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A perfusable 3D cell-matrix tissue culture chamber for in situ evaluation of nanoparticle vehicle penetration and transport.

ABSTRACT: A key factor in gene or drug therapy is the development of carriers that can efficiently reach targeted cells from a distal administration. In many gene/drug delivery studies, results obtained in 2D cultures fail to translate to similar results in vivo. In this work, we developed a perfusable 3D chamber for studying nanoparticle penetration and transport in cell-gel soft tissue cultures. The compartmented chamber is made of a polydimethylsiloxane (PDMS) top layer with the chamber features, created using micromachined lithography, bonded to a bottom glass coverslip. A solution of cells embedded in a hydrogel is loaded in the chamber between PDMS posts that serve as anchors to the cell-matrix at the gel-media interface. The chamber offers the following unique features: (i) rapid fabrication and simplicity in assembly, (ii) direct in situ cell imaging in a plane normal to the direction of flow or action, (iii) an easily configurable and controllable environment conducive cell culture under static or interstitial flow conditions, and (iv) facile recovery of live cells from chambers for post-experimental analysis. To assess the chamber, we delivered fluorescently labeled nanoparticles of three distinct sizes to cells-embedded Matrigels in the 3D chamber under flow and static conditions. Penetration of nanoparticles were enhanced under interstitial flow while live cell imaging and flow cytometry of recovered cells revealed particle size restrictions to efficient delivery. Although designed for delivery studies, the chamber is versatile and can be easily modified. Thus it may have broad applications for biological, tissue engineering, and therapeutic studies.

SUBMITTER: Ng CP

PROVIDER: S-EPMC2796126 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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A perfusable 3D cell-matrix tissue culture chamber for in situ evaluation of nanoparticle vehicle penetration and transport.

Ng Chee Ping CP Pun Suzie Hwang SH

Biotechnology and bioengineering 20080401 6

A key factor in gene or drug therapy is the development of carriers that can efficiently reach targeted cells from a distal administration. In many gene/drug delivery studies, results obtained in 2D cultures fail to translate to similar results in vivo. In this work, we developed a perfusable 3D chamber for studying nanoparticle penetration and transport in cell-gel soft tissue cultures. The compartmented chamber is made of a polydimethylsiloxane (PDMS) top layer with the chamber features, creat ...[more]

PMID: 17969174

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Project description:Devices for in vitro culture of three-dimensional (3D) skeletal muscle tissues have multiple applications, including tissue engineering and muscle-powered biorobotics. In both cases, it is crucial to recreate a biomimetic environment by using tailored scaffolds at multiple length scales and to administer prodifferentiative biophysical stimuli (e.g., mechanical loading). On the contrary, there is an increasing need to develop flexible biohybrid robotic devices capable of maintaining their functionality beyond laboratory settings. In this study, we describe a stretchable and perfusable device to sustain cell culture and maintenance in a 3D scaffold. The device mimics the structure of a muscle connected to two tendons: Tendon-Muscle-Tendon (TMT). The TMT device is composed of a soft (E ∼ 6 kPa) porous (pore diameter: ∼650 μm) polyurethane scaffold, encased within a compliant silicone membrane to prevent medium evaporation. Two tendon-like hollow channels interface the scaffold with a fluidic circuit and a stretching device. We report an optimized protocol to sustain C2C12 adhesion by coating the scaffold with polydopamine and fibronectin. Then, we show the procedure for the soft scaffold inclusion in the TMT device, demonstrating the device's ability to bear multiple cycles of elongations, simulating a protocol for cell mechanical stimulation. By using computational fluid dynamic simulations, we show that a flow rate of 0.62 mL/min ensures a wall shear stress value safe for cells (<2 Pa) and 50% of scaffold coverage by an optimal fluid velocity. Finally, we demonstrate the effectiveness of the TMT device to sustain cell viability under perfusion for 24 h outside of the CO2 incubator. We believe that the proposed TMT device can be considered an interesting platform to combine several biophysical stimuli, aimed at boosting skeletal muscle tissue differentiation in vitro, opening chances for the development of muscle-powered biohybrid soft robots with long-term operability in real-world environments.

Project description:BackgroundMulti-walled carbon nanotubes (MWCNT) have been shown to elicit the release of inflammatory and pro-fibrotic mediators, as well as histopathological changes in lungs of exposed animals. Current standards for testing MWCNTs and other nanoparticles (NPs) rely on low-throughput in vivo studies to assess acute and chronic toxicity and potential hazard to humans. Several alternative testing approaches utilizing two-dimensional (2D) in vitro assays to screen engineered NPs have reported conflicting results between in vitro and in vivo assays. Compared to conventional 2D in vitro or in vivo animal model systems, three-dimensional (3D) in vitro platforms have been shown to more closely recapitulate human physiology, providing a relevant, more efficient strategy for evaluating acute toxicity and chronic outcomes in a tiered nanomaterial toxicity testing paradigm.ResultsAs inhalation is an important route of nanomaterial exposure, human lung fibroblasts and epithelial cells were co-cultured with macrophages to form scaffold-free 3D lung microtissues. Microtissues were exposed to multi-walled carbon nanotubes, M120 carbon black nanoparticles or crocidolite asbestos fibers for 4 or 7 days, then collected for characterization of microtissue viability, tissue morphology, and expression of genes and selected proteins associated with inflammation and extracellular matrix remodeling. Our data demonstrate the utility of 3D microtissues in predicting chronic pulmonary endpoints following exposure to MWCNTs or asbestos fibers. These test nanomaterials were incorporated into 3D human lung microtissues as visualized using light microscopy. Differential expression of genes involved in acute inflammation and extracellular matrix remodeling was detected using PCR arrays and confirmed using qRT-PCR analysis and Luminex assays of selected genes and proteins.Conclusion3D lung microtissues provide an alternative testing platform for assessing nanomaterial-induced cell-matrix alterations and delineation of toxicity pathways, moving towards a more predictive and physiologically relevant approach for in vitro NP toxicity testing.

Dataset Information

A perfusable 3D cell-matrix tissue culture chamber for in situ evaluation of nanoparticle vehicle penetration and transport.

Publications

A perfusable 3D cell-matrix tissue culture chamber for in situ evaluation of nanoparticle vehicle penetration and transport.

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