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RETINA Publication-derived

Retinal Organoids for Drug Screening (No. 2)

Full SOP with Academic or a plan

Source Nakano et al. · 10.1016/j.stem.2012.05.009

Authors: Tokushige Nakano, Satoshi Ando, Nozomu Takata, Masako Kawada, Keiko Muguruma, Kiyotoshi Sekiguchi, Koichi Saito, Shigenobu Yonemura, Mototsugu Eiraku, Yoshiki Sasai Duration: 126 days 6 phases Cell source: Human ESC (KhES-1)

Abstract

This protocol describes the self-formation of optic cups and stratified neural retina tissues from human embryonic stem cells (hESCs) using a serum-free floating culture of embryoid body-like aggregates with quick re-aggregation (SFEBq). The resulting neural retina develops photoreceptor layers with functional cilia and can be cryopreserved for long-term storage and later differentiation.

Cell source
Human ESC (KhES-1)
Application
Developmental study, disease modeling platform, drug screening

Protocol overview

26 steps across 6 phases

Phase 1 · Initial aggregation and early retinal specification Days 0-12
  1. a Preparation of hESC aggregates in V-bottomed wells
  2. b Addition of differentiation medium and growth factor inhibitors
  3. c Addition of Matrigel from day 2
  4. d Maintain standard culture conditions for days 0-12
Phase 2 · Retinal progenitor enhancement (NR-selective pathway) Days 12-18
  1. a Removal of IWR1e and addition of pro-retinal signaling factors
  2. b Culture with FBS and SAG through day 18
  3. c Optional: verify retinal specification by immunostaining
Phase 3 · Optic cup formation (days 15-26) Days 15-26
  1. a Addition of Wnt agonist for balanced NR and RPE generation
  2. b Transfer aggregates to retina culture medium on day 18
  3. c Observation of optic vesicle formation
  4. d Optic cup invagination (days 19-26)
  5. e Continued culture and pigmentation of RPE
Phase 4 · Isolation and long-term culture of neural retina Days 18-126
  1. a Isolation of Rx::venus[+] neural retina epithelium on day 18
  2. b Culture in suspension with high oxygen conditions
  3. c Observation of epithelial eversion and stratification (days 18-30)
  4. d Retinal progenitor expansion (days 30-60)
  5. e Multilayered stratification and early photoreceptor accumulation (days 60-126)
Phase 5 · Acceleration of photoreceptor differentiation by Notch inhibition Days 29-43
  1. a Treatment with Notch inhibitor DAPT
  2. b Monitoring photoreceptor accumulation
  3. c Observation of tissue architectural changes
Phase 6 · Cryopreservation of neural retina epithelium Days 18-40 (or up to day 105)
  1. a Preparation of NR tissues for cryopreservation
  2. b Pretreatment with cryoprotectants
  3. c Vitrification freezing
  4. d Storage of frozen tissues
  5. e Thawing of frozen NR tissues
  6. f Post-thaw culture and evaluation

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Attribution

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

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