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Studies of corneas in vivo and in vitro
&ion Research Vol.21, pp 41 to 43 'ergamon PressLtd 1981.Printed in GreatBritam
II. Cornea STUDIES
OF CORNEAS
IN
BEATRICE Y. J. T. YUE
and
I/IV0
AND
IN
VITRO
JULES L. BAUM*
Department of Ophthalmology, Tufts University School of Medicine and New England Medical Center Hospital, 171 Harrison Avenue, Boston, MA 02111, U.S.A. Abstract-We
present our step-by-step procedure for the preparation of rabbit and human cornea1 endothelial cells in culture. We also compare biochemical differences between rabbit and human cornea1 stromal and endothelial cells in viuo and in vitro.
1. CELL
CULTURE
TECHNIQUES
tor at 37°C with 5% CO2 and 95% air. Rabbit cultures are fed fresh media with 100 pg/ml penicillin G for 3 consecutive days; then the penicillin is discontinued. Do not disturb the explants during feeding. No penicillin is used on human eyes. After start of incubation, cultures of human cells are not disturbed for 34 days to allow the explants to attach. After this initial period, cultures are fed with fresh media three times per week. Rabbit cultures show outgrowth within 2448 hr. With human cultures, time of first outgrowth varies from 3 to 5 days for young eyes and up to 2 weeks for old eyes. Rabbit cultures are trypsinized when outgrowth achieves a 25 cell dia. Human cultures are trypsinized when the cell diameter reaches 50 cells. Explants can be left in culture for further growth or used to start new cultures. The trypsinizing procedure requires two stock solutions (Microbiological Associates), a trypsin solution, 2.5% trypsin in Hank’s balanced salt solution (cat. No. 17-160), and versene 1:5000 in an isotonically buffered solution (cat. No. 17-717). A working solution is prepared by combining 1 ml of the trypsin solution and 9 ml of versene solution 1:5000. Rinse the culture twice with versene 1:5000 to remove media from the cells, approximately 2 ml per rinse. Next add 1.5 ml of the working solution (0.25% trypsin). Let stand for 2-3 min at 37°C. Pour off the excess trypsin solution, leaving approximately 0.5 ml. Check constantly under the phase microscope until the cells become rounded and refractile. Add 2-3 ml of complete media. Cells can be further suspended by gentle washing with a Pasteur pipette. For subculturing (passing), transfer aliquots of cell suspensions to new culture flasks. The size of the aliquot depends on the splitting ratio. Otherwise, leave cells in “old” culture flask. Leave cells undisturbed in 2-3 ml of media. Twenty-four hours later, change media and maintain culture by the same culture techniques.
Although we have published our technique for the preparation of rabbit (Perlman and Baum, 1974a) and human (Baum et al., 1979) cornea1 endothelial cells in culture, we have had requests for specific details of the technique. The following represents the updated stepby-step procedure. Process eyes in a laminar flow hood. Rinse the eyes well with approximately 50 ml of sterile saline solution. Next, soak the eyes for 6 min in Neosporin ophthalmic solution plus 50 pg/ml gentamicin. For rabbit eyes, also add 100 pg/ml penicillin G. The eyes should be placed in the smallest possible vessel so that the antibiotic solution covers the entire eye. After soaking, again rinse the eyes with 50ml of sterile saline. Excise the corneal-scleral shell using sterile technique. The excised cornea is then placed in a petri plate with complete media (Tables 1 and 2) with the endothelial side up. Under a dissecting microscope and using two pairs of jeweler’s forceps, gently loosen the endothelium-Descemet’s membrane around the edges of the cornea. Gently pull the endotheliumDescemet’s membrane from the remaining cornea. Usually this tissue is removed as a whole and, if removed correctly, is not contaminated with stromal cells. This is essentially the method described by Stocker et ul. (1958). Pass the endotheliumDescemet’s membrane through 2 petri plates containing complete media as washes. Do this gently. Do not drop or wash back and forth. This is done to remove any possible bacterial or fungal contamination which may have come from the epithelial side of the cornea. The endothelium-Descemet’s membrane explants are placed in T-30 flasks or petri plates with 1 ml or less of complete media. A small amount of media is critical for explant adhesion and growth. Check the cultures daily for evaporation. Add several drops of media as necessary. For rabbit cultures, usually 8 whole explants are used per culture. For human cultures, each explant is cut into 2-3 pieces, with a total of 4-6 explants per culture. Incubate in moist incuba-
2.
COMPARISON
OF BIOCHEMICAL
PERFORMED
STUDIES
ON CORNEAS
IN VIVO AND IN VITRO
a. Sti-oma
* Requests for reprints L. Baum, Tufts-New 171 Harrison Avenue,
should be addressed to Dr Jules England Medical Center Hospital, Boston, MA 02111, U.S.A.
Collagen and proteoglycan are the major structural components of the cornea. In cornea1 stroma, type I is 41
42
B. Y. J. T. YUE and J. L. BAUM Table
1. Final concentrations
in complete
media --
Supplies
from Microbiological
Associates
1. 2. 3. 4. 5. 6. 7. 8.
Eagle’s minimum essential medium (MEM) No. 12-125. 109; fetal bovine serum (No. 14-414). 5:; calf serum (No. 14-401). 2%; 200 mM glutamine (No. 17-60SF). O.O75Y, sodium bicarbonate (No. 17-613, 7.5”” stock stolution). 10 pgjml ~entamicin (10 mg/ml stock st~luti~n). 1.2 pg/ml amphotericin B (E.R. Squibb & Sons, inc.. Princeton, N.J.). l”,, non-essential amino acid mixture (No. 13-l14. 10 mM solution of each amino acid). 9. 2”,, essential amino acid mixture (No. 13-606, 50 x ). IO. Quality control: each new batch of serum and MEM is plated on agar to check for bacterial and fungal growth.
the main collagenous product (Freeman, 1978). A.B chains (Hong et al., 1978) and type III collagen (Schmut, 1978) have also been recovered. Studies of collagen synthesis il2 vitro. performed on confluent cultures of cornea1 stromal cells, generally agree with the studies in ciao. Type I collagen (Stoesser et (II., 1978; Yue et u/., 1979b), A,B chains (Yue er al., 197911) and type III collagen (Newsome et u1., presented at the Eleventh Cornea1 Research Conference in Boston, Sept., 1979) have been identified. Glycosaminoglycans synthesized by cultures of corneal stromal cells, however, differ considerably from those found in normal cornea (Conrad and Dorfman, 1974; Dahi ub nl., 1974; Gngdinger and SchwagerHiibner, 1975; Klintworth and Smith, 1976; Yue rr al., 1976, 1978, 1979a). The production of keratan sulfate in vitro. the principal glycosaminoglycan component of the normal cornea (Meyer er al., 1953; Anseth and Laurent, 1961; Salitermik-Girant and Berman, 1970). is minimal and heparan sulfate, and large amounts of dermatan sulfate are produced in addition to ~hondroitin 6- and chondroitin 4-sulfates, the normal products of cornea. Apart from the various sulfated glycosaminoglycans, hyaluronic acid is also produced by stromal cultures in significant amounts. These differences indicate that the metabolism of glycosaminoglycans is significantly affected by the environmental conditions of cell cultures.
Cornea1 endothelium both possess the aminoglycans (Maurice 1976, 1978) and collagen vitro
Table 2. Composition 1 ml 1ml 2 ml 5 ml 10 ml MEM 1 ml 0.1 ml 0.1 ml
in tjico and cells maintained in capacity to produce glycosand Riley, 1970; Yue et nE., (Kefalides, 1973; Perlman et of 100 ml complete
media
essential amino acids (50 x ). non-essential amino acids. glutamine. calf serum. fetal bovine serum.
up to IOOml. sodium bicarbonate (7.5”,, stock stolution). gentamicin (10 mg/ml stock solution). fungizone (1.25 mg/ml stock solution). --
al., 1974; Davison and Cannon, 1977; Sundar Raj et ul., 1979). Basement membrane produced in culture increases in thickness with age as in normal cornea and has hydroxyproline content similar to that found in Descemet’s membrane (Perlman and Baum, 1974b). There are, however, certain basic differences between the in cino and in rGtro systems. In tissue, human cornea1 endothelial cells form a hexagonal mosaic and do not divide by mitosis. In contrast, human endothelial cells divide and multiply in culture although the growth potential is age dependent (Baum et ui.. 1979). Mosaic-type cells are rarely seen in culture and may be indicative of unhealthy cells. A single basement membrane collagen (type IV) has been isolated from rabbit cornea1 endothelial cultures (Sundar Raj et al.. 1979). The levels of hydroxyproline and hydroxylysine are lower than those found in Descemet’s membrane (Kefalides, 1973). This suggests a possible modification due to culture conditions. The rate of collagen synthesis relative to total protein synthesis is independent of age of donor in normal human endotheiial cultures (Table 3). Preliminary analyses performed in our laboratory indicate that the collagen synthesized is extremely heterogenous. Among all the collagenous proteins, type I collagen
Table 3. Rate of collagen synthesis in cultures of normal human cornea1 endothelial cells Collagen synthesis rate relative to protein synthesis
Age of donor 2: months 5 months 1 yr 2 yr 2 yr. 2 months 13yr 43 yr 67 yr
I.00 1.10 1.35 1.70 I.20
I.30 1.10
---.
1.x0 -___-
Confluent monolayer cuitures were labeied with 5 $?/mi of (3H)proline for 24 hr in the presence of 75 {[g/ml of ascorbic acid. Experiments were performed as described by Perlman rr trl. (1974).
Studies of corneas in uiuo and in vitro has been identified in both the cell layer and the medium fraction of endothelial cultures (Yue, et al., unpublished data). This is in agreement with a previous study of Descemet’s membrane in vim (Davison and Cannon, 1977). Based on electron microscopy, Davison and Cannon (1977) have shown that Descemet’s membrane contains not only basement membrane collagen, but also several other types of collagen, including type I. c. Lattice dystrophy of the cornea Lattice cornea1 dystrophy is a localized amyloidosis as indicated by immunohistochemical (Bowen et al., 1970; Meretoja, 1972; Klintworth, 1967) and microscopic (Francois and Fehr, 1972; Francois et al., 1975) studies. The biochemical properties of amyloid fibrils accumulated in the cornea of patients have not yet been elucidated. Cultures of stromal cells derived from lattice dystrophic corneal- buttons’ have been obtained in our laboratory. However, no amyloid has been detected from the cultured cells or from the pooled and concentrated culture media. Congo red staining on all culture specimens was negative. No cross-reactivity was found against anti-amyloid protein by immunodiffusion and no amyloid fibrils could be demonstrated with electron microscopy (Yue, Baum, Skinner and Cohen, unpublished studies). Acknowledgements~This investigation was supported by Research Grant Number EY-01793 from the National Eye Institute, National Institutes of Health. REFERENCES
Anseth A. and Laurent T. C. (1961) Studies
on cornea1 polysaccharides I. Separation. Expl Eye Res. 1, 25-38. Baum J. L., Niedra R., Davis C. and Yue B. Y. J. T. (1979) Mass culture of human cornea1 endothelial cells. Archs Ophthalmol. 97, 1136-l 140. Bowen R. A., Hassard D. T. R., Wong V. G., DeLellis R. A. and Glenner G. G. (1970) Lattice dystrophy of the cornea as a variety of amyloidosis. Am. J. OphthalmoL 70, 822-825. Conrad G. W. and Dorfman A. (1974) Synthesis of sulfated mucopolysaccharides by chick cornea1 fibroblasts in vitro. Expl Eye Res. 18, 421433. Dahl I.-M., Johnsen W., Anseth A. and Prydz H. (1974) The synthesis of glycosaminoglycans by cornea1 stromal cells in culture. Expl Cell Res. 88, 193-197. Davison P. F. and Cannon D. J. (1977) Heterogeneity of collagens from basement membranes of lens and cornea. Expl Eye Res. 25, 129-137. Freeman I. L. (1978) Collagen polymorphism in mature rabbit cornea. Invest. OphthahnoL Visual Sci. 17, 171-l 77. Francois J. and Fehr J. (1972) Light microscopic and polarization optical study of lattice dystrophy of the cornea. Ophthalmologica 164, 1-18. Francois J., Hanssens M. and Teuchy H. (1975) Ultrastructural changes in lattice dystrophy of the cornea. Ophthal. Res. 7, 321-344. GnIdinger
M. C. and Schwager-Hubner
M. E. (1975) Bio-
43
synthesis of glycosaminoglycans by mammalian cornea1 epithelium and fibroblasts in vitro II. Approach to
specify the GAG from the two cell types. AIbrecht uon Graefes Arch. Klin. Exp. Ophthalmol. l%, 21-30. Hong B. S., Cannon D. J. and Davison D. F. (1978) Isolation and identification of a distinct collagen from several different tissues. Fed. Proc. 37, 1528. Kefalides N. A. (1973) Structure and biosynthesis of basement membranes. Int. Rev. Connect. Tissue Res. 6, 63-104. Klintworth G. K. (1967) Lattice cornea1 dystrophy. Am. J. Pathol. 50, 371-399. Klintworth G. K. and Smith C. F. (1976) A comparative study of extracellular sulfated glycosaminoglycans synthesized by rabbit cornea1 fibroblasts in organ and confluent cultures. Lab. Invest. 35, 258-263. Maurice D. M. and Riley M. W. (1970) The Cornea in the Biochemistry of the Eye (Edited by Graymore C. N.) pp. l-103. Academic Press, New York. Meretoja J. (1972) Comparative histopathological and clinical findings in eyes with lattice cornea1 dystrophy of two different types. Ophthalmologica 165, 15-37. Meyer K., Linker A., Davidson E. A. and Weissmair B. (1953) The mucopolysaccharides of bovine cornea. J. bio!. Chem. 205, 61 l-616. Perlman M. and Baum J. L. (1974a) The mass culture of rabbit cornea1 endothelial cells. Archs Ophthalmol. 92, 235-237. Perlman M. and Baum J. L. (1974b) Synthesis of a collagenous basal membrane by rabbit cornea1 endothelial cells in culture. Archs OphthalmoL 92, 238-239. Perlman M., Baum J. L. and Kay G. I. (1974) Fine structure and collagen synthesis activity of monolayer cultures of rabbit cornea1 endothelium. J. Cell Biol. 63, 306-311. Salitermik-Girant S. and Berman E. R. (1970) Biochemical of the cornea1 glycosaminoglycans. heterogeneity Ophthalmol. Res. 1, 94-108. Schmut 0. (1978) The organization of tissues of the eye by different collagen types. Albrecht v. Graefes Arch. K/in. Exp. Ophthafmol. 207, 189-199. Stocker F. W., Eiring A., Georgiade R. and Georgiade N. (1958) A tissue culture technique for growing cornea1 epithelial stromal and endothelial tissues separately. Am. J. Ophthalmol. 46, 294-298. Stoesser T. R., Church R. L. and Brown S. I. (1978) Partial characterization of human collagen and procollagen secreted by human cornea1 stromal fibroblasts in cell culture. Invest. Ophthalmol. Visual Sci. 17, 264-271. Sundar Raj C. V., Freeman I. L., Church R. L. and Brown S. I. (1979) Biochemical characterization of procollagencollagen synthesized by rabbit cornea1 endothelial cells in culture. Invest. OphthalmoL Visual Sci. 18, 75-84. Yue B. Y. J. T., Baum J. L. and Silbert J. E. (1976) The synthesis of glycosaminoglycans by cultures of rabbit cornea1 endothelial and stromal cells. Biochem. J. 158, 567-573. Yue B. Y. J. T., Baum J. L. and Silbert J. E. (1978) Syn-
thesis of glycosaminoglycans by cultures of normal human cornea1 endothelial and stromal cells. Invest. Ophthalmol Visual Sci. 17, 523-527. Yue B. Y. J. T., Baum J. L. and Silbert J. E. (1979a) The synthesis of glycosaminoglycans by cultures of cornea1 stromal cells from patients with keratoconus. J. din. Invest. 63, 545-551. Yue B. Y. J. T., Baum J. L. and Smith B. D. (1979b) Collagen synthesis by cultures of stromal cells from normal hiiman and keratoconus corneas. Biochem. Biophys. Rex Comm. 86,465472.
II. Cornea STUDIES
OF CORNEAS
IN
BEATRICE Y. J. T. YUE
and
I/IV0
AND
IN
VITRO
JULES L. BAUM*
Department of Ophthalmology, Tufts University School of Medicine and New England Medical Center Hospital, 171 Harrison Avenue, Boston, MA 02111, U.S.A. Abstract-We
present our step-by-step procedure for the preparation of rabbit and human cornea1 endothelial cells in culture. We also compare biochemical differences between rabbit and human cornea1 stromal and endothelial cells in viuo and in vitro.
1. CELL
CULTURE
TECHNIQUES
tor at 37°C with 5% CO2 and 95% air. Rabbit cultures are fed fresh media with 100 pg/ml penicillin G for 3 consecutive days; then the penicillin is discontinued. Do not disturb the explants during feeding. No penicillin is used on human eyes. After start of incubation, cultures of human cells are not disturbed for 34 days to allow the explants to attach. After this initial period, cultures are fed with fresh media three times per week. Rabbit cultures show outgrowth within 2448 hr. With human cultures, time of first outgrowth varies from 3 to 5 days for young eyes and up to 2 weeks for old eyes. Rabbit cultures are trypsinized when outgrowth achieves a 25 cell dia. Human cultures are trypsinized when the cell diameter reaches 50 cells. Explants can be left in culture for further growth or used to start new cultures. The trypsinizing procedure requires two stock solutions (Microbiological Associates), a trypsin solution, 2.5% trypsin in Hank’s balanced salt solution (cat. No. 17-160), and versene 1:5000 in an isotonically buffered solution (cat. No. 17-717). A working solution is prepared by combining 1 ml of the trypsin solution and 9 ml of versene solution 1:5000. Rinse the culture twice with versene 1:5000 to remove media from the cells, approximately 2 ml per rinse. Next add 1.5 ml of the working solution (0.25% trypsin). Let stand for 2-3 min at 37°C. Pour off the excess trypsin solution, leaving approximately 0.5 ml. Check constantly under the phase microscope until the cells become rounded and refractile. Add 2-3 ml of complete media. Cells can be further suspended by gentle washing with a Pasteur pipette. For subculturing (passing), transfer aliquots of cell suspensions to new culture flasks. The size of the aliquot depends on the splitting ratio. Otherwise, leave cells in “old” culture flask. Leave cells undisturbed in 2-3 ml of media. Twenty-four hours later, change media and maintain culture by the same culture techniques.
Although we have published our technique for the preparation of rabbit (Perlman and Baum, 1974a) and human (Baum et al., 1979) cornea1 endothelial cells in culture, we have had requests for specific details of the technique. The following represents the updated stepby-step procedure. Process eyes in a laminar flow hood. Rinse the eyes well with approximately 50 ml of sterile saline solution. Next, soak the eyes for 6 min in Neosporin ophthalmic solution plus 50 pg/ml gentamicin. For rabbit eyes, also add 100 pg/ml penicillin G. The eyes should be placed in the smallest possible vessel so that the antibiotic solution covers the entire eye. After soaking, again rinse the eyes with 50ml of sterile saline. Excise the corneal-scleral shell using sterile technique. The excised cornea is then placed in a petri plate with complete media (Tables 1 and 2) with the endothelial side up. Under a dissecting microscope and using two pairs of jeweler’s forceps, gently loosen the endothelium-Descemet’s membrane around the edges of the cornea. Gently pull the endotheliumDescemet’s membrane from the remaining cornea. Usually this tissue is removed as a whole and, if removed correctly, is not contaminated with stromal cells. This is essentially the method described by Stocker et ul. (1958). Pass the endotheliumDescemet’s membrane through 2 petri plates containing complete media as washes. Do this gently. Do not drop or wash back and forth. This is done to remove any possible bacterial or fungal contamination which may have come from the epithelial side of the cornea. The endothelium-Descemet’s membrane explants are placed in T-30 flasks or petri plates with 1 ml or less of complete media. A small amount of media is critical for explant adhesion and growth. Check the cultures daily for evaporation. Add several drops of media as necessary. For rabbit cultures, usually 8 whole explants are used per culture. For human cultures, each explant is cut into 2-3 pieces, with a total of 4-6 explants per culture. Incubate in moist incuba-
2.
COMPARISON
OF BIOCHEMICAL
PERFORMED
STUDIES
ON CORNEAS
IN VIVO AND IN VITRO
a. Sti-oma
* Requests for reprints L. Baum, Tufts-New 171 Harrison Avenue,
should be addressed to Dr Jules England Medical Center Hospital, Boston, MA 02111, U.S.A.
Collagen and proteoglycan are the major structural components of the cornea. In cornea1 stroma, type I is 41
42
B. Y. J. T. YUE and J. L. BAUM Table
1. Final concentrations
in complete
media --
Supplies
from Microbiological
Associates
1. 2. 3. 4. 5. 6. 7. 8.
Eagle’s minimum essential medium (MEM) No. 12-125. 109; fetal bovine serum (No. 14-414). 5:; calf serum (No. 14-401). 2%; 200 mM glutamine (No. 17-60SF). O.O75Y, sodium bicarbonate (No. 17-613, 7.5”” stock stolution). 10 pgjml ~entamicin (10 mg/ml stock st~luti~n). 1.2 pg/ml amphotericin B (E.R. Squibb & Sons, inc.. Princeton, N.J.). l”,, non-essential amino acid mixture (No. 13-l14. 10 mM solution of each amino acid). 9. 2”,, essential amino acid mixture (No. 13-606, 50 x ). IO. Quality control: each new batch of serum and MEM is plated on agar to check for bacterial and fungal growth.
the main collagenous product (Freeman, 1978). A.B chains (Hong et al., 1978) and type III collagen (Schmut, 1978) have also been recovered. Studies of collagen synthesis il2 vitro. performed on confluent cultures of cornea1 stromal cells, generally agree with the studies in ciao. Type I collagen (Stoesser et (II., 1978; Yue et u/., 1979b), A,B chains (Yue er al., 197911) and type III collagen (Newsome et u1., presented at the Eleventh Cornea1 Research Conference in Boston, Sept., 1979) have been identified. Glycosaminoglycans synthesized by cultures of corneal stromal cells, however, differ considerably from those found in normal cornea (Conrad and Dorfman, 1974; Dahi ub nl., 1974; Gngdinger and SchwagerHiibner, 1975; Klintworth and Smith, 1976; Yue rr al., 1976, 1978, 1979a). The production of keratan sulfate in vitro. the principal glycosaminoglycan component of the normal cornea (Meyer er al., 1953; Anseth and Laurent, 1961; Salitermik-Girant and Berman, 1970). is minimal and heparan sulfate, and large amounts of dermatan sulfate are produced in addition to ~hondroitin 6- and chondroitin 4-sulfates, the normal products of cornea. Apart from the various sulfated glycosaminoglycans, hyaluronic acid is also produced by stromal cultures in significant amounts. These differences indicate that the metabolism of glycosaminoglycans is significantly affected by the environmental conditions of cell cultures.
Cornea1 endothelium both possess the aminoglycans (Maurice 1976, 1978) and collagen vitro
Table 2. Composition 1 ml 1ml 2 ml 5 ml 10 ml MEM 1 ml 0.1 ml 0.1 ml
in tjico and cells maintained in capacity to produce glycosand Riley, 1970; Yue et nE., (Kefalides, 1973; Perlman et of 100 ml complete
media
essential amino acids (50 x ). non-essential amino acids. glutamine. calf serum. fetal bovine serum.
up to IOOml. sodium bicarbonate (7.5”,, stock stolution). gentamicin (10 mg/ml stock solution). fungizone (1.25 mg/ml stock solution). --
al., 1974; Davison and Cannon, 1977; Sundar Raj et ul., 1979). Basement membrane produced in culture increases in thickness with age as in normal cornea and has hydroxyproline content similar to that found in Descemet’s membrane (Perlman and Baum, 1974b). There are, however, certain basic differences between the in cino and in rGtro systems. In tissue, human cornea1 endothelial cells form a hexagonal mosaic and do not divide by mitosis. In contrast, human endothelial cells divide and multiply in culture although the growth potential is age dependent (Baum et ui.. 1979). Mosaic-type cells are rarely seen in culture and may be indicative of unhealthy cells. A single basement membrane collagen (type IV) has been isolated from rabbit cornea1 endothelial cultures (Sundar Raj et al.. 1979). The levels of hydroxyproline and hydroxylysine are lower than those found in Descemet’s membrane (Kefalides, 1973). This suggests a possible modification due to culture conditions. The rate of collagen synthesis relative to total protein synthesis is independent of age of donor in normal human endotheiial cultures (Table 3). Preliminary analyses performed in our laboratory indicate that the collagen synthesized is extremely heterogenous. Among all the collagenous proteins, type I collagen
Table 3. Rate of collagen synthesis in cultures of normal human cornea1 endothelial cells Collagen synthesis rate relative to protein synthesis
Age of donor 2: months 5 months 1 yr 2 yr 2 yr. 2 months 13yr 43 yr 67 yr
I.00 1.10 1.35 1.70 I.20
I.30 1.10
---.
1.x0 -___-
Confluent monolayer cuitures were labeied with 5 $?/mi of (3H)proline for 24 hr in the presence of 75 {[g/ml of ascorbic acid. Experiments were performed as described by Perlman rr trl. (1974).
Studies of corneas in uiuo and in vitro has been identified in both the cell layer and the medium fraction of endothelial cultures (Yue, et al., unpublished data). This is in agreement with a previous study of Descemet’s membrane in vim (Davison and Cannon, 1977). Based on electron microscopy, Davison and Cannon (1977) have shown that Descemet’s membrane contains not only basement membrane collagen, but also several other types of collagen, including type I. c. Lattice dystrophy of the cornea Lattice cornea1 dystrophy is a localized amyloidosis as indicated by immunohistochemical (Bowen et al., 1970; Meretoja, 1972; Klintworth, 1967) and microscopic (Francois and Fehr, 1972; Francois et al., 1975) studies. The biochemical properties of amyloid fibrils accumulated in the cornea of patients have not yet been elucidated. Cultures of stromal cells derived from lattice dystrophic corneal- buttons’ have been obtained in our laboratory. However, no amyloid has been detected from the cultured cells or from the pooled and concentrated culture media. Congo red staining on all culture specimens was negative. No cross-reactivity was found against anti-amyloid protein by immunodiffusion and no amyloid fibrils could be demonstrated with electron microscopy (Yue, Baum, Skinner and Cohen, unpublished studies). Acknowledgements~This investigation was supported by Research Grant Number EY-01793 from the National Eye Institute, National Institutes of Health. REFERENCES
Anseth A. and Laurent T. C. (1961) Studies
on cornea1 polysaccharides I. Separation. Expl Eye Res. 1, 25-38. Baum J. L., Niedra R., Davis C. and Yue B. Y. J. T. (1979) Mass culture of human cornea1 endothelial cells. Archs Ophthalmol. 97, 1136-l 140. Bowen R. A., Hassard D. T. R., Wong V. G., DeLellis R. A. and Glenner G. G. (1970) Lattice dystrophy of the cornea as a variety of amyloidosis. Am. J. OphthalmoL 70, 822-825. Conrad G. W. and Dorfman A. (1974) Synthesis of sulfated mucopolysaccharides by chick cornea1 fibroblasts in vitro. Expl Eye Res. 18, 421433. Dahl I.-M., Johnsen W., Anseth A. and Prydz H. (1974) The synthesis of glycosaminoglycans by cornea1 stromal cells in culture. Expl Cell Res. 88, 193-197. Davison P. F. and Cannon D. J. (1977) Heterogeneity of collagens from basement membranes of lens and cornea. Expl Eye Res. 25, 129-137. Freeman I. L. (1978) Collagen polymorphism in mature rabbit cornea. Invest. OphthahnoL Visual Sci. 17, 171-l 77. Francois J. and Fehr J. (1972) Light microscopic and polarization optical study of lattice dystrophy of the cornea. Ophthalmologica 164, 1-18. Francois J., Hanssens M. and Teuchy H. (1975) Ultrastructural changes in lattice dystrophy of the cornea. Ophthal. Res. 7, 321-344. GnIdinger
M. C. and Schwager-Hubner
M. E. (1975) Bio-
43
synthesis of glycosaminoglycans by mammalian cornea1 epithelium and fibroblasts in vitro II. Approach to
specify the GAG from the two cell types. AIbrecht uon Graefes Arch. Klin. Exp. Ophthalmol. l%, 21-30. Hong B. S., Cannon D. J. and Davison D. F. (1978) Isolation and identification of a distinct collagen from several different tissues. Fed. Proc. 37, 1528. Kefalides N. A. (1973) Structure and biosynthesis of basement membranes. Int. Rev. Connect. Tissue Res. 6, 63-104. Klintworth G. K. (1967) Lattice cornea1 dystrophy. Am. J. Pathol. 50, 371-399. Klintworth G. K. and Smith C. F. (1976) A comparative study of extracellular sulfated glycosaminoglycans synthesized by rabbit cornea1 fibroblasts in organ and confluent cultures. Lab. Invest. 35, 258-263. Maurice D. M. and Riley M. W. (1970) The Cornea in the Biochemistry of the Eye (Edited by Graymore C. N.) pp. l-103. Academic Press, New York. Meretoja J. (1972) Comparative histopathological and clinical findings in eyes with lattice cornea1 dystrophy of two different types. Ophthalmologica 165, 15-37. Meyer K., Linker A., Davidson E. A. and Weissmair B. (1953) The mucopolysaccharides of bovine cornea. J. bio!. Chem. 205, 61 l-616. Perlman M. and Baum J. L. (1974a) The mass culture of rabbit cornea1 endothelial cells. Archs Ophthalmol. 92, 235-237. Perlman M. and Baum J. L. (1974b) Synthesis of a collagenous basal membrane by rabbit cornea1 endothelial cells in culture. Archs OphthalmoL 92, 238-239. Perlman M., Baum J. L. and Kay G. I. (1974) Fine structure and collagen synthesis activity of monolayer cultures of rabbit cornea1 endothelium. J. Cell Biol. 63, 306-311. Salitermik-Girant S. and Berman E. R. (1970) Biochemical of the cornea1 glycosaminoglycans. heterogeneity Ophthalmol. Res. 1, 94-108. Schmut 0. (1978) The organization of tissues of the eye by different collagen types. Albrecht v. Graefes Arch. K/in. Exp. Ophthafmol. 207, 189-199. Stocker F. W., Eiring A., Georgiade R. and Georgiade N. (1958) A tissue culture technique for growing cornea1 epithelial stromal and endothelial tissues separately. Am. J. Ophthalmol. 46, 294-298. Stoesser T. R., Church R. L. and Brown S. I. (1978) Partial characterization of human collagen and procollagen secreted by human cornea1 stromal fibroblasts in cell culture. Invest. Ophthalmol. Visual Sci. 17, 264-271. Sundar Raj C. V., Freeman I. L., Church R. L. and Brown S. I. (1979) Biochemical characterization of procollagencollagen synthesized by rabbit cornea1 endothelial cells in culture. Invest. OphthalmoL Visual Sci. 18, 75-84. Yue B. Y. J. T., Baum J. L. and Silbert J. E. (1976) The synthesis of glycosaminoglycans by cultures of rabbit cornea1 endothelial and stromal cells. Biochem. J. 158, 567-573. Yue B. Y. J. T., Baum J. L. and Silbert J. E. (1978) Syn-
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