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Influence of human induced pluripotent stem cell-derived skin organoid-conditioned culture medium on the function of human dermal fibroblasts induced by high glucose

Source Zhixin et al., 2025 · Guangdong Medical University, The First Clinical Medical College · 10.3760/cma.j.cn501225

👤 Liu Zhixin, Qiu Kaizhen, He Jia, Wang Jingru, Liu Bilai, Xin Qi, Li Guiqiang, Chen Xiaodong ⏱ 92 days 📋 8 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of human iPSC-derived skin organoids and preparation of their conditioned culture medium (SO-CM) to evaluate effects on high glucose-induced dermal fibroblasts. The study demonstrates that SO-CM promotes proliferation and migration of fibroblasts while modulating inflammatory and growth factor expression, providing a therapeutic strategy for diabetic wound healing.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

26 steps across 8 phases

Human iPSC differentiation into skin organoids Days 0–92
  1. 1 iPSC dissociation and aggregate formation
  2. 2 First differentiation phase with Essential 6 medium
  3. 3 Second differentiation phase with LDN193189
  4. 4 Nutrient supplementation phase
  5. 5 Transfer to skin organoid culture medium (SOM) with Matrigel
  6. 6 Maturation phase without Matrigel
Preparation of conditioned media (Fb-CM and SO-CM) Days 1–3
  1. 1 Culture human fibroblasts to passage 5–6
  2. 2 Seed fibroblasts in SOM for conditioned medium preparation
  3. 3 Seed skin organoids in SOM for conditioned medium preparation
  4. 4 Collect culture supernatant (Fb-CM and SO-CM)
Cytokine analysis (ELISA) Day 1
  1. 1 ELISA detection of cytokines in SOM, Fb-CM, and SO-CM
High glucose fibroblast induction and cell culture experiments Days 1–10 (induction) + experiment days
  1. 1 Induce fibroblasts with high glucose
  2. 2 Seed high glucose-induced fibroblasts for proliferation assay
  3. 3 Randomize groups and treat with conditioned media (proliferation)
  4. 4 Seed high glucose-induced fibroblasts for migration assay
  5. 5 Perform cell scratch assay
  6. 6 Measure cell migration at 13 hours post-scratch
Ki67 immunofluorescence staining (24 h culture) After 24 h treatment
  1. 1 Perform immunofluorescence staining for Ki67
  2. 2 Microscopic observation and Ki67 quantification
Cell viability assay (CCK-8, 24 h culture) After 24 h treatment
  1. 1 Measure cell absorbance using CCK-8 kit
Reactive oxygen species (ROS) detection (48 h culture) After 48 h treatment
  1. 1 Seed and treat high glucose-induced fibroblasts for ROS assay
  2. 2 ROS detection with fluorescent probe
  3. 3 Microscopic observation and ROS quantification
Senescence detection using β-galactosidase staining (48 h culture) After 48 h treatment
  1. 1 Seed and treat high glucose-induced fibroblasts for senescence assay
  2. 2 β-galactosidase staining
  3. 3 Microscopic observation and senescence quantification

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Zhixin et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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