Development of acoustofluidic devices for bronchial epithelial cell sheet creation

Abstract

Epithelial cell (EC) sheet engineering is currently of interest for both regenerative medicine and drug research. Existing cell models for asthma research lack the complexity of heterogeneous tissue, while animal models provide complexity at the expense of molecular pathway accuracy via genetic drift. New models that are both complex and accurate are needed to improve efficacy testing and increase drug translation to the clinic. Acoustofluidic (AF) levitation is the latest, non-invasive cell manipulation technique, that uses a piezoelectric transducer and resonator chamber to create an ultrasonic standing wave field (USWF) to trap cells within a pressure node. AF devices have previously been used to engineer 3D cartilage for transplant, create hepatocyte spheroids, and spatially organise cells within an extracellular matrix to create a vascular co-culture model. This project proposes novel microfluidic AF device designs for EC sheet engineering, utilising inexpensive glass capillaries as bioreactors, with potential for scalability by operating multiple devices in sequence. Levitated EC sheets could be used to engineer scaffold-free, autologous, multilayered, retinal and myocardial constructs for transplant, or more replicative in vitro research models. Bronchial epithelial cells (BECs) were levitated to continue work by Dr Tait developing a direct-contact airway mucosa co-culture model using similar AF devices. The final developed AF devices were able to levitate single BECs for one hour to create singular or multiple BEC sheets which satisfied morphological and functional requirements, such that they behave as cohesive sheet layers that move collectively via plithotaxis. The formation of BEC sheets within AF devices was visualised using immunofluorescence (IF); the surface area was then calculated using ImageJ software. Focal plane adjustments while levitating and IF adherens junction (AJ) staining post-seeding into collagen-coated wells confirmed that BEC sheets were sufficiently planar. Successful adhesion of BEC sheets to collagen before fixation was used as a blunt measure of viability. Transepithelial electrical resistance (TER) of long term BEC sheet cultures was measured using chopstick electrodes and voltohmmeter. TER readings after 11 – 13 days were indicative of tight junction and barrier formation. This study presents the first successful application of an inexpensive, glass capillary-based AF device for EC sheet creation, with potential for higher EC sheet output and easier sheet handling and transfer via a microfluidic system. Further work is needed to confirm the morphology and functionality of successfully levitated EC sheets, as well as experimentation using additional cell types to demonstrate the versatility of AF devices.

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Identifiers

ORCID for Donna Davies: orcid.org/0000-0002-5117-2991

ORCID for Emily Swindle: orcid.org/0000-0003-3644-7747

ORCID for Peter Glynne-Jones: orcid.org/0000-0001-5684-3953

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