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2.
J Biotechnol ; 205: 24-35, 2015 Jul 10.
Article in English | MEDLINE | ID: mdl-25592049

ABSTRACT

Rational development of more physiologic in vitro models includes the design of robust and flexible 3D-microtissue-based multi-tissue devices, which allow for tissue-tissue interactions. The developed device consists of multiple microchambers interconnected by microchannels. Pre-formed spherical microtissues are loaded into the microchambers and cultured under continuous perfusion. Gravity-driven flow is generated from on-chip reservoirs through automated chip-tilting without any need for additional tubing and external pumps. This tilting concept allows for operating up to 48 devices in parallel in order to test various drug concentrations with a sufficient number of replicates. For a proof of concept, rat liver and colorectal tumor microtissues were interconnected on the chip and cultured during 8 days in the presence of the pro-drug cyclophosphamide. Cyclophosphamide has a significant impact on tumor growth but only after bio-activation by the liver. This effect was only observed in the perfused and interconnected co-cultures of different microtissue types on-chip, whereas the discontinuous transfer of supernatant via pipetting from static liver microtissues that have been treated with cyclophosphamide did not significantly affect tumor growth. The results indicate the utility and multi-tissue functionality of this platform. The importance of continuous medium circulation and tissue interaction is highlighted.


Subject(s)
Coculture Techniques/methods , Liver/cytology , Microfluidic Analytical Techniques/instrumentation , Spheroids, Cellular/cytology , Tissue Culture Techniques/methods , Animals , Antineoplastic Agents, Alkylating/pharmacology , Cell Survival/drug effects , Cells, Cultured , Coculture Techniques/instrumentation , Cyclophosphamide/pharmacology , HCT116 Cells/cytology , HCT116 Cells/drug effects , Humans , Microfluidic Analytical Techniques/methods , Rats , Spheroids, Cellular/drug effects , Tissue Culture Techniques/instrumentation
3.
J Lab Autom ; 20(3): 274-82, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25524491

ABSTRACT

In this article, we present a microfluidic platform, compatible with conventional 96-well formats, that enables facile and parallelized culturing and testing of spherical microtissues in a standard incubator. The platform can accommodate multiple microtissues (up to 66) of different cell types, formed externally by using the hanging-drop method, and enables microtissue interconnection through microfluidic channels for continuous media perfusion or dosage of substances. The platform contains 11 separate channels, and each channel has six tissue compartments. Primary rat liver tissues were cultured over 8 days, and multiple tumor tissues (HCT116) were exposed to various concentrations of 5-fluorouracil for platform characterization.


Subject(s)
Cell Culture Techniques , Lab-On-A-Chip Devices/statistics & numerical data , Liver/cytology , Microfluidics/methods , Spheroids, Cellular/cytology , Animals , Fluorouracil/pharmacology , HCT116 Cells , Humans , Liver/drug effects , Primary Cell Culture , Rats , Spheroids, Cellular/drug effects
4.
Nat Commun ; 5: 4250, 2014 Jun 30.
Article in English | MEDLINE | ID: mdl-24977495

ABSTRACT

Integration of multiple three-dimensional microtissues into microfluidic networks enables new insights in how different organs or tissues of an organism interact. Here, we present a platform that extends the hanging-drop technology, used for multi-cellular spheroid formation, to multifunctional complex microfluidic networks. Engineered as completely open, 'hanging' microfluidic system at the bottom of a substrate, the platform features high flexibility in microtissue arrangements and interconnections, while fabrication is simple and operation robust. Multiple spheroids of different cell types are formed in parallel on the same platform; the different tissues are then connected in physiological order for multi-tissue experiments through reconfiguration of the fluidic network. Liquid flow is precisely controlled through the hanging drops, which enable nutrient supply, substance dosage and inter-organ metabolic communication. The possibility to perform parallelized microtissue formation on the same chip that is subsequently used for complex multi-tissue experiments renders the developed platform a promising technology for 'body-on-a-chip'-related research.


Subject(s)
Cell Culture Techniques/methods , Microfluidics/instrumentation , Spheroids, Cellular/cytology , Cell Line, Tumor , Cell Proliferation , Humans , Microfluidics/methods
5.
Biotechnol Bioeng ; 110(2): 648-55, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22949074

ABSTRACT

Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated adherent or aggregate-based culture, consequently limiting robust scale-up of cell production, on-line control and optimization of culture conditions. We recently developed a method that enables continuous (non-serially dissociated) suspension culture-mediated reprogramming to pluripotency. Herein, we use this method to demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen-controlled perfusion culture. Cell densities of greater than 1 × 107 cells/mL were achieved after 7 days of expansion at a specific growth rate (µ) of 0.61 ± 0.1 day⁻¹ with a perfusion rate (D) of 5.0 day⁻¹. A twofold increase in maximum cell density (to greater than 2.5 × 107 cells/mL) was achieved when the medium dissolved oxygen was reduced (5% DO). Cell densities and viabilities >80% were maintained for extended production periods during which maintenance of pluripotency was confirmed by stable expression of pluripotency factors (SSEA-1 and Nanog), as well as the capacity to differentiate into all three germ layers. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable and intensified stirred suspension culture.


Subject(s)
Bioreactors , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Oxygen/chemistry , Oxygen/metabolism , Pluripotent Stem Cells/cytology , Analysis of Variance , Animals , Biotechnology , Cell Differentiation , Cell Survival , Glucose/metabolism , Homeodomain Proteins/metabolism , Lactic Acid/metabolism , Lewis X Antigen/metabolism , Mice , Nanog Homeobox Protein , Oxygen/analysis , Pluripotent Stem Cells/metabolism
6.
Nat Methods ; 9(5): 509-16, 2012 Mar 25.
Article in English | MEDLINE | ID: mdl-22447133

ABSTRACT

We describe derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension culture-reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor-expressing cells based on their differential survival and proliferation in suspension culture. Seamless integration of SiPSC reprogramming and directed differentiation enabled scalable production of beating cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step toward the development of robust PSC generation, expansion and differentiation technology.


Subject(s)
Cell Culture Techniques/methods , Fibroblasts/cytology , Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Animals , Cell Differentiation/physiology , Cellular Reprogramming/physiology , Chimera/physiology , Mice
7.
Nucleic Acids Res ; 40(5): e37, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22187155

ABSTRACT

Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.


Subject(s)
Food Additives/pharmacology , Gene Expression Regulation , Transcription, Genetic/drug effects , Transgenes , Vanillic Acid/pharmacology , Animals , CHO Cells , Caulobacter crescentus/genetics , Cell Line , Cricetinae , Cricetulus , Genetic Engineering , Homeostasis , Kinetics , Mice , Operator Regions, Genetic , Promoter Regions, Genetic , Transcriptional Activation
8.
J Control Release ; 131(3): 211-9, 2008 Nov 12.
Article in English | MEDLINE | ID: mdl-18755229

ABSTRACT

Technologies currently available for the controlled release of protein therapeutics involve either continuous or tissue-specific discharge from implants or engineered extracellular matrix mimetics. For some therapeutic applications the trigger-controlled release of protein cargo from a synthetic implant could be highly desirable. We have designed the CellEase technology, a two-component system consisting of cellulose sulfate (CS) poly-diallyldimethyl ammonium chloride (pDADMAC) capsules harboring mammalian sensor cells transgenic for trigger-inducible expression of an engineered secreted mammalian cellulase (SecCell). SecCell is a Bacillus subtilis-derived (1-4)-beta-glucanase, which was modified by replacing the N-terminal part of the bacterial enzyme with a murine Igkappa-chain V-12-C region-derived secretion signal. SecCell was engineered for doxycycline- or erythromycin-inducible expression to enable trigger-controlled secretion by mammalian cells. Detailed characterization of SecCell showed that it was glycosylated and efficiently secreted by a variety of mammalian sensor cells such that it could internally rupture CS-pDADMAC capsules within which the cells had been encapsulated. When SecCell was inducibly expressed in sender cells, that were co-encapsulated with producer cell lines expressing therapeutic proteins, the removal of relevant inducer molecules enabled the time-dependent discharge of these therapeutic proteins, the kinetics of which could be modified by varying the concentration of inducer molecules or the amount of encapsulated sender cells. SecCell's capacity to rupture CS-pDADMAC capsules exclusively internally also enabled the independent trigger-induced release of different proteins from two capsule populations harboring different inducible SecCell sensor cells. CellEase-based protein release was demonstrated in vivo using capsules implanted intraperitoneally into mice that enabled the doxycycline-controlled release of a model glycoprotein and accumulation in the bloodstream of treated animals. Trigger-induced breakdown of tissue-compatible implants which provide a timely controlled release of biologics may foster novel opportunities in human therapy.


Subject(s)
Allyl Compounds/chemistry , Biocompatible Materials/chemistry , Cellulase/pharmacokinetics , Cellulose/analogs & derivatives , Polymers/chemistry , Quaternary Ammonium Compounds/chemistry , Adenoviridae/genetics , Animals , Antigens, Polyomavirus Transforming/genetics , Bacillus subtilis/enzymology , CHO Cells , Capsules , Cell Line , Cell Line, Tumor , Cellulase/analysis , Cellulase/metabolism , Cellulose/chemistry , Cricetinae , Cricetulus , Delayed-Action Preparations/chemistry , Delayed-Action Preparations/pharmacokinetics , Glycosylation , HeLa Cells , Humans , Kidney/cytology
9.
BMC Biotechnol ; 7: 75, 2007 Nov 06.
Article in English | MEDLINE | ID: mdl-17986332

ABSTRACT

BACKGROUND: Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV) type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features. RESULTS: We designed recombinant adeno-associated virus (rAAV) vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (EON and EOFF) into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7). Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression. CONCLUSION: Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice.


Subject(s)
Dependovirus/genetics , Gene Expression/drug effects , Genetic Vectors/genetics , Macrolides/pharmacology , Transgenes/genetics , Animals , CHO Cells , Cell Line , Cell Line, Tumor , Cricetinae , Cricetulus , Erythromycin/pharmacology , Female , Green Fluorescent Proteins/blood , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mice , Microscopy, Fluorescence , Reproducibility of Results , Transfection/methods
10.
Biotechnol Bioeng ; 98(3): 655-67, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17461419

ABSTRACT

Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream. InXy and SeXy are highly sensitive, compact and robust reporter proteins, fully compatible with pre-existing marker genes and can be assayed in high-throughput formats using very small sample volumes.


Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Endo-1,4-beta Xylanases/genetics , Genes, Reporter/genetics , Kidney/physiology , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Cell Line , Endo-1,4-beta Xylanases/metabolism , Humans , Promoter Regions, Genetic/genetics
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