Ultrastructure of Human Organ-Cultured Cornea II. Stroma and
Epithelium
Diane L. Van Horn, PhD; Donald J. Doughman, Richard Lindstrom, MD; Robert A. Good, MD
The stroma and epithelium of human that had been stored in organ culture medium for 10 to 22 days at 37 C were examined by light and electron microscopy. Total corneal thickness was found to be doubled at ten days and there was no further increase even at 22 days. The posterior portion of the stroma was more hydrated than the anterior region. Stromal cells were reduced in number and normal-appearing cells were present only in superficial stroma. The epithelial basement membrane was irregular and thickened. Although the epithelium was reduced to three or four cells in thickness and the intercellular spaces were dilated, the epithelial cells contained normal subcellular organelles and appeared to be viable. corneas
MD; John E. Harris, MD; George E. Miller, MD;
weeks. Recent studies in our labora¬ tories have shown that the endothe¬ lial cells of human corneas maintain their ultrastructural integrity for up to 21 days of storage in organ cul¬ ture.'-' This paper describes the ul-
Materials and Methods
Fig 1.—Cross-section of human cornea stored in organ culture medium at 37 C for ten days. Irregular swelling (clear areas between collagen lamellae) is apparent in posterior stroma. Epithelium is only three to four cells in thickness. Descemet mem¬ brane and endothelium are at lower right (original magnification
86).
of donor corneal tis¬ in organ culture offers prom¬ the time of storage few days up to two or three
Preservati on extending sue
ise of from a
trastructural appearance of the epi¬ thelium and stroma in organ-cultured human corneas and is part of a con¬ tinuing and extensive evaluation of the effects of organ culture on corneal tissue.
Submitted for publication Nov 9, 1973. From the Research Service, Veterans Administration Center, Wood, Wisc, and the departments of ophthalmology and physiology (Dr. Van Horn), The Medical College of Wisconsin, Milwaukee; Department of Ophthalmology (Drs. Doughman, Harris, Miller, Lindstrom) University of Minnesota, Minneapolis; and Memorial Sloan-Kettering Cancer Center (Dr. Good), New York. Reprint requests to Research Service/151, Veterans Administration Center, Wood, WI 53193 (Dr. Van Horn).
Downloaded From: http://archopht.jamanetwork.com/ by a Western University User on 06/06/2015
stroma of eight examined by light and elec¬ tron microscopy. Some of the same corneas had been used in the previous studies of the endothelium, and the methods of organ culture and preparation of the tissue for electron microscopy were described in the
The
epithelium and
corneas were
previous papers.'"
Results
Light microscopy of human corneas stored in organ culture medium for different lengths of time (10 to 22 days) demonstrated similar changes in all of the corneas. Due to swelling of the stroma and resultant produc¬ tion of folds in Descemet membrane, central corneal thickness ranged from 0.8 to 1.2 mm. The posterior three quarters of the stroma appeared to be more hydrated than the superficial portion (Pig 1 and 2), and stromal cells were reduced in number and could be positively identified only in
superficial
stroma. The
epithelium
reduced in thickness and was composed of three to four cell layers
was
(Fig 1 and 2). Intercellular edema was apparent and the basal cells were flat
cuboidal rather than columnar (Fig 2). The superficial surface of the cornea was smooth. Electron microscopy of the super¬ ficial stroma showed that the regular arrangement of the collagen lamellae was maintained, and a few normalappearing stromal cells were present between some of the lamellae (Fig 3). In contrast, in the posterior stroma, the lamellae were separated by ede¬ matous spaces that contained rem¬ nants of disrupted stromal cells, an amorphous substance, and widebanded fibrils (Fig 4). or
Edematous spaces were also ob¬ served between the epithelial cells (Fig 5 and 6). The cells, however, con¬ tained their normal complement of organelles and were relatively normal in appearance except that the basal cells were flattened. The wing and su¬ perficial cells frequently contained ac¬ cumulations of glycogen, as did some basal cells. The superficial cells had numerous microvilli and microplicae. A few lipid granules were present in some epithelial cells. Desmosomes and hemidesmosomes were reduced in number and the basement membrane was frequently irregular and thick¬ ened, consisting of both basement membrane-like material and a more dense, finely filamentous material
(Fig 6).
15% dextran in our organ culture me¬ dium to maintain corneal clarity and, we had hoped, normal corneal thick¬ ness. Kuwahara et al have reported
Fig 4.—Remnants of stromal cell in ede¬ matous space in posterior stroma. Space between collagen lamellae is more than twice that occupied by normal stromal cell in previous Figure at same magnification. Space also contains amorphous sub¬ stance and wide-banded fibrils (original
magnification
8,500).
Comment
Although stromal thickness was considerably and irregularly in¬ creased in the organ-cultured corneas compared to control, nonstored tissue,
the
clear at the time removed from the organ they culture medium.1 The apparent dif¬ ferential hydration of anterior and posterior stroma that we observed may be a pressure-related artifact, since the corneas were stored with the epithelial side down. Some of the stromal cells in superficial stroma ap¬ peared normal and probably were metabolically active, but most of the cells had undergone irreversible de¬ generation. Ultrastructural changes that are considered to be irreversible include interruptions in the continu¬ ity of the membranes, formation of membranous whorls and blebs, con¬ siderable swelling of the matrix compartment of the mitochondria, dilatation and fragmentation of the cisternae of the endoplasmic reticulum, clumping of the nuclear chromatin, karyolysis, and karyorrhexis. ' In preliminary studies, we have shown that the epithelial intercellular edema and stromal hydration are cor¬ rected after transplantation of or¬ gan-cultured corneas in animals and the transplanted tissue returns to normal thickness. Nevertheless, it would be advantageous to maintain normal corneal thickness during stor¬ age in organ culture. We have used corneas were were
Fig 2.—Higher magnification of epithe¬ lium and superficial stroma. Basal epithe¬ lial cells are flattened or cuboidal and intercellular spaces are enlarged. Few stromal cells (arrows) can be identified in superficial stroma. Clear areas of hydra¬ tion are more prominent deeper in stroma (lower right) 270).
(original
magnification
Fig 3.—Normal stromal cell in superficial stroma. Note regular arrangement and close apposition of collagen to cell that lies stretched out between collagen lamellae (original magnification 8,500).
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Fig 5.—Epithelium of cornea stored in organ culture for 21 days. Intercellular spaces are enlarged, although desmosomes connect cells. Accumulations of glycogen (arrows) are present in cells. Basement membrane is irregular and thickened (original magnification x
9,700).
lipid from organ culture medium has previously been reported.7 Although the viability of the donor endothelium is of far greater impor¬ tance for the success of penetrating keratoplasty than is that of the epi¬ thelium, Thoft et al have recently in¬ dicated that the increasing use of do¬ nor corneal epithelium in cases of severe disturbances to epithelial and stromal integrity requires a reexamination of storage effects on the epi¬ thelial layer.8 The results of the pres¬ ent study indicate that organ culture may be a useful way of preserving epithelium for transplantation as well as preserving whole cornea for penetrating keratoplasty. This investigation was supported in part by grants EY 00027, EY 00625, and EY 00202 from the National Eye Institute, and by the Min¬
Eye Bank. The human donor corneas were supplied by the Minnesota Lions Eye Bank. nesota Lions
Key Words.—Cornea; corneal epithelium, corneal stroma; corneal tron
microscopy;
preservation;
elec¬
organ culture.
References 1. Summerlin WT, et al: The organ cultured An in vitro study. Invest Ophthalmol 12:176-180, 1973. 2. Doughman DJ, et al: Electron microscopic study of human organ cultured endothelium. Trans Am Ophthalmol Soc 71:304-328, 1973. 3. Doughman DJ, et al: The ultrastructure of human organ-cultured cornea. 1. Endothelium. Arch Ophthalmol 92:516-523, 1974. 4. Trump BF, Ginn FL: The pathogenesis of subcellular reaction to lethal injury, in Bajusz E, Jasmin G (eds): Methods and Achievements in cornea:
Fig 6.—Higher magnification of basal epithelial cell and thickened basement mem¬ brane in cornea stored in organ culture for ten days. Dense, finely fibrillar material, as well as basement membrane-like material, is present In thickened basement membrane. Epithelial cell contains desmosomes (D), hemidesmosomes (HD), tonofilaments, endoplasmic reticulum, and mitochondria. Intercellular space (IS) is dilated (original magnifi¬ cation 14,400). that storage of animal and human corneas up to one week at 0 C in a special tissue culture medium con¬
taining keratopolysulphate prevents stromal swelling and epithelial
edema.5 Further studies are now un¬ der way to reduce the hydration of the tissue by changes in our organ culture medium. All of the epithelial cells appeared to be viable, even in the corneas stored up to 22 days in organ culture.
Although the epithelial layer was only three to four cells in thickness, the cells contained normal organelles and had maintained their ability to control their intracellular volume, in¬ dicating that metabolic pumps were
still active.4 The cells showed few signs of degeneration, although many contained accumulations of glycogen and a few cells had taken up lipid. Su¬ perficial cells normally accumulate glycogen in vivo,6 and the uptake of
Downloaded From: http://archopht.jamanetwork.com/ by a Western University User on 06/06/2015
Experimental Pathology, Examples of Descriptive and Functional Morphology. Basel, Switzerland, S Karger AG, 1969, vol 4, pp 1-29. 5. Kuwahara Y, et al: Studies onthe long-term preservation of the cornea for penetrating keratoplasty. Acta Soc Ophthalmol Jap 40:225-314,
1965. 6. Hogan MJ, Alvarado JA, Weddell JE: The cornea, in The Histology of the Human Eye. Philadelphia, WB Saunders, 1971, pp 55-111. 7. Krey PR, etal: The human fetal synovium: Histology, fine structure and changes in organ culture. Arthritis Rheum 14:319-341, 1971. The biology of human donor 8. Thoft R, etal: cornea preservation. Presented at a meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Fla, 1973.
Epithelium
Diane L. Van Horn, PhD; Donald J. Doughman, Richard Lindstrom, MD; Robert A. Good, MD
The stroma and epithelium of human that had been stored in organ culture medium for 10 to 22 days at 37 C were examined by light and electron microscopy. Total corneal thickness was found to be doubled at ten days and there was no further increase even at 22 days. The posterior portion of the stroma was more hydrated than the anterior region. Stromal cells were reduced in number and normal-appearing cells were present only in superficial stroma. The epithelial basement membrane was irregular and thickened. Although the epithelium was reduced to three or four cells in thickness and the intercellular spaces were dilated, the epithelial cells contained normal subcellular organelles and appeared to be viable. corneas
MD; John E. Harris, MD; George E. Miller, MD;
weeks. Recent studies in our labora¬ tories have shown that the endothe¬ lial cells of human corneas maintain their ultrastructural integrity for up to 21 days of storage in organ cul¬ ture.'-' This paper describes the ul-
Materials and Methods
Fig 1.—Cross-section of human cornea stored in organ culture medium at 37 C for ten days. Irregular swelling (clear areas between collagen lamellae) is apparent in posterior stroma. Epithelium is only three to four cells in thickness. Descemet mem¬ brane and endothelium are at lower right (original magnification
86).
of donor corneal tis¬ in organ culture offers prom¬ the time of storage few days up to two or three
Preservati on extending sue
ise of from a
trastructural appearance of the epi¬ thelium and stroma in organ-cultured human corneas and is part of a con¬ tinuing and extensive evaluation of the effects of organ culture on corneal tissue.
Submitted for publication Nov 9, 1973. From the Research Service, Veterans Administration Center, Wood, Wisc, and the departments of ophthalmology and physiology (Dr. Van Horn), The Medical College of Wisconsin, Milwaukee; Department of Ophthalmology (Drs. Doughman, Harris, Miller, Lindstrom) University of Minnesota, Minneapolis; and Memorial Sloan-Kettering Cancer Center (Dr. Good), New York. Reprint requests to Research Service/151, Veterans Administration Center, Wood, WI 53193 (Dr. Van Horn).
Downloaded From: http://archopht.jamanetwork.com/ by a Western University User on 06/06/2015
stroma of eight examined by light and elec¬ tron microscopy. Some of the same corneas had been used in the previous studies of the endothelium, and the methods of organ culture and preparation of the tissue for electron microscopy were described in the
The
epithelium and
corneas were
previous papers.'"
Results
Light microscopy of human corneas stored in organ culture medium for different lengths of time (10 to 22 days) demonstrated similar changes in all of the corneas. Due to swelling of the stroma and resultant produc¬ tion of folds in Descemet membrane, central corneal thickness ranged from 0.8 to 1.2 mm. The posterior three quarters of the stroma appeared to be more hydrated than the superficial portion (Pig 1 and 2), and stromal cells were reduced in number and could be positively identified only in
superficial
stroma. The
epithelium
reduced in thickness and was composed of three to four cell layers
was
(Fig 1 and 2). Intercellular edema was apparent and the basal cells were flat
cuboidal rather than columnar (Fig 2). The superficial surface of the cornea was smooth. Electron microscopy of the super¬ ficial stroma showed that the regular arrangement of the collagen lamellae was maintained, and a few normalappearing stromal cells were present between some of the lamellae (Fig 3). In contrast, in the posterior stroma, the lamellae were separated by ede¬ matous spaces that contained rem¬ nants of disrupted stromal cells, an amorphous substance, and widebanded fibrils (Fig 4). or
Edematous spaces were also ob¬ served between the epithelial cells (Fig 5 and 6). The cells, however, con¬ tained their normal complement of organelles and were relatively normal in appearance except that the basal cells were flattened. The wing and su¬ perficial cells frequently contained ac¬ cumulations of glycogen, as did some basal cells. The superficial cells had numerous microvilli and microplicae. A few lipid granules were present in some epithelial cells. Desmosomes and hemidesmosomes were reduced in number and the basement membrane was frequently irregular and thick¬ ened, consisting of both basement membrane-like material and a more dense, finely filamentous material
(Fig 6).
15% dextran in our organ culture me¬ dium to maintain corneal clarity and, we had hoped, normal corneal thick¬ ness. Kuwahara et al have reported
Fig 4.—Remnants of stromal cell in ede¬ matous space in posterior stroma. Space between collagen lamellae is more than twice that occupied by normal stromal cell in previous Figure at same magnification. Space also contains amorphous sub¬ stance and wide-banded fibrils (original
magnification
8,500).
Comment
Although stromal thickness was considerably and irregularly in¬ creased in the organ-cultured corneas compared to control, nonstored tissue,
the
clear at the time removed from the organ they culture medium.1 The apparent dif¬ ferential hydration of anterior and posterior stroma that we observed may be a pressure-related artifact, since the corneas were stored with the epithelial side down. Some of the stromal cells in superficial stroma ap¬ peared normal and probably were metabolically active, but most of the cells had undergone irreversible de¬ generation. Ultrastructural changes that are considered to be irreversible include interruptions in the continu¬ ity of the membranes, formation of membranous whorls and blebs, con¬ siderable swelling of the matrix compartment of the mitochondria, dilatation and fragmentation of the cisternae of the endoplasmic reticulum, clumping of the nuclear chromatin, karyolysis, and karyorrhexis. ' In preliminary studies, we have shown that the epithelial intercellular edema and stromal hydration are cor¬ rected after transplantation of or¬ gan-cultured corneas in animals and the transplanted tissue returns to normal thickness. Nevertheless, it would be advantageous to maintain normal corneal thickness during stor¬ age in organ culture. We have used corneas were were
Fig 2.—Higher magnification of epithe¬ lium and superficial stroma. Basal epithe¬ lial cells are flattened or cuboidal and intercellular spaces are enlarged. Few stromal cells (arrows) can be identified in superficial stroma. Clear areas of hydra¬ tion are more prominent deeper in stroma (lower right) 270).
(original
magnification
Fig 3.—Normal stromal cell in superficial stroma. Note regular arrangement and close apposition of collagen to cell that lies stretched out between collagen lamellae (original magnification 8,500).
Downloaded From: http://archopht.jamanetwork.com/ by a Western University User on 06/06/2015
Fig 5.—Epithelium of cornea stored in organ culture for 21 days. Intercellular spaces are enlarged, although desmosomes connect cells. Accumulations of glycogen (arrows) are present in cells. Basement membrane is irregular and thickened (original magnification x
9,700).
lipid from organ culture medium has previously been reported.7 Although the viability of the donor endothelium is of far greater impor¬ tance for the success of penetrating keratoplasty than is that of the epi¬ thelium, Thoft et al have recently in¬ dicated that the increasing use of do¬ nor corneal epithelium in cases of severe disturbances to epithelial and stromal integrity requires a reexamination of storage effects on the epi¬ thelial layer.8 The results of the pres¬ ent study indicate that organ culture may be a useful way of preserving epithelium for transplantation as well as preserving whole cornea for penetrating keratoplasty. This investigation was supported in part by grants EY 00027, EY 00625, and EY 00202 from the National Eye Institute, and by the Min¬
Eye Bank. The human donor corneas were supplied by the Minnesota Lions Eye Bank. nesota Lions
Key Words.—Cornea; corneal epithelium, corneal stroma; corneal tron
microscopy;
preservation;
elec¬
organ culture.
References 1. Summerlin WT, et al: The organ cultured An in vitro study. Invest Ophthalmol 12:176-180, 1973. 2. Doughman DJ, et al: Electron microscopic study of human organ cultured endothelium. Trans Am Ophthalmol Soc 71:304-328, 1973. 3. Doughman DJ, et al: The ultrastructure of human organ-cultured cornea. 1. Endothelium. Arch Ophthalmol 92:516-523, 1974. 4. Trump BF, Ginn FL: The pathogenesis of subcellular reaction to lethal injury, in Bajusz E, Jasmin G (eds): Methods and Achievements in cornea:
Fig 6.—Higher magnification of basal epithelial cell and thickened basement mem¬ brane in cornea stored in organ culture for ten days. Dense, finely fibrillar material, as well as basement membrane-like material, is present In thickened basement membrane. Epithelial cell contains desmosomes (D), hemidesmosomes (HD), tonofilaments, endoplasmic reticulum, and mitochondria. Intercellular space (IS) is dilated (original magnifi¬ cation 14,400). that storage of animal and human corneas up to one week at 0 C in a special tissue culture medium con¬
taining keratopolysulphate prevents stromal swelling and epithelial
edema.5 Further studies are now un¬ der way to reduce the hydration of the tissue by changes in our organ culture medium. All of the epithelial cells appeared to be viable, even in the corneas stored up to 22 days in organ culture.
Although the epithelial layer was only three to four cells in thickness, the cells contained normal organelles and had maintained their ability to control their intracellular volume, in¬ dicating that metabolic pumps were
still active.4 The cells showed few signs of degeneration, although many contained accumulations of glycogen and a few cells had taken up lipid. Su¬ perficial cells normally accumulate glycogen in vivo,6 and the uptake of
Downloaded From: http://archopht.jamanetwork.com/ by a Western University User on 06/06/2015
Experimental Pathology, Examples of Descriptive and Functional Morphology. Basel, Switzerland, S Karger AG, 1969, vol 4, pp 1-29. 5. Kuwahara Y, et al: Studies onthe long-term preservation of the cornea for penetrating keratoplasty. Acta Soc Ophthalmol Jap 40:225-314,
1965. 6. Hogan MJ, Alvarado JA, Weddell JE: The cornea, in The Histology of the Human Eye. Philadelphia, WB Saunders, 1971, pp 55-111. 7. Krey PR, etal: The human fetal synovium: Histology, fine structure and changes in organ culture. Arthritis Rheum 14:319-341, 1971. The biology of human donor 8. Thoft R, etal: cornea preservation. Presented at a meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Fla, 1973.
