IN VITRO Volume13, No. 5, 1977 Allrightsreserved9
CULTURE
OF RAT RETINAL PIGMENT EPITHELIUM ROSS B. E D W A R D S
Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, Massachusetts 02114
SUMMARY A method of preparing monolayer cultures of retinal pigment epithelium from normal pigmented neonatal rats is described. Critical features include incubating the eyes in balanced salt solution and treating with trypsin before dissecting the eyes. The tissue also has been cultured from RCS rats with inherited retinal degeneration. Since the pigment epithelium has been shown to be the primary site of action of the gene for retinal dystrophy in the RCS rat, the method should be useful in studying the defect(s) associated with this mutation.
Key words: retinal pigment epithelium; RCS rat; retinal dystrophy. INTRODUCTION
and then transferred to culture medium F-10 (8) (Microbiological Associates) supplemented with 20% fetal bovine serum, 50/~g per ml gentamicin and 100/ag per ml kanamycin, pH 7.2 to 7.6. An incision was made, using a dissecting microscope, in the globe just posterior to the ora serrata and extended circumferentiaUy; the anterior portion of the globe was discarded. The remaining eyecup with adherent pigment epithelium was peeled gently from the neural retina with two pairs of forceps. The neural retina was dissected free at the optic nerve, and for convenience during the subsequent dissection, the eyecup was bisected. With a fine dissecting knife or jeweler's forceps, the intact pigment epithelial layer was teased free from Bruch's membrane. Pooled sheets of pigment epiMATERIALS AND METHODS thelial cells were rinsed 3 to 4 times in calciumCultures were prepared from Long-Evans rats and magnesium-free saline F containing antibio(Charles River Breeding Laboratories) and from tics and 0.1 mM ethylenediaminetetraacetic acid RCS-p § pigmented rats with inherited retinal de- (EDTA) and then incubated in 0 . I % trypsin (pregeneration ~6). Animals were 6 to 8 days old and pared in the same solution) 8 to l0 rain at 37~ weighed an average of 11 to 12 g. Eyes were enuc- The tissue was dissociated into a single-cell susleated under sterile conditions and immersed in pension by repeated gentle pipetting. Two volsaline F (7), a balanced salt solution, pH 8.0 to umes of culture medium were added to one vol8.4, to which 50 #g per ml gentamicin and 100 #g ume of cell suspension, and the cells were pelleted per ml kanamycin had been added. After remov- by centrifugation for 5 min at 70 • g. Cells were ing loosely adherent connective tissue, the intact resuspended in culture medium, counted in a globes were rinsed two to three times and allowed hemacytometer and seeded at a density of 25,000 to soak in the same solution at room temperature to 30,000 per cm 2 in 17-mm Multiwell plastic for 6 to 24 hr. Intact globes were incubated at dishes (Falcon). Cultures were maintained at 37~ for 45 min in filter-sterilized 0.1% trypsin 37.5~ in 5% CO2 in air; medium was changed iDifco 1:250) dissolved in saline F, pH 8.0 to 8.1, every 2 to 3 days. The seeding efficiency was 301 The retinal pigment epithelium participates in the maintenance of photoreceptor cells by phagocytizing the tips of rod outer segments ~I). In the Royal College of Surgeons ~RCS) rat with inherited retinal degeneration, the pigment epithelium fails to perform this function (2-4). The primary defect resides in the pigment epithelium rather than in photoreceptor or other cell types (5). Comparative studies of cultured retinal pigment epithelium from normal and RCS rats may help to determine the biochemical defect(s) in the RCS tissue. This report describes a method of preparing such cultures.
302
EDWARDS
determined by counting the cells which were attached to the culture surface at 24 hr in vitro and dividing that value by the number of initially seeded cells. For counting, cultures were fixed in situ with 4% formaldehyde and stained with 1% toluidine b l u e - l % sodium borate. Cells were counted in 20 randomly chosen microscopic fields in each of two to four replicate cultures for each time point. Results are expressed as the mean number of cells per cm 2 of culture surface. Protein was measured by the Folin reaction ~9). RESULTS Cells comprising freshly removed intact sheets of pigment epithelium are hexagonal in shape, forming the cobblestone pattern of the tissue seen in situ. The second trypsinization yields a suspension of spherical or asymmetrically dumbbellshaped cells which appear highly refractile under phase-contrast optics. Seeding efficiency varies between 30 and 60%. Typical cells are shown in
Figs. 1 and 2. The majority are binudeate and contain numerous perinuclear pigment granules. Mitoses are commonly seen; the number of cells increases until the cultures become confluent at 5 to 10 days in vitro, at which time the cell density is about 55,000 cells per cm 2 (Fig. 3). Protein content for typical confluent cultures was 27 to 37/~g protein per cm 2. The RCS-p § strain of rats with inherited retinal degeneration showed no differences from normal Long-Evans rats with respect to applicability of the method of tissue isolation or viability and appearance of the cells. The population of such cultures is very nearly 100% pigment granule-containing cells when care is taken to separate properly sheets of pigment epithelium from other tissue layers and to remove fragments of nonpigmented tissue before the second trypsinization; these cultures contained no pigment-free cells which were detectable by phase-contrast microscopy. Cultures have been subeultured serially for two passages; such secondary cultures have not been characterized.
FIG. 1. Pigmentepithelial cell of a 5-day-old culture prepared from Long-Evans rats. Note the double nuclei and perinuclear pigment granules. Phase-contrast optics, x830.
RAT RETINAL PIGMENT EPITHELIUM
f/?
303
FIG. 2. Confluent 9-day culture prepared from RCS-p§ rats, illustrating the predominance of binucleate cells and perinuclear distribution of pigment granules. Phase-contrast optics, x350. 60
|
50
4O x
_o
2O
Days in Culture
FIG. 3. Cell density as a function of time in culture. The two curves represent different sets of replicate cultures prepared from Long-Evans rats; each point is the mean + S.D. of two to four cultures.
DISCUSSION Initial attempts to prepare cultures of rat retinal pigment epithelium by previously developed methods (10-12} were not successful. Yields were poor, and the cells were contaminated with fibroblasts and other nonpigmented cells. The pigment epithelial cells were injured when the neural retina was separated from the eyecup; fragments of the pigment epithelial apical processes were seen adhering to the neural retina. In order to overcome these problems, attempts were made to remove the neural retina without damaging the apical processes of the pigment epithelium and to isolate intact sheets of pigment epithelium free of other cell types. These objectives were achieved by the following: (a} selection of a developmental stage early in rod outer segment formation (5 to 12 days of age} (13) to minimize adhesion between photoreeeptor cells and retinal pigment epithelium; (b} incubation for at least 6 hr in balanced salt solution, which improves the
304
EDWARDS
reproducibility of the method; and (c) trypsinization of the intact globe before incising the eye to reduce the adhesion between the neural retina and pigment epithelium. Isolated sheets of pigment epithelium resemble the tissue as it appears in situ in the rat (14). The cells in culture are binucleate, as is characteristic of rat retinal pigment epithelial cells (14}, and resemble pigment epithelial cells cultured from other species ( 10-12). Both the Long-Evans and RCS-p § cells are metabolically active. The cells divide in vitro and rapidly incorporate "C-L-leucine into protein (R. Edwards, unpublished results). Cultures actively accumulate the aminosulfonic acid, taurine, against a concentration gradient at a much higher rate than scleral-choroidal fibroblasts cultured from rat eyes under identical conditions (15); the cells retain this capacity for at least 15 days in vitro and 5 days after becoming confluent, the longest periods tested. Therefore such cultures should be useful for studies on the biochemistry and metabolism of isolated retinal pigment epithelium. In particular, the method presented here permits investigations of the cell type known to be the primary site of action of the gene for retinal dystrophy in RCS rats.
REFERENCES 1. Young, R. W., and D. Bok. 1969. Participation of the retinal pigment epithelium in the rod outer segment renewal process. J. C e l l Biol. 42: 392-403. 2. Herron, W. L., B. W. Riegel, O. E. Myers, and M. L. Rubin. 1969. Retinal dystrophy in the rat a pigment epithelial disease. Invest. Ophthalmol. 8: 595-604. 3. Bok, D., and M. O. Hall. 1971. The role of the pigment epithelium in the etiology of inherited re-
-
tinal dystrophy in the rat. J. Cell Biol. 49: 664-682. 4. LaVail, M.M., R. L. Sidman, and D. O'Neil. 1972. Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal degeneration. J. Cell Biol. 53: 185-209. 5. Mullen, R. J., and M. M. LaVail. 1976. Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras. Science 192: 799-801. 6. LaVail, M. M., R. L. Sidman, and C. O. Gerhardt. 1975. Congenic strains of RCS rats with inherited retinal dystrophy. J. Hered. 66: 242-244. 7. Puck, T.T., S.J. Cieciura, and A. Robinson. 1958. Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. J. Exp. Med. 108: 945-956. 8. Ham, R. G. 1963. An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp. Cell Res. 29: 515-526. 9. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 10. Cahn, R. D., H. G. Coon, and M. B. Cahn. 1967. Cell culture and cloning techniques. In: F. Wilt, and N. K. WessellslEds.}, Methods in Developmental Biology. Crowell-Collier, New York, pp. 493-530. 11. Albert, D. M., M. O. M. Ts'o. and A. S. Rabson. 1972. In vitro growth of pure cultures of retinal pigment epithelium. A r c h . Ophthalmol. 88: 63-69. 12. Mannagh, J., D. V. Arya, and A. R. Irvine, Jr. 1973. Tissue culture of human retinal pigment epithelium. Invest. Ophthalmol. 12: 52-64. 13. Weidman, T. A., and T. Kuwabara. 1968. Postnatal development of the rat retina. Arch. Ophthalmol. 79: 470-484. 14. Ts'o, M. O. M., and E. Friedman. 1967. The retinal pigment epithelium. I. Comparative histology. Arch. Ophthalmol. 78: 641-649. 15. Edwards, R. B. 1977. Accumulation of taurine by cultured retinal pigment epithelium of the rat. Invest. Ophthalmol. 16: 201-208.
The author thanks Eugene F. McCarthy, Jr. for providing suggestions in developing the methods. This work was supported by grants from the National Retinitis Pigmentosa Foundation, Baltimore, Maryland, and the George Gund Foundation, Cleveland, Ohio. Breeding pairs of RCS-p + rats were provided through National Institutes of Health Research Contract EY-3-2133 to Dr. R. L. Sidman.
CULTURE
OF RAT RETINAL PIGMENT EPITHELIUM ROSS B. E D W A R D S
Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, Massachusetts 02114
SUMMARY A method of preparing monolayer cultures of retinal pigment epithelium from normal pigmented neonatal rats is described. Critical features include incubating the eyes in balanced salt solution and treating with trypsin before dissecting the eyes. The tissue also has been cultured from RCS rats with inherited retinal degeneration. Since the pigment epithelium has been shown to be the primary site of action of the gene for retinal dystrophy in the RCS rat, the method should be useful in studying the defect(s) associated with this mutation.
Key words: retinal pigment epithelium; RCS rat; retinal dystrophy. INTRODUCTION
and then transferred to culture medium F-10 (8) (Microbiological Associates) supplemented with 20% fetal bovine serum, 50/~g per ml gentamicin and 100/ag per ml kanamycin, pH 7.2 to 7.6. An incision was made, using a dissecting microscope, in the globe just posterior to the ora serrata and extended circumferentiaUy; the anterior portion of the globe was discarded. The remaining eyecup with adherent pigment epithelium was peeled gently from the neural retina with two pairs of forceps. The neural retina was dissected free at the optic nerve, and for convenience during the subsequent dissection, the eyecup was bisected. With a fine dissecting knife or jeweler's forceps, the intact pigment epithelial layer was teased free from Bruch's membrane. Pooled sheets of pigment epiMATERIALS AND METHODS thelial cells were rinsed 3 to 4 times in calciumCultures were prepared from Long-Evans rats and magnesium-free saline F containing antibio(Charles River Breeding Laboratories) and from tics and 0.1 mM ethylenediaminetetraacetic acid RCS-p § pigmented rats with inherited retinal de- (EDTA) and then incubated in 0 . I % trypsin (pregeneration ~6). Animals were 6 to 8 days old and pared in the same solution) 8 to l0 rain at 37~ weighed an average of 11 to 12 g. Eyes were enuc- The tissue was dissociated into a single-cell susleated under sterile conditions and immersed in pension by repeated gentle pipetting. Two volsaline F (7), a balanced salt solution, pH 8.0 to umes of culture medium were added to one vol8.4, to which 50 #g per ml gentamicin and 100 #g ume of cell suspension, and the cells were pelleted per ml kanamycin had been added. After remov- by centrifugation for 5 min at 70 • g. Cells were ing loosely adherent connective tissue, the intact resuspended in culture medium, counted in a globes were rinsed two to three times and allowed hemacytometer and seeded at a density of 25,000 to soak in the same solution at room temperature to 30,000 per cm 2 in 17-mm Multiwell plastic for 6 to 24 hr. Intact globes were incubated at dishes (Falcon). Cultures were maintained at 37~ for 45 min in filter-sterilized 0.1% trypsin 37.5~ in 5% CO2 in air; medium was changed iDifco 1:250) dissolved in saline F, pH 8.0 to 8.1, every 2 to 3 days. The seeding efficiency was 301 The retinal pigment epithelium participates in the maintenance of photoreceptor cells by phagocytizing the tips of rod outer segments ~I). In the Royal College of Surgeons ~RCS) rat with inherited retinal degeneration, the pigment epithelium fails to perform this function (2-4). The primary defect resides in the pigment epithelium rather than in photoreceptor or other cell types (5). Comparative studies of cultured retinal pigment epithelium from normal and RCS rats may help to determine the biochemical defect(s) in the RCS tissue. This report describes a method of preparing such cultures.
302
EDWARDS
determined by counting the cells which were attached to the culture surface at 24 hr in vitro and dividing that value by the number of initially seeded cells. For counting, cultures were fixed in situ with 4% formaldehyde and stained with 1% toluidine b l u e - l % sodium borate. Cells were counted in 20 randomly chosen microscopic fields in each of two to four replicate cultures for each time point. Results are expressed as the mean number of cells per cm 2 of culture surface. Protein was measured by the Folin reaction ~9). RESULTS Cells comprising freshly removed intact sheets of pigment epithelium are hexagonal in shape, forming the cobblestone pattern of the tissue seen in situ. The second trypsinization yields a suspension of spherical or asymmetrically dumbbellshaped cells which appear highly refractile under phase-contrast optics. Seeding efficiency varies between 30 and 60%. Typical cells are shown in
Figs. 1 and 2. The majority are binudeate and contain numerous perinuclear pigment granules. Mitoses are commonly seen; the number of cells increases until the cultures become confluent at 5 to 10 days in vitro, at which time the cell density is about 55,000 cells per cm 2 (Fig. 3). Protein content for typical confluent cultures was 27 to 37/~g protein per cm 2. The RCS-p § strain of rats with inherited retinal degeneration showed no differences from normal Long-Evans rats with respect to applicability of the method of tissue isolation or viability and appearance of the cells. The population of such cultures is very nearly 100% pigment granule-containing cells when care is taken to separate properly sheets of pigment epithelium from other tissue layers and to remove fragments of nonpigmented tissue before the second trypsinization; these cultures contained no pigment-free cells which were detectable by phase-contrast microscopy. Cultures have been subeultured serially for two passages; such secondary cultures have not been characterized.
FIG. 1. Pigmentepithelial cell of a 5-day-old culture prepared from Long-Evans rats. Note the double nuclei and perinuclear pigment granules. Phase-contrast optics, x830.
RAT RETINAL PIGMENT EPITHELIUM
f/?
303
FIG. 2. Confluent 9-day culture prepared from RCS-p§ rats, illustrating the predominance of binucleate cells and perinuclear distribution of pigment granules. Phase-contrast optics, x350. 60
|
50
4O x
_o
2O
Days in Culture
FIG. 3. Cell density as a function of time in culture. The two curves represent different sets of replicate cultures prepared from Long-Evans rats; each point is the mean + S.D. of two to four cultures.
DISCUSSION Initial attempts to prepare cultures of rat retinal pigment epithelium by previously developed methods (10-12} were not successful. Yields were poor, and the cells were contaminated with fibroblasts and other nonpigmented cells. The pigment epithelial cells were injured when the neural retina was separated from the eyecup; fragments of the pigment epithelial apical processes were seen adhering to the neural retina. In order to overcome these problems, attempts were made to remove the neural retina without damaging the apical processes of the pigment epithelium and to isolate intact sheets of pigment epithelium free of other cell types. These objectives were achieved by the following: (a} selection of a developmental stage early in rod outer segment formation (5 to 12 days of age} (13) to minimize adhesion between photoreeeptor cells and retinal pigment epithelium; (b} incubation for at least 6 hr in balanced salt solution, which improves the
304
EDWARDS
reproducibility of the method; and (c) trypsinization of the intact globe before incising the eye to reduce the adhesion between the neural retina and pigment epithelium. Isolated sheets of pigment epithelium resemble the tissue as it appears in situ in the rat (14). The cells in culture are binucleate, as is characteristic of rat retinal pigment epithelial cells (14}, and resemble pigment epithelial cells cultured from other species ( 10-12). Both the Long-Evans and RCS-p § cells are metabolically active. The cells divide in vitro and rapidly incorporate "C-L-leucine into protein (R. Edwards, unpublished results). Cultures actively accumulate the aminosulfonic acid, taurine, against a concentration gradient at a much higher rate than scleral-choroidal fibroblasts cultured from rat eyes under identical conditions (15); the cells retain this capacity for at least 15 days in vitro and 5 days after becoming confluent, the longest periods tested. Therefore such cultures should be useful for studies on the biochemistry and metabolism of isolated retinal pigment epithelium. In particular, the method presented here permits investigations of the cell type known to be the primary site of action of the gene for retinal dystrophy in RCS rats.
REFERENCES 1. Young, R. W., and D. Bok. 1969. Participation of the retinal pigment epithelium in the rod outer segment renewal process. J. C e l l Biol. 42: 392-403. 2. Herron, W. L., B. W. Riegel, O. E. Myers, and M. L. Rubin. 1969. Retinal dystrophy in the rat a pigment epithelial disease. Invest. Ophthalmol. 8: 595-604. 3. Bok, D., and M. O. Hall. 1971. The role of the pigment epithelium in the etiology of inherited re-
-
tinal dystrophy in the rat. J. Cell Biol. 49: 664-682. 4. LaVail, M.M., R. L. Sidman, and D. O'Neil. 1972. Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal degeneration. J. Cell Biol. 53: 185-209. 5. Mullen, R. J., and M. M. LaVail. 1976. Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras. Science 192: 799-801. 6. LaVail, M. M., R. L. Sidman, and C. O. Gerhardt. 1975. Congenic strains of RCS rats with inherited retinal dystrophy. J. Hered. 66: 242-244. 7. Puck, T.T., S.J. Cieciura, and A. Robinson. 1958. Genetics of somatic mammalian cells. III. Long-term cultivation of euploid cells from human and animal subjects. J. Exp. Med. 108: 945-956. 8. Ham, R. G. 1963. An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp. Cell Res. 29: 515-526. 9. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 10. Cahn, R. D., H. G. Coon, and M. B. Cahn. 1967. Cell culture and cloning techniques. In: F. Wilt, and N. K. WessellslEds.}, Methods in Developmental Biology. Crowell-Collier, New York, pp. 493-530. 11. Albert, D. M., M. O. M. Ts'o. and A. S. Rabson. 1972. In vitro growth of pure cultures of retinal pigment epithelium. A r c h . Ophthalmol. 88: 63-69. 12. Mannagh, J., D. V. Arya, and A. R. Irvine, Jr. 1973. Tissue culture of human retinal pigment epithelium. Invest. Ophthalmol. 12: 52-64. 13. Weidman, T. A., and T. Kuwabara. 1968. Postnatal development of the rat retina. Arch. Ophthalmol. 79: 470-484. 14. Ts'o, M. O. M., and E. Friedman. 1967. The retinal pigment epithelium. I. Comparative histology. Arch. Ophthalmol. 78: 641-649. 15. Edwards, R. B. 1977. Accumulation of taurine by cultured retinal pigment epithelium of the rat. Invest. Ophthalmol. 16: 201-208.
The author thanks Eugene F. McCarthy, Jr. for providing suggestions in developing the methods. This work was supported by grants from the National Retinitis Pigmentosa Foundation, Baltimore, Maryland, and the George Gund Foundation, Cleveland, Ohio. Breeding pairs of RCS-p + rats were provided through National Institutes of Health Research Contract EY-3-2133 to Dr. R. L. Sidman.
