Development of a dynamic in vitro stretch model of the alveolar interface with aerosol delivery.

We describe the engineering design, computational modeling, and empirical performance of a moving air-liquid interface (MALI) bioreactor for the study of aerosol deposition on cells cultured on an elastic, porous membrane which mimics both air-liquid interface exposure conditions and mechanoelastic motion of lung tissue during breathing. The device consists of two chambers separated by a cell layer cultured on a porous, flexible membrane. The lower (basolateral) chamber is perfused with cell culture medium simulating blood circulation. The upper (apical) chamber representing the air compartment of the lung is interfaced to an aerosol generator and a pressure actuation system. By cycling the pressure in the apical chamber between 0 and 7 kPa, the membrane can mimic the periodic mechanical strain of the alveolar wall. Focusing on the engineering aspects of the system, we show that membrane strain can be monitored by measuring changes in pressure resulting from the movement of media in the basolateral chamber. Moreover, liquid aerosol deposition at a high dose delivery rate (>1 µl cm-2 min-1 ) is highly efficient (ca. 51.5%) and can be accurately modeled using finite element methods. Finally, we show that lung epithelial cells can be mechanically stimulated under air-liquid interface and stretch-conditions without loss of viability. The MALI bioreactor could be used to study the effects of aerosol on alveolar cells cultured at the air-liquid interface in a biodynamic environment or for toxicological or therapeutic applications.

Efficient bioactive delivery of aerosolized drugs to human pulmonary epithelial cells cultured in air-liquid interface conditions.

In inhalation therapy, drugs are deposited as aerosols onto the air-facing lung epithelium. The currently used in vitro cell assays for drug testing, however, typically dissolve drugs in the medium, completely covering the cells, which represents an unphysiological drug application scenario. Although physiologically realistic in vitro cell culture models of the pulmonary air-blood barrier are available, reliable, easy-to-handle, and efficient technologies for direct aerosol-to-cell delivery are lacking. Here, we introduce the Air-Liquid Interface (ALI) Cell Exposure-Cloud (ALICE-CLOUD) technology, which uses principles of cloud motion for fast and quantitative delivery of aerosolized liquid drugs to pulmonary cells cultured under realistic ALI conditions. Aerosol-to-cell delivery proved to be highly efficient, reproducible, and rapid when using aerosolized fluorescein as surrogate drug. As a proof-of-concept study for the ALICE-CLOUD, we performed functional efficacy studies with the U.S. Food and Drug Administration-approved proteasome inhibitor, Bortezomib, a novel candidate drug for inhalation therapy. Aerosolized Bortezomib had a pronounced anti-inflammatory effect on human epithelial lung cells (A549), as indicated by a significant reduction of (TNFα-induced) IL-8 promoter activation. Importantly, cell-based therapeutic efficacy of aerosolized Bortezomib under ALI conditions was similar to that under dissolved and nonaerosolized submerged conditions, but with faster uptake kinetics. Our data indicate that the ALICE-CLOUD is a reliable tool for aerosolized drug screening with cells cultured under ALI conditions, which combines ease of handling with rapid, efficient, and dosimetrically accurate drug-to-cell delivery. This may pave the way for screening of inhalable drugs under physiologically more relevant and, hence, potentially more predictive conditions than the currently used submerged cell culture systems.

In vitro development of engineered muscle using a scaffold based on the pressure-activated microsyringe (PAM) technique.

The development of new human skeletal muscle tissue is an alternative approach to the replacement of tissue after severe damage, for example in the case of traumatic injury, where surgical reconstruction is often needed following major loss of natural tissue. Treatment to date has involved the transfer of muscle tissue from other sites, resulting in a functional loss and volume deficiency of donor sites. Approaches that seek to eliminate these problems include the relatively new solution of skeletal muscle engineering. Here there are two main components to consider: (a) the cells with their regenerative potential; and (b) the polymeric structure onto which cells are seeded and where they must perform their activities. In this paper we describe well-defined two- and three-dimensional polymeric structures able to drive the myoblast process of adhesion, proliferation and differentiation. We examine a series of polymers and protein adhesions with which to functionalize the structures, and cell-seeding methods, with a view to defining the optimal protocol for engineering skeletal muscle tissue. All polymer samples were tested for their mechanical and biological properties, to support the validity of our results in the real context of muscle tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

Polymeric Microporous Nanofilms as Smart Platforms for in Vitro Assessment of Nanoparticle Translocation and Caco-2 Cell Culture.

The study of nanomaterial translocation across epithelial barriers is often hindered by the low permeability of transwell membranes to nanoparticles. To address this issue ultra-thin poly(L-lactic acid) nanofilms with zero tortuosity micropores were developed for use in nanoparticle passage tests. In this study we demonstrate that microporous polymeric nanofilms allow a significantly higher passage of silver nanoparticles in comparison with commercial membranes normally used in transwell inserts. A reliable procedure for collecting free-standing nanofilms which enables their manipulation and use in lab-on-chip systems is described. We also demonstrate the cytocompatibility of porous nanofilms and their ability to sustain the adhesion and proliferation of Caco-2 cells. Ultra-thin microporous membranes show promise as low-cost nanomaterial screening tools and may be used as matrices for the development of bioengineered systems for mimicking the intestinal epithelium.

A bioreactor with an electro-responsive elastomeric membrane for mimicking intestinal peristalsis.

This study describes an actuated bioreactor which mimics the pulsatile contractile motion of the intestinal barrier using electro-responsive elastomers as smart materials that undergo deformation upon electrical stimulation. The device consists of an annular dielectric elastomer actuator working as a radial artificial muscle able to rhythmically contract and relax a central cell culture well. The bioreactor maintained up to 4 h of actuation at a frequency of 0.15 Hz and a strain of 8%-10%, to those of the cyclic contraction and relaxation of the small intestine. In vitro tests demonstrated that the device was biocompatible and cell-adhesive for Caco-2 cells, which formed a confluent monolayer following 21 days of culture in the central well. In addition, cellular adhesion and cohesion were maintained after 4 h of continuous cyclic strain. These preliminary results encourage further investigations on the use of dielectric elastomer actuation as a versatile technology that might overcome the limitations of commercially available pneumatic driving systems to obtain bioreactors that can cyclically deform cell cultures in a biomimetic fashion.