Skip to content
Back to browse
SKIN Publication-derived

Cutaneous Tuberculosis Skin Organoids

Full SOP with Academic or a plan

Source Yue et al., 2025 · Academy of Military Medical Sciences, Beijing, China · 10.1038/s41467-025-65848-z

Authors: Liang Yue, Yating Liang, Pengfei Zhong, Leiming Fang, Zhimin Yun, Qisheng Su, Chaofan Li, Meida Xiang, Jie Zhang, Jiru Wang, Leixin Ge, Chengjun Wu, Hebing Chen, Xiaochen Qiu, Xiaochen Bo, Yingxia Tan Duration: 125 days 8 phases Cell source: Human iPSC

Abstract

This protocol describes the generation of hair-bearing skin organoids (SKOs) from human induced pluripotent stem cells (hiPSCs) and their use as an in vitro model for cutaneous tuberculosis (CTB) pathogenesis. Following Mtb infection of mature SKOs, the model recapitulates key pathological features of CTB, including extracellular matrix remodeling, fibrosis, and alterations in cell populations, providing a platform for investigating CTB mechanisms and testing antifibrotic therapeutics.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

41 steps across 8 phases

Phase 1 · hiPSC Culture and Maintenance Ongoing until differentiation
  1. a Prepare culture plates with Matrigel coating
  2. b Culture hiPSCs in mTeSR1 medium
  3. c Passage hiPSCs at ~80% confluency
Phase 2 · Skin Organoid Differentiation (Days 0-45) Days -1 to 45
  1. a Prepare single-cell suspension and aggregate formation
  2. b Day -1: Dilute Y27632
  3. c Day 0: Initiate differentiation with E6SFB medium
  4. d Day 3: Supplement with LDN and increase bFGF
  5. e Day 6: Partial medium exchange
  6. f Day 9: Half-medium replacement
  7. g Day 12: Transfer to maturation plates
  8. h Day 15: Initial half-medium replacement
  9. i Days 18-45: Maintenance with periodic medium changes
Phase 3 · Skin Organoid Maturation and Stabilization (Days 45-125) Days 45-125
  1. a Days 45-125: Continue maturation with adjusted medium changes
  2. b Quality assessment at day 125
Phase 4 · Mycobacterium tuberculosis Preparation Before infection (ongoing culture)
  1. a Culture Mtb H37Rv to mid-exponential phase
  2. b Harvest and prepare bacterial suspension
Phase 5 · Mycobacterium tuberculosis Infection of SKOs Days 0-15 post-infection (or as needed)
  1. a Pre-infection culture preparation
  2. b Transfer organoids and prepare for infection
  3. c Add Mtb inoculum
  4. d Remove extracellular bacteria
  5. e Continue culture post-infection
  6. f Collect organoids at specified timepoints
Phase 6 · Pharmacological Inhibitor Treatment (optional) Concurrent with infection (days 0-9)
  1. a Prepare inhibitor stocks
  2. b Simultaneous infection and inhibitor treatment
  3. c Collect treated organoids
Phase 7 · Sample Collection and Tissue Processing At designated timepoints
  1. a Fix organoids for histology
  2. b Dehydrate and embed in paraffin
  3. c Section paraffin blocks
  4. d Collect tissue for RNA/protein extraction
  5. e Prepare single-cell suspension for scRNA-seq
Phase 8 · Downstream Analyses (Histology, qPCR, Immunostaining, Western Blot, ELISA, scRNA-seq) Immediately after sample collection
  1. a Hematoxylin & Eosin (H&E) staining
  2. b Acid-Fast (Ziehl-Neelsen) staining for Mtb detection
  3. c Sirius Red staining for collagen quantification
  4. d Masson's Trichrome staining for fibrosis assessment
  5. e Immunohistochemistry (IHC) for protein detection
  6. f Immunofluorescence (IF) staining
  7. g Real-time quantitative PCR (qPCR) for mRNA quantification
  8. h Western blotting for protein phosphorylation and expression
  9. i ELISA for MMP quantification
  10. j Single-cell RNA sequencing (scRNA-seq) library preparation and sequencing
  11. k scRNA-seq data processing and analysis

Full SOP

Full SOPs need an account and a plan

Create an account, then get free Academic access (for verified academics) or choose a plan. Plans include step-by-step procedures, reagent concentrations, QC checklists and batch record templates.

Create an account

Already registered? Log in

Attribution

This SOP was written by Organthis based on the published method in Yue et al., 2025. Once the originating laboratory has confirmed it, the SOP is marked Verified.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence