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BRAIN Publication-derived

Brain Organoids for Disease and Development (2017)

Full SOP with Academic or a plan

Source Lancaster et al., 2017 · IMBA—Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna, Austria · 10.1038/nbt.3906

Authors: Madeline A Lancaster, Nina S Corsini, Simone Wolfinger, E Hilary Gustafson, Alex W Phillips, Thomas R Burkard, Tomoki Otani, Frederick J Livesey, Juergen A Knoblich Duration: 60 days 3 phases Cell source: Human ESC (H9, H1) and Human iPSC

Abstract

This protocol describes the generation of engineered cerebral organoids (enCORs) from human pluripotent stem cells using PLGA microfilaments as internal floating scaffolds combined with Matrigel-based basement membrane reconstitution. enCORs reproducibly generate elongated forebrain tissue with polarized cortical plate formation, radial glial organization, and neuronal migration patterns characteristic of in vivo cortical development.

Cell source
Human ESC (H9, H1) and Human iPSC
Application
Disease modeling and developmental study of forebrain cortical development

Protocol overview

16 steps across 3 phases

Phase 1 · Embryoid Body Formation and Initial Culture Day 0-6
  1. a Prepare and disperse PLGA microfilaments
  2. b Prepare hPSC single-cell suspension
  3. c Prepare embryoid body media
  4. d Seed cells onto microfilaments (enCOR method)
  5. e Culture embryoid bodies (day 0-3)
  6. f First media change (day 3)
  7. g Transfer to neural induction at day 5-6
Phase 2 · Neural Induction and Forebrain Patterning Day 6-20
  1. a Neural induction culture
  2. b Matrigel embedding at day 11-12
  3. c Switch to improved differentiation media–A (IDM-A) and add CHIR99021
  4. d Move to orbital shaker (days 18-20)
Phase 3 · Cortical Development and Basement Membrane Reconstitution Day 20-60+
  1. a Switch to improved differentiation media+A (IDM+A) at day 20
  2. b Add dissolved Matrigel at day 40
  3. c Continued culture and monitoring (day 40-60+)
  4. d Optional: Matrix metalloprotease inhibitor treatment (alternative approach)
  5. e Optional: Pure ECM component testing (purified laminin, entactin, collagen)

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Attribution

This SOP was written by Organthis based on the published method in Lancaster et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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