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02995nam a2200265Ia 4500 |
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10.3791-63875 |
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|a 1940087X (ISSN)
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|a Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells
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|b Journal of Visualized Experiments
|c 2022
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|z View Fulltext in Publisher
|u https://doi.org/10.3791/63875
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|a In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as "matrix"), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs. © 2022 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.
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|a Abo, K.M.
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|a Alber, A.B.
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|a Alysandratos, K.-D.
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|a Berthiaume, K.
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|a Burgess, C.L.
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|a Hix, O.T.
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|a Huang, J.
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|a Kotton, D.N.
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|a Merritt, C.
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|a Minakin, K.
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|a Werder, R.B.
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|a Wilson, A.A.
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|t Journal of Visualized Experiments
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