Survey of previously described microscale

1.3.3.1 Microscale devices applied to respiratory biology

Several microfabricated platforms have been developed and applied to respiratory biology inquiries. Reports of these platforms were consulted for their potential for use with primary AT1 cells. Because they can be patterned so easily and with such a high degree of control over their dimensions, a number of platforms utilize cell-lined microchannels to examine the role of shear stresses on cell attachment and vitality. Propagation of liquid plugs down the microchannels simulated the opening of occluded airways, providing data relevant

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to studies of pulmonary surfactant dysfunction or depletion.177-179 Building on these past works, a study by Douville et al. created a microfluidic model of the alveolus in which sheets of primary murine alveolar cells or cells from the human immortalized alveolar epithelial A549 cell line were grown on a flexible membrane of PDMS subjected to a migrating ALI.180 This study demonstrated the role of migrating ALIs in worsening the cellular damage caused by cyclic stretch/compression as compared to cells that were entirely submerged or air- exposed when stretched, results relevant to research on ventilator-induced lung injury.

Additionally, a seminal study by the Ingber Group engineered a “lung-on-a-chip” device that featured a microporous membrane separating two microchannels.181 On one side of the membrane, a sheet of immortalized endothelial cells were exposed to a fluid filled microchannel. The opposite side of the membrane was covered with sheets of cells from either small airway NCI H-441 or alveolar A549 cell lines, creating an ALI across the membrane. A noteworthy feature of this device was the pair of microchannels that laterally bordered the membrane: reducing or normalizing air pressure to these side channels created stretch or relaxation forces that simulated the expansion of the pulmonary tissues that occurs during inspiration.

Several of the studies cited above are surveyed in a review of efforts to study

respiratory physiology on chip-based platforms.182 This review reiterates the need for an ALI in culture of pulmonary cells as a way to recapitulate true in vivo architecture. Several

attempts to create a co-culture model of the alveolar-capillary barrier, typically via culture of alveolar and endothelial cell lines on opposite sides of a porous membrane, were discussed. Of note, this review includes coverage of chip-based research efforts at extracorporeal

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membrane oxidation (ECMO), a direct therapeutic end goal not falling within the physiology- and pharmacology-oriented research goals of this dissertation.

While useful advances, none of these devices are suited for research with primary AT1 cells, for at least one of a variety of reasons: incompatibility with primary cell use, designs that do not feature permeable supports, or designs that do not appropriately replicate the physicochemical features of the alveolar architecture. For example, while the “lung-on-a- chip” represents an impressive engineering advance, its use of sheets of cells grown atop a membrane with large, 10 µm-diameter pores does not sufficiently replicate the alveolar microenvironment, which features smaller physical concavities approximately 250 µm in diameter whose basement membrane has submicron-scale porosity.1, 183 The microfluidic alveolar model developed by Douville et al. has similar shortfalls: sheets of cells 6 mm in diameter, grown on an impermeable membrane 100 µm thick, do not replicate the alveolar microenvironment seen by an AT1 cell. Given the shortcomings of these otherwise laudable devices as they relate to AT1 culture, other examples of microtechnology that utilize

permeable membranes were explored.

1.3.3.2 Microscale devices utilizing a permeable support

To date there has been some success in combining microscale technologies with porous or otherwise permeable materials, even if they have not been used in respiratory biology studies. Porous membranes are used most commonly in microfluidic setups

fabricated with PDMS, and a number of studies focused on methods in which to incorporate the membranes into the fabricated devices. A common design for these systems involves the fabrication of devices using stacked PDMS layers with a microporous membrane sandwiched in the middle, creating separate PDMS flow channels separated by the membrane. The

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membranes and the PDMS layers are most commonly bonded either indirectly using a thin PDMS mortar layer or directly via the organosilane 3-aminopropyltriethoxysilane

(APTES).184-186 However, most of the studies cited above bond only the membrane edges, at the macroscopic scale, to the borders of PDMS layers. A literature review did not locate any studies that involve bonding membranes to PDMS films with a microscale pattern, likely due in one part to the relative difficulty in making through-hole arrays in such films in the first place and due in another part to difficulty in establishing uniform seals around each microwell that eliminate communicating gaps between them. Indeed, the smallest area of membrane exposed was still 4 mm2.187 Use of “microfluidic stickers” presents an alternative method that is chemically distinct from the sandwich methods described above, but nearly identical in general concept.188

Studies employing the PDMS-membrane sandwich method have used the membrane in a microfluidic setup in such varied applications as establishing standing chemical gradients to which cells can be exposed without the harsh shearing forces of high flow rates.188-190 In a general sense, seeding cells onto one side of the membrane allows for single-stream

perfusion at relatively slow rates – or even static culture – that do not introduce shearing stresses to the cell. On the other side of the membrane, flows from two entering fluid

streams, one of which carries a solute of interest, meet in the middle. This interface allows a standing chemical gradient of the solute to be set up along an axis orthogonal to the flow direction in the middle of the channel, with the extent of the gradient controlled by the flow rates. Because the cells are separated from these higher flow rates by the membrane, shearing forces are spared. A second category of applications for this kind of microfluidic setup is that of a plasma filter. Both Aran et al. and Son et al. describe use of the membrane for isotonic

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and hemolysis-free filtration of plasma from whole blood.191-192 By using membranes with pores too small for red blood cells, whole blood infused into the system could be efficiently separated into two compartments, with one compartment retaining erythrocytes and

leukocytes and the other containing only plasma, with low shear stress and minimal erythrocyte hemolysis.

Other reports utilize the membrane to recapitulate the polarization seen in vivo of certain types of non-respiratory epithelial cells. For example, Jang and Suh created a

microfluidic “kidney-on-a-chip” using PDMS bonded to a porous membrane on which sheets of primary rat inner medullary collecting duct (IMCD) cells were cultured.193-194 The

McGuigan Group successfully patterned MDCK and retinal epithelial cells into

microcolonies atop commercially-available porous substrates.195 A device fabricated as an extension of “lung-on-a-chip” technology, described in the previous section, was used to grow cells from an immortalized colon adenocarcinoma-derived cell line, hence the term “gut-on-a-chip.”196

Alternatively, insertion of cylindrical electrodes on each side of the membrane allows measurement of the transepithelial electrical resistance (TEER) of a monolayer of cells cultured on the membrane.187 This concept was later extended in a microfluidic model of the blood-brain barrier that incorporated electrodes to measure TEER into the device.197

A few devices do attempt to introduce micropatterns onto porous membranes. The Takayama Group developed a method to microstamp PDMS that could pattern microwells 5 µm in height and as small as 50 µm in diameter atop a porous polyester membrane.198 Two reports – one of which used a combination of electrospun polyblend fibers and soft

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combined microscale patterning with a unique permeable surface, but fabrication of these devices requires both large manpower and highly specialized equipment.199-200 A similar requirement for specialized equipment is one drawback to a recent report describing the ability to thermoform polyester or polycarbonate into films with 3D topographies.201 The fabricator must have ready access to not only a specialty thermoforming apparatus, but also to a heavy ion accelerator in order to track-etch pores into the final product.

Despite the many examples presented, these platforms have inherent limitations that prevent their immediate use or adaptation for AT1 cells. Chief among these limitations is an inability to pattern the membranes themselves, either directly or via the enclosures of a microfluidic device to the specifications needed by a device for primary AT1 culture. Indeed, the McGuigan Group’s cell colony micropatterns and the Takayama Group’s orthogonally- oriented microchannel array represent the works most relevant to culture of primary AT1 cells, but each of these two methods has detractions: the McGuigan Group’s micropatterns appear to be limited to features larger than approximately 250 µm (comparative AT1

diameter is 50-100 µm), and the Takayama Group’s PDMS microstamps were so thin (5 µm in height) that cells readily migrated out of the microwells and spread over the entire

surface.195, 198

1.3.4 Design guidelines for a microscale device applicable to AT1 cells