Early Phase Cultures Research Articles

Thrombopoietin stimulates ex vivo expansion of mature neutrophils in the early stages of differentiation

We examined the effects of thrombopoietin (TPO) in combination with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte colony-stimulating factor (G-CSF) on the proliferation and differentiation of human neutrophils. Purified CD34(+) hematopoietic progenitor cells were cultivated with SCF, IL-3, and G-CSF for 7 days (early phase), and thereafter nonadherent cells were further cultivated for 9 days with G-CSF alone (late phase). A large number of highly selected neutrophils (>95%) was obtained on day 16. We compared the expansion capacity in the presence or absence of TPO in each culture phase. The significantly larger number of neutrophils was obtained in the presence of TPO in the early culture phase. The number of expanded cells plateaued at day 16. Ultimately, a 550-fold increase in the number of neutrophils was achieved. These neutrophils gained the ability to respond effectively with chemotaxis and superoxide release, and were appropriately primed by G-CSF, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and IL-1beta for enhanced release of O(2)(-). The responsiveness of these cells was identical to that of peripheral blood neutrophils. However, TPO did not accelerate the maturation of neutrophils supported by G-CSF in the late phase of culture. Furthermore, priming effects and triggering effects of TPO on the production of superoxide metabolites from peripheral blood neutrophils were not observed. These results suggest that TPO regulates the proliferation and differentiation of neutrophils in the early stages, but not the late stages, of differentiation.

A chemically defined culture of VEGFR2+ cells derived from embryonic stem cells reveals the role of VEGFR1 in tuning the threshold for VEGF in developing endothelial cells

Vascular endothelial growth factor (VEGF) is a major growth factor for developing endothelial cells (ECs). Embryonic lethality due to haploinsufficiency of VEGF in the mouse highlighted the strict dose dependency of VEGF on embryonic vascular development. Here we investigated the dose-dependent effects of VEGF on the differentiation of ES cell-derived fetal liver kinase 1 (Flk-1)/VEGF receptor 2(+) (VEGFR2(+)) mesodermal cells into ECs on type IV collagen under a chemically defined serum-free condition. These cells could grow even in the absence of VEGF, but differentiated mostly into mural cells positive for alpha-smooth muscle actin. VEGF supported in a dose-dependent manner the differentiation into ECs defined by the expression of VE-cadherin, platelet-endothelial cell adhesion molecule 1 (PECAM-1)/ CD31, CD34, and TIE2/TEK. VEGF requirement was greater at late than at early phase of culture during EC development, whereas response of VEGFR2(+) cells to VEGF-E, which is a virus-derived ligand for VEGFR2 but not for Flt-1/VEGFR1, was not dose sensitive even at late phase of culture. Delayed expression of VEGFR1 correlated with increased dose dependency of VEGF. These results suggested that greater requirement of VEGF in the maintenance than induction of ECs was due to the activity of VEGFR1 sequestering VEGF from VEGFR2 signal. The chemically defined serum-free culture system described here provides a new tool for assessing different factors for the proliferation and differentiation of VEGFR2(+) mesodermal cells.

Suppressive Effect of Iron on Concanavalin A‐Induced Multinucleated Giant Cell Formation by Human Monocytes

Immune dysfunction in patients with iron overload has been reported. Iron disturbed CD2 expression on T‐cells, cell‐mediated immunity by Th1 cells and monocyte functions including phagocytosis and natural killer activity. In the present study, we examined the effects of iron and desferrioxamine (DFX, an iron chelator) on generation of multinucleated giant cells (MGC) by human monocytes in vitro. Human monocytes were isolated from venous blood and cultured with concanavalin A (Con A) stimulation with additives, ferric citrate (Fe‐citrate) or sodium citrate (Na‐citrate) or DFX for 4 days. The cells were fixed and subjected to Wright staining. MGC formation was observed under light microscopy. Con A induced MGC formation in a dose‐dependent manner, and reached a plateau after 3 days of incubation. MGC formation was suppressed when Con A‐stimulated monocytes were cultured with the co‐addition of Fe‐citrate but not Na‐citrate only in the early phase of culture (less than 24 hours). DFX also suppressed MGC formation in a dose‐dependent manner. Using flow cytometry analysis, the co‐addition of Fe‐citrate significantly suppressed CD18 (ß2 integrin) and CD54 (ICAM‐1) but not CD11a (α integrin) expression on Con A‐stimulated monocytes. Iron supressed the generation of MGC by human monocytes in vitro. These observations suggested that iron might affect MGC generation by down‐regulation of adhesion molecule expression on monocytes.

Regulation of YKL-40 production by human articular chondrocytes.

YKL-40 (human cartilage glycoprotein 39) is one of the most abundant proteins secreted by cultured chondrocytes. The objectives of the present study were to identify regulators of YKL-40 production in cartilage and chondrocytes and to map the localization of YKL-40 in chondrocytes. Human articular chondrocytes and cartilage explants (obtained from subjects at autopsy, from a tissue bank, and from osteoarthritis [OA] patients undergoing total joint replacement surgery) were stimulated with cytokines, growth factors, and other agents. YKL-40 expression was analyzed by Northern blot and polymerase chain reaction. YKL-40 secretion into the media was determined by enzyme-linked immunosorbent assay. YKL-40 production increased to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury. Spontaneous YKL-40 release was higher in OA than in normal cartilage explant cultures. In chondrocyte monolayer cultures, interleukin-1beta (IL-1beta) and transforming growth factor beta (TGFbeta) decreased the levels of secreted YKL-40, and this was associated with a reduction in YKL-40 messenger RNA levels. IL-1beta, but not TGFbeta, reduced YKL-40 production in cartilage explant cultures. Media from explants treated with cycloheximide had no detectable YKL-40, suggesting that the released protein was newly synthesized. Immunofluorescence microscopy showed YKL-40 staining in the Golgi system of the chondrocytes, but YKL-40 could not be detected in the extracellular matrix. The spontaneous increase in the production of YKL-40 in the early phase of culture appears to represent a cellular response to changes in the extracellular matrix environment. This, coupled with the profound suppressive effects of IL-1beta and TGFbeta on YKL-40 production, identifies a novel regulatory pattern for this major chondrocyte-derived protein.

Apoptosis, rather than oncosis, is the predominant mode of spontaneous death of isolated adult rat cardiac myocytes in culture.

A number of isolated adult cardiomyocytes dies within a few days of culture and the mode of death has recently been suggested to be apoptosis, based on its association with the appearance of DNA fragmentation. However, morphological evidence is still lacking and precise analysis, including quantification, has not been performed. Adult rat ventricular cardiomyocytes isolated by enzymatic dissociation were incubated for 7 days in a serum-free medium (the rapid attachment model) and after various incubation periods, both attached and floating cells were counted and classified based on combined criteria of morphology and membrane permeability (dye exclusion): type 1, rod cells with intact membranes; type 2, non-rod cells with intact membranes; and type 3, non-rod cells with ruptured membranes. The number of both rod-shaped and dye-excluding cells decreased with the incubation period. After 7 days culture, the number of residual cells decreased to 12% of the initial value. Electron microscopy identified type 1 cells as viable, type 2 cells as viable or apoptotic, and type 3 as undergoing oncosis (primary necrosis) or secondary post-apoptotic necrosis. Ultrastructural morphometry revealed that oncotic cell death occurred predominantly during the early phase of culture whereas the more abundant apoptotic cell death occurred throughout the culture period. In conclusion, although both apoptotic and oncotic death occur in the natural course of adult rat cardiomyocytes in short-term culture, apoptosis is more predominant. Because of the high incidence of spontaneous cell death predominantly via apoptosis, this information is important for the interpretation of studies using this cell type in culture.

Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells

CD14-positive monocytes obtained from human peripheral blood were cultured with GM-CSF and IL-4. During the early culture phase immature dendritic cells (DCs) developed which not only expressed CD1a, HLA-DR and CD86, but also expressed the endothelial cell markers von Willebrand factor (vWF), VE-cadherin and VEGF receptors Flt-1 and Flt-4. Further maturation of DCs was achieved by prolonged cultivation with TNFalpha. These cells showed typical DC morphology and like professional antigen-presenting cells (APCs) expressed CD83 and high levels of HLA-DR and CD86. However, if immature DCs were grown with VEGF, bFGF and IGF-1 on fibronectin/vitronectin-coated culture dishes, a marked change in morphology into caudated or oval cells occurred. In the presence of these angiogenic growth factors the cultured cells developed into endothelial-like cells (ELCs), characterized by increased expression of vWF, KDR and Flt-4 and a disappearance of CD1a and CD83. Addition of IL-4 and Oncostatin M also increased VE-cadherin expression, and the loosely adherent cells formed clusters, cobblestones and network-like structures. vWF- expressing ELCs mainly originated from CD1a-positive cells, and VEGF was responsible for the decrease in the expression of the DC markers CD1a and CD83. In mixed leukocyte cultures, mature DCs were more potent APCs than ELCs. Moreover, Ac-LDL uptake, and the formation of tubular structures on a plasma matrix was restricted to ELCs. These results suggest that in the presence of specific cytokines immature DCs have the potential to differentiate along different lineages, i.e. into a cell type resembling ELCs.

Growth Characteristics and Morphogenesis of the Liverwort Marchantia paleacea Suspension Cells.

Suspension cultured cells of the liverwort Marchantia paleacea var. diptera (MpK) were investigated to clarify the relationship between morphogenesis and the mode of utilization of nitrogen sources in different media. MpK cells grew vigorously in a Murashige and Skoog's liquid medium with 4% glucose in the light condition. Thallus regeneration from cultured cells occurred in culture medium with 0.5% glucose and 0.1% CaCO3 or 10mM malic acid. Addition of CaCO3 or malic acid to the medium was necessary to prevent the pH of the medium dropping rapidly to a very acidic value over the duration of the culture. The cause for the extreme acidification of the medium was that the cells regenerating thalli preferentially utilize ammonium as a nitrogen source without the depletion of nitrate in the early phase of culture.

Collagen gel immobilization: a useful cell culture technique for long-term metabolic studies on human hepatocytes.

1. Primary cultures of human hepatocytes have already been employed in various applications for the study of xenobiotic metabolism. Most of these approaches were performed either on freshly isolated cells or on short-term primary cultures. Standard culture techniques do not maintain functional stability of P450 enzymes for > 1 week in vitro. 2. The aim of this study was to demonstrate the beneficial effect of an easy to apply, extracellular matrix configuration on the long-term performance of cultured human liver cells. Light microscopical examination of the cultures indicated that the cells remained viable over 1 month. As revealed by electron microscopy, hepatocytes exhibited bile canaliculi and desmosomes and were rich in mitochondria and endoplasmatic reticulum, indicating metabolic activity. 3. An early culture phase (3 days after isolation) could be described with decreasing DNA content of the cultures, peak values of alanine-amino-transferase (ALAT), and increasing albumin synthesis. After this adaptive period stable levels for DNA content and albumin synthesis were noted; ALAT returned to low values. 4. Functional activity was monitored by measurements of P450 1A1-dependent O-demethylation of p-nitroanisole to p-nitrophenol, which appeared to be constant over 3 weeks and weakly inducible by 1 mM phenobarbital. Another set-up examined conjugation of acetaminophen at subtoxic concentrations: acetaminophen was metabolized to its glucuronide and sulphate; 3-(glutathione-S-yl)-acetaminophen was not detected. Almost identical metabolism was found, comparing day 3 with 16 of culture. 5. We concluded that collagen gel immobilization not only provides mechanical support to cultured hepatocytes, but also supports long-term differentiated function of the cells for metabolic studies.

Role of LFA-1/ICAM-1 in interleukin-2-stimulated lymphocyte proliferation.

Major adhesion routes between lymphoid cells involve the receptor/ligand pairs LFA-1/ICAM-1 and CD2/LFA-3, in addition to VLA or CD44 molecules. In this study we evaluated the role of these adhesion receptors in the proliferative response of lymphoid cells to interleukin-2 (IL-2). Blocking studies were performed with a panel of monoclonal antibodies (mAb) directed against these adhesion molecules. Selective inhibition of recombinant (r)IL-2-induced cell proliferation was observed with mAb directed against the alpha or beta subunit of LFA-1 or to its ligand ICAM-1. Interestingly, rIL-2-induced proliferation was also inhibited by NKI-L16, and anti-1 alpha antibody known to enhance cell-cell interaction. Resting lymphocytes were preferentially susceptible to the inhibition, particularly in an early phase of culture and when stimulated with a relatively low dose of rIL-2. By using mAb that specifically could block distinct rIL-2 activation pathways, LFA-1/ICAM-1 interaction was found to be required for p55 IL-2 receptor (IL-2R)-mediated interaction of rIL-2 with its high-affinity receptor, but not for p75 IL-2R-mediated responses. Furthermore, it was shown that the rIL-2 response of T lymphocytes, but not of natural killer cells, was dependent on LFA-1/ICAM-1 interaction. This suggests that LFA-1/ICAM-1 interaction is required for an optimal rIL-2 response of cells capable of IL-2 secretion. Our data provide evidence for the hypothesis that adhesion receptor-directed release of IL-2 may result in a locally high concentration of IL-2 that triggers high-affinity IL-2R signaling and up-regulates p55 IL-2R to enhance cytokine responsiveness.

Combination of epidermal growth factor and insulin is required for multicellular spheroid formation of rat hepatocytes in primary culture.

We showed that the combination of epidermal growth factor (EGF) and insulin is an essential supplement to Williams' #E medium for the formation of floating multicellular spheroids in primary culture of rat hepatocytes. Isolated hepatocytes assembled to form floating multicellular spheroids within 96 h through transient assembly of monolayer islands within the initial 24 h in dishes coated with liver-derived proteoglycans. However, the assembly of multicellular spheroids was severely suppressed in the absence of either EGF or insulin. The reduction of spheroid assembly was correlated with decreased attachment and subsequent decreased formation of monolayer islands within 24 h. The minimum amounts of EGF and insulin required for the formation of floating spheroids were 1 ng/ml and 0.4 microgram/ml, respectively. These results suggest that the enhancement of hepatocyte attachment provided by the combination of EGF and insulin during the early phase of culture is required for the formation of floating spheroids.

Cellular mechanism for norepinephrine suppression of pineal photoreceptor-like cell differentiation in rat pineal cultures

Although the rat pineal is an endocrine organ and has no photoreceptor activity, pineals from neonatal rats contain cells that can differentiate into rod-like cells with rhodopsin immunoreactivity (Rho-I), when cultured in vitro. Norepinephrine (NE) reduces the number of Rho-I cells in a dose-dependent manner and has a considerable effect even at 20 n M. When cultured in vitro, pineals removed up to Postnatal Day 4 differentiated into Rho-I cells to the same extent as did those removed at Day 1 (neonatal), but those removed at Day 5 showed a sharp reduction in the number of differentiated Rho-I cells. This suggests that either pineal cells in situ lose their potential to differentiate by Day 5 or the subpopulation of cells involved normally disappears in pineals older than Day 5. The effect of NE was examined in cultures of neonatal pineals by administering it for 1 or 2 days at different stages during a 9-day culture period. NE was most effective when present in the culture medium at an early culture phase and was not efficacious if present only later than Culture Day 7. This indicates that presumptive pineal photoreceptors may become sensitive to NE only for a limited period and that once they are exposed to NE within this period they are irreversibly affected, possibly to degenerate. These cells are similarly and severely affected by potassium ion concentrations as low as 15 m M, suggesting that NE may act at the adrenoreceptor to modify the membrane properties. Serotonin-immunoreactive cells, another cell type (endocrine) found in the cultures, appeared to be regulated by NE by a separate mechanism. NE suppresses process extension by serotonin cells in a reversible manner, and KCl does not have this effect. These findings further evidence that neurotransmitters may have essential roles, other than the transmission of signals, in modulating the developing nervous system.

Stimulation by 1,25-Dihydroxyvitamin D 3 of in vitro mineralization induced by osteoblast-like MC3T3-E1 cells

Although vitamin D is essential for mineralization of bone, it is as yet unclear whether vitamin D has a direct stimulatory effect on the bone mineralization process. In the present study, the effect of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] on in vitro mineralization mediated by osteoblast-like MC3T3-E1 cells was examined. MC3T3-E1 cells continued to grow after they reached confluency, and DNA content and alkaline phosphatase activity increased linearly until about 16 days of culture, whereas 45Ca accumulation into cell and matrix layer remained low. After this period, DNA content plateaued, and 45Ca accumulation increased sharply. Histological examination by von Kossa staining revealed that calcium was accumulated into extracellular matrix. In addition, needle-shaped mineral crystals similar to hydroxyapatite crystals could be demonstrated in between collagen fibrils by electron microscopy. Thus, MC3T3-E1 cells differentiate in vitro into cells with osteoblastic phenotype and exhibit mineralization. When MC3T3-E1 cells were treated with 1,25(OH) 2D 3 at this stage of culture, there was a dose-dependent stimulation of 45Ca accumulation by 1,25(OH) 2D 3, and a significant stimulation of 45Ca accumulation was observed with 3 × 10 −10M 1,25(OH) 2D 3. Although 1,25(OH) 2D 3 enhanced alkaline phosphatase activity and collagen synthesis at the early phase of culture, it did not affect any of these parameters at the late phase when 1,25(OH) 2D 3 stimulated mineralization. Neither 24,25-dihydroxyvitamin D 3 nor human PTH(1–34) affected mineralization in the presence or absence of 1,25(OH) 2D 3. These results demonstrate that 1,25(OH) 2D 3 stimulates matrix mineralization induced by osteoblastic MC3T3-E1 cells, and are consistent with the possibility that 1,25(OH) 2D 3 has a direct stimulatory effect on bone mineralization process. The mechanism of how 1,25(OH) 2D 3 exerts these effects remains to be clarified.

Inhibition of callus development from protoplasts of Lycopersicon peruvianum by extracellular polysaccharides of Clavibacter michiganensis subsp. michiganensis

Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato, produces phytotoxic extracellular polysaccharides (EPS). To evaluate the potential use of EPS in a selection system at cellular level, its effects were studied on protoplasts of susceptible and partially resistant genotypes of the wild tomato species Lycopersicon peruvianum. The number of protoplasts that developed into minicalli was used as a parameter for toxic effect. Protoplasts of the partially resistant genotypes were not, or only slightly, affected by the EPS. The development of minicalli from protoplasts of some susceptible genotypes was significantly inhibited by EPS, however no absolute correlation between susceptibility at plant level and sensitivity of protoplasts to EPS was found. A putative effect of the phytotoxic EPS on plant cell membranes was tested for protoplasts of a bacterial canker sensitive L. peruvianum genotype. This was studied by measuring sucrose uptake, depending on membrane-bound processes, in the presence or absence of EPS. Since no alteration in sucrose accumulation was measured, apparently EPS has no direct effect on the membranes of EPS sensitive protoplasts during the early phase of culture. More information about the mode of action of EPS and the insensitivity of protoplasts of some genotypes is necessary, before considering application of EPS for in vitro selection.

Morphological characteristics and ganglioside composition of substratum adhesion sites in a hepatoma cell line (CMH5123 cells) during different phases of growth

This study compares the ganglioside composition of tissue culture substrate-attached material (SAM) with that of cell bodies in a line of transformed hepatocytes derived from the minimal deviation Morris hepatoma 5123c (CMH5123 cells). We examined both confluent cultures (late-phase cultures) and cells which were allowed to attach for only 3 h (early-phase cultures). We also determined to what extent ganglioside compositions of SAM and cell bodies from early- and late-phase cultures of CMH5123 cells are affected by the block of complex ganglioside biosynthesis induced by treatment with chelating agents (EGTA+EDTA). The morphological characteristics of SAM were monitored by scanning electron microscopy during the different steps of this study. In early-phase cultures, SAM was composed of fragments of filopodia and small vesicles probably representing newly formed substratum adhesion sites. In contrast, SAM of late-phase cultures was made up of large pools of membranous material resulting from the breakage of thick retraction fibers connecting the cell body with broad, mature adhesion sites. SAM of early-phase cultures yielded ganglioside profiles with a higher content of GM1 and GD1 a than those of cell bodies, while in late-phase cultures there was no difference between SAM and cell body gangliosides. When cells were grown in the presence of chelating agents, SAM of earlyphase cultures was composed of vesicles and filopodial fragments similar to those found in early-phase cultures grown in regular media; these morphological features also appeared in SAM of confluent cultures (in contrast to the membranous material characteristic of latephase cultures grown in regular media). In early-phase cultures grown in the presence of chelating agents, gangliosides of SAM were enriched in complex homologs relative to their content in cell bodies. These ganglioside characteristics were also found in SAM of confluent cultures grown in the presence of chelating agents, reflecting the presence of newly formed adhesion sites. On the basis of these results, we may conclude that the molecular assembly of newly formed adhesion sites implies the preferential distribution of several surface components involved in cell adhesion, including complex gangliosides.

Effect of heptaminol AMP amidate, a new nucleotide derivative, on in vitro humoral immunity.

Heptaminol AMP amidate (HAA), a newly developed derivative of 5'-AMP, was found to potentiate the in vitro primary humoral immune response against T cell-dependent antigen, sheep red blood cells, when HAA was present in the early phase of spleen cell culture. Such a potentiating effect was not found against T cell-independent antigens such as lipopolysaccharide (LPS), trinitrophenylated (TNP)-LPS and TNP-Ficoll. The pattern of HAA-mediated immunopotentiation was similar to that of dibutyryl cyclic AMP. When HAA was added to the culture simultaneously with theophylline and imidazole, the immunopotentiating effect of HAA was further augmented and suppressed, respectively. The present results suggested that HAA-mediated immunopotentiation might be in some way related to the intracellular level of cyclic nucleotides in the early phase of culture.

Effect of Heptaminol AMP Amidate, a New Nucleotide Derivative, on In Vitro Humoral Immunity

Heptaminol AMP amidate (HAA), a newly developed derivative of 5’-AMP, was found to potentiate the in vitro primary humoral immune response against T cell-dependent antigen, sheep red blood cells, when HAA was present in the early phase of spleen cell culture. Such a potentiating effect was not found against T cell-independent antigens such as lipopolysaccharide (LPS), trinitro-phenylated (TNP)-LPS and TNP-Ficoll. The pattern of HAA-mediated immuno-potentiation was similar to that of dibutyryl cyclic AMP. When HAA was added to the culture simultaneously with theophylline and imidazole, the immunopotentiating effect of HAA was further augmented and suppressed, respectively. The present results suggested that HAA-mediated immunopotentiation might be in some way related to the intracellular level of cyclic nucleotides in the early phase of culture.

Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions

Specific differentiated gene expression and the morphology of adult rat hepatocytes can be maintained for as long as 8 weeks in vitro only when they are cultured in the presence of biliary epithelial cells; when primary hepatocytes are cultured alone, they lose these functions within 2 to 3 days. We obtained evidence suggesting that contact between hepatocytes and biliary epithelial cells is necessary for maintaining hepatocyte functions. We examined whether junctional communication between and among hepatocytes and biliary epithelial cells is required for long-term maintenance of hepatocyte functions, using a dye-transfer method, in three co-cultures: (1) hepatocytes and biliary epithelial cells prepared from Sprague-Dawley rats; (2) hepatocytes from Sprague-Dawley rats and epithelial cells of the IAR 20 line, originally established from BDVI rats; and (3) hepatocytes from BDVI rats and IAR 20 epithelial cells. The established epithelial cell line (IAR 20) and early-passage cultures of biliary epithelial cells maintained hepatocyte-specific functions in culture for 40 and 70 days, respectively, but the latter induced more stable maintenance of albumin secretion. Hepatocytes cultured alone lost their characteristic morphology within 5 to 8 days, and almost no dye transfer was observed. In co-cultures, the capacity of biliary epithelial cells to communicate among themselves remained relatively high throughout the culture period, whereas hepatocytes showed almost no junctional communication at an early phase of culture and first began to communicate after 2 weeks, communication capacity increasing for at least the next 10 days of culture. The most notable finding was that there was no dye transfer between hepatocytes and biliary epithelial cells in any co-culture system. These results suggest that the maintenance of hepatocyte-specific functions requires intercellular contact but probably not gap-junctional communication between hepatocytes and biliary epithelial cells. This system is useful for studying heterotypic cell-cell interactions and the control of gene expression.

The differentiation in vitro of the neural crest cells of the mouse embryo

A culture method for neural crest cells of mouse embryo is described. Trunk neural tubes were dissected from 9-day mouse embryos and explanted in culture dishes. The developmental potential of mouse neural crest in vitro was shown to be essentially similar to that of avian neural crest. In the mouse, however, melanocytes always appeared in association with the epithelial sheet close to the explant. Neural crest cells surrounding the epithelial sheet, which probably migrated from the neural tubes in the early culture phase, never differentiated into melanocytes. The bimodal behaviour of mouse crest cells seems to be due to the heterogenous potency of the crest cells and the interaction of these cells with the surrounding microenvironment. This culture system is well suited for various experiments including the analysis of gene control on the differentiation of neural crest cells.

The Production of Interleukin 2 as a Measure of Reactivity in Mixed Lymphocyte Culture

Interleukin 2 (IL2) produced in vitro in mixed lymphocyte cultures was measured by induction of growth in cells dependent on IL2 for proliferation. The amount of IL2 in early cultures (24 and 48 hours) clearly differentiated the degree of HLA-D locus identity between various individuals. Very low amounts of IL2 were produced in cultures between HLA-D identical subjects. The use of IL2 measurement offers a method in the early culture phase for predicting the T cell proliferation which is dependent on the presence of IL2.

Effects of non-steroidal anti-inflammatory drugs on lymphocyte activation.

Guinea-pig lymph node lymphocytes were stimulated with mitogen (phytohaemagglutinin) in vitro and lymphocyte activation was measured by tritiated thymidine incorporation (DNA synthesis). Inclusion of non-steroidal anti-inflammatory drugs (NSAIDs) in the culture medium at therapeutic concentrations, frequently exerted an inhibitory effect. Such inhibition could not be attributed to the ability of these drugs to inhibit cyclo-oxygenase or lipoxygenase enzymes. Inhibition by salicylates was not associated with cytotoxic or cytopathic effects, since inhibition was only evident when the drugs were included in the early phase of culture. Other NSAIDs exhibited varying degrees of toxicity, which in some instances may account for observed inhibition. The effects on lymphocyte activation of selective inhibitors of pathways of arachidonic acid metabolism, do not support the proposition that the generation of prostaglandins, thromboxanes, leukotrienes or related compounds is an obligatory step during lymphocyte activation.