HDGF enhances VEGF­dependent angiogenesis and FGF­2 is a VEGF­independent angiogenic factor in non­small cell lung cancer.

Non­small cell lung cancer (NSCLC) accounts for over 80% of all diagnosed lung cancer cases. Lung cancer is the leading cause of cancer­related deaths worldwide. Most NSCLC cells overexpress vascular endothelial growth factor­A (VEGF­A) which plays a pivotal role in tumour angiogenesis. Anti­angiogenic therapies including VEGF­A neutralisation have significantly improved the response rates, progression­free survival and overall survival of patients with NSCLC. However, the median survival of these patients is shorter than 18 months, suggesting that NSCLC cells secrete VEGF­independent angiogenic factors, which remain unknown. We aimed to explore these factors in human NSCLC cell lines, A549, Lu99 and EBC­1 using serum­free culture, to which only EBC­1 cells could adapt. By mass spectrometry, we identified 1,007 proteins in the culture supernatant derived from EBC­1 cells. Among the identified proteins, interleukin­8 (IL­8), macrophage migration inhibitory factor (MIF), galectin­1, midkine (MK), IL­18, galectin­3, VEGF­A, hepatoma­derived growth factor (HDGF), osteopontin (OPN), connective tissue growth factor (CTGF) and granulin (GRN) are known to be involved in angiogenesis. Tube formation, neutralisation and RNA interference assays revealed that VEGF­A and HDGF function as angiogenic factors in EBC­1 cells. To confirm whether VEGF­A and HDGF also regulate angiogenesis in the other NSCLC cell lines, we established a novel culture method. NSCLC cells were embedded in collagen gel and cultured three­dimensionally. Tube formation, neutralisation and RNA interference assays using the three­dimensional (3D) culture supernatant showed that VEGF­A and HDGF were not angiogenic factors in Lu99 cells. By gene microarray in EBC­1 and Lu99 cells, we identified 61 mRNAs expressed only in Lu99 cells. Among these mRNAs, brain­derived neurotrophic factor (BDNF), fibroblast growth factor­2 (FGF­2) and FGF­5 are known to be involved in angiogenesis. Tube formation and neutralisation assays clarified that FGF­2 functions as an angiogenic factor in Lu99 cells. These results indicate that HDGF enhances VEGF­dependent angiogenesis and that FGF­2 is a VEGF­independent angiogenic factor in human NSCLC cells.

Extracellular small non-coding RNA contaminants in fetal bovine serum and serum-free media.

In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for in vitro studies is advocated to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EV-depleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA (snRNA, Y RNA, snoRNA, and piRNA) were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media.

Transcriptomic changes in CHO cells after adaptation to suspension growth in protein-free medium analysed by a species-specific microarray.

Chinese Hamster Ovary (CHO) cells are the preferred cell line for production of biopharmaceuticals. These cells are capable to grow without serum supplementation, but drastic changes in their phenotype occur during adaptation to protein-free growth, which typically include the change to a suspension phenotype with reduced growth rate. A possible approach to understand this transformation, with the intention to counteract the reduction in growth by targeted supplementation of protein-free media, is gene expression profiling. The increasing availability of genome-scale data for CHO now facilitates quests for a better understanding of metabolic pathways and gene networks. So far, systematic large-scale expression profiling in CHO cells by microarray was limited due to lack of publicly available array designs and limitations of alternative approaches. Based on the recent release of CHO and Chinese Hamster genome sequences, including an annotated RefSeq genome, we have constructed a publicly available microarray design for effective genome-scale expression profiling. The design employed microarray probes optimized for uniformity, sensitivity, and specificity, with probe properties computed using the latest thermodynamic models. We validated the platform in an analysis of gene expression changes in response to serum-free adaptation. The observed effects on the lipid metabolism as well as on nucleotide synthesis were used to successfully select media supplements that were able to increase growth rate.

A high-throughput colorimetric-assay for monitoring glucose consumption by cultured trophoblast cells and placental tissue.

Diabetes mellitus is associated with abnormalities in placental structure and function and significant pregnancy complications. In vitro studies to investigate the effect of hyperglycaemia on trophoblast structure and function require an accurate, inexpensive and reliable assay to monitor the concentration of glucose in culture medium. We have modified and validated an existing protocol for use with a 96-well microplate. This provides a specific, high-throughput assay which accurately measures culture media glucose concentrations between 7 and 30 mM, without spectral interferences by phenol red or sera. Use of this assay revealed that the concentration of glucose in BeWo cell cultures remains stable for 48 h. In contrast placental explants rapidly consume glucose thus the concentration in culture media significantly decreases over 12 h necessitating more frequent replenishment in order to maintain the desired concentration. We therefore advise researchers to monitor glucose concentrations in in vitro investigations modelling the effect of diabetes mellitus on placental structure and function.

Retinoic acid stability in stem cell cultures.

It has been reported that retinoids, such as retinoic acid (RA) and retinol (ROL), dissolved in aqueous solutions are susceptible to oxidative damage when exposed to light, air, and relatively high temperatures, conditions that are normal for culturing stem cells. Thus, questions arise regarding the interpretation of results obtained from studies of mouse embryonic stem cells exposed to retinoids because their isomerization state, their stability in culture conditions, and their interactions with other potential differentiation factors in growth media could influence developmental processes under study. Media samples were supplemented with retinoids and exposed to cell culture conditions with and without mouse embryonic stem cells (mESC), and retinoids were extracted and analyzed using HPLC. To determine whether retinoids are stable in media supplemented with fetal bovine serum (FBS) or in chemically-defined, serum-free media, mESC adapted to each type of growth media were investigated. Studies reported here indicate there was little loss or isomerization of at-RA, 9-cis-RA, 13-cis-RA, or ROL in cell cultures grown in serum-supplemented media when cell cultures were maintained in the dark and manipulated and observed under yellow light. In contrast, the stability of both at-RA and ROL were determined to be greatly reduced in serum-free media as compared with serum-supplemented media. Addition of 6 mg/ml bovine serum albumin was found to stabilize retinoids in serum-free media. It was also determined that ROL is less stable than RA in cell culture conditions.

Downstream processing of Vero cell-derived human influenza A virus (H1N1) grown in serum-free medium.

A downstream processing was examined for Vero cell-derived human influenza virus (H1N1) grown in serum free medium. Vero cell banks were established in serum free medium and characterized according to regulatory requirements. Serum free Vero cells were grown on Cytodex 3 microcarriers in 5L bioreactor and infected with influenza A virus (A/New Caledonia/99/55). The harvests were processed with the sequence of inactivation, clarification, anion exchange chromatography (DEAE FF), Cellufine Sulfate Chromatography (CSC) and size exclusion chromatography (Sepharose 6FF). Host cell DNA (hcDNA) was mainly removed with DEAE FF column and CSC by 40 and 223 fold, respectively. Most of Vero cell proteins were eliminated in CSC and Sepharose 6FF unit operation by about 13 fold. The overall scheme resulted in high recovery of hemagglutinin (HA) activity and the substantial removal of total protein, host protein and DNA. The total protein content and DNA content per 15 µg HA protein in final product was 89 µg and 33 pg, respectively, which complied with regulatory requirements for single strain influenza vaccines. SDS-PAGE analysis and Western blotting confirmed the purity of the final product. In conclusion, the suggested downstream process is suitable for the purification of microcarrier-based cell-derived influenza vaccine.

SV40-transformed human corneal keratocytes: optimisation of serum-free culture conditions.

Aiming at the replacement of animal experiments in eye irritation testing, we have established a multilay ered cornea model comprising the co-culture of all three corneal cell types. It was the objective of this study to optimise serum-free culture conditions to preserve both growth and phenotype of an SV40-immortalised human corneal keratocyte cell line (HCK). Our results revealed that HCK continue to proliferate in both monolayer cultures as well as after seeding in a collagen matrix and resemble primary corneal keratocytes in morphology and functional characteristics under defined serum-free conditions. Furthermore, HCK were shown to transform into activated corneal fibroblast phenotypes in response to serum and TGF(beta)1. In summary, HCK cells mimic their in vivo (primary) precursors, both in sustaining the quiescent keratocyte phenotype (serum-starved conditions) and in responding to growth factor stimulation. Hence, this cell line may provide a useful tool to study the toxicity and wound healing response of corneal keratocytes in vitro.

Release kinetics of intact and degraded troponin I and T after irreversible cell damage.

PURPOSE: We characterized the release kinetics of cardiac troponin I and T in relation to lactate dehydrogenase (LDH) from cardiomyocytes before and after the transition from reversible to irreversible cell damage. METHODS: Cardiomyocytes were exposed to mild metabolic inhibition (1 mmol/L sodium azide) to induce a necrotic cell death process that is characterized by a reversible (0-12 h) and irreversible phase (12-30 h). At various time intervals cells and media were collected and analyzed for LDH activity, intact cTnI and cTnT, and their degradation products. RESULTS: During the first 12 h of metabolic inhibition, cell viability was unchanged with no release of intact cTnI and cTnT nor their degradation products. Between 12 and 30 h of azide treatment, cardiomyocytes showed progressive cell death accompanied by release of intact cTnI (29 kDa), intact cTnT (39 kDa), four cTnI degradation products of 26, 20, 17 and 12 kDa, and three cTnT degradation products of 37, 27 and 14 kDa. Possibly due to degradation, there is progressive loss of cTnI and cTnT protein that is obviously undetected by the antibodies used. CONCLUSIONS: Metabolic inhibition of cardiomyocytes induces a parallel release of intact cTnI and cTnT and their degradation products, starting only after onset of irreversible cardiomyocyte damage.

Optimized mouse ES cell culture system by suspension growth in a fully defined medium.

Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time efficiency and typically requires only 20 min of effective hands-on time per week. This protocol maintains a very high degree of pluripotent cells partly by mechanical separation of spontaneously differentiating cells. mES cells can be cultured for extended periods (>6 months) without the loss of pluripotency markers. High passage (>20) adherent mES cultures containing contaminating differentiated cells can be rescued and enriched in undifferentiated ES cells.

[Serum-free knockout SR medium supports the short-time viability of mouse spermatogonial stem cells].

Spermatogonial stem cells (SSCs), the only stem cells that are capable of transmitting genetic information to subsequent generations in adult animals,have abilities to self-renew and differentiate into spermatozoa. Therefore, SSCs are not only the study object of stem cell biology, but also the valuable resource of in vitro spermatogenesis, gene analysis and functional genomics. In the present study, we selected the mouse SSCs from mice on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders by differential adherence selection in DMEM/F12 containing 5% FBS, and used a serum-free defined medium prepared with Knockout SR basal medium to culture SSCs. Our results showed that enriched SSCs could be maintained for a short of time and form colonies, but the proliferation of SSCs was unconspicuous, suggesting that some factors that are detrimental to the self-renewal of SSCs possibly existed in the Knockout SR basal medium. However, the use of KSR medium, which was widely used in the culture of embryonic stem cells (ESCs), in the SSC maintenance was the first time, and our results indicated that the Knockout SR basal medium don't appropriate for the long-time culture of SSCs.

Extracellular and transmembrane region of a podocalyxin-like protein 1 fragment identified from colon cancer cell lines.

Regulated intramembrane proteolysis of membrane proteins has been shown to play an important role in cell differentiation and in the pathogenesis of diseases. The aim of the present study was to identify novel peptides generated by intramembrane proteolysis. The peptides were identified in serum-free cultured (SFC) media from various cell lines by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). A 2315-Da peptide found only in medium from SFC colon cancer cell lines was identified and shown to consist of a portion of both the extracellular and transmembrane regions of human podocalyxin-like 1. This protein fragment was not found in lung or pancreatic cancer cell lines by immunoprecipitation-SELDI tests using an antibody specific to this fragment, suggesting that this human podocalyxin-like protein 1 fragment may be unique to colon cancer cell lines.

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells.

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.

Micro-quantitation of lipids in serum-free cell culture media: a critical aspect is the minimization of interference from medium components and chemical reagents.

Lipids (fatty acids) at a concentration range of 10-100 microg/L are essential components included in most serum-free cell culture medium formulations. A gas chromatography/mass spectrometry (GC/MS) method for the micro-quantitation of lipids, determined as fatty acid methyl esters (FAMEs), in complex serum-free cell culture media was developed. The interference of derivatizing reagents, extraction solvents and medium additives in the micro-quantitation of lipids was also examined. The results show that the concentration of fatty acids such as palmitic and stearic acids detected in derivatizing reagents or extraction solvents was in the range of 10-230 microg/L. Tween-80, a surfactant and medium additive, produced nearly 20 FAMEs alone when methylated using a derivatizing agent. Moreover, the surfactant Pluronic F-68, a medium additive, interfered in the FAME recovery. Procedures, which include use of low volumetric ratio of reagent to medium and precipitation of the above surfactants, were developed to minimize background FAMEs to levels which do not significantly affect the quantitation of medium lipids and to diminish the interference caused by Pluronic F-68. Fatty acid concentrations in several complex serum-free culture media were quantitated by this method and were very close to the values indicated in their formulations.

Mass spectrometric high-throughput analysis of serum-free conditioned medium from cancer cell lines.

Proteins secreted by cancer cells may be useful as tumor markers. We analyzed constituents produced by cells grown in serum-free conditioned medium to avoid confounding factors present in serum. Mass spectrometric techniques were used to obtain expression patterns of constituents between 2000 and 15000 Da from as little as 1 microl of crude sample. This protocol can produce distinctive mass profiles from 16 cell lines within 1 h. Thus, differential display by mass spectrometry will expedite the discovery of peptides specifically secreted by particular cells. These data illustrate the advantage of this strategy over the conventional approach of electrophoretic separation of serum samples.

Secretion of prosaposin, a multifunctional protein, by breast cancer cells.

Western blotting and immunodetection with three antibodies were used to probe conditioned media of breast cancer cells (MDA231, MDA435, MCF-7) for prosaposin, a lysosomal protein that occurs in milk. It was readily detected in media from these cells, and from that of an sv40-transformed mammary epithelial cell, HBL100, but not from medium of human neural tumor cells (SK-N-MC). In cultures of MCF-7 cells, the prosaposin pattern of secretion over time closely resembled that of procathepsin D, another lysosomal protein occurring in milk. Supplementing medium with 17beta-estradiol (0. 1-100 nM) dose dependently increased secretion of both proteins after 48 h without changes in cell viability. The influence of 17beta-estradiol on secretion could play a role in the trophic activity of prosaposin in cellular differentiation and cell death protection. In concert with other lysosomal proteins in the tumor environment, such as procathepsin D, prosaposin may be a factor in eliminating barriers to tumor metastasis by facilitating hydrolysis of membrane glycolipids. The number of milk proteins known to be secreted by breast cancer cells is growing. There is evidence that at least some of these may be secreted in an endocrine manner in the normal, non-lactating breast.

Production of alph-amylase in acid cheese whey culture media with automatic pH control

The influence of aeration and automatic pH control on the production of alpha-amylase by a strain of "Bacillus subtilis" NRRL 3411 from acid cheese whey was studied. Tests were carried out in a rotary shaker and in mechanically stirred ferments. Alpha-maylase was analysed according to DUN's method. Oxygen absorption rate was determined by Cooper's method. Cell oxygen demand was determined as oxygen consumption in a Warburg respirometer. The level of dissolved oxygen was mesured by means of a galvanic silver-lead electrode. Results suggest the possibility of industrial use of acid cheese whey as a carbon source for alpha-amylase production, since the yiels was similar to that produced with lactose. The highest alpha-amylase levels 100,000 DUN/ml units were not attained at highr aeration rates -431 mLO2/L.h-. The indicated value correspond to a 96 h process with automatic pH enzyme production was directly related to growth in the form of cell aggregates.

Derivation and application of monoclonal antibodies recognizing several epitopes on bovine serum albumin.

Three (AB-3, AB-4 and AB-6) monoclonal antibodies (mAb) to bovine serum albumin (BSA) were derived and characterized for their physicochemical and immunological properties. AB-3 recognized an epitope distinct from epitopes recognized by AB-4 and AB-6 as determined by binding inhibition assay. AB-4 and AB-6 mAbs recognized similar but not identical epitopes on BSA. Based on the antigenic specificity, we applied these mAbs to quantitative analysis of BSA in medium and to depletion of BSA from culture medium containing fetal calf serum (FCS). For quantitative analysis, we employed a sandwich enzyme-linked immunosorbent assay (ELISA) using biotinylated AB-3, solid-phase of AB-6 and an avidin-biotin-peroxidase complex system. This assay was highly sensitive and quantitative in the range of BSA concentration at 10 to 1,500 ng/ml. To deplete BSA from medium, we prepared affinity-gel coupled to AB-6. Repeated treatment of FCS-containing medium with the affinity-gel efficiently depleted BSA from the medium. The depletion capacity was 0.74 to 1.0 moles of BSA/mole of coupled mAb.

Protein-free culture of Vero cells: a substrate for replication of human pathogenic viruses.

A protein-free chemically defined medium designated PFEK-1 was developed for culture of VERO cells on polyvinyl formal (PVF) culture surface without serum or other macromolecular supplements. VERO cells proliferated in PFEK-1 medium on PVF surface to a similar extent as cells in serum-supplemented medium without previous adaptation from serum-containing conditions. The protein-free culture infected with coxsackievirus B4, herpes simplex virus types 1 and 2, measles virus and poliovirus types 1, 2 and 3 developed viral titers comparable to those found in conventionally grown cells. The results demonstrated that VERO cells in protein-free culture provide a sensitive substrate for the production of human pathogenic viruses which are not contaminated by serum or other protein factors usually added to a culture medium.

High-level expression of cytokine-induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: purification and production of blocking antibodies.

Significant levels of cytokine-induced neutrophil chemoattractant (CINC) were found in serum-free medium conditioned by a highly metastatic rat cell line, RC20. To study CINC's role in inflammation and metastasis, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits. CINC was a potent chemoattractant for rat neutrophils (EC-50 0.5 nM). A fusion protein of glutathione-S-transferase and CINC (GST-CINC) was produced in E. coli. Anti-CINC polyclonal IgG was purified from immune goat and rabbit sera by protein A and GST-CINC affinity chromatography. Both goat and rabbit anti-CINC antibody preparations at 4 micrograms/mL (an 11-fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat neutrophil chemotaxis assay. These antibodies have been used to develop a sensitive immunoassay for CINC. The availability of large amounts of affinity-purified blocking anti-CINC antibody will allow investigations into the role played by CINC in rodent inflammation models and in the metastasis of RC20 cells.

Characterization of an eosinophilic leukemia cell differentiation factor (ELDF) produced by a human T cell leukemia cell line, HIL-3.

An adult T cell leukemia cell line, HIL-3, constitutively secretes a factor which induces the phenotypical and functional eosinophilic differentiation of a human eosinophilic leukemia cell line, EoL-1. Biochemical characteristics of the factor, termed eosinophilic leukemia cell differentiation factor (ELDF), were examined. ELDF was precipitated by 35 to 65% saturated ammonium sulfate from the culture supernatants of HIL-3 cells (HIL-3 sup). ELDF was eluted in a peak corresponding to a molecular weight of 30 to 40 kd by gel filtration. Isoelectric focusing in the Rotofor showed that ELDF had isoelectric points of 5 to 6. ELDF was trypsin-sensitive and stable to heat treatment at 65 degrees C for 30 minutes but labile at 80 degrees C or pH lower than 3. Half of the activity adhered to lentil-lectin but not to Con-A, indicating that a part of ELDF is glycoprotein with an N-linked carbohydrate moiety, which did not seem to be essential for ELDF activity. The biochemical characteristics of ELDF and blocking experiments using cytokine-specific neutralizing antibodies suggest that ELDF is different from gamma-interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-5 (IL-5) and interleukin-2 (IL-2), which may exist in HIL-3 sup, and that ELDF may be a previously unrecognized leukemia differentiation factor.