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Protocol for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue.

ABSTRACT: Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility. For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023).¹.

SUBMITTER: Castaneda DC

PROVIDER: S-EPMC10696416 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Protocol for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue.

Castaneda Diana Cadena DC Jangra Sonia S Yurieva Marina M Martinek Jan J Callender Megan M Coxe Matthew M Choi Angela A García-Bernalt Diego Juan J Wu Te-Chia TC Marches Florentina F Chaussabel Damien D García-Sastre Adolfo A Schotsaert Michael M Williams Adam A Palucka Karolina K

STAR protocols 20231121 4

Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol d ...[more]

PMID: 37991921

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Project description:BackgroundAlthough biological resources are essential for basic and preclinical research in the oncological field, those of sarcoma are not sufficient for rapid development of the treatment. So far, some sarcoma cell lines have been established, however, the success rate was low and the established sarcoma types were frequently biased. Therefore, an efficient culture method is needed to determine the various types of sarcomas. Organoid culture is a 3-dimentional culture method that enables the recapitulation of the tumor microenvironment and the success rate reported is higher than the 2-dimentional culture. The purpose of this study was to report our newly established organoids from human epithelioid sarcoma using the air-liquid interface organoid culture method.MethodsWe treated 2 patients with epithelioid sarcoma in our institute. The remaining sarcoma specimens after surgical resection were embedded in collagen type 1 gels according to the air-liquid interface organoid culture method. After serial passages, we xenografted the organoids to NOD-scid IL2Rgnull (NSG) mice. Using the developed tumors, we performed histological and genomic analyses to compare the similarities and differences with the original epithelioid sarcoma from the patient.ResultsOrganoids from the epithelioid sarcoma could be serially cultured and maintained in collagen type 1 gels for more than 3 passages. Developed orthotopic tumor xenografts were detected in the NSG mice. After the process was repeated severally, the patient derived organoid lines from the epithelioid sarcoma were established. The established organoids showed loss of integrase interactor 1 expression with polymerase chain reaction and immunohistochemical analyses. The xenografted organoids of the epithelioid sarcoma had histologically similar phenotypes with the original tumor and genetically resembled it to some degree.ConclusionsThe present study demonstrated 2 novel established organoid models of epithelioid sarcoma, and our organoid models could be used to investigate the molecular pathogenesis and develop a novel treatment.

Dataset Information

Protocol for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue.

Publications

Protocol for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue.

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