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RETINA Publication-derived

Retinal 2D Cell Cultures for Drug Screening

Full SOP with Academic or a plan

Source Ohlemacher et al. · Indiana University-Purdue University Indianapolis, Department of Biology · 10.1002/9780470151808.sc01h08s32

Authors: Sarah K. Ohlemacher, Clara L. Iglesias, Akshayalakshmi Sridhar, David M. Gamm, Jason S. Meyer Duration: 90 days 6 phases Cell source: Human pluripotent stem cells (hPSCs)

Abstract

This protocol describes a stepwise differentiation method to efficiently generate diverse retinal cell types from human pluripotent stem cells. Cells are transitioned through neural induction medium and retinal differentiation medium, forming three-dimensional optic vesicle-like structures that yield all major retinal cell types including ganglion cells, retinal pigment epithelium, and photoreceptors within 90 days of differentiation.

Cell source
Human pluripotent stem cells (hPSCs)
Application
Generation of retinal cell types for disease modeling, drug screening, and developmental studies

Protocol overview

59 steps across 6 phases

Phase 1 · Enzymatic Passaging of Human Pluripotent Stem Cells Day 0 (ongoing maintenance)
  1. a Coat 6-well plates with Matrigel
  2. b Prepare wells with mTeSR1 medium
  3. c Prepare conical tubes for cell collection
  4. d Mark areas of spontaneous differentiation
  5. e Remove differentiated cells
  6. f Treat cells with dispase
  7. g Monitor cell detachment
  8. h Wash cells with DMEM/F12
  9. i Detach colonies by forceful pipetting
  10. j Transfer cells to conical tubes
  11. k Settle and aspirate cells
  12. l Resuspend cells in mTeSR1
  13. m Plate cell suspension
  14. n Agitate plates for even distribution
  15. o Change medium daily
Phase 2 · Generation of Embryoid Bodies for Differentiation Day 0-3
  1. a Resuspend cells in mTeSR1:NIM mixture
  2. b Transition cells to neural induction medium (Day 0-1)
  3. c Transition to 1:3 mTeSR1:NIM (Day 2)
  4. d Switch to complete NIM (Day 3 onwards)
Phase 3 · Induction to a Primitive Anterior Neuroepithelial Fate Day 7-16
  1. a Collect embryoid bodies
  2. b Prepare 6-well plates with NIM
  3. c Resuspend EBs in NIM
  4. d Add FBS for cell attachment
  5. e Agitate plates for even distribution
  6. f Remove FBS-containing medium
  7. g Maintain cells in NIM until Day 16
Phase 4 · Differentiation of Primitive Anterior Neuroepithelial Cells to Retinal Pigmented Epithelial Fate Day 16-60
  1. a Switch to Retinal Differentiation Medium (RDM)
  2. b Identify RPE populations for microdissection
  3. c Microdissect RPE cells
  4. d Transfer RPE cluster to coated coverslip
  5. e Allow RPE to attach overnight
  6. f Add RDM with growth factors
  7. g Monitor RPE dedifferentiation and expansion
  8. h Switch to RDM without growth factors for maturation
Phase 5 · Differentiation and Long-Term Maintenance of Retinal Progenitor Cells Day 16-30
  1. a Lift cells from plate to form neurospheres
  2. b Collect cell aggregates
  3. c Resuspend in RDM
  4. d Transfer to 60 mm dish
  5. e Identify retinal neurospheres by morphology
  6. f Manually enrich retinal neurospheres
  7. g Transfer enriched neurospheres to fresh dish
  8. h Tilt plate to collect neurospheres
  9. i Transfer medium containing neurospheres
  10. j Rinse plate for remaining neurospheres
  11. k Transfer remaining medium to conical tube
  12. l Settle neurospheres
  13. m Resuspend in fresh RDM
Phase 6 · Induction of Retinal Progenitors to Specific Retinal Subtypes Day 40-90
  1. a Prepare neurospheres for dissociation
  2. b Gather neurospheres into 1.5 mL tube
  3. c Remove excess medium
  4. d Add accutase for dissociation
  5. e Incubate at 37°C
  6. f Agitate cells to aid dissociation
  7. g Repeat dissociation if needed
  8. h Centrifuge cell suspension
  9. i Resuspend in RDM
  10. j Plate on coated coverslips
  11. k Allow cells to adhere overnight
  12. l Add medium for continued culture or fix for analysis

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Attribution

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

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