To study the effects of serum-free murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of bone marrow endothelial cells, mBMEC-CM was collected and ultrafiltrated by Centriprep-10. The retentate of mBMEC-CM [molecular weight (MW)>10 kDa] and the filtrate of mBMEC-CM (MW<10 kDa) were obtained. The effect of bone marrow conditioned media, their components and exogenous cytokines on the formation of endothelial cell colonies were observed. The effect of bone marrow conditioned media, their components and exogenous cytokines on the proliferation of murine bone marrow endothelial cells were determined by [(3)H]-thymidine incorporation. The method of hybridizing to the Atlas cDNA array was used to determine the expression of cytokine mRNAs in bone marrow endothelial cells. The results obtained are as follows: vWF was expressed in bone marrow endothelial cells. The original mBMEC-CM and MW>10 kDa component of mBMEC-CM promoted the proliferation of bone marrow endothelial cell colonies and increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. The MW<10 kDa component did not affect the production of endothelial cell colonies and did not increase [(3)H]-thymidine incorporation of endothelial cells. Six cytokines (IL-6, IL-11, SCF, GM-CSF, VEGF, bFGF) promoted the proliferation of bone marrow endothelial cell colonies. VEGF, bFGF and SCF increased [(3)H]-thymidine incorporation of bone marrow endothelial cells. According to the results of the Atlas cDNA array, GM-CSF,TGF-beta,BMP-2, bFGF, SCF, endothelin-2, thymosin beta10, MSP-1, connective tissue GF, PDGF-A chain, MIP-2 alpha, PlGF, neutrophil activating protein ENA-78, INF-gamma, IL-1, IL-6, IL-13, IL-11, inhibin-alpha mRNAs were expressed in endothelial cells. These results suggest that murine bone marrow endothelial cell conditioned medium promotes the proliferation of bone marrow endothelial cells.
Animals ; Bone Marrow Cells ; cytology ; Cell Line ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; pharmacology ; Culture Media, Serum-Free ; pharmacology ; Endothelial Cells ; cytology ; Hematopoiesis ; physiology ; Mice

This study was aimed to investigate the effect of angiotensin II on differentiation of cord blood CD34+ cells into megakaryocytes in vitro. The CD34+ cells from eight fresh umbilical cord blood samples sorted by a high-gradient magnetic cell sorting system (MACS) were cultured in serum-free culture medium containing thrombopoietin (TPO) 50 ng/ml, IL-3 10 ng/ml, stem cell factor (SCF) 50 ng/ml and different concentrations of angiotensin II (0, 50, 100, 1000 microg/ml) for 14 days. Mononuclear cells (MNC) were counted by automatic cell analyzer. Cultured CD41+ cell and platelet counts in cultured system, and cell cycle were analyzed by flow cytometry. CD41 specific monoclonal antibody staining was observed by immunofluorescence microscopy. The results showed that as compared with the control group, the number of MNC not increased significantly (P > 0.05), but the number of CD41+ cells and platelets increased significantly in treatment group (P < 0.05). Cell cycle analysis revealed that the amounts of 4N cells increased and apoptosis cells obviously existed in treatment group (P < 0.05). After fluorescence staining, more CD41+ cells of different sizes were observed by means of fluorescence microscopy in both groups. It is concluded that angiotensin II can induce the cord blood CD34+ cells to differentiate towards megakaryocyte, and enhance the function of megakaryocyte to produce platelet.
Angiotensin II ; pharmacology ; Antigens, CD34 ; analysis ; Cell Differentiation ; drug effects ; Cells, Cultured ; Culture Media, Serum-Free ; Fetal Blood ; cytology ; Humans ; Megakaryocytes ; cytology ; Stem Cell Factor ; pharmacology ; Thrombopoietin ; pharmacology

The presence of serum in a culture medium makes it impossible to identify whether changed cellular functions are directly caused by a manipulation itself or mediated by a component in serum. Madin Darby canine kidney cells can survive in a serum-free medium for about 48 h. We took this advantage to examine whether low K(+)-induced up-regulation of Na,K-ATPase requires serum. We found that serum was essential for low K(+) to induce an increase in Na,K-ATPase binding sites as quantified by ouabain factor binding assays. In an attempt to identify which component was critical, we screened EGF, IGF1, PGE1 and transferrin to identify which one can replace serum. We discovered that transferrin was the single most important factor that mimicked about 80% to 90% of the effect of serum. Transferrin potentiated the effect of low K(+) on the Na,K-ATPase binding sites in a time- and dose-dependent manner. Furthermore, transferrin was also required for low K(+)-induced increase in alpha(1)-promoter activity, alpha(1)- and beta(1)-subunit protein abundance of the Na,K-ATPase. In the presence of transferrin, low K(+) enhanced cellular uptake of iron approximately by 70%. Inhibition of intracellular iron activity by deferoxamine (30 micromol/L) abrogated the effect of low K(+). We conclude that stimulation of the Na,K-ATPase by low K(+) is critically dependent on transferrin. The effect of transferrin is mediated by increased iron transport.
Animals ; Cell Line ; Culture Media, Serum-Free ; Dogs ; Kidney ; cytology ; Potassium ; metabolism ; pharmacology ; Sodium-Potassium-Exchanging ATPase ; genetics ; metabolism ; Transferrin ; metabolism ; pharmacology

AIMTo investigate the influence and mechanisms of 17beta-estradiol on the CTP: phosphorylcholine cytidylyltransferase (CCT) activity from cultured lung explants without serum.

METHODSWe detected the amount of [M-14C] choline incorporation into phosphatidylcholine so as to reflect CCT activity by liquid scintillation.

RESULTS(1) 17beta-estradiol increased the CCT activity in dose-dependence and time-dependence. (2) Both the protein kinase C inhibitor H-7 and calmodulin antagonist W-7 abolished the stimulatory effect of 17beta-estradiol (3 x 10(-6) mol/L) on the CCT activity.

CONCLUSION17beta-estradiol can increase CCT activity in cultured lung explants, its mechanism is related to protein kinase C and calmodulin.

Animals ; Calmodulin ; metabolism ; Choline-Phosphate Cytidylyltransferase ; metabolism ; Culture Media, Serum-Free ; Estradiol ; pharmacology ; In Vitro Techniques ; Lung ; drug effects ; enzymology ; Male ; Protein Kinase C ; metabolism ; Rats ; Rats, Wistar

AIMTo investigate the protective effects of adenovirus-mediated hepatocyte growth factor (Ad-HGF) on injury of rat cortex neurons induced by in vitro serum-free culture.

METHODSFlow cytometry was used to assay the transfection rate of rat cortex neurons infected by adenovirus-mediated green fluorescent protein(Ad-GFP) at different multiplicity of infection (MOI) to find out the best MOI in experiment. ELISA was used to elucidate the expression patterns of cortex neuron. Neutral red stain and PI-Hoechst 33342 double stain were used to compare the viability of cortex neurons, which were cultured in serum-free medium for 6 h, 12 h, 24 h and 48 h respectively, among the Ad-HGF transfected group, the Ad-GFP transfected group and the control group.

RESULTSIt was found that when MOI was 50 PFU per cell, a transfection rate as high as 99.3% was maintained and Ad-HGF was able to express in cortex neurons effectively and persistently. In addition, the death rate and apoptotic rate of cortex neurons (infected 2 hours after seeding) cultured in serum-free medium for 12 h in Ad-HGF transfected group was significantly lower than that in both the Ad-GFP group and the control group (P < 0.05).

CONCLUSIONAd-HGF plays a protective role against in vitro serum-free culture induced injury on rat cortex neurons infected 2 hours after seeding. Though its effects on rat cortex neurons infected 5 days after seeding are not so remarkable, Ad-HGF also has the potential to protect cortex neurons from serum-free culture induced injury.

Adenoviridae ; genetics ; Animals ; Animals, Newborn ; Cells, Cultured ; Cerebral Cortex ; drug effects ; Culture Media, Serum-Free ; Hepatocyte Growth Factor ; genetics ; pharmacology ; Neurons ; drug effects ; Rats ; Rats, Wistar ; Transfection

The effect of lysophosphatidic acid (LPA), with a wide range of its different concentrations, upon cultured mouse cortical neurons was assessed by electrophoresis of DNA fragments, HO33342 and TUNEL stainings, and also by ultrastructural examination at times. The results showed that administration of LPA at lower concentrations (0.1-30 micromol/L) dose-dependently protected cortical neurons from apoptosis that was induced by deprivation of serum from the cultural medium, while 50 micromol/L or higher concentrations of LPA failed to show this effect; and moreover, the concentrations higher than 50 micromol/L induced apoptosis in neurons cultured in serum-containing complete medium. These results suggest that a moderate concentration of LPA may play as a survival factor in apoptotic cortical neurons, while an excessive level of LPA induces apoptosis in neurons cultured in complete medium.
Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; cytology ; Culture Media, Serum-Free ; Lysophospholipids ; pharmacology ; Mice ; Neurons ; cytology

BACKGROUNDIn vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering.

METHODSBovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum (FBS) medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype (re-differentiation) and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining.

RESULTSChondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium.

CONCLUSIONThese findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.

Animals ; Cartilage, Articular ; cytology ; Cattle ; Cell Dedifferentiation ; Cells, Cultured ; Chondrocytes ; cytology ; physiology ; Culture Media, Serum-Free ; Fibronectins ; pharmacology ; Real-Time Polymerase Chain Reaction ; SOX9 Transcription Factor ; genetics

The aim of the present study was to examine the functional changes that occur when a rabbit carotid artery is cultured in serum-free medium. In endothelium (EC)-intact arteries cultured under serum-free conditions, acetylcholine (ACh)-induced relaxation responses were partially, yet significantly, reduced when compared with freshly isolated arteries. After pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, application of ACh resulted in a significant contraction in organ cultured arteries. The amplitude of the ACh-induced contractions increased with the duration of culture. In EC-denuded arteries cultured under serum-free conditions, ACh induced responses similar to those in EC-intact arteries pretreated with L-NAME. Furthermore, ACh caused a significant increase in intracellular Ca2+ concentration ([Ca2+]i) in EC-denuded arteries cultured under serum-free condition for 7 days. There was little change in either [Ca2+]i or tension in freshly isolated carotid rings. There was no difference in sodium nitroprusside-induced relaxation responses between fresh and cultured arteries. These results suggest that prolonged culture of carotid arteries under serum-free conditions changes the functional properties of vascular reactivity in rabbit carotid arteries.
Time Factors ; Rabbits ; Organ Culture Techniques/*methods ; Nitroprusside/pharmacology ; NG-Nitroarginine Methyl Ester/metabolism/pharmacology ; *Muscle Contraction ; Models, Statistical ; Dose-Response Relationship, Drug ; Culture Media, Serum-Free/metabolism ; Carotid Arteries/*drug effects/metabolism/*pathology ; Calcium/metabolism ; Animals ; Acetylcholine/*pharmacology

With suspension adapted recombinant Chinese hamster ovary (CHO) cell lines 11G-S expressing human pro-urokinase (pro-UK) as the object of study, a serum-free medium for the cultivation of recombinant CHO cells in suspension was formulated by using Plackett-Burman design and response surface methodology. The two-level Plackett-Burman design was used to evaluate the effect of 10 medium supplements on the growth of the 11G-S cells in suspension culture. Among the 10 medium supplements, insulin, transferrin, and putrescine were identified as the most significant factors (P < 0.05). The response surface methodology with three factors and three levels was used to determine the optimal levels of these factors. And a serum-free medium, SFM-CHO-S for recombinant CHO cells suspension culture was formulated. The maximum cell density of 11G-S cells in SFM-CHO-S in suspension batch culture reached 4.12 x 10(6) cells/mL with a maximum pro-UK activity at 5614 IU/mL, which was superior to the commercial serum-free medium for recombinant CHO cells.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Genetic Engineering ; Insulin ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Transferrin ; pharmacology ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics

This study was aimed to investigate the protocol in vitro to incubate the dendritic cell (DC) derived from peripheral blood monocytes using serum-free medium X-VIVO 20. Peripheral blood monocytes from healthy donors were treated with 100 ng/ml GM-CSF and 500 U/ml IL-4, respectively. After cultivation for 6 days, they were treated with 100 ng/ml calcium ionophore A23187. After cultivation for 24 hours the cellular morphology was observed under invert microscope, the surface markers were analyzed by flow cytometry, the proliferation of allogenetic T cells was detected by MTT colorimetry, the specific cytotoxicity of T cells primed with DC was examined by MTT assay. The results showed that in all three groups with serum-free, fetal calf serum (FCS) and human AB serum mediums, cells displayed characteristic morphological features of DC. Simultaneously CD14 expression was decreased, and CD83, HLA-DR and CDw123 expression were increased on these cells. In addition, DCs cultured with these methods could evidently stimulate the proliferation of allogenetic T cell. As compared with the two controls of serum containing groups, the cultured cells in the serum-free groups showed almost the same allo-stimulatory capability and cellular morphology and surface markers, and T lymphocytes primed with the culture-derived DC exhibited the similar killing activity to K562 (P > 0.05). It is concluded that there is no significance in DC numbers, morphology, epitope and ability to stimulate the proliferation of allogenetic T cells between DC induced by serum-free X-VIVO 20 medium and DC induced by serum-contained medium. DC cultured and induced by serum-free medium is worth using in practice widely.
Cell Culture Techniques ; Culture Media, Serum-Free ; Dendritic Cells ; cytology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Monocytes ; cytology ; Recombinant Proteins ; T-Lymphocytes ; cytology ; T-Lymphocytes, Cytotoxic