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LUNG Publication-derived

Lung Cancer Tumouroids for Drug Screening

Full SOP with Academic or a plan

Source Kim et al., 2019 · Asan Institute for Life Sciences, Seoul, South Korea · 10.1038/s41467-019-11867-6

Authors: Minsuh Kim, Hyemin Mun, Chang Oak Sung, Eun Jeong Cho, Hye-Joon Jeon, Sung-Min Chun, Da Jung Jung, Tae Hoon Shin, Gi Seok Jeong, Dong Kwan Kim, Eun Kyung Choi, Seong-Yun Jeong, Alison M. Taylor, Sejal Jain, Matthew Meyerson, Se Jin Jang Duration: 21 days 7 phases Cell source: Patient-Derived Lung Cancer Tissues (Multiple Histological Subtypes)

Abstract

This protocol describes the establishment and culture of lung cancer organoids (LCOs) and normal bronchial organoids (NBOs) from patient-derived lung tissues. LCOs maintain the histological, genetic, and phenotypic characteristics of original tumors while enabling rapid patient-specific drug screening and therapeutic response prediction in a 3D culture system.

Cell source
Patient-Derived Lung Cancer Tissues (Multiple Histological Subtypes)
Application
Disease modeling and drug screening

Protocol overview

43 steps across 7 phases

Phase 1 · Tissue Preparation and Quality Control Day 0-1
  1. a Collect and transport tissue samples
  2. b Wash tissue samples
  3. c Section tissue samples
  4. d Dissociate tissue and prepare cell suspension
  5. e Triturate and filter cell suspension
  6. f Centrifuge and prepare quality control sample
Phase 2 · Lung Cancer Organoid (LCO) Culture Initiation Day 1-7
  1. a Prepare MBM (Minimum Basal Medium)
  2. b Seed LCOs in Matrigel
  3. c Add culture medium
  4. d Incubate and observe organoid formation
  5. e Change medium every 4 days
Phase 3 · LCO Passaging and Expansion Day 8-21 and beyond
  1. a Determine passaging time point
  2. b Harvest organoids from Matrigel
  3. c Wash organoid pellet
  4. d Dissociate organoids to single cells
  5. e Inactivate trypsin and centrifuge
  6. f Wash and resuspend cells
  7. g Solidify Matrigel and add medium
  8. h Continue culture and monitor growth
Phase 4 · Normal Bronchial Organoid (NBO) Culture Day 1-7 and beyond
  1. a Prepare MBM + WNA medium
  2. b Seed NBOs in Matrigel
  3. c Add MBM + WNA medium
  4. d Incubate and monitor organoid formation
  5. e Passage NBOs using same protocol as LCOs
Phase 5 · Cryopreservation and Banking Passage 3-4
  1. a Determine organoid size for banking
  2. b Harvest and clean organoids
  3. c Prepare freezing medium
  4. d Resuspend organoids in freezing medium
  5. e Aliquot into cryogenic vials
  6. f Freeze organoids gradually
  7. g Store in gas phase nitrogen tank
Phase 6 · Organoid Thawing and Viability Assessment As needed for experiments
  1. a Retrieve and thaw organoid vial
  2. b Transfer to culture medium
  3. c Centrifuge and resuspend
  4. d Seed thawed organoids
  5. e Add medium and assess viability
Phase 7 · Drug Sensitivity Testing Passage 2+, Days 10-16
  1. a Prepare organoids for drug screening
  2. b Prepare organoid suspension for seeding
  3. c Allow organoid formation
  4. d Prepare and add drug solutions
  5. e Monitor organoid response
  6. f Measure cell viability
  7. g Calculate IC50 values

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Attribution

This SOP was written by Organthis based on the published method in Kim et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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