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Human iPSC-derived retinal organoids

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Receiving and maintaining retinal organoids

General Notes

  • Organoids will be shipped in 5ml microcentrifuge tubes in their cell culture medium
  • All work with live organoids should be performed in a Class II laminar flow tissue culture hood using aseptic technique
  • Organoids are to be maintained in a humidified tissue culture incubator (37°C, 5% CO2, 20% O2)
  • We recommend allowing at least 2 days for retinal organoids to acclimatise before assaying
  • Cell culture medium needs to be stored at 4°C for up to 1 month and pre-warmed to room temperature prior to use

Materials and reagents provided by Axol Bioscience

  • 3 ml sterile Pasteur pipettes (x3)
  • 96-well plate for culturing organoids
  • Organoid maintenance medium

Materials and Reagents provided by the user

  • For all customers
    • Fetal Bovine Serum (FBS) (recommended Fisher-Scientific, product code: 10082147 or 15383681)

For organoids day 120 or less

  • Retinoic acid (recommended Sigma-Aldrich, product code: R2625)

Packaging

  • The organoids and the media are delivered in two separate QuickSTAT CREDO boxes with temperature monitor

DO NOT DISPOSE OF THE BOX OR THE MONITOR.

  • QuickSTAT will organize pick up and return of the boxes and monitor.

 

Procedure

  1. Preparation of organoid maintenance medium
  2. Please add 10% (v/v) FBS to organoid maintenance medium upon receipt of the organoids.

For customers receiving less than 50 organoids, add 15ml FBS to 135ml maintenance medium. For customers receiving more than 50 organoids, add 30ml FBS to 270ml maintenance medium.

  1. For customers receiving organoids day 120 or less: prepare and store aliquots of retinoic acid following the manufacturer’s guidelines. Protect aliquots from light and do not re-freeze. For every feed, transfer the required volume of maintenance media to a sterile tube/bottle and supplement with retinoic acid at a final concentration of 0.5 uM. Feed organoids with maintenance media supplemented with retinoic acid until they reach 120 days of differentiation. Organoids older than day 120 should be fed with maintenance media only.
  2. Transfer each individual organoid to a well of the provided 96-well plate by following the procedure described below:
  3. Using a Pasteur pipette, transfer each of the organoids into separate wells of the plate, one organoid per well b. Avoid direct contact of the Pasteur pipette with the organoids: allow the organoids to be carried within the medium c. Let the organoids settle with gravity (this should take ~ 1 minute)
  4. Using a P200 pipette, remove as much medium as possible but ensure the organoids remain covered

Note: avoid touching the organoids as it may damage them

  1. Add 200 μL of fresh organoid maintenance medium to each well containing the organoids
  2. Replenish cell culture medium by removing half of the medium and adding 100 μL of fresh medium per well
  3. For customers receiving organoids older than day 120: Cell culture medium should be replenished twice a week (e.g. Monday and Friday) b. For customers receiving organoids day 120 or less: cell culture medium should be replenished three times a week (e.g Monday, Wednesday and Friday) Note: for organoids that are day 120 or less, supplement maintenance medium with fresh retinoic acid (please see Step 1b).

Technical sheet

Specifications

Format Cell types Donor
10 retinal organoids per 5 ml microfuge tube

150 ml of optimized cell culture

medium per 100 organoids

2 x 96 well plates per 100

organoids

2 x Pasteur pipettes

 Cone and Rod photoreceptors

Retinal ganglion cells (RGCs)

Bipolar cells

Horizontal cells

Amacrine cells

Müller glial cells

 Human, male, 68-year-old

Readouts

  • Immunofluorescence analyses
  • Gene expression by RT-qPCR
  • Transcriptomic analysis by single-cell RNA sequencing
  • Cytotoxicity assays
  • Cytokine release
  • Flow cytometry
  • Electron microscopy

Gene expression in retinal organoids through different stages

Cell Type Gene Timepoint of appearance Timepoint of peak expression
Retinal ganglion cells MATH5 (ATOH7) d30-d180 d60
Retinal ganglion cells BRN3 (POU4F2) d30-d210 d60
Horizontal and amacrine cells TFAP2A d30-d210 d150
Horizontal and amacrine cells PROX1 d30-d210 d150
Bipolar cells GRIK1 d30-d210 d150
Bipolar cells CADPS d30-d210 d150
Photoreceptors RP11VS d60-d210 d210
Photoreceptors RBP3 d60-d210 d210
Photoreceptors IMPG1 d120-d210 d210
Photoreceptors CRX d60-d210 d210
Cone photoreceptors OPN1SW d120-d210 d210
Cone photoreceptors OPN1MW d150-d210 d210
Cone photoreceptors OPN1LW d120-d210 d210
Cone photoreceptors ARR3 d60-d210 d180
Cone photoreceptors RXRG d60-d210 d150
Rod photoreceptors RHO d120-d210 d210
Rod photoreceptors NRL d90-d210 d180
RPE RPE65 d60-d210 d210
Muller glia RLBP1 d90-d210 d210
Muller glia CRYM d30-d210 d210

Expected protein expression in retinal organoids at d150 and d180

Cell Type Cell marker Protein expression at d150 Protein expression at d180 Protein localization at d150-d180
Photoreceptors RCVRN Yes Yes ONL
Retinal ganglion cells SNCG and HuC/D Yes Yes INL/GCL
Cone photoreceptors OPN1MW/LW * Yes ONL
Rod photoreceptors RHO * Yes ONL
Bipolar cells PKC-a ** Yes INL
Amacrine cells AP-2a Yes Yes INL
Horizontal cells PROX1 Yes Yes INL
Muller glia CRALBP Yes Yes All layers

* small number of developing rods and cones ** Expressed at transcriptional level