Isolation, culture and identification of primary Müller cells from the macula and peripheral human retina

Human Primary Muller Cell (huPMCs) Culture SOP

MEDIA REQUIRED:

DMEM (Gibco, Cat#10569-010) without FBS

Complete medium: DMEM containing 10% FBS and 1% Penicillin-Streptomycin

Trypsin-EDTA (TrypLE^TM, Invitrogen Cat#12563-029, 1×): Concentrations of Trypsin-EDTA are kept confidential by the company

T25-cell culture flasks (Corning cat#3289)

EQUIPMENT REQUIRED:

(sterile) forceps; scissors; hemostatic forceps; gauzes; tubes; Petri dishes; 1ml syringes and 18G needles; glass pipettes.

PROCEDURES:

Day 0:

1. Collect 1 cm² of human retina in a sterile tube containing 5ml DMEM without FBS.
2. Wrap the tube with foil and kept it in 4 ºC fridge overnight.

Day 1:

1. Pre-warm the Trypsin-EDTA digestion solution and DMEM medium supplemented with 10% FBS and 1% Penicillin-Streptomycin in a 37°C water bath before the experiment. 
2. Transfer the tissue to a new sterile tube containing 5ml pre-warmed TrypLE to digest the retina. Wrap the tube with foil and incubate in a CO2 incubator at 37°C for 60 min.
3. Transfer the digested retina into 60mm petri dish containing 10 ml complete medium. 
4. Cut the tissue into small pieces (around 1 mm²) in the complete medium under a dissecting microscope in the dissection hood.
5. Transfer the retinal pieces into T25-cell culture flasks using sterile glass pipettes with a minimum volume of DMEM medium.
6. Bend the sterile 18G needle to 90˚ angle using hemostatic forceps. Separate the dissected retinal pieces to prevent clumping using the angled needle.
7. Use 18G angled needle to nail the retinal pieces onto the bottom of T-25 flasks to enhance the success rate of primary human Muller cell culture. Add 2 ml complete medium into each flask. Avoid washing off the attached retina pieces.
8. Place the flask vertically in the cell culture incubator (CO₂, 37°C) for 15 minutes to dry a little bit and allow better attachment of retinal pieces to the bottom of T-25 flasks.
9. Place the T-25 flasks to a normal position. The medium (2ml) is enough to cover the bottom of the flask and would minimize the chance of tissue detachment during culture.
10.  Minimize the disturbance of the flasks.

Day 7:

1. Add another 2ml complete medium in each flask.
2. Minimize the disturbance of the flasks.

From Day 14:

1. Change the medium with 4ml fresh complete medium.
2. The maintenance of huPMCs requires medium change twice a week.
3. Primary Muller cells usually grow out of retinal pieces in 2-4 weeks. 

Category