A protocol for isolation and culturing of mouse primary postmitotic photoreceptors and isolation of extracellular vesicles

Summary

Here, we present a protocol for isolating and culturing mouse photoreceptors in a minimal, chemically defined medium free from serum. We describe steps for retina dissection, enzymatic dissociation, photoreceptor enrichment, cell culture, extracellular vesicles (EVs) enrichment, and EV ultrastructural analysis. This protocol, which has been verified for cultured cells derived from multiple murine strains, allows for the study of several aspects of photoreceptor biology, including EV isolation and nanotube formation.

For complete details on the use and execution of this protocol, please refer to Kalargyrou et al. (2021).¹

Graphical abstract

Highlights

• •Preparation of primary postmitotic photoreceptor precursor cultures from murine retinae
• •Enrichment of primary photoreceptor-derived extracellular vesicles
• •Steps for ultrastructural analysis of EVs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for isolating and culturing mouse photoreceptors in a minimal, chemically defined medium free from serum. We describe steps for retina dissection, enzymatic dissociation, photoreceptor enrichment, cell culture, extracellular vesicles (EVs) enrichment, and EV ultrastructural analysis. This protocol, which has been verified for cultured cells derived from multiple murine strains, allows for the study of several aspects of photoreceptor biology, including EV isolation, and cell-cell interactions such as nanotubes.

Before you begin

The protocol below has been used to study the intercellular exchange of proteins and lipids between photoreceptors in an isolated in vitro system.¹ It can be utilized for a plethora of studies such as EV enrichment, cell-cell interactions, and morphological analysis of photoreceptor extensions, such as neurites and NTs.

The day prior to dissection/dissociation, autoclave the necessary tools and dry them at 40°C and prepare all reagents needed prior to retinal dissections and dissociations, as detailed below.

Institutional permissions

All experiments described have been conducted in accordance with the Policies on the Use of Animals and Humans in Neuroscience Research, revised and approved by the ARVO Statement for Use of Animals in the Ophthalmic Research, and under the regulation of the UK Home Office Animals (Scientific Procedures) Act 1986. Briefly, rodents were maintained on a standard 12/12-h light dark cycle, housed in same sex groups or sustained breeding pairs wherever possible and provided with fresh bedding and nesting material and food and water ad libitum. Both male and female mice were used without discrimination. Photoreceptors from the following mouse lines have been successfully isolated and cultured using this protocol: C57BL/6J (wild type, wt) (Harlan), Nrl.Gfp (GFP specifically expressed in rod photoreceptors in the retina; kind gift of A. Swaroop, University of Michigan, USA),² Nrl.Cre.³ All mice were kept as homozygotes and were maintained in the animal facility at King’s College London. Please note that to our knowledge any murine animal line may be used for this method. Users are reminded that they should acquire the necessary permissions from the relevant institutions.

Reagent set-up for papain dissociation set up with Worthington neural dissociation kit

Timing: 30 min

Use Worthington Neural Dissociation Kit, Lorne Laboratories, UK; cat. no. LK003153; kit is based on process described in Worthington, Huettner and Baughman, 1986.⁴

• 1.Albumin-ovomucoid inhibitor (Worthington kit): reconstitute vial in 32 mL of Earle’s Balanced Salt Solution (EBSS) (Worthington).
• 2.DNase solution (Worthington kit): reconstitute vial in 0.5 mL of EBSS.
• 3.Papain solution (Worthington kit): reconstitute vial in 5 mL of EBSS.
• 4.Resuspension buffer: 2.7 mL EBSS, 300 μLs reconstituted albumin-ovomucoid inhibitor, and 50 μL of DNase solution.
• 5.Dissociation solution: 5 mL reconstituted Papain solution (Solution Z; Worthington) supplemented with 0.25 mL reconstituted DNase (Solution Y; Worthington).

CRITICAL: EBSS solution should be at pH (7.2–7.6) to yield orange-red stock solutions of all the kit components. EBSS may be aliquoted in 5 mL volumes in 15 mL Falcon tubes and store at 4°C (fridge) to protect reagent from pH changes. Store all reconstituted kit components in aliquots at 4°C (fridge) for over a month.

Prepare all other reagents for the photoreceptor primary cultures

Timing: 1–2 h

• 6.30% BSA stock solution (for recipe see materials and equipment).
• 7.Taurine (50 mM) stock solution (for recipe see materials and equipment).
• 8.PDL coating solution (0.1 mg/ml) (for recipe see materials and equipment).
• 9.Fibronectin coating solution (0.04 mg/mL) (for recipe see materials and equipment).
• 10.Photoreceptor medium (for recipe see materials and equipment and Table 1).
• 11.Dissection medium (for recipe see materials and equipment).
• 12.MACS staining buffer (for recipe see materials and equipment).

Table 1.

Time of dissociation per developmental timepoint

Developmental stage	Papain incubation
P0-P2	10′
P3-P5	15′
P6-P8	20′
P11+	30′

Coat the culture surface with PDL/fibronectin as follows

Timing: 2 h

• 13.Select the appropriate dish for culturing the cells, depending on the application. For example, for multiple conditions imaging or viability assays use 96 well plate.
• 14.Charge the plastic or glass tissue culture surfaces with 0.4 mL of PDL/well in a 6 well plate.
• 15.Incubate PDL solution for 5′ in room temperature inside the TC hood.
• 16.Discard the PDL solution.
• 17.Wash twice for 5 min inside the hood, at room temperature with TC grade sterile dH₂O.
• 18.Allow the surface to fully dry in the TC hood (10–30′) and then proceed to fibronectin coating.
• 19.Coat the surface with fibronectin solution 0.4 mL/well of a 6 well plate.
• 20.Incubate the culture dishes in the incubator at 37°C (3 h-o/n). Use the same volume of fibronectin solution as for the PDL.

For extracellular vesicles ultrastructural analysis prepare following reagents

• 21.Methyl cellulose Stock Solution 2%.
• 22.Methylcellulose for EV staining.
• 23.Uranyl acetate solution 2%.
• 24.Sucrose 20%.
• 25.PFA 4% solution.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Primary anti-mouse-CD73 APC-conjugated antibody (1:100)	Miltenyi Biotec	130-102-586
Anti-rhodopsin (1:2,000)	Merck KGaA- Sigma-Aldrich	O4886
Anti-recoverin (1:3,000)	Merck KGaA	AB5585
Anti-TSG101 (1:1,000)	BD Biosciences	612696
Anti-LAMP1 (1:1,000)	Abcam	[1D4B] (ab25245)
Anti-GM130 (1:1,000)	Cell Signaling Technology	12480
Anti-Alix (1:1,000)	Cell Signaling Technology	2171
Goat anti-rabbit HRP (1:5,000)	Thermo Fisher Scientific	31460
Goat anti-mouse HRP (1:7,000)	Thermo Fisher Scientific	31430
Goat anti-mouse 633 (1:400)	Thermo Fisher Scientific	A-21052
Goat anti-rabbit 546 (1:400)	Thermo Fisher Scientific	A-11035
Chemicals, peptides, and recombinant proteins
Anti-APC-Microbeads (1:50)	Miltenyi Biotec	130-090-855
SiR-tubulin probe	Cytoskeleton, Inc.	CY-SC002
SiR-actin probe	Cytoskeleton, Inc.	SC001
SUPERase·In RNase inhibitor	Thermo Fisher Scientific	AM2694
RNase A, DNase, and protease-free (10 mg/mL)	Thermo Fisher Scientific	EN0531
Proteinase K	Thermo Fisher Scientific	25530–015
Taurine	Merck KGaA - Sigma-Aldrich	T869
BSA tissue culture (TC) grade sterile	Merck KGaA - Sigma-Aldrich	A9418
Poly-D-lysine (PDL)	Merck KGaA - Sigma-Aldrich	P6407-5MG
Fibronectin solution	Merck KGaA - Sigma-Aldrich	F1141
Antibiotics (Pen/Strep)	Merck KGaA - Sigma-Aldrich	P4458-100ML
EBSS	Thermo Fisher Scientific	24010043
PBS	Thermo Fisher Scientific	10010031
HEPES 1 M	Thermo Fisher Scientific	15630080
UltraPure 0.5 M EDTA, pH 8.0	Merck KGaA - Sigma-Aldrich	15575020
Trypan blue solution 0.4%, liquid, sterile-filtered, suitable for cell culture	Merck KGaA	T8154-100ML
Sterile TC grade dH2O	Merck KGaA	W3500-500ML
ITS (0.01 mg/mL recombinant human insulin, 0.0055 mg/mL human transferrin [substantially iron-free], and 0.005 μg/mL sodium selenite)	Merck KGaA - Sigma-Aldrich	I3146-5ML
Brain Phys neuronal medium, no phenol red	STEMCELL Technologies, Inc.	05791
RIPA buffer	Merck KGaA - Sigma-Aldrich	R0278
Protease and phosphatase inhibitor tablets	Thermo Fisher Scientific	A32959
Paraformaldehyde (PFA)	Merck KGaA - Sigma-Aldrich	158127
Glutaraldehyde, 25% aqueous solution	Merck KGaA - Sigma-Aldrich	354400
Uranyl acetate (depleted uranium)	SPI-Chem	6159-44-0
Methyl cellulose	Merck KGaA - Sigma-Aldrich	182312500
Experimental models: Organisms/strains
C57BL/6J (wild type, wt) mice or any wt strain (pups at postnatal day P3-P8 developmental stage, any sex)	Harlan	C57BL/6J
Nrl.Gfp mice or other transgenic lines (pups at postnatal day P3-P8 developmental stage, any sex)	Kind gift of A. Swaroop, University of Michigan, USA	Akimoto et al., 2006
Other
LS MACS columns with plungers	Miltenyi Biotec	130-042-401
MACS MultiStand	Miltenyi Biotec	130-042-303
MidiMACS separator	Miltenyi Biotec	130-042-302
Stericup Millipore 0.22 mm	Millipore	S2GPU05RE
Hydrophobic PVDF transfer membrane for western blotting	Millipore	IPVH00010
Hemocytometer - counting chamber Neubauer	Laboquip	0640030
Plastic Petri dishes 35 mm diameter	Thermo Fisher Scientific	121V
Transfer pipettes, individually wrapped-sterile 5 mL	VWR International	612-1685P
Falcon tubes round-bottom polystyrene test tubes with cell strainer snap cap, 5 mL	Fisher Scientific	08-771-23
Sterile TC grade Falcon tubes 15 mL	Greiner Bio-One International GmbH	188271
Sterile TC grade Falcon tubes 50 mL	Greiner Bio-One International GmbH	227261
Precision general purpose baths (any brand)	Thermo Fisher Scientific	TSGP02
Nunc cell-culture treated 6-well dishes	Thermo Fisher Scientific	140685
Pipettes - Eppendorf Research plus (1000 μL) or equivalent	Eppendorf	3124000121
Pipettes - Eppendorf Research plus (200 μL) or equivalent	Eppendorf	3123000055
Pipettes - Eppendorf Research plus (20 μL) or equivalent	Eppendorf	3123000039
Pipette tips sterile with filter epT.I.P.S. Motion pipette tips 1000 μL or equivalent	Eppendorf	0030015258
Pipette tips sterile with filter epT.I.P.S. Motion pipette tips 300 μL or equivalent	Eppendorf	0030015231
Pipette tips sterile with filter epT.I.P.S. Motion pipette tips 10 μL or equivalent	Eppendorf	0030015193
Pipette tips sterile with filter epT.I.P.S. Motion pipette tips 50 μL or equivalent	Eppendorf	0030015215
Standard benchtop centrifuge for 70 × g and 260 × g spin	Any brand
Sterile tissue culture hood	Any brand
Tissue culture incubator	Any brand
Routine stereo microscope with internal light source at the base	Leica Microsystems	M50
Routine upright light microscope	Leica Microsystems	DM750
Confocal microscope equipped with live imaging chamber with temperature and CO2 control	Leica Microsystems or Zeiss or equivalent	Leica TCS SP8 or Zeiss LSM700 Airy Scan
Centrifuge 5804/5804 R or equivalent	Beckman Coulter	5804/ 5804 R
Centrifuge 5804/5804 R, swing bucket rotor or equivalent	Beckman Coulter	Rotor A- 4-44
Centrifuge 5804/5804 R, high speed, fixed angle rotor or equivalent	Beckman Coulter	Rotor F-34-6-38
Thin wall ultra-clear tube 25 × 89 mm - 50 Pk 38.5 mL, open-top or equivalent	Beckman Coulter	344058
Ultracentrifuge Beckman Coulter rotor fixed angle or equivalent	Beckman Coulter	Type 70 Ti or Type 50.2 Ti
Ultracentrifuge Beckman Coulter swinging bucket or equivalent	Beckman Coulter	SW 32 Ti or SW 28
Ultracentrifuge Optima XPN-90 - IVD (Biosafe) or equivalent	Beckman Coulter	B10052
Formvar carbon EM grids	Agar Scientific	AGS162
Transmission electron microscope or equivalent fitted with a digital camera	JEOL, Japan	JEOL JEM 1010 80 kV
Dynamic light scattering equipment-Zetasizer	Malvern, UK	Zetasizer Nano ZS
Microfiltration blotting device, includes 96-well Bio-Dot apparatus, or other appropriate	Bio-Rad	1706545
Imaging System MP ChemiDoc, or other appropriate chemiluminescence imaging equipment	Bio-Rad	12003154
Curved forceps - Jewelers forceps	John Weiss	0101374SS
Bonn toothed forceps	Duckworth & Kent, Ltd.	2-110E
Troutman-Barraquer toothed forceps	Duckworth & Kent, Ltd.	2-132E
Kelman-McPherson tying forceps	Duckworth & Kent, Ltd.	2-529E
Sterile Becton Dickinson Microlance needles 21G needles x 1.5″ x 100	Any brand

Materials and equipment

Recipes of stock solutions

• •30% BSA stock solution: Dissolve 15 g of BSA in 500 mL BrainPhys Neuronal Medium phenol red free. Filter with Stericup Millipore 0.22 mm and store at the fridge for up to 1 month or aliquot in 5 mL volumes in 15 mL Falcon tubes and freeze for long term storage in −20°C.
• •Taurine (50 mM) stock solution: Dissolve 31.29 mg Taurine (Merck KGaA- Sigma-Aldrich, cat. no. T869) in sterile conditions in 5 mL sterile PBS. Make sure the solution is homogenous and filter sterilize via vacuum using Stericup Millipore 0.22 mm (Millipore, cat no. S2GPU05RE). Store aliquots of taurine solution in 5 mL volumes in 15 mL Falcon tubes and freeze for long term storage in −20°C.
• •PDL coating solution (0.1 mg/mL): Reconstitute in sterile dH₂O according to manufacturer’s instructions (concentration). Add 50 mL of sterile tissue culture grade water to 5 mg of Poly-D-lysine. Store at 4°C, solution is stable for long term.
• •Fibronectin coating solution (0.04 mg/mL): Dilute 2 mg of liquid Fibronectin in 50 mL of sterile TC grade PBS. Store at 4°C, solution is stable for long term.
• •Methyl cellulose Stock Solution 2%: Heat 196 mL of distilled water to 90°C and add 4 g methyl cellulose powder at a magnetic stirrer, while stirring at 100 rpm. Immediately transfer solution in cold-room area and continue slow stirring overnight at 4°C. Allow solution in the fridge for 3 days, bring volume to 200 mL.
• •Methylcellulose for EV staining: Use 9 parts 2% methyl cellulose and 1 part of 2% uranyl acetate.
• •Uranyl acetate solution 2%: Dissolve 0.2 g of uranyl acetate in 10 mL distilled water. Filter with 0.22 μm PVDF filters and store in a plastic bottle at 4°C. Radioactive materials require special handling. Make sure you follow Institutional Radio safety guidelines.
• •Sucrose 20%: Dissolve 10 g of Sucrose and dissolve in 50 mL EM grade PBS. Store at 4°C
• •PFA 4% solution: Dissolve 4 g of PFA in 90 mL PBS (Gibco) at 65°C while stirring. Add 1 N NaOH in drops until the solution becomes clear. Bring volume to 100 mL with PBS, allow the solution to cool down at 25°C and filter with 0.22 μm PVDF filters. Store in aliquots at −20°C. Once aliquots thawed keep in 4°C for no longer than a week.
• •Sir-Actin or Sir-Tubulin live imaging staining: Dilute probe in photoreceptor cell culture medium without phenol red, at a concentration of 0.01 nmol (1:5000 dilution) supplement the probe medium mix with 1 nmol Verapamil (1:1000 dilution). Note that Verapamil is included in the staining kit. Prepare staining solution fresh every time and incubate the cells for 19 h at 37°C, 5% CO₂. The following day remove the staining solution and replace with fresh medium.

Photoreceptor chemically defined medium	Final concentrations	Volume
BrainPhys Neuronal Medium no phenol red STEMCELL Technologies Inc.	Up to 50 mL	45.65 mL
HEPES (1 M)	15 mM	0.750 mL
100× ITS (1.0 mg/mL recombinant human insulin, 0.55 mg/mL human transferrin (substantially iron-free), and 0.5 μg/mL sodium selenite)	Diluted from 100× to 1×	0.500 mL
Taurine (50 mM)	1 mM	1 mL
BSA (30%)	1%	1.6 mL
Antibiotics Pen/ Strep 1000 mg/ml (Sigma)	Diluted from 100× to 1×	0.500 mL

∗Aliquot Photoreceptor medium and store at −20°C, medium should be used within 1 week if kept at 4°C. Use within a week once complete medium is prepared.

Dissection buffer	Final concentrations	Volume
DMEM/F12	Up to 50 mL	48.20 mL
HEPES (1 M)	15 mM	0.750 mL
Taurine (50 mM)	1 mM	1 mL
DNase (from Worthington kit)	0.5 U/mL	0.05 mL

∗Aliquot and store at −20°C, medium should be used within 1 week if kept at 4°C. Use within a week once thawed.

MACS staining buffer	Final concentrations	Volume
PBS	Up to 50 mL	46.87 mL
HEPES (1 M)	15 mM	0.750 mL
30% BSA	0.5%	0.83 mL
EDTA (0.5 M)	2 mM	1.5 mL
DNase (from Worthington kit)	0.5 U/mL	0.05 mL

∗Preferably, make MACS staining/wash buffer fresh on the day but can be used for up to 1 week, if kept at 4°C.

Step-by-step method details

Postnatal retina dissection

Timing: 1 h

At this step, neural retinas are dissected from the enucleated eyes. Dissections can be performed outside TC hood, provided all surfaces are wiped down with 70% ethanol and good hygiene maintained. This is a very critical step as shorter dissection times and retention of neural retinal tissue integrity yield higher returns of viable cells after enrichment. Please refer to Figure 2 panels(i-iii) for visual representation of the dissection process.

• 1.Pre-warm dissociation solution in a water-bath (37°C) 10–15 min prior the dissection step.
• 2.Euthanize an appropriate number of pups according to Institutional and regional regulations and spray the heads with 70% Ethanol.

Note: For example, for 4 pups of post-natal day (P)8 the starting retinal cell number should be ∼52 × 10⁶, and the expected recovery of rod photoreceptors at the end of this protocol is ∼32 × 10⁶

• 3.Once ethanol dries, open both eyelids, and enucleate the eyes with blunt end forceps and puncture the cornea with a sterile needle.
• 4.Carefully transfer the eyes in 5 mL of fresh dissection medium in a Falcon tube (15 mL) on ice.

Note: Try to maintain parts of the optic nerve or some remaining fat around the orbit; this will facilitate the excision of the neuroretina without damage, by providing tissue to use to hold the eye stable during retinal isolation.

• 5.Allow the eyes to sink at the bottom of the tube and decant the dissection medium.

CRITICAL: Total time for enucleation/dissection should be kept to a minimum (< 3 min per retina).

• 6.
  Transfer 1 eye at a time with a wide tipped transfer pipette.

  • a.
    Place the eyes in a clean 30 mm plastic dish, placed under a stereomicroscope equipped with a light source.
  • b.
    Add <200 μL drop of fresh sterile dissection medium per eye.
  • c.
    Keep dissection medium to <200 μL per eye to avoid unnecessary movement of the excised neural retina.
• 7.Hold the eye with curved forceps and insert a pair of fine forceps in the puncture hole made previously into the cornea-sclera area.
• 8.
  Use the second pair of fine forceps and unpeel the limbus in as few movements as possible.

  • a.
    Extract the neuroretina from the eyecup by inserting blunt-end forceps into the subretinal space and by carefully separating the surrounding layers of the sclera, freeing the neuroretina from the retinal pigment epithelium (RPE).
  • b.
    Try to avoid touching the retina during excision to minimize damaging the photoreceptors.
  • c.
    If explant culture is required, the lens could be maintained. If dissociation is required, it is mandatory to remove the lens.
• 9.Cut the tip of the plastic Pasteur pipette using a pair of scissors and transfer the isolated neural retina using a plastic Pasteur pipette to a 15 mL Falcon tube with 5 mL fresh dissection buffer.
• 10.Repeat for all eyes (no need to add additional dissection media with subsequent transfers of individual retinas to the collection tube).

Note: Perform the dissections, sacrificing one pup at a time and maintain all eyes on ice inside a firmly closed 15 mL Falcon tube with 5 mL Dissection buffer.

Figure 2.

Representative morphologies of primary photoreceptors in culture

(A) Representative live imaging of P7 Nrl.Gfp^+ve primary postmitotic photoreceptor precursor cells at day 3 in culture live bright-field wide-field imaging versus (B) live confocal imaging in low and (C) high magnifications. Red arrows; cell processes in bright-field microscopy, white arrows; cell processes in confocal microscopy, white arrowheads; inner segment-like extensions. Scale bars: 50 μm in low magnification, 20 μm in high magnification. Green = Nrl.Gfp^+ve.

(D–F) Representative confocal images of P7 wild-type primary postmitotic photoreceptor precursor cells at day 3 in culture fixed and immunostained with Recoverin (magenta) and DAPI (nuclei, white). White arrows; cell processes with confocal microscopy. Scale bar 50 μm in low magnification (d&e) and 20 μm in high magnification (F). Magenta = recoverin; Gray = nuclei.

(G–I) Representative live confocal images of P7 Nrl.Gfp^+ve primary postmitotic photoreceptor precursor cells at day 3 in culture stained with sir-Tubulin probe (red). Asterisk; tubulin rich nanotube connecting two Nrl.Gfp^+ve cells.

Retina dissociation

Timing: 1 h

Perform the Enzymatic dissociation of neuroretinas based on a papain dissociation system. This step should take place in sterile conditions, according to manufacturer’s instructions⁴ (https://www.worthington-biochem.com/products/papain-dissociation-system/manual) with some minor modifications.^1,4 Please refer to Figure 2, (panels iv & v) for visual representation of the dissociation process.

• 11.Inside a hood, decant the dissection buffer and replace with 1 mL of prewarm dissociation buffer for ∼0.1 mL.
• 12.Firmly close the tube and place it back in the water-bath for dissociation.

Note: Adjust volume appropriately for more/fewer retinas.

• 13.Incubate the retinas with the dissociation solution for the appropriate length of time for the respective developmental time point (see Table 2) with discontinuous agitation every 5 min.

Note: For example, for x8-12 P6-8 murine retinas, incubate for 20 min and agitate 3–4 times.

• 14.Use a wide tip pipette to gently triturate the retinal tissue 5 times without generating bubbles. Place the tube back in the water bath for 5 min to complete digestion.

Note: To generate the wide tip cut ∼2 cm of the end of the plastic 1 mL tip with scissors.

CRITICAL: It is important to not use a thinner tip or over-triturate the retinal tissue as this may impact on cell viability.

• 15.Centrifuge the dissociated tissue at 260 × g for 5 min at 25°C.
• 16.Discard the supernatant, carefully, and reconstitute the pellet by mild trituration with a 1000 μL tip with 300–500 μL of resuspension buffer.

Note: For example, for 12 x P8 murine retinas resuspend in 350 μL resuspension buffer.

• 17.Layer the dissociated retinas, carefully, over 1 mL of ovomucoid-inhibitor solution (use a 15 mL Falcon tube), angling the tube during the layering process to minimize mixing.
• 18.Centrifuge at 70 × g for 10 min at 25°C.
• 19.Discard the supernatant and gently resuspend the cell pellet in 1 mL MACS buffer.

Note: The dissociated retinal cells will be in the pellet at the bottom of the tube, broken membranes will be at the interface between the ovomucoid solution and the resuspension media.

• 20.Assess cell number and viability using a small sample and with Trypan blue.

CRITICAL: It is important that the viability should not fall below 80% at this step.

Note: For mixed retinal cultures, STOP here and plate the cells in photoreceptor medium at a final density not exceeding 80,000 live cells/mm² in PDL/fibronectin pre-coated glass or plastic bottom plates.

Photoreceptor enrichment and culture

Timing: 2 h

Photoreceptor enrichment is performed by established methods of magnetic immune-isolation, based on CD73 expression, as described by Eberle et al., and Lakowski et al.^5,6,7 Please refer to Figure 1, panels 6–11 for visual representation of the enrichment process.

Figure 1.

Step by step photoreceptor cells enrichment

(i-iii) Representative images of protocol dissection steps 1–9; (iv-v) Representative images of papain dissociation steps 10–17 layering pre (iv) and post centrifugation (v); Representative images of photoreceptor cell enrichment steps 18–29: (vi-vii) labeling with CD73-APC and anti-APC beads, (viii-x) Magnetic immune-isolation, (xi) anticipated pellet of CD73+ve cell fraction post MAC-sorting.

This step should take place in sterile conditions.

• 21.Adjust the volume of MACS buffer supplemented with anti-CD73-APC antibody according to the formula: 1 x 10⁷ cells / 100 μL MACS buffer / 2 μL of CD73-APC.
• 22.Incubate at 4°C for 30 min with discontinuous agitation.
• 23.Top up the cells with 5 mL MACS buffer and wash by centrifugation at 260 × g for 5 min at 25°C.
• 24.Decant supernatant and resuspend in 1 x 10⁷ cells / 80 μL MACS buffer/ 20 μL of anti-APC magnetic beads.
• 25.Incubate at 4°C for 30 min with discontinuous agitation.
• 26.Dilute cells further in 1 mL of MACS buffer and place them in LC-Column, attached firmly in the magnetic stand and connected to a tube to collect flow through.

Note: At this step, negative cells (unstained photoreceptors and other retinal cells) can be collected and used for other applications or discarded. It is recommended that the negative collection tube is retained until completion (see troubleshooting) before discarding.

• 27.Wash the column with 5 mL of MACS buffer, collected in the same tube.
• 28.Remove the LC column from the magnetic stand and place it on top of a 15 mL Falcon tube in a rack.
• 29.Wash the column with 5 mL of MACS buffer to collect the CD73-positive photoreceptors.

Note: Use the plunger to add gentle positive pressure while cells are passing through the column.

Note: The positive cell fraction is collected at this step. Cells are pulled down with the beads. We recommend discarding the first drop to achieve higher purity as this may include remaining impurities. Notice, due to the color of the beads, the first drops will be a light shade of brown. See Figure 1, panels 8–11.

• 30.Collect the flow-through and further dilute in 5 mL Photoreceptor Medium.
• 31.Wash by centrifugation at 260 × g for 5 min at 25°C.
• 32.Asses the viability and plate photoreceptors at a final density not lower than 90,000 cells/mm² in PDL/ fibronectin pre-coated glass bottom plates.
• 33.Incubate cells at 37°C, 5% CO₂.
• 34.Remove and replace the medium every 3–4 days.

Photoreceptor extracellular vesicles enrichment

Timing: 5–6 h

Extracellular vesicles (EVs) are lipid bilayer rounded structures that may be secreted by cells through various mechanisms. Regardless of the mechanism of production and release, they can be found in, and enriched from, tissue culture supernatants and tissue matrixes. Here, EV isolation was based on the sequential ultracentrifugation protocol described by Thery et al., (2006),⁸ with the modifications proposed by Kowal et al. (2016).⁹

CRITICAL: Prior proceeding to EV isolation make sure the cells are viable by assessing the morphologies under a light microscope and performing a viability test (Trypan Blue is adequate). Keep a record of the morphologies observed and sacrifice one well of cells to confirm that viability is above 80% to proceed with EV enrichment.

Note: To generate adequate numbers of 100 K small EVs for characterization and experimentation, a pool of three independent cultures required at least 90∗10⁶ photoreceptor cells.

• 35.Allow the cells to attach to the culture dishes for at least 19–24 h post plating.
• 36.The next day remove culture medium and replace with fresh medium.
• 37.Collect the photoreceptor medium from culture day 3, 5, 7 for EV enrichment.
• 38.Immediately centrifuge cell supernatant at 350 × g for 10 min at 4°C to remove cell debris.

Note: Use centrifuge 5804/ 5804 R, Rotor A- 4-44.

Note: This step can be performed with a standard benchtop centrifuge. Keep the supernatant on ice.

• 39.Discard the pellet and carefully transfer the supernatant to a new Falcon tube by pipetting without disturbing the pellet.

Note: The procedure can be performed at 25°C.

• 40.Centrifuge cell supernatant then at 2,000 × g for 20 min at 4°C. This step can be performed with a standard benchtop centrifuge.

Note: Use centrifuge 5804/ 5804 R, Rotor F-34-6-38, high speed, fixed angle, or equivalent.

• 41.Proceed by keeping the supernatant on ice.

Note: Large EVs and apoptotic bodies, or 2 K pellet, can be retrieved from this step.

• 42.Transfer supernatant into a clear 15 mL Falcon tube and centrifuge at 10,000 rpm for 60 min at 4°C.

Note: Medium EVs and apoptotic bodies, or 10 K pellet, can be retrieved from this step.

• 43.Filter remaining supernatant with 0.22 μm PVDF filter (Millipore)
• 44.Transfer the filtered supernatant into ultra-centrifuge tubes (30 mL, Beckman)
• 45.Centrifuge at 4°C, for 3 h at 25,000 rpm on a SW 32Ti rotor 38 mL tubes (100 K).
• 46.Discard the supernatant in one motion and drain the tubes in tissue paper inside a TC hood.

CRITICAL: Small EVs or 100 K pellets will be retrieved from this step.

• 47.100 K pellets should be reconstituted in 0.1 mL of EM grade sterile ice-cold PBS.
• 48.Keep 100 K pellets on ice for 1 h shaking before any use.

Note: The total volume of EVs obtained will be increased to ∼0.150 mL after agitation as the remaining not drained medium will also contribute to the sample volume.

CRITICAL: EV samples can be stored at 4°C for two weeks or −80°C for up to 2 months. For in vivo experimentation do not freeze-thaw EV preparations.

• 49.Post collection of 100 K EV samples, pool 3 or more dissociations together for ultrastructural and molecular analysis

Note: EV samples can be stored at 4°C for two weeks or −80°C for up to 2 months. For in vivo experimentation do not freeze-thaw EV preparations, store at 4°C and use within 2 weeks.

• 50.
  For each EV enrichment experiment, separate the EVs in aliquots.

  • a.
    Use 0.05 mL EV sample for molecular characterization with Dot-blots/Western Blots or PCR;
  • b.
    Use 0.01 mL of sample for particle analysis with Dynamic Light Scattering (DLS) instrument or other available equipment such as Nanoparticle tracking analyzer (NTA);
  • c.
    Use 0.02 mL for Ultrastructural analysis should also take place to confirm EV population with EM;
  • d.
    Use the remaining 0.07 mL sample for in vivo or in vitro testing of biological functions of EVs.

CRITICAL: It is mandatory to characterize the EV content prior any in vivo or in vitro experimentation with EV preps. Preferably within 48 h of isolation as the EV samples tend to aggregate.¹⁰ For more information, please see ISEV position papers for minimal requirements for publication.^11,12

Photoreceptor extracellular vesicles ultrastructural analysis

The ultrastructural analysis will confirm the presence of extracellular vesicles in the preparations and should be performed for every EV enrichment. This step was performed as described in Thery et al., 2006⁸ with some modifications described below.

• 51.Utilize 0.02 mL of the retrieved 100 K pellets in EM grade PBS.
• 52.Dilute the sample 1:1 ratio with a fixation buffer containing 4% PFA/10% Sucrose, resulting in 0.04 mL of EV sample in 2% PFA/5% Sucrose in PBS.
• 53.Fix the samples inside a fume hood for 10 min in 25°C.
• 54.Allow EV samples to be absorbed by Formvar-carbon coated EM grids 20 min-19 h in 20–25°C, by forming a drop on the one side of the EM grid.
• 55.Wash, by inverting the grid -using fine forceps- on drops of 0.05 mL of PBS on parafilm.

CRITICAL: Inverting the grid refers to the side of the EM grid that the EVs have been absorbed. For all steps after absorbing the EVs the EM grid should be facing the drops (inverted).

• 56.Allow the drop for 5 min 20–25°C.
• 57.Repeat PBS wash once more for 5 min 20–25°C.
• 58.Post fix the grid on a drop of 0.05 mL 1% Glutaraldehyde solution for 5 min 20–25°C.
• 59.Wash 3 times (2 min each) by transferring the grid on 0.05 mL drops of ddH₂O formed in parafilm.
• 60.Transfer the EM grid on 0.05 mL drop of 1% uranyl-acetate solution (UA) (pH = 7) for 5 min 25°C.
• 61.Wash 3 times (2 min each) by inverting the grid in tissue paper and by adding 0.05 mL of ddH₂O.
• 62.Add methyl-cellulose staining solution (pH 4) and incubate for 10 min on ice.
• 63.Allow grids to air dry for 5 min 25°C

Note: At this step the EM grid side that has absorbed the EVs should be facing upwards.

• 64.Store samples in grid storage boxes.
• 65.Analyze EM grids using a JEOL 1010 Transmission Electron Microscope (80 kV), fitted with a digital camera for image capture, or another equivalent electron microscope.

CRITICAL: Prior the molecular characterization of the encapsulated cargo of EVs the samples should be pretreated with Proteinase K, if protein cargo is to be analyzed and Proteinase K followed by RNase A, if RT-qPCR analysis is required.

Extracellular vesicles enzymatic treatment prior molecular analysis

The molecular analysis of the EV cargo will provide important information of molecules that reside inside the vesicle lumen. During the ultracentrifugation of EVs genetic material and or other proteins can precipitate and aggregate along with the vesicles, resulting in unspecific proteins and RNA species in EV preps, it is therefore critical to eliminate any non-specific to EV molecules when analyzing the EV cargo. For further information readers are referred to Norman et al., 2021.¹³

Before proceeding to molecular analysis of EVs, perform an enzymatic pretreatment as described below.

• 66.Add 100 μg proteinase K in the 0.05 mL EV pellet and incubate for 15 min 37°C.

Note: Heat inactivation of Proteinase K at 90°C for 10 min is optional.

• 67.Add 10 ng/μL RNase A and further incubate for 15 min at 37°C.
• 68.Inhibit RNase using 1 μL of RNase Inhibitor (stock concentration 20 U/μL) post RNase treatment at 37°C for 10 min.
• 69.Keep samples on ice for further use.

CRITICAL: Extension of the time of Proteinase K and RNase enzyme incubation may result in sample evaporation.

For further information in Dot Blot analysis and DLS analysis readers are referred to Kalargyrou et al., 2021¹ and Wang et al., 2021.¹⁰ If more molecular characterization is required, then readers should refer to.⁸

Expected outcomes

Establishing a robust primary culture system for postmitotic photoreceptor precursors is mandatory to permit the enrichment of EVs from the culture medium and to investigate the potential for physical connections, nanotubes, between photoreceptors in a 2D system.

The protocol described herein (Graphical Abstract) incorporates steps to enrich postmitotic primary photoreceptors based on previously published reports.^5,6,7,14 Briefly, neuroretina tissue is obtained between postnatal day (P)4-8 via dissection. The tissue is then enzymatically dissociated using papain - if mixed retinal cultures are required, the user can stop at this step and plate the dissociated cells. Otherwise, dissociated retinas are enriched for photoreceptors based on positive selection for the cell surface marker, CD73, with magnetic sorting. The established rod photoreceptor cultures are viable for up to 30 days in a chemically defined medium in the absence of serum or supplements.

Primary post mitotic photoreceptor cells extend processes that are visible within the first three days of culture as seen in bright-field imaging (Figure 2A arrows). In order to study photoreceptor processes subtypes (neuronal processes, inner segment like buds and nanotubes) higher magnification and confocal imaging is required (Figure 2B compared to 2c). Representative examples of photoreceptors cells extending processes with super resolution confocal microscopy are provided in Figure 2C. Most cells extend thin processes to the substratum (Figure 2C arrows) and less are presented with bud like protrusions resembling inner segments (Figure 2C arrowheads). CD73 MAC-sorted cells in culture express the photoreceptor marker recoverin along their cell soma and processes (Figures 2D–2F). Photoreceptor processes can be visualized with cytoplasmic GFP driven by Nrl -rod photoreceptor specific² -promoter (Figure 2C) and recoverin immune staining (Figure 2F arrows). For the study of photoreceptor-photoreceptor cell connections like nanotubes live imaging and labeling of the cytoskeleton is required (Figures 2H–2J). Representative example of super resolution live imaging of tubulin cell-cell connections is provided herein (Figures 2H–2J asterisk).

Photoreceptors release extracellular vesicles (EV) in culture. Here we provide a representative example of TEM analysis of the 100 K pellet from a pool of three independent EV enrichments. PR 100 K EVs (Figure 3A arrows; Figure 3B) are ∼120 nm diameter “donut” shaped vesicles with a lipid bilayer as revealed by negative staining with Uranyl acetate. Note that the EV preps also show membranes of various origin -potentially from broken vesicles- therefore pretreatment with Proteinase K and RNase prior the analysis of their cargo content is necessary. Dot blot analysis of the photoreceptor EV cargo of three independent isolations reveals variability in the presence of rhodopsin protein, which may reflect the developmental stage of photoreceptors (Figure 3C). The presence of recoverin in evident in all 100 K preps. Alix, an important protein for EV cargo sorting^15,17 is present in low amounts in all EV preps and LAMP1 a lysosomal/endocytic marker is present in two out of three 100 K EV preps.

Figure 3.

Representative TEM images of primary photoreceptor derived extracellular vesicles and dot blot characterization

(A) Representative TEM wide field image of 100 K EV pellet derived from wild-type photoreceptor cultures; Scale bar = 500 nm (B) Representative TEM microphotograph image of 100 K EV derived from wild-type photoreceptor cultures. Scale bar = 200 nm (C) Representative Dot blot of 100 K EV pellet (each dot represents three individual EV isolations, derived from 40∗10⁶ cells from (1) P6 Nrl.Gfp^+ve, (2) P8 wild-type and (3) P4 Nrl.Gfp^+ve photoreceptors. Photoreceptor markers = Recoverin, Rhodopsin; EV markers = Alix, Lamp1.

Limitations

In contrast to previous reports, our method can permit the isolation and long-term maintenance of rod photoreceptors without the addition of any neurotrophic stimuli. Moreover, the absence of both ECM-based matrix and serum in the culture media permits the enrichment of high purity EVs. The cultured cells continue to mature in vitro and express markers of maturing photoreceptors such as rhodopsin. Whilst the basic morphological characteristics of post-mitotic photoreceptors are retained using this method, the cultured rods do not form an outer segment, a highly specialized cilium.^16,18,19 However, they do extend neurites and nanotubes and, as noted above, nascent segment-like termini.^1,20 The absence of outer segment formation in a 2D culture is not unexpected; indeed, it has proved challenging to achieve fairly rudimentary outer segments in the more intact 3D environment of retinal organoids in the absence of RPE, and after transplantation of photoreceptor precursors in vivo.

Troubleshooting

Problem 1

Cells clumping during dissociation process (related to step 12).

Potential solution

• •Dissection took longer than expected, ensure proficiency of dissections.
• •Double the volume of DNase during dissociation/staining.
• •Use fresh DNase and avoid freeze-thaw cycles for enzymes.
• •Please follow the table of dissociation timings per developmental time point.

Problem 2

Increased amount of cell impurities (related to step 27).

Potential solution

• •At early developmental stages P0-P3 the expression levels of CD73 are low. Therefore, doubling the amount of antibody at the immune isolation step (step 18) is recommended.
• •That might also represent a failure at enrichment step (see problem 4).

Problem 3

Too many RPE cells in the cell prep prior (related to step 12).

Potential solution

• •The presence of RPE might be seen as cell prep clumping (step 12) or cell prep is brown prior anti-APC beads addition. Filter cells through a mesh 80 μm prior proceeding to immune staining.
• •During dissections (step 7), try to remove as much RPE as possible.

Problem 4

Cells not adhering to the plate (related to steps 27–29).

Potential solution

• •Always make fresh coating solutions on the day of dissection and do not leave coating on for >19 h.
• •Make sure you avoid using low binding TC dishes.

Problem 5

Cells not making processes (see Figure 4).

Figure 4.

Examples of healthy versus not healthy photoreceptor cultures

Representative bright-field microscopy photos of primary photoreceptor precursor cells of P7 developmental stage at day 3 in culture extending processes (A) compared to (B) cells possibly dying and not extending processes. Scale bar 20 μm.

Potential solution

• •Always triturate gently the cells. If processes are not visible during the first culture day, wait until day 3 and check cells under both light microscope and confocal.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Rachael A. Pearson, rachael.pearson@kcl.ac.uk.

Technical contact

Aikaterini A Kalargyrou, akalargyrou@meei.harvard.edu

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate/analyze [datasets/code].