates (cores) formed during first 3–4 days of clonogenic cultures. MB-derived primitive mesenchymal cells have potential to differentiate into mesenchymal stromal/stem cells (MSCs), pericytes, and smooth muscle cells. In this review, we summarize the specification and developmental potential of MBs, emphasize features that distinguish MBs from other mesenchymal progenitors described in the literature and discuss the value of these findings for identifying molecular pathways leading to MSC and vasculogenic cell specification, and developing cellular therapies using MB-derived progeny.     

Growth and sporulation of Entomophthora virulenta Hall and Dunn in discontinuous liquid culture]

Fermented cultures of Entomophthora virulenta showed an exponential phase with a specific growth rate of 0,14 h-1. The deceleration phase was 4 times longer than the exponential phase. Sexual sporulation occurred at the end of the deceleration phase. A technique of medium replacement demonstrated that any lack of nutritive balance induced the sporulation.     

Primary tissue cultures from organ fragments: a simplified method.

Organ fragments washed in Ca++ and Mg++--free saline, treated with trypsin and placed directly into culture flasks adhered within seconds to the vessel surface. If the fragments were suspended in culture medium before they were added to the flasks, they did not adhere. This technique permits the rapid attachment and subsequent growth of the primary tissue cultures.     

Adenosine triphosphatase ofAspergillus nidulans: Stimulation by aminoacids

Summary Ca²⁺-dependent ATPase ofAspergillus nidulans was found to be stimulated by aminoacids in vitro. Both histidine and arginine stimulated the enzyme more effectively than the aromatic aminoacids. Supplementation of the growth medium with basic or aromatic aminoacids increased the enzyme activity in vivo 2–6-fold. The enhanced activity observed in these cultures in vivo was not mediated through the synthesis of new isoenzyme, as observed in proteinenriched cultures, but appeared to be through the activation of enzyme activity.     

Regression of genetically determined polycystic kidney disease in murine organ culture

Summary Cystic kidneys from the mutant CPK strain of C57BL/6J mice were cultured in serum-free organ culture. During 120 h of incubation in chemically-defined medium, CPK cystic tubular changes underwent complete regression. Environmental factors regulate the expression of genetically determined polycystic kidney disease in this model.     

Regression of genetically determined polycystic kidney disease in murine organ culture

Cystic kidneys from the mutant CPK strain of C57BL/6J mice were cultured in serum-free organ culture. During 120 h of incubation in chemically-defined medium, CPK cystic tubular changes underwent complete regression. Environmental factors regulate the expression of genetically determined polycystic kidney disease in this model.     

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: effect of nafenopin and electroporation

Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive beta-oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes. In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. 'Whorl formation' by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.