Isolation and culture of porcine muller cells

1. Porcine eyes were harvested from pentobarbital-anesthetized animals, each weighing 30 to 40 pounds, and transported to the laboratory in ice-cold saline containing antibiotics.

2. Each eye was hemisected with a circumferential cut posterior to the ora serrata, and the retinas were removed essentially intact after dissection at the optic nerve.

3. Retinas were transferred to ice-cold primary cell solution, washed several times with additional medium to remove loosely adherent material, and stored on ice until use.

4. Cooled retinas were transferred to 10 ml of primary cell solution containing 17 U/ml papain preactivated for 30 minutes with 2 mM cysteine and 1 mM ethylenediaminetetraacetic acid at 34°C.

5. Digestion was for 60 minutes at 34°C, after which the digestion medium was removed and the retina washed twice with fresh primary cell solution.

6. The retinas were then incubated in primary cell solution containing 150 U/ml of deoxyribonuclease I for an additional 30 minutes at 34°C.

7. The retinas were dissociated by trituration (repeated passage) of the tissue through a borosilicate tissue culture pipette (10 ml).

8. After 10 cycles through the pipette, the tissue fragments were permitted to settle for 10 minutes, and the top 8 ml of supernatant was removed and stored on ice.

9. This procedure was repeated five to six times until the retina was almost completely dissociated, leaving primarily vascular elements and fibrous material.

10. Phase-contrast microscopic examination of the individual supernatants revealed that the cellular composition varied with each trituration.

11. Typically, the first three supernatants contained large numbers of photoreceptors but relatively few morphologically recognizable Muller cells, and they were discarded.

12. Generally, fractions 4 to 6 were enriched for Muller cells.

13. Supernatants with the highest numbers of Muller cells were combined for further processing.

14. Cells were pelleted, resuspended in primary cell solution containing 10% fetal bovine serum and layered on a 10-ml continuous density gradient composed of 0% to 50% Percoll in normal saline (0.9% wt/vol NaCl) for separation at 500g for 5 minutes.

15. Optimal purification was achieved by sequential centrifugation through two gradients.

16. After recovering from the second density gradient, cells were resuspended in growth medium composed of primary cell culture medium containing 20 mM HEPES, 10% fetal bovine serum, and 1% Penn/Strep/Fungisone and were introduced into culture.

17. Cells were maintained at 37°C in a humidified atmosphere composed of 5% CO2 and 95% air.

18. Routine cell culture was in 75-cm2 tissue culture flasks coated with 0.1mg/ml monomeric type I collagen dissolved in 0.012 M HC1.

References
1. Guidry C. Isolation and characterization of porcine Müller cells. Myofibroblastic dedifferentiation in culture. Invest. Ophthalmol. Vis. Sci. 1996; 37: 740-752.
2. Serge Picaud, Bikash Pattnaik, David Hicks, Valérie Forster, Valérie Fontaine, José Sahel and Henri Dreyfus. GABAA and GABAC receptors in adult porcine cones: evidence from a photoreceptor–glia co-culture model. Journal of Physiology. 1998; 513: 33-42.