Skip to content
Back to browse
RETINA Publication-derived

Retinal 2D Cell Cultures for Developmental Studies (2012)

Full SOP with Academic or a plan

Source Phillips et al., 2012 · Waisman Center, University of Wisconsin-Madison · 10.1167/iovs.11-9313

Authors: M. Joseph Phillips, Kyle A. Wallace, Sarah J. Dickerson, Michael J. Miller, Amelia D. Verhoeven, Jessica M. Martin, Lynda S. Wright, Wei Shen, Elizabeth E. Capowski, E. Ferda Percin, Enio T. Perez, Xiufeng Zhong, Maria V. Canto-Soler, David M. Gamm Duration: 108 days 7 phases Cell source: Human iPSC (Blood-derived T lymphocytes)

Abstract

This protocol describes the generation of optic vesicle-like structures (OVs) from blood-derived human induced pluripotent stem cells (TiPSCs) and their differentiation into multiple neuroretinal cell types including photoreceptors, ganglion cells, and amacrine cells. The protocol produces stratified retinal laminae capable of expressing synaptic markers critical for intercellular communication.

Cell source
Human iPSC (Blood-derived T lymphocytes)
Application
Retinal cell differentiation and developmental study

Protocol overview

29 steps across 7 phases

Phase 1 · T-Cell Activation and Retroviral Transduction Day 0-2
  1. a Blood collection and PBMC isolation
  2. b Thaw PBMCs and resuspend
  3. c T-cell activation with OKT3 and IL-2
  4. d Retroviral transduction
  5. e Post-transduction incubation
Phase 2 · iPSC Colony Expansion Day 2-20
  1. a Transfer to MEF feeder layers
  2. b Transition to conditioned medium
  3. c Identify and isolate iPSC colonies
  4. d Hand-pick and propagate iPSC colonies
Phase 3 · Embryoid Body Formation and Neural Induction Day 0-16 (relative to differentiation start)
  1. a Lift colonies and form embryoid bodies
  2. b Replace medium with neural induction medium
  3. c Transfer aggregates to laminin-coated plates
  4. d Mechanically lift neural clusters
Phase 4 · Retinal Differentiation and Optic Vesicle Isolation Day 16-20
  1. a Culture in retinal differentiation medium
  2. b Identify and isolate optic vesicles
  3. c Pool isolated OVs
Phase 5 · Retinal Neuron Differentiation and Maturation Day 20-108
  1. a Continue culture in RDM
  2. b Partial dissociation and plating for differentiation studies
  3. c Monitor marker expression over time
Phase 6 · Cryosectioning and Laminar Structure Analysis Day 20-105 (as needed)
  1. a Fix free-floating OVs
  2. b Cryosection OVs
  3. c Perform immunocytochemistry on sections
Phase 7 · Pluripotency and Genetic Characterization (Quality Control) Performed on established iPSC lines
  1. a Genomic DNA PCR for retroviral integration
  2. b RT-PCR for transgene silencing and endogenous gene activation
  3. c Short tandem repeat (STR) analysis
  4. d G-banding karyotype analysis
  5. e Teratoma analysis
  6. f Flow cytometry for pluripotency markers
  7. g Immunocytochemistry for pluripotency markers

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 Phillips et al., 2012. 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