Low Cell Density Cultures Research Articles

Identification of Autocrine Growth Factors Secreted by CHO Cells for Applications in Single-Cell Cloning Media

Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However, regulatory authorities have discouraged the use of FBS to reduce the risk of contamination by adventitious agents from animal-derived components. In this study, we demonstrated how a complementary mass spectrometry-based shotgun proteomics strategy enabled the identification of autocrine growth factors in CHO cell-conditioned media, which has led to the development of a fully defined single-cell cloning media that is serum and animal component-free. Out of 290 secreted proteins that were identified, eight secreted growth factors were reported for the first time from CHO cell cultures. By supplementing a combination of these growth factors to protein-free basal media, single cell growth of CHO cells was improved with cloning efficiencies of up to 30%, a 2-fold improvement compared to unsupplemented basal media. Complementary effects of these autocrine growth factors with other paracrine growth factors were also demonstrated when the mixture improved cloning efficiency to 42%, similar to that for the conditioned medium.

FRI0032 Tophus formation limits neutrophil-induced inflammation

BackgroundIn the presence of sodium uric acid precipitates as monosodium urate (MSU)1. In kidney and joints MSU induces renal calculi and gouty arthritis, respectively. The latter is characterized by red,...

Indicators of replicative damage in equine tendon fibroblast monolayers

BackgroundSuperficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it.ResultsWe found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents.Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress.ConclusionsResearchers using tendon fibroblast monolayers should assess their “health” using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled.

BiotecVisions 2012, August

BiotecVisions 2012, August

Rapid DNA, RNA and protein extraction protocols optimized for slow continuously growing yeast cultures

Conventional extraction protocols for yeast have been developed for relatively rapid-growing low cell density cultures of laboratory strains and often do not have the integrity for frequent sampling of cultures. Therefore, these protocols are usually inefficient for cultures under slow growth conditions or of non-laboratory strains. We have developed a combined mechanical and chemical disruption procedure using vigorous bead-beating that can consistently disrupt yeast cells (> 95%), irrespective of cell cycle and metabolic state. Using this disruption technique coupled with quenching, we have developed DNA, RNA and protein extraction protocols that are optimized for a large number of samples from slow-growing high-density industrial yeast cultures. Additionally, sample volume, the use of expensive reagents/enzymes, handling times and incubations were minimized. We have tested the reproducibility of our methods using triplicate/time-series extractions and compared these with commonly used protocols or commercially available kits. Moreover, we utilized a simple flow-cytometric approach to estimate the mitochondrial DNA copy number. Based on the results, our methods have shown higher reproducibility, yield and quality.

Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

γ-Heptalactone is an endogenously produced quorum-sensing molecule regulating growth and secondary metabolite production by Aspergillus nidulans

Microbes monitor their population density through a mechanism termed quorum sensing. It is believed that quorum-sensing molecules diffuse from the microbial cells and circulate in the surrounding environment as a function of cell density. When these molecules reach a threshold concentration, the gene expression of the entire population is altered in a coordinated manner. This work provides evidence that Aspergillus nidulans produces at least one small diffusible molecule during its growth cycle which accumulates at high cell density and alters the organism's behaviour. When added to low-density cell cultures, ethyl acetate extracts from stationary phase culture supernatants of A. nidulans resulted in the abolition of the lag phase, induced an earlier deceleration phase with 16.3% decrease in the final cell dry weight and resulted in a 37.8% increase in the expression of ipnA::lacZ reporter gene construct, which was used as a marker for penicillin production compared to non-treated controls. The bioactive molecule present in the stationary phase extract was purified to homogeneity and was identified by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy to be γ-heptalactone. This study provides the first evidence that A. nidulans produces γ-heptalactone at a high cell density and it can alter the organism's behaviour at a low cell density. γ-Heptalactone hence acts as a quorum-sensing molecule in the producing strain.

Harvesting Nannochloris oculata by inorganic electrolyte flocculation: Effect of initial cell density, ionic strength, coagulant dosage, and media pH

Process variables affecting harvesting efficiency of Nannochloris oculata by AlCl3 flocculation such as, cell density, ionic strength, coagulant dosage, media pH, and cell surface charge were investigated. Initial cell density and coagulant dosage had a significant effect on the removal efficiency; however, levels of ionic strength tested were not significant. Best flocculation conditions of investigated variables were: 0.0016ng of AlCl3/cell, 3.0×107cell/mL, and pH 5.3. Removal efficiency at optimum conditions and salt concentrations of: 0, 15, and 30g/L NaCl was 96, 98, and 97 %, respectively. Low cell density cultures ∼106cell/mL, required five times greater AlCl3 dosage to achieve the same removal efficiency. Destabilization of algal cultures using 0.0032ng of AlCl3/cell was observed by reducing the zeta potential to −22mV. Acidification with HCl for conducting flocculation at pH 5.3 could be a significant cost burden unless is mitigated by selecting a low-buffering-capacity media.

Xylose isomerase improves growth and ethanol production rates from biomass sugars for both Saccharomyces pastorianus and Saccharomyces cerevisiae

The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion.

Improved enrichment and proteomic identification of outer membrane proteins from a Gram-negative bacterium: Focus on Caulobacter crescentus

Efforts to characterize proteins found in the outer membrane (OM) of Gram-negative bacteria have been steadily increasing due to the promise of expanding our understanding of fundamental bacterial processes such as cell adhesion or cell wall biogenesis as well as the promise of finding potential vaccine- or drug-targets for virulent bacteria. We have developed a mass spectrometry-compatible experimental strategy that resulted in increased coverage of the OM proteome of a model organism, Caulobacter crescentus. The specificity of the OM enrichment step was improved by using detergent solubilization of the protein pellet, low-density cell culture conditions, and a surface-layer deficient cell line. Additionally, efficient gel-assisted digestion, high-resolution RP/RP-MS/MS, and rigorous bioinformatic analysis led to the identification of 234 proteins using strict identification criteria (≥ two unique peptides per protein; peptide false discovery rate <2%). Eighty-four of the detected proteins were predicted to localize to the OM or extracellular space. These results represent ~70% coverage of the predicted OM/extracellular proteome of C. crescentus. This analytical approach, which considers important experimental variables not previously explored in published OM protein studies, can be applied to other OM proteomic endeavors "as is" or with slight modification and should improve the large-scale study of this especially challenging subproteome.

Impact of linoleic acid supplementation on lovastatin production in Aspergillus terreus cultures

This work proposes a novel approach for enhancing the yield of lovastatin in Aspergillus terreus cultures by exploiting linoleic acid-derived signalling molecules, which are potentially involved in fungal cell-cell communication. High-performance liquid chromatography analysis revealed that production of lovastatin was enhanced up to 1.8-fold upon exogenous addition of the oxylipin precursor linoleic acid to low cell density cultures of A. terreus. Real-time PCR analysis showed that supplementation of linoleic acid also resulted in an increase in transcriptional levels of lovastatin biosynthetic genes lovB and lovF, indicating a transcriptional control of fatty acids (linoleic acid) on these genes in A. terreus. This study therefore demonstrates for the first time the potential of an oxylipin molecule as an enhancer of a fungal secondary metabolite production with positive impact for industrial exploitation.

Low density cell culture of locust neurons in closed-channel microfluidic devices

Microfluidic channel systems were fabricated out of polydimethylsiloxane (PDMS) and used as culture vessels for primary culture of neurons from locust thoracic ganglia. In a biocompatibility study it was shown that cell adhesion and neuronal cell growth of locust neurons on uncoated PDMS was restricted. Coating with concanavalin A improved cell adhesion. In closed-channel microfluidic devices neurons were grown in static-bath culture conditions for more than 15 days. Cell densities of up to 20 cells/channel were not exceeded in low-density cultures but we also found optimal cell growth of single neurons inside individual channels. The first successful cultivation of insect neurons in closed-channel microfluidic devices provides a prerequisite for the development of low density neuronal networks on multi electrode arrays combined with microfluidic devices.

Interleukin-6 is an autocrine growth factor for folliculostellate-like TtT/GF mouse pituitary tumor cells

Within the pituitary, folliculostellate (FS) cells are considered to regulate the intercellular communication between endocrine cells by paracrine mechanisms. One of the possible paracrine factors involved, could be interleukin-6 (IL-6) which is produced by these cells. Since IL-6 has been shown to be a growth factor of pituitary cells, we have determined whether IL-6 can also influence FS cell proliferation. To test this, a FS cell-like mouse pituitary cell line (TtT/GF cells) was used that exhibits most characteristics of normal FS cells. Under serum-free conditions the proliferation of TtT/GF cells is critically dependent on the initial seeding density: cells seeded at low density do not grow at all whereas cells seeded at high density proliferate with maximal doubling times of 34 hrs. Mouse IL-6 (mIL-6) stimulated only low density cell cultures in a dose and time dependent manner. For cells seeded at high density, exogenously added IL-6 may have failed to stimulate growth because of endogenously produced mIL-6. Conditioned medium from TtT/GF cells, in which mIL-6 concentrations up to 1017 pg/ml were measured, stimulated the proliferation of TtT/GF cells, indicating an autocrine growth stimulatory mechanism. A neutralizing mIL-6 antibody partially suppressed the growth of TtT/GF cells seeded at high density and partially reduced the growth stimulatory activity of TtT/GF conditioned medium. Thus, IL-6 is one but not the only factor that is involved in the autocrine growth stimulatory loop. The relevance of this mechanism for normal FS cells and its physiological consequence needs to be elucidated.

Pro-Photoreceptor Activity of Chickneurogenin1

Better understanding of photoreceptor fate specification may lead to efficient production of photoreceptors for cell replacement studies. The authors investigated the role of proneural bHLH gene neurogenin1 (ngn1) in photoreceptor genesis using the chick retina. In situ hybridization was used to delineate the spatial and temporal pattern of ngn1 expression. RCAS retrovirus was used to drive overexpression of ngn1 in retinal cells, and siRNA was used to reduce ngn1 expression in loss-of-function experiments. Chick ngn1 was transiently expressed during early phases of retinal neurogenesis, from embryonic day (E)3 to E6, with cells expressing ngn1 confined to the apical side of the retinal neuroepithelium. The time window and the anatomic location of ngn1 expression coincided with photoreceptor genesis and differed from those of other transiently expressed proneural bHLH genes, such as ash1, ath3, ath5, and ngn2. Most ngn1-expressing cells lacked BrdU incorporation and lacked phosphorylated histone H3. In low-density cell culture, ngn1 overexpression increased neuroD expression and expanded the photoreceptor population but reduced the ganglion population. Treatment of dissociated retinal cells with siRNA against ngn1 mRNA specifically reduced the photoreceptor population. Overexpression of ngn1 in the retina reduced the expression of ash1, ath5, chx10, and ngn2. The data suggest that ngn1 participates in a complex transcriptional network and may play a role in guiding a progenitor cell to the photoreceptor pathway.

Sparse plating increases the heterogeneity of proliferative potential of fibroblasts

High heterogeneity of proliferative potential in the cultures of diploid human fibroblasts was reported in many studies. It was generally believed that the heterogeneity of proliferative potential of human fibroblasts reflects the unevenness of their senescence. However we show here that immortalized (telomerized) human fibroblasts obey the same rule. Up to 50% of these cells rapidly ceased to proliferate when plated at low density in contrast to usual conditions of mass culture where at least 98% of these cells keep on proliferating. Initially, we proposed that the appearance of non-dividing or slow-dividing cells in low-density cell culture experiments could be caused by cell damage due to the experimental setup. Indeed, lowering of oxygen level and addition of conditioned medium improved colony formation, but there were a large number of non-proliferating cells (13–20%). When we sparsely plated cells on a feeder layer of cells of certain density, the portion of non-proliferating cells decreased to 2%, i.e. became the same as in mass culture. Thus, the heterogeneity of proliferative potential is partially a result of the adverse effect of low cell density.

Up-regulation of vimentin expression in low-density malignant glioma cells as immediate and late effects under irradiation and temozolomide treatment

Nervous system tumors are one of the leading causes of cancer related death. Specific mechanisms facilitating the invasive behavior of gliomas remain obscure. Advanced simulation models of the in vivo response to therapy conditions should potentially improve malignant glioma treatment. Expressional profiling of vimentin--one of reliable pro-invasive tumor makers--in those simulation models was the goal of this study, in order to estimate a pro-invasive response of surviving malignant glioma cells under clinically relevant therapeutic conditions. Human U87-MG malignant glioma cells were used. These cells are characterized by the wild p53-phenotype, which is relevant for the majority of primary malignant glioblastomas. Experimental design foresaw the cells to undergo either irradiation or chemo-treatment with temozolomide alone, or combined treatment. Expression profiling of vimentin was performed by quantitative "Real-Time"-PCR under all treatment conditions simulating diverse tumor regions. Here we demonstrated that vimentin expression patterns in human malignant glioma cells strongly depend on cellular density, algorithms of drug delivery and chemo/radio treatment. Substantial differences were recognized between immediate and late therapy effects. Significant increase in vimentin expression levels was detected particularly in low-density cell cultures under durable treatment with constant concentration levels of temezolomide. Simulation of variable intratumoral regional conditions (central intratumoral regions vs. disseminated malignant cells in peripheral regions) demonstrated differential response of vimentin expression in malignant glioma cell cultures treated under clinically relevant conditions. Slight ebbing of expression levels as late effects of the treatment in confluent cultures may correspond to necrotic processes clinically observed in central intratumoral regions. Contrary, in disseminated malignant cells of peripheral regions therapy resulted in vimentin-inducing effects. This is in agreement with the clinical observations of an increased aggressiveness and malignancy grade of post-operatively chemo/radio-treated malignant gliomas.

A perforated CMOS microchip for immobilization and activity monitoring of electrogenic cells

CMOS-based microelectrode systems offer decisive advantages over conventional micro-electrode arrays, which include the possibility to perform on-chip signal conditioning or to efficiently use larger numbers of electrodes to obtain statistically relevant data, e.g., in pharmacological drug screening. A larger number of electrodes can only be realized with the help of on-chip multiplexing and readout schemes, which require integrated electronics. Another fundamental issue in performing high-fidelity recordings from electrogenic cells is a good electrical coupling between the cells and the microelectrodes, in particular, since the recorded extracellular signals are in the range of only 10–1000 µV. In this paper we present the first CMOS microelectrode system with integrated micromechanical cell-placement features fabricated in a commercial CMOS process with subsequent post-CMOS bulk micromachining. This new microdevice aims at enabling the precise placement of single cells in the center of the electrodes to ensure an efficient use of the available electrodes, even for low-density cell cultures. Small through-chip holes have been generated at the metal-electrode sites by using a combination of bulk micromachining and reactive-ion etching. These holes act as orifices so that cell immobilization can be achieved by means of pneumatic anchoring. The chip additionally hosts integrated circuitry, i.e., multiplexers to select the respective readout electrodes, an amplifier with selectable gain (2×, 10×, 100×), and a high-pass filter (100 Hz cut-off). In this paper we show that electrical signals from most of the electrodes can be recorded, even in low-density cultures of neonatal rat cardiomyocytes, by using perforated metal electrodes and by applying a small underpressure from the backside of the chip. The measurements evidenced that, in most cases, about 90% of the electrodes were covered with single cells, approximately 4% were covered with more than one cell due to clustering and approximately 6% were not covered with any cell, mostly as a consequence of orifice clogging. After 4 days of culturing, the cells were still in place on the electrodes so that the cell electrical activity could be measured using the on-chip circuitry. Measured signal amplitudes were in the range of 500–700 µV, while the input-referred noise of the readout was below 15 µVrms (100 Hz–4 kHz bandwidth). We report on the development and fabrication of this new cell-biological tool and present first results collected during the characterization and evaluation of the chip. The recordings of electrical potentials of neonatal rat cardiomyocytes after several days in vitro, which, on the one hand, were conventionally cultured (no pneumatic anchoring) and, on the other hand, were anchored and immobilized, will be detailed.

Indole signalling contributes to the stable maintenance of Escherichia coli multicopy plasmids

The efficient transmission of multicopy plasmids to daughter cells at division requires that a high copy number is maintained. Plasmid multimers depress copy number, thereby causing instability. Various mechanisms exist to counter multimerization and thus ensure stable maintenance. One well-studied example is the multimer resolution system of the Escherichia coli plasmid ColE1 which carries a recombination site (cer) at which multimers are resolved to monomers by the XerCD recombinase. A promoter within cer initiates synthesis of a short transcript (Rcd) in multimer-containing cells. The Rcd checkpoint hypothesis proposes that Rcd delays cell division until multimer resolution is complete. We have identified tryptophanase (which catabolizes tryptophan to pyruvate and indole) as an Rcd binding protein. Furthermore, the stabilization of multicopy plasmids by Rcd is shown to be tryptophanase dependent, and a tryptophanase-deficient strain is resistant to growth inhibition by Rcd overexpression. Rcd increases the affinity of tryptophanase for its substrate tryptophan which causes increased indole production by cells in low-density cultures. Thus Rcd-mediated stabilization of multicopy plasmids is dependent upon indole acting as a signalling molecule. This is an novel role for this molecule which previously has been implicated in quorum sensing-like processes at high cell density.

Death receptor‐3 mediates apoptosis in human osteoblasts under narrowly regulated conditions

We previously reported that a soluble form of the TNF-family receptor death receptor-3 (DR3) is expressed in osteoblasts. DR3 regulates death or differentiation in other tissues, and DR3 ligands occur in bone, but the function of DR3 in the osteoblast was unknown. We studied the expression of DR3 and the effects crosslinking antibodies to DR3 or of natural DR3 ligands in human osteoblasts. Western analysis showed that nontransformed osteoblasts and the MG63 osteosarcoma cell line produce both soluble decoy receptor and transmembrane isoforms of DR3. Cell surface labeling showed that low and high DR3-expressing osteoblast populations occur. Verification of by cloning showed a point mutation in DR3 from MG63 cells. Activation of DR3 by antibody crosslinking or with DR3 ligands caused apoptosis in osteoblasts and in MG63 cells, but only in low-density cell cultures. In dense cultures apoptosis did not occur, but nuclear factor-kappaB nuclear translocation was observed under some conditions. Crosslinking of DR3 in high-density MG63 cultures blocked expression of bone matrix elements. DR3 activation in high-density nontransformed osteoblasts had only minor effects on cell maturation. We conclude that DR3 activation can mediate apoptosis in osteoblasts. Its activity is, however, highly restricted by its soluble ligand-binding isoform and possibly also by alternate survival signals. In the presence of survival signals, DR3 may affect cell maturation although effects on differentiation were clearly seen only in the MG63 transformed cell line.

Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli

The intein-mediated purification system has the potential to significantly reduce the recovery costs of industrial recombinant proteins. The ability of inteins to catalyze a controllable peptide bond cleavage reaction can be used to separate a recombinant protein from its affinity tag during affinity purification. Inteins have been combined with a chitin-binding domain to serve as a self-cleaving affinity tag, facilitating highly selective capture of the fusion protein on an inexpensive substrate--chitin (IMPACT) system, New England Biolabs, Beverly, MA). This purification system has been used successfully at a lab scale in low cell density cultures, but has not been examined comprehensively under high-cell density conditions in defined medium. In this study, the intein-mediated purification of three commercially relevant proteins expressed under high-cell density conditions in E. coli was studied. Additionally, losses during the purification process were quantified. The data indicate that the intein fusion proteins expressed under high cell density fermentations were stable in vivo after induction for a significant duration, and the intein fusion proteins could undergo thiol or pH and temperature initiated cleavage reaction in vitro. Thus, the intein-mediated protein purification system potentially could be employed for the production of recombinant proteins at the industrial-scale.