On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

The inaccessibility of living bone marrow (BM) hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human BM-on-a-chip (BM chip) supports the differentiation and maturation of multiple blood cell lineages over 4 weeks while improving CD34+ cell maintenance, and that it recapitulates aspects of BM injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation, as well as BM recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34+ cells and BM-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that BM chips containing cells from patients with the rare genetic disorder Shwachman-Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil maturation abnormality. As an in vitro model of haematopoietic dysfunction, the BM chip may serve as a human-specific alternative to animal testing for the study of BM pathophysiology.

Biomimetic smoking robot for in vitro inhalation exposure compatible with microfluidic organ chips.

Exposure of lung tissues to cigarette smoke is a major cause of human disease and death worldwide. Unfortunately, adequate model systems that can reliably recapitulate disease biogenesis in vitro, including exposure of the human lung airway to fresh whole cigarette smoke (WCS) under physiological breathing airflow, are lacking. This protocol extension builds upon, and can be used with, our earlier protocol for microfabrication of human organs-on-chips. Here, we describe the engineering, assembly and operation of a microfluidically coupled, multi-compartment platform that bidirectionally 'breathes' WCS through microchannels of a human lung small airway microfluidic culture device, mimicking how lung cells may experience smoke in vivo. Several WCS-exposure systems have been developed, but they introduce smoke directly from above the cell cultures, rather than tangentially as naturally occurs in the lung due to lateral airflow. We detail the development of an organ chip-compatible microrespirator and a smoke machine to simulate breathing behavior and smoking topography parameters such as puff time, inter-puff interval and puffs per cigarette. Detailed design files, assembly instructions and control software are provided. This novel platform can be fabricated and assembled in days and can be used repeatedly. Moderate to advanced engineering and programming skills are required to successfully implement this protocol. When coupled with the small airway chip, this protocol can enable prediction of patient-specific biological responses in a matched-comparative manner. We also demonstrate how to adapt the protocol to expose living ciliated airway epithelial cells to smoke generated by electronic cigarettes (e-cigarettes) on-chip.

Human Lung Small Airway-on-a-Chip Protocol.

Organs-on-chips are microfluidic cell culture devices created using microchip manufacturing techniques that contain hollow microchannels lined by living cells, which recreate specialized tissue-tissue interfaces, physical microenvironments, and vascular perfusion necessary to recapitulate organ-level physiology in vitro. Here we describe a protocol for fabrication, culture, and operation of a human lung "small airway-on-a-chip," which contains a differentiated, mucociliary bronchiolar epithelium exposed to air and an underlying microvascular endothelium that experiences fluid flow. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin rigid porous membrane; this requires less than 1 day to complete. Next, primary human airway bronchiolar epithelial cells isolated from healthy normal donors or patients with respiratory disease are cultured on the porous membrane within one microchannel while lung microvascular endothelial cells are cultured on the opposite side of the same membrane in the second channel to create a mucociliated epithelium-endothelium interface; this process take about 4-6 weeks to complete. Finally, culture medium containing neutrophils isolated from fresh whole human blood are flowed through the microvascular channel of the device to enable real-time analysis of capture and recruitment of circulating leukocytes by endothelium under physiological shear; this step requires less than 1 day to complete. The small airway-on-a-chip represents a new microfluidic tool to model complex and dynamic inflammatory responses of healthy and diseased lungs in vitro.

Matched-Comparative Modeling of Normal and Diseased Human Airway Responses Using a Microengineered Breathing Lung Chip.

Smoking represents a major risk factor for chronic obstructive pulmonary disease (COPD), but it is difficult to characterize smoke-induced injury responses under physiological breathing conditions in humans due to patient-to-patient variability. Here, we show that a small airway-on-a-chip device lined by living human bronchiolar epithelium from normal or COPD patients can be connected to an instrument that "breathes" whole cigarette smoke in and out of the chips to study smoke-induced pathophysiology in vitro. This technology enables true matched comparisons of biological responses by culturing cells from the same individual with or without smoke exposure. These studies led to identification of ciliary micropathologies, COPD-specific molecular signatures, and epithelial responses to smoke generated by electronic cigarettes. The smoking airway-on-a-chip represents a tool to study normal and disease-specific responses of the human lung to inhaled smoke across molecular, cellular and tissue-level responses in an organ-relevant context.