Skip to content
Back to browse
RETINA Publication-derived

Retinal Organoids for Disease Modelling (2015)

Full SOP with Academic or a plan

Source Kuwahara et al., 2015 · RIKEN Center for Developmental Biology, Kobe, Japan · 10.1038/ncomms7286

Authors: Atsushi Kuwahara, Chikafumi Ozone, Tokushige Nakano, Koichi Saito, Mototsugu Eiraku, Yoshiki Sasai Duration: 150 days 7 phases Cell source: Human ESC (hESC; KhES-1 line; Venus-knock-in reporter lines Rx::Venus, Rx::AcGFP, Crx::Venus)

Abstract

This protocol describes a three-dimensional self-organizing culture method for human embryonic stem cells (hESCs) that generates stratified neural retina with an adjacent retinal pigment epithelium, forming a ciliary margin-like growth zone. The method combines selective retinal differentiation using transient BMP4 treatment with an induction-reversal culture approach, producing tissue with functional stem cell niches that expand retinal progenitors peripherally.

Cell source
Human ESC (hESC; KhES-1 line; Venus-knock-in reporter lines Rx::Venus, Rx::AcGFP, Crx::Venus)
Application
Developmental study; Disease modeling; Stem cell niche formation in retinal tissue

Protocol overview

28 steps across 7 phases

Phase 1 · hESC maintenance and preparation Ongoing until differentiation
  1. a hESC culture on feeder layer
  2. b Passage hESCs
Phase 2 · Initial aggregation and BMP4 treatment Day 0 to Day 24
  1. a Prepare hESC aggregates (SFEBq culture)
  2. b Add BMP4 on Day 6
  3. c Half-dilute BMP4 every 3 days
  4. d Assess neural retina formation by day 24
Phase 3 · Long-term NR culture (condition 1) Day 18 to Day 150+
  1. a Transfer aggregates to non-adhesive culture dish
  2. b Culture in NR-differentiation medium
  3. c Monitor NR maturation
Phase 4 · Induction-reversal culture for NR–RPE co-generation Day 18 to Day 60+
  1. a Transfer aggregates for RPE induction (day 18)
  2. b Apply induction medium (RPE-induction phase)
  3. c Switch to reversal medium (NR-differentiation phase)
  4. d Select two-domain aggregates
  5. e Continue long-term culture of two-domain aggregates
Phase 5 · Characterization of ciliary margin-like zone Day 50 to Day 150
  1. a Assess ciliary margin markers by immunostaining
  2. b Verify progenitor dynamics by nucleotide analogue labeling
  3. c Confirm presence of de novo NR generation
Phase 6 · Retinosphere formation from CM-like tissue Day 60 to Day 85+
  1. a Dissect CM-like tissue from two-domain aggregates
  2. b Dissociate tissue into single cells
  3. c Plate cells for retinosphere formation
  4. d Assess primary sphere formation
  5. e Generate secondary spheres
  6. f Differentiate retinospheres
Phase 7 · Optional: SSEA1 cell sorting and retinosphere formation Day 60 to Day 85
  1. a Dissect and dissociate CM-like tissue
  2. b Immunostain for SSEA1 (FACS or MACS)
  3. c Sort SSEA1-positive and SSEA1-negative cells
  4. d Plate sorted cells for retinosphere formation
  5. e Assess sphere formation and differentiation

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 Kuwahara et al., 2015. 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