Skip to content
Back to browse
BRAIN Publication-derived

Parkinson's Disease Midbrain 2D Cell Cultures

Full SOP with Academic or a plan

Source Doi et al., 2020 · Center for iPS Cell Research and Application, Kyoto University · 10.1038/s41467-020-17165-w

Authors: Daisuke Doi, Hiroaki Magotani, Tetsuhiro Kikuchi, Megumi Ikeda, Satoe Hiramatsu, Kenji Yoshida, Naoki Amano, Masaki Nomura, Masafumi Umekage, Asuka Morizane, Jun Takahashi Duration: 60 days 12 phases Cell source: Patient-Derived iPSC (Parkinson's disease)

Abstract

This protocol describes the GMP-grade manufacturing of dopaminergic progenitor cells (DAPs) derived from clinical-grade human induced pluripotent stem cells (iPSCs) for pre-clinical evaluation of safety and efficacy in Parkinson's disease models. The protocol includes in vitro characterization, genomic/epigenetic analysis, tumorigenicity assessment, and efficacy validation in rodent and primate models.

Cell source
Patient-Derived iPSC (Parkinson's disease)
Application
Disease modeling, drug screening, and cell-based therapy

Protocol overview

74 steps across 12 phases

Phase 1 · iPSC Stock Establishment and Master Cell Bank (MCB) Production Day 0 to Day 60 (approx.)
  1. a Peripheral Blood Collection and Mononuclear Cell Isolation
  2. b Episomal Reprogramming of Peripheral Blood Cells
  3. c Primary iPSC Colony Selection and Expansion
  4. d Quality Control of Primary Cell Stock (PCS)
  5. e Freeze Primary Cell Stock (PCS) at Passage 4
  6. f Thaw PCS, Expand, and Generate Secondary Cell Stock (SCS)
  7. g Thaw SCS, Passage Twice, and Generate Master Cell Bank (MCB)
  8. h Quality Control Testing of MCB003
Phase 2 · iPSC Thawing and Pre-differentiation Maintenance Day 0 to Day 12 (pre-differentiation culture)
  1. a Thaw MCB Vial and Recover iPSCs
  2. b Plate Thawed iPSCs on iMatrix-Coated Culture Vessels
  3. c Expand iPSCs for Two Passages
Phase 3 · In Vitro Dopaminergic Progenitor Differentiation (Days 0-30) Day 0 to Day 30
  1. a Dissociate iPSCs and Initiate Differentiation
  2. b Add BMP Inhibitor LDN193189 (Days 0-12)
  3. c Add ALK5 Inhibitor A8301 (Days 0-6)
  4. d Add FGF8 and Purmorphamine (Days 1-6)
  5. e Add Wnt Pathway Activator CHIR99021 (Days 3-12)
  6. f Daily Media Changes (Days 0-12)
  7. g Monitor Cell Morphology (Days 0-12)
Phase 4 · CORIN+ Cell Sorting (Day 12-13) Day 12-13
  1. a Dissociate Day-12 Cultures into Single Cells
  2. b Prepare Cells for Flow Cytometry Staining
  3. c Stain with Anti-CORIN Antibody
  4. d Prepare Cell Sorter and Set Gates
  5. e Sort CORIN+ Cells
  6. f Quality Control: Post-Sort CORIN+ Purity Assessment
Phase 5 · Post-Sort Differentiation in Aggregate Spheres (Days 13-30) Day 13 to Day 30
  1. a Plate Sorted CORIN+ Cells into U-Shaped 96-Well Plates
  2. b Culture Aggregate Spheres (Days 13-30)
  3. c Quality Control: In Vitro Characterization at Day 26
  4. d Extended Culture to Day 30 (Optional, for Transplantation)
  5. e Collect and Formulate Day-30 Spheres for Transplantation
Phase 6 · In Vitro Functional Characterization (Days 26-56) Day 26 to Day 56
  1. a Extend Culture on Adhesive Substrate for Dopamine Measurement
  2. b Dopamine Measurement by LC-MS/MS (Day 56)
  3. c Whole-Cell Patch-Clamp Electrophysiology (Day 42-56)
Phase 7 · Genomic, Epigenomic, and Single-Cell Analyses Day 0, 12, and 26
  1. a Collect Cell Samples for Genomic Analysis
  2. b Carry out complete genome sequencing (WGS) and complete exome sequencing (WES).
  3. c Analyze Mutations in Cancer-Related Genes
  4. d Copy Number Variation (CNV) Detection
  5. e Residual Plasmid Detection by qPCR
  6. f DNA Methylation Analysis
  7. g Single-Cell Gene Expression Analysis
Phase 8 · In Vivo Safety and Tumorigenicity Assessment in Mice (Brain Injection) Week 0 to Week 52
  1. a Animal Preparation and Stereotactic Surgery (Week 0)
  2. b Animal Groups and Study Design
  3. c Weekly Monitoring of General Health and Behavior (Weeks 0-52)
  4. d Pharmacological Testing (Modified Irwin Test)
  5. e Ophthalmological Examination
  6. f Clinical Pathology Testing (Weeks 4, 12, 26, 52)
  7. g Necropsy and Organ Weight Assessment (Week 52)
  8. h Histopathological Analysis of Brain
  9. i Immunohistochemistry for Graft Analysis
  10. j Systemic Organ Histopathology
  11. k Data Analysis and Statistical Comparison
Phase 9 · Teratoma Formation Assay (Subcutaneous Injection in Mice) Week 0 to Week 26
  1. a Prepare Cell Suspensions and Spike Controls
  2. b Surgical Injection into Subcutaneous Space
  3. c Animal Groups and Observation
  4. d Tumor Size Monitoring
  5. e Necropsy and Tissue Collection (Week 26 or at Tumor Threshold)
  6. f Histopathological Analysis of Subcutaneous Tissue
  7. g Data Analysis: Teratoma Incidence and Minimal Residual iPSC Detection
Phase 10 · Testicular Teratoma Formation Assay (Optional) Week 0 to Week 16
  1. a Prepare Cell Suspension and Surgical Injection
  2. b Observation for Tumor Formation (Weeks 0-16)
  3. c Necropsy and Histopathological Analysis
Phase 11 · Efficacy Study in 6-OHDA-Lesioned Rats Week 0 (lesioning) to Week 20 (endpoint)
  1. a 6-OHDA Lesioning of Rats
  2. b Cell Transplantation into Striatum
  3. c Behavioral Assessment: Methamphetamine-Induced Rotation
  4. d Study Groups and Sample Size
  5. e Statistical Analysis: Two-Way ANOVA
  6. f Terminal Necropsy and Brain Tissue Collection (Week 20)
  7. g Immunohistochemistry: Tyrosine Hydroxylase and FOXA2 Co-staining
  8. h Immunohistochemistry: Additional Cell Type Characterization
Phase 12 · Efficacy Study in MPTP-Treated Non-Human Primates Week 0 (MPTP lesioning) to Week 26 (terminal endpoint)
  1. a MPTP Lesioning of Monkeys
  2. b Cell Transplantation into Putamen
  3. c Immunosuppression
  4. d Neuroimaging: MRI Scanning
  5. e Study Duration and Terminal Necropsy
  6. f Histopathological Analysis of Primate Brain

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 Doi et al., 2020. 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