Continuous Cell Culture Research Articles

Smallpox: a disease and a weapon

Smallpox: a disease and a weapon

Derivation, growth and applications of human embryonic stem cells.

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass cells of blastocysts with the potential to maintain an undifferentiated state indefinitely. Fully characterised hES cell lines express typical stem cell markers, possess high levels of telomerase activity, show normal karyotype and have the potential to differentiate into numerous cell types under in vitro and in vivo conditions. Therefore, hES cells are potentially valuable for the development of cell transplantation therapies for the treatment of various human diseases. However, there are a number of factors which may limit the medical application of hES cells: (a) continuous culture of hES cells in an undifferentiated state requires the presence of feeder layers and animal-based ingredients which incurs a risk of cross-transfer of pathogens; (b) hES cells demonstrate high genomic instability and non-predictable differentiation after long-term growth; and (c) differentiated hES cells express molecules which could cause immune rejection. In this review we summarise recent progress in the derivation and growth of undifferentiated hES cells and their differentiated progeny, and the problems associated with these techniques. We also examine the potential use of the therapeutic cloning technique to derive isogenic hES cells.

Do Toxoplasma gondii RH strain tachyzoites evolve during continuous passage?

Aim: To examine three lineages of Toxoplasma gondii RH strain in terms of performance in the dye test, culture, and gene expression. Methods: Historical data (culture growth and performance in...

Arrays of horizontally-oriented mini-reservoirs generate steady microfluidic flows for continuous perfusion cell culture and gradient generation

This paper describes the use of arrays of horizontally-oriented reservoirs to deliver liquids through microchannels at a constant flow rate over extended periods of time (hours to days). The horizontal orientation maintains a constant hydraulic pressure drop across microfluidic channels even as the volumes of liquids within the reservoirs change over time. For a given channel-reservoir system, the magnitude of the flow velocity depends linearly on the height difference between reservoirs. The simple structure and operation mechanism make this pumping system versatile. A one-inlet-one-outlet system was used to continuously deliver media for perfusion cell culture, and an array of inlet reservoirs coupled to an outlet reservoir via microchannels was used to drive flows of multiple laminar streams. The parallel pumping scheme conveniently generated various smooth and step concentration gradients, and allowed evaluation of the effect of colchicine on myoblasts. Since the reservoir arrays are configured to be compatible with commercialized multichannel pipettors designed for 96 well plate handling, this simple pumping scheme is envisioned to be broadly useful for medium to high throughput microfluidic perfusion cell culture assays, cell migration assays, multiple laminar flow drug tests, and any other applications needing multiple microfluidic streams.

Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays

We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology.

골수 유래 기질 줄기세포의 탐식작용 매개성 케모카인 수용체 발현 연구

To design gene deliver systems which can deliver higher amounts of genes into stem cells, we studied the expression of receptors involved in the receptor-mediated endocytosis of bone marrow stromal stem cells. Bone marrow was isolated from ICR mice, and bone marrow stromal stem cells were isolated based on their plastic adherence property. Several culture conditions were screened for effective and continuous culture of marrow stromal stem cells. MesenCult medium was finally used to cultivate marrow stromal stem cells in vitro. As candidate receptors, various chemokine receptors were studied. Both bone marrow cells ad marrow-derived stromal stem cells showed expression of CC chemokine receptors (CCR) and CXC chemokine receptors (CXCR). Marrow stromal stem cells showed higher expression of CCR5 ad CXCR4 chemokine receptors as compared to other types of chemokine receptors. Moreover, though the expression of chemokine receptors generally decreased in most chemokine receptors with the cultivaton of marrow stromal stem cells, CCR5 and CXCR4 chemokine receptors retained the higher level of receptor expressions over prolonged periods. These results suggest that the ligands exhibiting specific binding to CCR5 or CXCR4 might be used to modify gene delivery systems for increased levels of receptor-mediated gene delivery into stromal stem cells.

Relief of oxidative stress by ascorbic acid delays cellular senescence of normal human and Werner syndrome fibroblast cells.

Primary human cells have a definite life span and enter into cellular senescence before ceasing cell growth. Oxidative stress produced by aerobic metabolism has been shown to accelerate cellular senescence. Here, we demonstrated that ascorbic acid, used as an antioxygenic reagent, delayed cellular senescence in a continuous culture of normal human embryonic cells, human adult skin fibroblast cells, and Werner syndrome (WS) cells. The results using human embryonic cells showed that treatment with ascorbic acid phospholic ester magnesium salt (APM) decreased the level of oxidative stress, and extended the replicative life span. The effect of APM to extend the replicative life span was also shown in normal human adult cells and WS cells. To understand the mechanism of extension of cellular life span, we determined the telomere lengths of human embryonic cells, both with and without APM treatment, and demonstrated that APM treatment reduced the rate of telomere shortening. The present results indicate that constitutive oxidative stress plays a role in determining the replicative life span and that suppression of oxidative stress by an antioxidative agent, APM, extends the replicative life span by reducing the rate of telomere shortening.

Continuous culture of novel mitochondrial cells lacking nuclei

We isolated stable cell lines, designated as mitochondrial cells, from cybrids obtained by fusing mitochondria-less HeLa cells with platelets from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy. The cells contain a pathogenic point mutation, T9176C, in the mitochondrial DNA. Hematoxylin–eosin staining, confocal fluorescent microscopy and flow cytometry in fixed or living cells showed that the majority of these mitochondrial cells lack nuclear DNA and nuclei, but contain active mitochondria. Despite the absence of nuclear DNA, these cells can be continuously generated in culture. Therefore, it is likely that they arise from the minority of cells which possess a nucleus.

Localization and ecological significance of oroidin and sceptrin in the Caribbean sponge Agelas conifera

The Caribbean sponge Agelas conifera was found to produce a mixture of previously described bromopyrrole alkaloids of which oroidin ( 1) and sceptrin ( 2) were predominant. This sponge harboured large populations of heterotrophic bacteria but no photosynthetic symbionts (cyanobacteria). However, 1 and 2 were not associated with the bacteria but with the sponge cells as shown by their distribution in enriched cell fractions obtained by differential centrifugation and Ficoll density gradients. Spherulous cells, found in great abundance in the sponge ectosome, were assumed to be involved in the production of 1 and 2. The target compounds were detected, although in small amounts, in short-term cultures of sponge cells, validating the possibility of a continuous cell culture source. Laboratory assays showed that organic sponge extracts affected the behaviour of the coral Madracis mirabilis in causing closure and retraction of the polyps at concentrations of the combined compounds 1 and 2 (1:3.3) as low as 0.7 mg/l (0.0125% of the concentration in whole sponges). At higher concentrations (1.4 mg/l) no recovery of the polyps occurred. The extracts, at almost natural concentrations of 1 and 2, deterred feeding by the predatory reef fish Stegastis partitus, supporting other reported research. In field experiments, wounding induced a sharp increase of 1 and 2 in the sponge tissues but prolonged predator exclusion by caging and forced confrontation with coral neighbours did not yield measurable changes in 1 and 2 concentrations. All sponges were found to release measurable amounts of bromopyrrole alkaloids in seawater conditioned for 30 min. Crude and fractionated sponge extracts and pure sceptrin ( 2) were active against bacteria, yeast and filamentous fungi. Taken together, these results support a role of oroidin ( 1) and sceptrin ( 2) in defence mechanisms against predators and possibly against space competitors and invading and fouling organisms.

Nitric oxide partially controls Coxiella burnetii phase II infection in mouse primary macrophages.

In most primary or continuous cell cultures infected with the Q-fever agent Coxiella burnetii, bacteria are typically sheltered in phagolysosome-like, large replicative vacuoles (LRVs). We recently reported that only a small proportion of mouse peritoneal macrophages (PMPhi) infected with a nonvirulent, phase II strain of C. burnetii developed LRVs and that their relative bacterial load increased only slowly. In the majority of infected PMPhi, the bacteria were confined to the small vesicles. We show here that nitric oxide (NO) induced by the bacteria partially accounts for the restricted development of LRVs in primary macrophages. Thus, (i) PMPhi and bone marrow-derived macrophages (BMMPhi) challenged with phase II C. burnetii produced significant amounts of NO; (ii) the NO synthase inhibitors aminoguanidine and N-methyl-L-arginine reduced the production of NO and increased the frequency of LRVs (although the relative bacterial loads of individual LRVs did not change, the estimated loads per well increased appreciably); (iii) gamma interferon (IFN-gamma) or the NO donor sodium nitroprusside, added to BMMPhi prior to or after infection, reduced the development and the relative bacterial loads of LRVs and lowered the yield of viable bacteria recovered from the cultures; and (iv) these effects of IFN-gamma may not be entirely dependent on the production of NO since IFN-gamma also controlled the infection in macrophages from inducible NO synthase knockout mice. It remains to be determined whether NO reduced the development of LRVs by acting directly on the bacteria; by acting on the traffic, fusion, or fission of cell vesicles; or by a combination of these mechanisms.

Modulation of Cell Cycle for Enhancement of Antibody Productivity in Perfusion Culture of NS0 Cells

A prolonged period of high productivity at high cell density is desirable for industrial production of biopharmaceuticals. Previous efforts have shown that cessation of cell proliferation in low cell density culture results in increased productivity. We report here further results on multigenic manipulation of cell cycle and apoptosis to enhance productivity at high cell density. The NS0 6A1/4-9F myeloma cell line, which constitutively expresses a chimeric IgG4 antibody and inducibly expresses the p21(CIP1) cyclin-dependent kinase inhibitor has been further engineered to constitutively overexpress the Y28 mutant Bcl-2 anti-apoptotic protein. The effects of overexpression of p21(CIP1) and Bcl-2 on cell proliferation, cell viability, and antibody production has been investigated in batch and continuous perfusion cultures. In both cultures the p21(CIP1) protein arrested cell proliferation, confirming the previous results in low-density culture of 4-fold increase in antibody production, whereas mutant Bcl-2 expression has not resulted in any significant improvement in cell viability of arrested cells. This study demonstrates that it is possible to enhance the productivity of relatively high-density continuous mammalian cell cultures by arresting the cell cycle in G1 phase.

Toxoplasma gondii from liquid nitrogen for continuous cell culture:methods to maximise efficient retrieval

This study aims to increase the efficiency of continuous growth of Toxoplasma gondii in HeLa cells from tachyzoite stocks frozen in liquid nitrogen. Freezing and retrieval of tachyzoites for continuous cell culture requires more stringent protocols than those published for animal culture. The freezing and retrieval conditions are optimised so that a quality harvest (≥ 1 x 106 tachyzoites/mL, ≥ 90% viability) can be produced using T. gondii recovered from liquid nitrogen as fast and reliably as possible. Retrieval success rate increased from 36% to 100%. An improved freezing procedure using chilled reagents and freshly harvested parasites, and adoption of an effective recovery protocol with retrieval of 3 x 107 tachyzoites into 75cm2 flasks, change of maintenance media after six hours and subsequent blind passage all contributed to this success. The result is faster and more dependable production of T. gondii for diagnostic and experimental use.

Cultivation of an Ovine Strain of Ehrlichia phagocytophila in Tick Cell Cultures

Ehrlichia phagocytophila (previously known as Cytoecetes phagocytophila) which causes tick-borne fever ( TBF ) in sheep and pasture fever in cattle in the UK and mainland Europe is transmitted by the temperate hard tick Ixodes ricinus. The disease in sheep is characterized by fever, leucopenia and immunosuppression. Studies on the pathogenesis and other aspects of the disease have been hampered because the organism has not been cultivated in continuous or primary cell culture systems. This paper describes the first successful cultivation of a European isolate of E. phagocytophila in two continuous cell lines, IDE8 and ISE6, derived from the temperate hard tick Ixodes scapularis. Once adapted to tick cell cultures the organism was serially sub-cultured in new cells by transferring small portions of infected cell suspension every 2 to 3 weeks. The identity of the organism was confirmed by polymerase chain reaction (PCR), with primers specific to the granulocytic ehrlichiae. Sequence analysis of the PCR products amplified from infected tick cells were shown to be identical with those amplified from the blood of sheep infected with the same strain of E. phagocytophila. A susceptible sheep inoculated with a third passage of the tick cell-adapted E. phagocytophila reacted with fever and rickettsiaemia 5 days later, thus satisfying Koch's postulates.

Herpes simplex virus type 1 origins of DNA replication play no role in the regulation of flanking promoters.

Herpes simplex virus (HSV) exhibits altered gene regulation in neuronal compared to nonneuronal tissues. It has been hypothesized that initiation of DNA synthesis at the viral origins of replication (oriS and oriL) is a critical step in the upregulation of transcriptional activity of flanking divergent promoters, thereby increasing productive gene expression in neurons. Notably, oriS is flanked by the immediate-early (IE) ICP4 and ICP22/47 promoters, and oriL is flanked by the early (E) UL29 and UL30 promoters. To test this hypothesis further, a series of constructs were generated in which these promoters were placed upstream of luciferase genes. In addition, DNA replication origins were deleted in the context of these promoter constructs. All cassettes were recombined into the viral genome of HSV type 1 strain KOS at a site distal to its native origins. Recombinant reporter expression was monitored in vitro and in vivo to determine the role of viral origins of DNA replication in the regulation of their flanking promoters. Reporter gene expression was unaffected by the presence or absence of oriS or oriL, with the exception of a twofold increase in ICP22/47 promoter activity in the absence of oriS. DNA synthesis inhibitors resulted in a decrease of both IE- and E-promoter activity in primary cells but not continuous cell cultures. Reporter activity was readily assayed in vivo during acute infection and reactivation from latency and was also sensitive to DNA synthesis inhibitors. In all assays, reporter gene expression was unaffected by the presence or absence of either oriS or oriL. These data support the requirement of DNA synthesis for full viral gene expression in vivo but suggest that the origin elements play no role in the regulation of their flanking promoters.

An assay for quantification of white spot syndrome virus using a capture ELISA

Keywords: capture ELISA, diagnosis, quantification,WSSV.White spot syndrome virus (WSSV) is the causativeagent of a shrimp viral disease which has severelyimpacted most of the shrimp farming regions ofsouth-eastern Asia and presently overshadows allother disease agents as the leading cause ofproduction losses in Asia (Flegel 1997). AlthoughWSSV has been known since 1993, viral quantifi-cation in infected animals was hindered by the lackof a continuous cell culture system for shrimp.Recently, an in vitro system using primary shrimplymphoid cells was used for the quantification ofthe Chinese baculo-like virus (CBV, now known asWSSV) from the haemolymph and gill samples ofexperimentally infected Penaeus stylirostris (Tapay,Lu, Gose, Nadala, Brock & Loh 1997). Acompetitive polymerase chain reaction (PCR)method was developed for quantification of theWSSV genome and successfully applied in analy-sing changes in virus quantity in the haemolymphduring the course of infection (Tang & Lightner2000). A monoclonal antibody (MAb), designated6E1, recognizing an epitope on a major envelopeprotein, VP 28 of WSSV, has been produced andcharacterized (Shih, Wang, Tan & Chen 2001). Inthe present note, we describe the development of adouble antibody sandwich, i.e. capture enzyme-linked immunosorbent assay (ELISA) combiningMAb 6E1 with polyclonal antibodies affinity-purified from WSSV-immunized rat sera. Themethod was applied to monitor changes in con-centration of WSSV proteins in the haemolymphduring the progression of white spot syndrome.The WSSV used in this study was obtained frominfected P. japonicus collected from Ilan in north-eastern Taiwan. The head soft tissues were homo-genized at 4 C in TNE buffer [50 mm Tris,100 mm NaCl, 1 mm ethylene diamine tetraaceticacid (EDTA), pH 7.4] and ultracentrifuged topurify WSSV as described previously (Shih et al.2001). An aliquot of tissue homogenate was dilutedwith TNE buffer and used as the inoculum for thesubsequent infection trial. The purified WSSV wasassayed for protein using a protein assay reagent(BIORAD Laboratories, NY, USA). Adult Wistarrats were immunized and boosted 3 weeks laterwith 100 lg purified WSSV. Immunoglobulin G(IgG) was purified from pooled rat sera using aHiTrap protein A HP column (Pharmacia Biotech,Uppsala, Sweden). The MAb 6E1, isotyped toIgG

A Homogeneous High Throughput Nonradioactive Method for Measurement of Functional Activity of Gs-Coupled Receptors in Membranes

A method is described for measuring the activity of G(s)-coupled receptors in a nonradioactive homogeneous membrane-based assay. This method has several major advantages over currently used methods for measuring functional activity of G(s)-coupled receptors. The assay is high throughput (>150,000 data points/day using a single reader). Dimethyl sulfoxide tolerance is high ( approximately 10%). Compared to complex cell-based assays, there is limited potential for nonspecific compound action. This resulted in low compound hit rates in robustness screening, where hit rates from a simulated screen were 1.0% (antagonist screen) and 0.1% (agonist screen). No continuous cell culture is required for the assay, reducing cell culture overheads and allowing the screen to run every day. Automation is simple and requires no temperature- or humidity-controlled incubation. No radioactivity is required. The method relies on measurement of cyclic AMP (cAMP) generation by fluorescence polarization assay using commercially available reagents. Membranes (1-2 microg protein per well, containing anti-cAMP antibody) are transferred to 384-well plates containing 1 microl test compound. For antagonist screens, agonist is added 15 min later. After 30 min incubation at room temperature, one further assay reagent (fluorescein-cAMP in a buffer containing detergent) is added. The signal may be read after 1 h and is stable for greater than 12 h. Typical Z' for the assay is approximately 0.5.

Chinese medicine induces neurite outgrowth in PC12 mutant cells incapable of differentiation.

During continuous culture of neural PC12 cells, we obtained a drug-hypersensitive PC12 mutant cell that showed high stimulation of neurite outgrowth by various drugs. When several Chinese medicines such as shu-jing-huo-xie-tang and Wu-Ling-San were provided to these PC12 mutant cells, the frequency of nerve growth factor (NGF)-induced neurite outgrowth increased approximately 30-fold compared to NGF alone. Neurite outgrowth induced by NGF in PC12 cells is accompanied by sustained activation of mitogen-activated protein kinase (MAPK); however, these Chinese medicines did not induce MAPK activity. The findings thus indicate that certain Chinese medicines may induce neurite outgrowth by a novel mechanism which is distinct from the NGF-activated pathway in PC12 mutant cells.

Proteomic investigation of metabolic shift in mammalian cell culture.

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down-regulated, and the others were up-regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down-regulated, and the precursor of the 23 kDa subunit of NADH-ubiquinone oxidoreductase, which was up-regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl-terminal hydrolase, which is involved in protein turnover and degradation, was also up-regulated in the metabolically altered cells. The remaining down-regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up-regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up-regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.

Forced periodic operations of continuous recombinant cell cultures subject to antibiotic selection pressure

Sustained production of recombinant proteins encoded by extrachromosomal plasmid DNA over long duration is not possible due to reversion of recombinant cells to unproductive plasmid-free cells. Application of antibiotic selection pressure, the most effective measure to overcome this problem, at a constant rate is prohibitively expensive. Nonlinearities in kinetics of recombinant bioprocesses provide an opportunity to possibly improve the time-average performance of these by periodic forcing of feed conditions. This possibility is investigated in this article using the generalized π-criterion for continuous recombinant cell cultures subject to periodic variations in one or more inputs (feed parameters), viz., dilution rate and feed concentrations of antibiotic and limiting substrate. The two types of antibiotic action considered are: (i) death of plasmid-free cells and (ii) repression of growth of plasmid-free cells. Pertinent analytical observations, which are applicable to continuous processes in general, are made about operations involving periodic forcing of multiple inputs. Where appropriate, analytical expressions are obtained for optimum amplitude ratios, optimum phase differences, and the corresponding maximum improvement in the process performance. The frequency intervals where an improvement in culture performance is guaranteed are identified via numerical illustrations for the two antibiotic types. These illustrations demonstrate that an increase in the number of feed parameters perturbed leads to broadening of the regions in the multidimensional parameter space where forced periodic operations are superior to steady-state operation. The π-criterion is also employed to investigate if forced periodic operation may enable retention of recombinant cells under conditions where such retention is not possible in a steady-state operation.

Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera: Noctuidae).

Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several cells lines were obtained from these P. unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo. The three major midgut cell types were present in the cultures, including stem (regenerative), columnar, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7-8 wk. After approximately six passages, the cell density in T-flasks appeared to be somewhat constant, reaching 10(3)-10(4) cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these cells with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.