SKIN Publication-derived

ALI Tumor Organoids Preserving Native Immune Microenvironment

Source Neal et al. · Stanford University, Stanford, USA · 10.1016/j.cell.2018.11.021

👤 Neal JT, Li X, Zhu J, Giangarra V, Grzeskowiak CL, Ju J, Liu IH, Chiou SH, Salahudeen AA, Smith AR, Deutsch BC, Liao L, Zember AJ, Zhao F, Karlsson K, Schultz LM, Mber TJ, Grandber CC, Kuo CJ ⏱ 30 days 📋 9 phases 🧫 Patient-Derived (Multiple Cancer Types Including Melanoma)

Abstract

Landmark ALI organoid culture method preserving native tumor-infiltrating lymphocytes and stromal cells for up to 30 days. Enables checkpoint blockade testing recapitulating clinical responses across multiple cancer types including melanoma.

Cell source

Patient-Derived (Multiple Cancer Types Including Melanoma)

Application

Immunotherapy Testing with Native TME

Protocol overview

40 steps across 9 phases

ALI Plate Preparation Day -1 to 0

1 Prepare reconstituted collagen matrix
2 Prepare ALI insert plates with bottom collagen layer
3 Solidify bottom collagen layer

Human PDO Tissue Preparation and Primary Plating Day 0

1 Prepare tumor tissue
2 Resuspend tissue in collagen
3 Layer tissue-collagen mixture on top of pre-solidified collagen
4 Prepare organoid culture medium
5 Add medium to outer dish and initiate ALI culture

PDO Culture Maintenance (Days 1–30) Days 1–30

1 Culture organoids at standard conditions
2 Replace medium every 3–4 days
3 Optional: Supplement with IL-2 to preserve immune cells
4 Monitor for necrotic tissue and assess viability

PDO Passage and Serial Propagation Days 14–30 and beyond

1 Dissociate organoids using collagenase IV
2 Wash dissociated cells in fetal bovine serum
3 Replate dissociated tissue into new ALI collagen gels
4 Confirm viability with serial passage (P1)
5 Manage fibroblast overgrowth (if applicable)

Cryopreservation and Recovery Variable (after P0 or P1)

1 Assess organoid suitability for cryopreservation
2 Dissociate and prepare organoid suspension
3 Freeze organoids slowly to –80°C, then transfer to liquid nitrogen
4 Thaw and recover organoids

Anti-PD-1/PD-L1 Immune Checkpoint Blockade Treatment (Human PDOs) Days 0–7 (treatment window)

1 Establish PDOs and prepare for treatment
2 Prepare anti-PD-1 antibody (nivolumab) solution
3 Replace organoid culture medium with antibody-containing medium
4 Incubate organoids with antibody for 7 days
5 Harvest organoids for analysis (see Phases 7–9 for downstream assays)

FACS Analysis of Immune Cells Day 7 (post-treatment)

1 Dissociate organoids to single cells
2 Prepare single-cell suspension for FACS
3 Stain cells with antibody cocktail for human PDO TILs
4 Wash and acquire cells by FACS
5 Quantify immune cell expansion and activation markers

qRT-PCR Analysis of TIL Activation Markers Day 7 (post-treatment)

1 Sort TILs by FACS and extract RNA
2 Perform reverse transcription
3 Perform quantitative real-time PCR (qRT-PCR) for activation markers
4 Analyze and report qRT-PCR results

Annexin V Apoptosis Assay for Tumor Cytotoxicity Day 7 (post-treatment)

1 Dissociate organoids to single cells
2 Pre-gate on tumor epithelial cells
3 Stain with Annexin V and 7-AAD
4 Acquire cells and identify apoptotic populations
5 Interpret results and report tumor cytotoxicity

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Neal et al.. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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