AND RETINA CULTURE
vitreous is loosened until the retina separates from the vitreous body. At this stage, you should have a complete retina that is shaped like a hemisphere with a small hole at the site of the optic disk. With practice the procedure will require about 1 min.
 C u l t u r e o f M a m m a l i a n R e t i n a l P i g m e n t Epithelium and Neural Retina
By Ross B. EDWARDS The purpose of this article is to describe methods of preparing primary monolayer cultures of mammalian retinal pigment epithelium (PE) and neural retina. Additional methods for culturing these tissues include organ culture of whole eyes of frog embryos, ~ adult tree toad, z mouseP "4 and ratS-7; suspension cultures of neural retinal cells from embryonic chicks'a; cell lines derived from human retinoblastoma~°'H; organ cultures of human 12"13and bovinC 3 PE-choroid complex; explant culture of human '4 and rat x5 PE; and monolayer cultures of embryonic chick neural retina, 8"~6 and PE. 17 General methods of tissue culture have been covered in this series TM and elsewhere? 9'2° 1 j. G. Hollyfield and P. Witkovsky, J. Exp. Zool. 189, 357 (t974). 2 j. C. Besharse, R. O. Terrill, and D. A. Dunis, Invest. Ophthalrnol. Visual Sci. 19, 1512 (1980). 3 R. L. Sidman, Dis. Nerv. Syst. 22, 14 (1961). 4 M. Tamai, J. Takahashi, T. Noji, and K. Mizuno, Exp. Eye Res. 26, 581 (1978). 5 M. M. LaVail and W. Hild, Z. Zellforsch. Mikrosk. Anat. 114, 557 (1971). 6 A. I. G o l d m a n and P. J. O'Brien, Science 201, 1023 (1978). 7 M. T a m a i and P. J. O'Brien, Exp. Eye Res. 28, 399 (1979). 8 j. E. Morris and A. A. Moscona, Science 167, 1736 (1970). s G. Ramirez and N. W. Seeds, Dev. Biol. 60, 153 (1977). 10 T. W. Reid, D. M. Albert, A. S. R a b s o n , P. Russell, J. Craft, E. W. Chu, T. S. Tralka, and J. L. Wilcox, J. Natl. Cancer Inst. 53, 347 (1974). 11 R. C. McFall, T. W. Sery, and M. M a k a d o n , Cancer Res. 37, 1003 (1977). 12 M. O. M. T ' s o , D. Albert, and L. E. Z i m m e r m a n , Invest. Ophthalmol. 12, 554 (1973). 13 L. F e e n e y and R. N. Mixon, Exp. Eye Res. 22, 533 (1976). 14 D. M. Albert, M. O. M. T ' s o , and A. S. Rabson, Arch. Ophthalmol. 88, 63 (1972). 15 R. B. Edwards and R. B. Szamier, Science 197, 1001 (1977). 16 T. S. Okada, Y. Itoh, K. Watanabe, and G. Eguchi, Dev. Biol. 45, 318 (1975). 17 R. D. Cahn, H. G. Coon, and M. B. Cahn, in " M e t h o d s in Developmental Biology" (F. H. Wilt and N. K. Wessells, eds.), p. 493. CroweU (Collier), N e w York, 1967. 18 W. B. Jakoby and I. H. Pastan, eds., " M e t h o d s in E n z y m o l o g y , " Volume 58. A c a d e m i c Press, N e w York, 1979. 19 j. Paul, "Cell and Tissue C u l t u r e , " 5th ed. C h u r c h i l l - L i v i n g s t o n e , Edinburgh and London, 1975. 20 p. F. K r u s e , Jr. and M. K. Patterson, Jr., eds., " T i s s u e Culture: M e t h o d s and Applicat i o n s . " Academic Press, N e w York, 1973. METHODS IN ENZYMOLOGY, VOL. 81
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181981-7
ISOLATION OF ORGANELLES AND MEMBRANES
Unmodified Puck's: Puck's saline F 21 containing 50/zg/ml gentamicin and 100/xg/ml kanamycin Calcium-enriched Puck's: unmodified Puck's with 0.3 mM CaCI~ Ca 2+- and Mg2÷-free Puck's: unmodified Puck's free of calcium and magnesium EDTA-containing Puck's: Ca 2÷- and Mg2+-free Puck's with 0.1 mM or 10 mM ethylenediaminetetraacetic acid (EDTA) as specified in the text Trypsin solutions (prepared fresh on day of use): trypsin (1:250, Difco, Detroit, MI) at the specified concentrations is mixed with the appropriate balanced salt solution (see text) for 5-10 min. The pH is adjusted to 7.9-8.1 with 0.1 N NaOH or 0.3 N HCI, followed by filter sterilization Culture medium: For rat PE and neural retina, F-10 defined medium2z is supplemented with 20% fetal bovine serum, 50/.~g/ml gentamicin, and 100/zg/ml kanamycin, pH 7.4-7.8. The same medium with an additional 5.5 g glucose per liter2z is used for human PE Scalpel blade, disposable, Bard-Parker no. 11 (pointed) Microdissection scissors, curved, 6-mm blades Dissection scissors, curved, 20-mm blades Forceps, blunt tipped Dissection microscope Retinal Pigment Epithelium Neonatal Rat PE. The method of preparing monolayer cultures of rat PE has been described in detail elsewhere 24,25 and will therefore be only briefly outlined here. Remove eyes from 6- to 8-day-old pigmented rats and allow them to soak overnight in calcium-enriched Puck's. Incubate the eyes in an enzyme mixture (1 mg/ml trypsin and 70 units/ml collagenase in calcium-enriched Puck's) for 45 min at 37°. Under a dissecting microscope remove the anterior segment and the neural retina and tease the intact sheets of PE from each eyecup. Trypsinize the pooled PE for 12 min with 1 mg/ml trypsin in Puck's with 0.1 mM EDTA and dissociate the cells with gentle pipetting. Add an equal volume of culture medium and centrifuge. Resuspend the pellet in medium and seed the cells at 21 T. T. Puck, S. J. Cieciura, and A. R o b i n s o n , J. Exp. Med. 108, 945 (1958). 22 R. G. H a m , Exp. Cell Res. 29, 515 (1963). 23 j. M a n n a g h , personal c o m m u n i c a t i o n (1974). 24 R. B. E d w a r d s , In Vitro 13, 301 (1977). 25 R. B. E d w a r d s , Vision Res. 21, 147 (1981).
AND RETINA CULTURE
25,000/cm 2 of culture surface in plastic tissue culture dishes. The method is equally applicable to normal pigmented rats and to pigmented RCS rats with hereditary retinal dystrophy. With practice the method can also be used to isolate and culture PE from albino rats. Cultured rat PE has been used to study the phagocytosis of rod outer segments 15.26,27 and taurine uptake .28 P o s t n a t a l H u m a n P E . The following is a modification of the method of Mannagh et al. 29 Eyes should be enucleated as soon as possible after death and stored at 0 - 4 ° on moistened gauze in a capped sterile bottle (i.e., normal eye bank storage conditions). Eyes should not be used for at least 8 hr after death, since removal of the neural retina at earlier times results in damage to the PE. Best results are obtained when cultures are prepared within 30 hr after death, although useful yields of viable cells may be obtained up to at least 72 hr after death. Rinse the globe in two changes of unmodified Puck's and place it on a dry sterile towel or gauze. With a pointed scalpel blade make an incision 3 - 5 mm posterior to the limbus just anterior to the ora serrata, which appears as a faint dark ring beneath the sclera. Extend the incision circumferentially with curved scissors and remove the anterior segment. To remove the vitreous and most of the neural retina, grasp the optic nerve stub with large forceps and, while holding the globe with the exposed surface of the vitreous facing downward, shake out the vitreous with a vigorous abrupt downward motion. This method minimizes injury to the PE and Br0ch's membrane and best maintains the adherence of B A c h's membrane to the sclera. If BrOch's membrane should become separated from the sclera, carefully place it back into position with forceps. Remove any remaining neural retina with forceps. Place the eyecup on a bed of cotton in a widemouth jar such that the plane defined by the ora serrata is horizontal. With a sterile Pasteur pipette, vigorously rinse the exposed PE twice with Puck's containing 0.1 mM EDTA to remove dead PE cells, contaminating cells, and retinal debris. Viable PE cells will remain attached to Briich's membrane. Rinse gently three more times. Add a sufficient volume (2-3 ml) of 2.5 mg/ml trypsin in Puck's with 0.1 mM EDTA to cover about two-thirds of the PE. To prevent contamination of the PE cells by choroidal cells do not allow solution to spill into the choriocapillaris. Cap the jar and incubate the eyecup at 37° for 30 min. Pipette the trypsin solution against Briich's membrane to dislodge the PE cells. Remove the cell suspension and add fresh trypsin solution to the eyecup. Repeat this incubation and harvestzn M. O. Hall, Science 202, 526 (1978). 27 R. B. Edwards and S. Bakshian, Invest. Ophthalmol. Visual Sci. 19, 1184 (1980). 28 R. B. Edwards, Invest. Ophthalmol. Visual Sci. 16, 201 (1977). 29 j. Mannagh, D. V. Arya, and A. R. Irvine, Jr., Invest. Ophthalmol. 12, 52 (1973).
ISOLATION OF ORGANELLES AND MEMBRANES
ing procedure until all the PE is removed, usually 2-2.5 hr. Immediately after the PE suspension of each incubation is removed from the eyecup, add to the suspension an equal volume of culture medium, centrifuge at room temperature, remove the supernatant, and resuspend the pellet in 0.5-1.0 ml of medium. Pool and count the resuspended PE cells; note, however, that the first 30-min incubation usually produces fewer cells and relatively more debris than subsequent incubations. Seed the cells in plastic tissue culture dishes at a density of 10,000-150,000 cells/cm z of culture surface. Maintain the cultures in a water-saturated atmosphere of 95% air and 5% COs. Allow the cells to remain undisturbed for at least 2 and preferably 3-4 days before changing medium; thereafter, change the medium every 2-4 days. Cultures may be rinsed free of debris with a sterile pipette. For eyes from donors less than 25-30 years of age, the sclera often collapses after removal of the vitreous. In such cases the anterior rim of the sclera may be pinned with dissecting needles to the inner surface of a hollowed-out block of silicon rubber. To subculture a confluent culture of primary human PE, rinse the cells three times with unmodified Puck's and once with Ca 2+- and Mg~+-free Puck's. Add 1 mg/ml trypsin in Puck's with 0.1 mM EDTA. Incubate at room temperature 2-3 min, then pipet the solution until all the cells are removed, but no longer than 4-5 min of total time in the initial trypsin incubation. Remove the suspended cells and immediately add to the suspension an equal volume of culture medium. Repeat the trypsin incubation if necessary. Centrifuge, count, and seed the cells at 50,000/cm 2 of culture surface. The foregoing method has also been successfully used to isolate and culture PE from the dog, monkey, and cow. In general, eyes from animals other than human or cow require that the sclera be supported with needies. Cultured human PE has been used to study the synthesis of glycosaminoglycans.30 Neural Retina The following method for newborn (within 12 hr of birth) rat retina is based on the method of Okada e t a l . 31 for fetal human retina. Decapitate each rat and quickly dip the head in 70% ethanol. Enucleate and completely immerse the eyes in unmodified Puck's. Under a dissecting microscope puncture each eye just posterior to the ora serrata with a pointed .a0 R. B. Edwards, Invest. Ophthalmol. Visual Sci. 17, ARVO Suppl., 124 (1978). .al T. S. Okada, K. Yasuda, M. Hayashi, Y. Hamada, and G. Eguchi, Dev. Biol. 60, 305 (1977).
scalpel. Extend the incision circumferentially with microdissection scissors and discard the anterior segment, a2 Allow the posterior eyecups to soak for a few minutes until the neural retina can be gently peeled from the eyecup free of adherent pigment epithelium with two pairs of forceps. The use of pigmented rats greatly facilitates the detection of pigment epithelial contamination. Cut the optic nerve to release the retina, transfer the retinas to a sterile tube, and rinse the retinas four times with Ca 2÷- and Mg2+-free Puck's. Incubate up to 12 retinas in 1 ml of Puck's with 10 mM EDTA for 2-3 min at 37°, then begin dissociating the cells by gently pipetting the tissue through a widemouthed plastic pipette until the tissue is broken into small clumps (4-5 additional minutes). Continue the pipetting with a smallmouthed pipette (e.g., a sterile cotton-plugged Pasteur pipette with a fire-polished tip) until the retina has been incubated a total of 15 min in 10 mM EDTA. Add 3 ml of trypsin solution (1 mg/ml in Ca 2÷and Mg2÷-free Puck's). Continue pipetting for 2 more min. Add an equal volume (4 ml) of culture medium. Allow any remaining tissue fragments to settle, then remove and centrifuge the cell suspension. Discard the supernatant, resuspend the pelleted cells in culture medium, and seed the cells at the desired density (up to 7 × 105 per cm 2 of culture surface) in plastic tissue culture dishes. Maintain the cultures at 37° in a water-saturated atmosphere of 95% air and 5% CO2. Change medium every 2-4 days. To avoid dislodging the cells from the culture surface, do not pour medium directly onto the cells. Cultures have been maintained for at least 2 weeks under these conditions. 32See Ref. 25 for illustrationof this technique.
 I s o l a t i o n a n d P u r i f i c a t i o n o f S q u i d R h a b d o m s
By YuJt KITO, TAKAHARU SEKI, and FRANCES M. HAGINS In the cephalopod eye the retina is arranged so that the receptors face the incident light. Each receptor cell is divided by a narrow neck into two distinct zones. The nucleus is in that part of the cell adjacent to the sclera, whereas the segment of the cell extending toward the center of the eye contains the rhodopsin-bearing membranes or rhabdoms. Thus this later part of the cell is functionally comparable to the rod outer segment in the vertebrate eye. Indeed, it is often referred to as the rod outer segment even though anatomically these segments form the inner layer in the cephalopod retina. The central core of the cells of the rhabdomeric layer is
METHODS IN ENZYMOLOGY, VOL. 81
Copyright ~ 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181981-7