Abstract
Stem cell technologies, especially patient‐specific, induced stem cell pluripotency and directed differentiation, hold great promise for changing the landscape of medical therapies. Proper exploitation of these methods may lead to personalized organ transplants, but to regenerate organs, it is necessary to develop methods for assembling differentiated cells into functional, organ‐level tissues. The generation of three‐dimensional human tissue models also holds potential for medical advances in disease modeling, as full organ functionality may not be necessary to recapitulate disease pathophysiology. This is specifically true of lung diseases where animal models often do not recapitulate human disease. Here, we present a method for the generation of self‐assembled human lung tissue and its potential for disease modeling and drug discovery for lung diseases characterized by progressive and irreversible scarring such as idiopathic pulmonary fibrosis (IPF). Tissue formation occurs because of the overlapping processes of cellular adhesion to multiple alveolar sac templates, bioreactor rotation, and cellular contraction. Addition of transforming growth factor‐β1 to single cell‐type mesenchymal organoids resulted in morphologic scarring typical of that seen in IPF but not in two‐dimensional IPF fibroblast cultures. Furthermore, this lung organoid may be modified to contain multiple lung cell types assembled into the correct anatomical location, thereby allowing cell‐cell contact and recapitulating the lung microenvironment. Our bottom‐up approach for synthesizing patient‐specific lung tissue in a scalable system allows for the development of relevant human lung disease models with the potential for high throughput drug screening to identify targeted therapies. Stem Cells Translational Medicine 2017;6:622–633
Highlights
This unit describes a protocol for the generation of bioengineered lung-like tissue known as the lung organoid
The main function of the lung organoid is to serve as a platform for 3D cell culture and pulmonary disease modeling
Organoid formation occurs over the course of several days depending heavily on the initial seeding conditions and the included cell types
Summary
This unit describes a protocol for the generation of bioengineered lung-like tissue known as the lung organoid. This process creates a cavity within the PDMS-cast petri dish with a volume of 2 ml This protocol is used to synthesize alginate beads using an electrostatic droplet generator (Figure 2). Beads can be stored for four weeks before use This protocol is used to coat alginate beads in collagen I and in poly(dopamine) to begin the process of creating 3D lung organoids. Once all excess collagen I is removed, place the beads back on ice. Begin preparing poly (dopamine) solution by taking the 50-ml tube with the dopamine powder and pipette in 50 ml of Tris buffer. 15-ml tube containing alginate beads (Basic Protocol 3) 2-ml custom-built PDMS-cast petri dish (see Basic Protocol 1) 10% bleach Cells cultured in relevant medium. 7. Pipette PDMS-cast petri dish contents into a 15-ml conical tube and allow the cell-coated beads to sediment. 500 ml DMEM/F12, 50/50 (1×; Corning) 50 ml HI fetal bovine serum (Life Technologies) 5 ml GlutaMAX (100×; Life Technologies) 5 ml of 10 mM MEM Non-Essential Amino Acid Solution
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