Days Of Continuous Culture Research Articles

Time-course characterization of whole-transcriptome dynamics of HepG2/C3A spheroids and its toxicological implications

Physiologically relevant in vitro models are a priority in predictive toxicology to replace and/or reduce animal experiments. The compromised toxicant metabolism of many immortalized human liver cell lines grown as monolayers as compared to in vivo metabolism limits their physiological relevance. However, recent efforts to culture liver cells in a 3D environment, such as spheroids, to better mimic the in vivo conditions, may enhance the toxicant metabolism of human liver cell lines. In this study, we characterized the dynamic changes in the transcriptome of HepG2/C3A hepatocarcinoma cell spheroids maintained in a clinostat system (CelVivo) to gain insight into the metabolic capacity of this model as a function of spheroid size and culture time. We assessed morphological changes (size, necrotic core), cell health, and proliferation rate from initial spheroid seeding to 35 days of continuous culture in conjunction with a time-course (0, 3, 7, 10, 14, 21, 28 days) of the transcriptome (TempO-Seq, BioSpyder). The phenotypic characteristics of HepG2/C3A growing in spheroids were comparable to monolayer growth until ∼Day 12 (Day 10–14) when a significant decrease in cell doubling rate was noted which was concurrent with down-regulation of cell proliferation and cell cycle pathways over this time period. Principal component analysis of the transcriptome data suggests that the Day 3, 7, and 10 spheroids are pronouncedly different from the Day 14, 21, and 28 spheroids in support of a biological transition time point during the long-term 3D spheroid cultures. The expression of genes encoding cellular components involved in toxicant metabolism and transport rapidly increased during the early time points of spheroids to peak at Day 7 or Day 10 as compared to monolayer cultures with a gradual decrease in expression with further culture, suggesting the most metabolically responsive time window for exposure studies. Overall, we provide baseline information on the cellular and molecular characterization, with a particular focus on toxicant metabolic capacity dynamics and cell growth, of HepG2/C3A 3D spheroid cultures over time.

Effects of chicken manure extract on the directed cultivation of bio-bait Chlorella vulgaris-rotifer (Brachionus plicatilis) and their fatty acid content

Chicken manure is rich in various nutrients, and it is used to improve the nutritional status of microalgae, which subsequently affects the production and nutrition of rotifers. The investigation of the effect of chicken manure on Chlorella and rotifer cultivation is therefore critical for practical production. Here, to investigate the effects of chicken manure extract (CME) on the continuous culture of the biological bait “Chlorella-rotifer”, different concentrations of CME (Groups 1–5: 0, 5, 10, 15, and 20 mL·L−1, respectively) were added to the culture medium for cultivating Chlorella vulgaris, assessed the growth of C. vulgaris, and the optimal amount of CME for culturing Chlorella was determined; and the effects of the cultured Chlorella as food for rotifer cultivation and fatty acid composition of Chlorella and rotifers were analyzed. In 12 days of continuous culture, no significant difference in Chlorella population density was noted among G3, G4, and G5 (P > 0.05). Following completion of the culturing period, the specific growth rate of Chlorella in G3 (0.14 ± 0.01) and G5 (0.14 ± 0.01) showed no significant difference (P > 0.05), and chlorophyll a (Chl a) content in G3 (3.79 ± 0.06 mg·mL−1) was significantly higher than that in G5 (3.30 ± 0.12 mg·mL−1) (P < 0.05). Hence, we chose G3-cultured Chlorella for rotifer cultivation. In the 120-h rotifer cultivation period, the longevity of G3-bred rotifers (G-CME) (15.06 ± 0.82 d) was significantly more than that of the control group (G-BG11) (13.13 ± 1.27 d) (P < 0.05), and the offspring number of rotifer in G-CME (24.75 ± 3.45) was significantly higher than that in G-BG11 (17.75 ± 3.06) (P < 0.05). Rotifer population density and fecundity were significantly improved by G-CME than by G-BG11. Regarding fatty acid composition, the principal component analysis showed that Chlorella cultured by CME (CME-C) and BG11 (BG11-C) and rotifers that used them as diet (CME-R and BG11-R, respectively) exhibited a variation in principal components, with LC-PUFAs (12.66% ± 0.42%) and n-6 LC-PUFAs (3.99% ± 0.17%) being significantly higher in CME-R than in BG11-R (LC-PUFAs: 9.76% ± 0.81%, n-6 LC-PUFAs: 2.17% ± 0.32%) (P < 0.01). In conclusion, CME can promote the growth and alter the fatty acid composition of Chlorella, thus indirectly improving the growth condition and fatty acid content of rotifers; the optimal amount of CME for the continuous cultivation of the bio-bait “Chlorella-rotifer” was 10 mL·L−1.

A novel, scalable, and modular bioreactor design for dynamic simulation of the digestive tract.

In vitro gut model systems permit the growth of gut microbes outside their natural habitat and are essential to the study of gut microbiota. Systems available today are limited by a lack of scalability and flexibility in the mode of operation. Here, we describe the development of a versatile bioreactor module that can be easily adjusted for culture size and capable of sensing and controlling of environmental parameters such as pH control of culture medium, rate of influx and efflux of the culture medium, and aerobic/anaerobic atmosphere. Bioreactor modules can be operated as single units or linked in series to construct a model of a digestive tract with multiple compartments to allow the growth of microbiota in vitro. We tested the growth of synthetic and natural bacterial communities in a multicompartment continuous dynamic culture model simulation of the mammalian gut. The distal compartments of a sterile system inoculated with the synthetic bacterial community at the proximal module attained a stable bacterial density by 24 h, and all the genera present in the inoculum were firmly established in the distal modules simulating the large intestine at 5 days of continuous culture. A natural bacterial community simultaneously inoculated into the distal modules attained a stable bacterial composition at the phylum level by Day 7 of continuous culture. The findings illustrate the utility of the system to culture mixed bacterial communities which can be used to study the collective biological activities of the cultured microbiota in the absence of host influence.

Laser assisted hatching of human embryos at fertilization check, on day 1 of culture, allows for continuous uninterrupted time-lapse incubation of 5+ days, while producing hatching blastocysts as expected

Objective Human embryos must fully hatch from the Zona Pellucida (ZP) for implantation. Furthermore, studies show that implantation rates increase when the embryo has low energy expenditure. Extended culture of embryos to the blastocyst stage does not allow the natural process of hatching to occur, thus necessitating assisted hatching (AH) to breach the ZP for optimal embryo development. This AH is generally performed on day 3 of culture, however this necessitates interrupting any continuous culture of embryos during incubation from day 1 to 7, especially during Time-Lapse. We set out to determine if human blastocysts possess the ability to reach the hatching stage during 5+ days of continuous culture after laser AH is performed two days earlier than standard; at the Day 1 fertilization check. Design A retrospective data analysis at a single large private fertility center to analyze blastocyst laser hatching performed on Day 1 when embryos undergo 5+ days of continuous culture in a Time-Lapse incubator. Materials and methods Oocytes were retrieved and inseminated using standard clinical practice protocols. 16-18 hours post insemination, the day 1 fertilization check was performed. Embryos with abnormal fertilization (3PN and MPN) were donated for research and underwent AH using a Hamilton Thorne Lykos Laser at a Pulse setting of 290µs per our protocol. The laser hatched embryos were placed into a Time-Lapse Embryoscope in order to monitor their ability to form blastocysts and to undergo hatching. Results A total of 41 abnormally fertilized embryos were cultured for 5+ days, with only 12 embryos undergoing blastulation, which is an expected rate for abnormal embryo development. All 12 embryos that formed blastocysts, hatched successfully (100%). Initial hatching was observed as early as 95 hours post insemination (3.96 days of culture) and the latest observed at 140 hours (5.83 days) with a mean time to start hatching of 112.31 hours (4.68 days). Conclusions This study demonstrates that laser AH performed on day 1 at the fertilization check can produce hatching blastocysts at the expected rate. This allows practices to place embryos into Time-Lapse incubation with completely uninterrupted continuous culture for 5+ days, until the desired hatching blastocyst stage has been achieved. Disclosure None Funding None

Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines.

BackgroundChinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells.ResultsIn this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95–100% of productivity after 40 days of continuous culture (~ 48–56 generations).ConclusionsSuspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.How to cite: Li J, Wei S, Cao C, et al. Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.07.002.

Abstract 35: 3D in vitro system for immuno-oncology: Real-time imaging of drug delivery, tumoroids, and immune cell activity

Abstract A Liquid Like Solid (LLS) 3D culture system in a convenient microtiter plate format enables long-duration culture of patient derived microtumors (&amp;gt;30 days), in situ confocal imaging, 3D cytotoxic drug studies, inclusion of T cells, and measurements of T cell migration, infiltration, and killing. Introduction: Cancer is a disease in 3-dimensions and there is a desperate need for new tools and infrastructure to study immuno-oncology treatment methods in 3D. Fabrication of microtumors using 3D printing in LLS media comprised of soft granular microgels (3-5 μm crosslinked polyacrylamide (PAA) microgel particles: 7.5% PAA) facilitate precise arrangement of detailed assemblies of extra-cellular matrix components and cells (1), including: epithelial cells (breast ATCC MCF10A), stem cells (bone marrow MSC), cancer cells (breast ATCC MCF7, prostate ATCC-PC3, osteosarcoma ATCC-MG63, melanoma ATCC-A375, primary glioblastoma, and osteosarcoma), fibroblasts (ThermoFisher dermal fibroblasts C0045C), endothelial cells (ThermoFisher HUVECs C0035C), and CD4+/CD8+ T cells (PBMC and Jurkat E6-1). Methods: Long term culture was enabled through the design, development, and validation of a modular perfusion system that uses passive negative pressure within a 96-well microtiter plate format to transport liquid growth medium, drugs (doxorubicin and puromycin), antibodies (aPD1 J43 clone, aCD3, aCD28), growth factors, FBS, and metabolic waste without disturbing the spatial organization and positioning of the experiments (i.e. 3D orientation is preserved and the packed bed of microgel particles remain solid). The perfusion velocities are precisely controlled to between 1-100 nm/s by setting the negative pressure, and complete exchange of liquid media can be tailored from hours to days. Results: Real-time imaging in these 3D assays is performed using in situ confocal microscopy under controlled perfusion. Cell motility, adhesion, and dynamic rearrangement of fibroblasts and endothelial cells within a 3D co-culture of microtumors evolved over the first 72 hours. Immunohistochemistry with Ki-67 and PCNA staining indicated active cell proliferation of the tumoroids after 28 days of continuous culture. Tracking of activated CD8+ T cells revealed super-diffusive motion in the presence of 3D tumors within a range of 250 µm. Activated T cell migration speeds have been measured to be between 1.3 and 2.0 μm/s in the 3D LLS media, and preliminary estimates of T cell migration forces are on the order of 1 nN. Conclusions: This integrated system of 3D bioprinting, perfusion culture plates, and confocal microscopy enables in situ 3D studies of cancer biology, immunotherapy, and drug treatment regimens and provides unique insights and measurements of immune cell invasion dynamics in 3D microtumors.

Influence of irrigation with microalgae-treated biogas slurry on agronomic trait, nutritional quality, oxidation resistance, and nitrate and heavy metal residues in Chinese cabbage

Biogas slurry (BS) is a main byproduct of biogas production that is commonly used for agricultural irrigation because of its abundant nutrients and microelements. However, direct application of BS may cause quality decline and nitrate and heavy metal accumulation in crops. To address this issue, a microalgae culture experiment and an irrigation experiment were performed to evaluate the removal efficiencies of nutrients and heavy metals from diluted BS by microalgae Scenedesmus sp. and to investigate the effects of irrigation with microalgae-treated BS (MBS-25, MBS-50, MBS-75, and MBS-100) on nutritional quality, oxidation resistance, and nitrate and heavy metal residues in Chinese cabbage. After 8 days of continuous culture, a ratio of 1/1 for BS/tap water mixture (BS-50) was the optimal proportion for microalgal growth (3.73 g dry cell L−1) and efficient removal of total nitrogen (86.1%), total phosphorus (94.3%), COD (87.5%), Cr (50%), Pb (60.7%), and Cd (59.7%). The pH in MBS-50 medium recovered to the highest level in a shorter period of time and accelerated the gas stripping of ammonia nitrogen and the formation of insoluble phosphate and metals, which partly contributed to the high removal efficiencies. MBS irrigation significantly promoted crop growth; improved nutritional quality, edible taste, and oxidation resistance; and reduced nitrate and heavy metal residues in Chinese cabbage at a large scale. Therefore, microalgae culture was beneficial to reduce negative impacts of BS irrigation in crop growth and agricultural product safety. This study may provide a theoretical basis for the safe utilization of BS waste in agricultural irrigation.

A Rotating Bioreactor for Scalable Culture and Differentiation of Respiratory Epithelium.

Respiratory epithelium is difficult to grow in vitro, as it requires a well-maintained polarizing air-liquid interface (ALI) to maintain differentiation. Traditional methods rely on permeable membrane culture inserts, which are difficult to work with and are ill-suited for the production of large numbers of cells, such as the quantities required for cell-based clinical therapies. Herein, we investigate an alternative form of culture in which the cells are placed on a porous substrate that is continuously rolled, such that the monolayer of cells is alternately submerged in media or apically exposed to air. Our prototype bioreactor is reliable for up to 21 days of continuous culture and is designed for scale-up for large-scale cell culture with continuous medium and gas exchange. Normal human bronchial epithelial (NHBE) cells were cultured on an absorbent substrate in the reactor for periods of 7, 14, and 21 days and were compared to static controls that were submerged in media. Quantification by immunohistochemistry and quantitative PCR of markers specific to differentiated respiratory epithelium indicated increased cilia, mucous production, and tight junction formation in the rolled cultures, compared to static. Together with scanning electron microscopy and paraffin histology, the data indicate that the intermittent ALI provided by the rolling bioreactor promotes a polarized epithelial phenotype over a period of 21 days.

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell-cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.

A Metabolic Prototype for Eliminating Tryptophan From The Genetic Code

We set out to reduce the chemical constitution of a living organism to 19 amino acids. A strain was constructed for reassigning the tryptophan codon UGG to histidine and eliminating tryptophan from Escherichia coli. Histidine codons in the gene for an essential enzyme were replaced with tryptophan codons and the restoration of catalytic activity by missense suppressor His-tRNA bearing a CCA anticodon was selected. We used automated cultivation to assess the stability of this genetic construct during evolution. Histidine to tryptophan mutation at codon 30 in the transketolase gene from yeast and its cognate suppressor tRNA were stably propagated in a tktAB deletant of E. coli over 2500 generations. The ratio of histidine misincorporation at tryptophan sites in the proteome increased from 0.0007 to 0.03 over 300 days of continuous culture. This result demonstrated that the genetic code can be forced to evolve by permanent metabolic selection.

Clinical Application of &lt;em&gt;Sleeping Beauty&lt;/em&gt; and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of &lt;em&gt;Sleeping Beauty&lt;/em&gt; and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase

During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.

Role of trapped air in the formation of cell-and-protein micropatterns on superhydrophobic/superhydrophilic microtemplated surfaces

Air trapped within the interstices of TiO2 nanotube surfaces bearing superhydrophobic/superhydrophilic microtemplated domains controls formation of protein micropatterns but not cell micropatterns. Protein binding from either bovine-serum albumin (BSA) or fetal-bovine serum (FBS) solutions to superhydrophobic domains is blocked in the presence of trapped air, leading to clear protein binding contrast between superhydrophilic and superhydrophobic domains. Protein binds to superhydrophobic domains when air is displaced by sonication, leading to more protein binding to superhydrophobic domains than to superhydrophilic, with concomitantly blurred protein binding contrast. The overall contrast obtained in formation of cell (hFOB1.19, MG63, and HeLa) micropatterns depends on the cell type and protein composition of the fluid phase. All cell types preferentially attach to superhydrophilic domains from each fluid phase tested (FBS, BSA, and basal media containing no protein). All cell types do not attach to superhydrophobic domains from FBS solutions, with-or-without trapped air, creating a visually-obvious cell attachment pattern. However, cells attached to superhydrophobic domains from basal media suspensions, with-or-without trapped air, creating a blurred cell attachment pattern. Cell attachment from BSA-containing solutions gave mixed results depending on cell type. Thus, trapped air does not necessarily block cell attachment as has been suggested in the literature. Rather, cell attachment is controlled by interfacial tensions between cells, surfaces, and fluid phases in a manner that can be understood in terms of the Dupré work-of-adhesion formulation. Cell attachment patterns developed within the initial attachment phase persist for up to two days of continuous culture but overgrow thereafter, with-or-without trapped air, showing that trapped air does not block cell overgrowth over time of continuous culture.

Relationship among growth curve, nutrient consumption and genetic transformation efficiency of 'Albariño' ( Vitis vinifera ) cell suspensions

Embryogenic cell suspensions of grapevine ( Vitis sp.) have been proposed as the best target to approach genetic transformation challenges. However, optimal phase and growth period of cell suspensions for successful gene transfer have not been investigated. Here, a step by step protocol to initiate and establish cell suspensions of 'Albarino' ( V. vinifera ) in only 4 weeks is presented. Growth kinetics, cell viability and nutrient consumption (phosphates and nitrates) as well as the number of transient transgenic events (using the uidA reporter gene) were studied in 'Albarino' cell suspensions grown for an 18-days period. Based on biomass growth, the exponential phase of cell suspensions was reached between days 3 to 6. Nutrient uptake results point to the exhaustion of phosphate in the culture medium at day 6. Moreover, the highest number of transgenic events after biolistic bombardment was obtained from cell suspensions grown for 6 days (4032 ± 695 blue spots), compared to 12 and 18 days of continuous culture. Plant regeneration percentage varied depending on the age of the culture and the selected embryo type. In conclusion, this paper shows for the first time the relationship between growth curve and nutrient consumption of embryogenic cell suspension with efficiency in genetic transformation and plant regeneration of grapevine.

Optimization of growth and production of toxins by three dinoflagellates in photobioreactor cultures

A bioreactor system for biotoxin production was appraised against traditional methods of growing dinoflagellate cultures. In an optimised bioreactor culture (5.4 L) operated in batch mode, growth of Karenia selliformis was more efficient than in 15-L bulk carboy culture in terms of growth rate (μ = 0.07 day−1 versus 0.05 day−1) and growth maximum (G max, 169.106 versus 41.106 cells L−1). Maximal gymnodimine concentration (1200 μg L−1) in bioreactor culture was 8-fold higher than in bulk carboy culture, and the yield per cell (pg cell−1) was 2-fold higher. Similarly the bioreactor batch culture of Alexandrium ostenfeldii performed more efficiently than carboy cultures in terms of growth rate (1.6-fold higher), growth maximum (15-fold higher) and desmethyl C spirolide (SPX-desMe-C) yield (5-fold higher [μg L−1], though the yield [pg cell−1basis] was lower). When bioreactor cultures of K. selliformis were operated in continuous mode, the yield of gymnodimine was substantially higher than a carboy or the bioreactor run in batch mode to growth max (793 μg day−1 over 58 days in continuous culture was achieved versus an average of 60 μg day−1 [carboy over 40 days] or 249 μg day−1 [batch mode] over 26 days). Likewise in continuous bioreactor cultures of A. ostenfeldii run over 25 days, the yield of SPX-desMe-C (29 μg day−1) was substantially higher than in same cultures run in batch mode or carboys (10.2 day−1 and 7.7 μg day−1 respectively). Similarly 5.4 L bioreactor batch cultures of K. brevisulcata reached 3.8-fold higher cell densities than carboy cultures, and when operated in continuous mode, the brevisulcatic acids were more efficiently produced than in batch culture (12 μg day−1 versus 7 μg day−1). When the bioreactor system was upscaled to 52 L, the maximum cell densities and toxin yields of K. brevisulcata cultures were somewhat less than those achieved in the smaller reactor, which was attributed to reduced light penetration.

Persistence of Resistance Plasmids Carried by Beta-HemolyticEscherichia coliWhen Maintained in a Continuous-Flow Fermentation System Without Antimicrobial Selection Pressure

It is thought that antimicrobial resistance imposes a fitness cost on bacteria, so that a reduction in antimicrobial use may reduce the incidence of resistant bacteria. The objectives of the present study were to determine (1) whether multidrug resistant (MDR) Escherichia coli field strains with different plasmid profiles show disparate plasmid loss when grown over time without selection pressure; (2) whether the number of plasmids present in the cell affects growth. Nine β-hemolytic E. coli strains from swine (n=8) and cattle (n=1) were grown in separate continuous-flow vessels for 36 days without antimicrobial selection. Populations were enumerated on brain heart infusion agar and brain heart infusion agar with tetracycline on days 2, 5, 8, 15, 22, 29, and 36. Growth rates, plasmid profiles and susceptibility profiles of the strains were compared, and day 36 isolates (n=40, five for each MDR strain) were compared with their corresponding day 0 strains. Plasmid content of the nine field strains ranged from zero to eight with sizes from 3.2 to 165 kb. Changes in susceptibility profiles of day 36 isolates were observed among 20% (8 of 40) of the isolates. MDR E. coli largely maintained their original plasmid profiles, replicon types, and susceptibility profiles over 36 days of continuous culture. There was no significant difference in maximum specific growth rate among strains when compared with the plasmid-free strain or when day 36 isolates were compared with their own day 0 strain. This suggests that there is little fitness cost in the maintenance of multiple plasmids of various sizes under the conditions of this study. Other strategies rather than merely reducing antimicrobial usage are needed to combat the emergence of MDR bacteria.

Evaluation of Pluripotency Gene Expression in Mouse Embryonic Stem Cell Cultured on the Human Feeder Layer

Embryonic stem (ES) cells are derived from the pluripotent inner cell mass (ICN) cells of blastocysts with the potential to maintain an undifferentiated state indefinitely. The derivation process involves plating of the blastocysts on mouse embryonic fibroblast (MEF) and expansion of the outgrowth in to established ES cell line. ES cell are capable of unlimited self-renewal by symmetric division and differentiated cells to all primitive embryonic germ layers. The capacity of ES cells to differentiate in to almost all the cell types of human body highlights their potential to play a promising role in cell replacement therapies for treatment of human diseases. In this study, MEFs have been replaced with human mesenchymal stem cells (hMSCs). C4 mES cell (mouse embryonic stem cell line) colonies are cultured on inactivated hMSCs amplified ≥ 600-folds during the 30 days of continuous culture. The longest continuous expansion of C4 mES cells on hMSC was 30 passages. In this study the gene expression for Oct-4, Nanog, Rex1, Brachyury, LIF, LIFR, TERT, B2M, Stat3, Sox2, Fgf4 in mES cells using reverse transcriptase polymerase chain reaction (RT-PCR) and in which genes expression for Stat3, Sox2, Fgf4 genes was negative whilst the gene expansion for Oct-4, Nanog, Rex1, Brachyury, LIF, LIFR, TERT, B2M genes was positive. There was also a karyotype analysis for ES which showed normal result. The immunocytochemical analysis of Oct4 transcriptional factor for ES cells was made which showed positive result for this factor. These genes may be novel candidates to play critical roles in the regulation of ES Cell pluripotency and self-renewal.

A microfluidic device for continuous cancer cell culture and passage with hydrodynamic forces

We demonstrate a novel and robust microfluidic chip with combined functions of continuous culture and output of PC-3 prostate cancer cells. With digital controls, polydimethylsiloxane (PDMS) flexible diaphragms are able to apply hydrodynamic shear forces on cultures, detaching a fraction of attached cancer cells from the surface for output while leaving others for reuse in subsequent cultures. The fractions of detached cells and remaining cells can be precisely controlled. The system has not only the advantages of small size, high cell culture efficiency, and digital control, but also of simple fabrication at low cost, easy operation and robust performance. The chip performs 9 passages during 30 days of continuous culture and shows promise as a durable design suitable for long-term cell output.

The generation of stable, high MAb expressing CHO cell lines based on the artificial chromosome expression (ACE) technology

The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable high expressing transfectants is normally laborious and time consuming. To improve this process, the ACE System has been developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for the targeted transfection of single or multiple genes and eliminates the need for random integration into native host chromosomes. To illustrate the utility of the ACE System in generating stable, high expressing cell lines, CHO based candidate cell lines were generated to express a human monoclonal IgG1 antibody. Candidate cell lines were generated in under 6 months and expressed over 1 g/L and with specific productivities of up to 45 pg/cell/day under non-fed, non-optimized shake flask conditions. These candidate cell lines were shown to have stable expression of the monoclonal antibody for up to 70 days of continuous culture. The results of this study demonstrate that clonal, stable monoclonal antibody expressing CHO based cell lines can be generated by the ACE System rapidly and perform competitively with those cell lines generated by existing technologies. The ACE System, therefore, provides an attractive and practical alternative to conventional methods of cell line generation.