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Tissue culture of rat pigment epithelium on different supporting media
0042.6989/81/0101-Ol27Kl2.00/0
VisionResearch Vol.21,pp.127to 132 Pergamon PressLtd 1981. Printedm GreatBritain
TISSUE
CULTURE OF RAT PIGMENT DIFFERENT SUPPORTING MICHAEL
0. HALL
EPITHELIUM MEDIA
ON
DEBORAH S. QUON
and
The Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. Abstract-We have used tissue culture to study the process of phagocytosis of rod outer segments (ROS) by normal and dystrophic rat pigment epithelial (PE) cells. These cells in culture retain some of their in uiuocapabilities, viz. (a) the ability to distinguish between light and dark adapted ROS (Hall, 1978); (b) PE cells from dystrophic rats have a reduced phagocytic capability in comparison with PE cells from normal rats (Hall, 1979). However, we do not know whether these functions are partially reduced in tissue culture, or to what extent other in uiuoproperties are adversely affected by removing the cells from the eye. We have thus studied the growth of PE cells on different supporting media in order to find conditions under which these cells morphologically and functionally resemble PE cells in oivo. As a first approximation, we have compared the parameters of plating efficiency, morphological appearance, and the ability to phagocytize and digest ROS, of PE cells grown on different media. Not all parameters have
been measured for each supporting medium.
GENERAL CONSIDERATIONS
We have used the general technique described by Edwards (1977) to isolate and culture PE cells from normal Long Evans rats, and from the dystrophic (rdy-p+) strain described by LaVail et nl. (1975). Cells are isolated from animals ranging in age from 6 to 10 days old. Attempts to culture cells from older animals have met with limited success. When strict adherence is paid to the isolation conditions, the yield of cells from Long Evans rats is 40,000 to 50,000 cells/ eye, while the yield from rdy-p+ rats is about 25,000 cells/eye. Cells are seeded at a density of 40,000/cm2, with a plating efficiency of 10 to 100% depending on the supporting medium used. Cells from normal and dystrophic rats behave identically on each of the supporting media studied.
GROWTH ON DIFFERENT SUPPORTING MEDIA (a) Plastic
tissue
culture dishes
When freshly isolated PE cell suspensions are seeded in plastic multiwell plates (16 mm dia.), SO-100% of the cells will attach within 24 hr. Such a culture will form a confluent monolayer in 4-7 days. Cells cover the surface of the plate both by spreading and by cell division. When cells are continuously exposed to 3H-thymidine from seeding until a confluent monolayer is formed, 62 & 8% of the cells show labeled nuclei. Some of the cells undergo no division at all, even though they attach, spread and appear to make contacts with neighboring cells. The presence of epidermal growth factor (l&30 ng/ml) does not enhance the growth of these cells. Pigment epithelial cells grown on plastic assume a flattened morphology (Fig. l), often projecting long pseudopod-like extensions to contact adjacent cells.
After a few days in culture, a disorganized extracellular matrix is secreted beneath the cells (Fig. 2). These PE cells are capable of phagocytizing isolated ROS, yet they show very few surface microvilli (Figs 2 and 3), which never approximate the number seen in uiuo. It is possible that the flat, extended.shape assumed by these cells does not easily allow for the projection of plasma membrane into microvilli and that as a consequence, the rate of phagocytosis measured in vitro is only a fraction of that occurring in uiuo. (b) Plastic disks and Milliporejlters In order to allow easier handling and manipulation of cell monolayers, particularly for microscopy, we have grown PE cells on Thermanox plastic disks (Lux Scientific Corp, Newbury Park, CA), which are compatible with the organic solvents and embedding media used in light and electron microscopy. The growth characteristics of the PE cells are identical on this support to those obtained when the cells are seeded in plastic dishes. For studies of the chemistry of the plasma membrane, it is often necessary to grow cells on a supporting medium which allows access of probes to the basal as well as to the apical surface of the cell. We have thus applied the method described by Misfeldt et al. (1976) to the growth of PE cells on Millipore filters (Millipore Corp, Bedford, MA). The number of cells seeded must be increased to 100,000/cm2 as the plating efficiency is about 50% on this support. Growth and division of the cells is slower on these filters than on plastic, but confluent monolayers are formed when cells are seeded at a high density. (c) Glass tissue culture chambers In order to visualize within PE cell monolayers, 127
fluorescent labeled ROS we have grown these cells
128
M. 0. HALL and D. S. QUON
Fig.
1, Light micrograph
of PE cells grown on plastic. The cells are flattened and extended. A phagocytized ROS is seen within a cell (arrow). n = nucleus (x 1600).
on glass supports (Lab-Tek Products, Naperville, IL). The plating efficiency is lower than on plastic (S&75%) and it is thus necessary to increase the density of cells seeded/cm2 in order to obtain a confluent monolayer within a reasonable time This support provides the best surface for subsequent light microscope examination of the monolayer, and for visualization of inclusions within the PE cells.
(d) Collagen membranes Polymerized
collagen
matrices
have been used as
supports for the growth of numerous cell types. We have examined a method described by Emerman and Pitelka (1977) for growing PE cells on a floating collagen membrane (FCM). Although we have had difficulty in routinely culturing PE cells on this type of support, the appearance of the cells most closely resembles the morphology of PE cells in oiuo, and we are thus striving to develop this as the routine method for culturing PE cells. Collagen membranes are prepared by neutralizing a solution of rat tail collagen and Hams F-10 medium
Fig. 2. Electron micrograph of PE cells grown on plastic. The cells were incubated with a ROS suspension for 6 hr, washed and returned to the incubator for a further 24 hr. Phagosomes (arrows) present within the cell have undergone digestion to,different degrees. The surface of the cell is essentially devoid of microvilli. A loose extracellular matrix (em) is present beneath the cell ( x 21.000).
Different
Fig. 3. Scanning electron sion, followed by removal
supports
for pigment
epithelium
micrograph of a monolayer of PE cells after incubation with a ROS suspenof the ROS. The cell surfaces exhibit a regular array of short microvilli, which also delineate the outlines of each cell ( x 1700).
Fig. 4. Electron micrograph of a portion of a PE cell monolayer grown on a floating collagen membrane (FCM). Numerous microvilli (mv) are seen on the apical surface of the cell. The PE cell is richly endowed with pigment granules (pg), rough endoplasmic reticulum (rer) and mitochondria (m). A discrete “basal lamina” (arrows) can be seen beneath the cell ( x 9100).
129
M. 0. HALL and D. S. QUON
Fig. 5. Higher
magnification
of the apical surfaces of two adjacent PE cells grown Numerous microvilii (mv) are present (x 24,000).
on a FCM.
Fig. 6. Higher magnification of the basal surface of a PE cell grown on a FCM. The “basal lamina” (arrows) closely follows the contours of the cell membrane. The osmiophilic granules over and adjacent to the pigment granules are artifacts ( x 20,000).
Different supports for pigment epithelium (10 x ), with sterile NaOH. The collagen polymerizes in a few minutes at room temperature. Prior to seeding, the membranes are washed with growth medium. Cells should be seeded at a density of 150,000/cm2, as the plating efficiency is always low (about 10% or less). Cell attachment is also slow, thus the plates are left undisturbed in the incubator for 3 days and the medium is not changed until 4 or 5 days after seeding. When confluent islands of cells are formed, the collagen membrane is gently released from its attachment to the plastic, and allowed to float in the growth medium. It is at this point that the cells change from the typical morphology seen when they are grown on any of the above supports, to that shown in Figs 4-6. Upon release from the plastic, the FCM progressively shrinks, presumably due to the tractional forces exerted upon it by the cells. At the same time the cells assume a rounder shape and show a more regular and polarized distribution of subcellular organelles than is seen when these cells are grown on solid supports. The most obvious changes occur at the apical and basal surfaces of the cell. Apically, the number of microvilli increases dramatically (Figs 4 and S), more closely resembling the appearance of the cell surface in uioo. Rather than the short, sparsely distributed microvilli seen when PE cells are grown on plastic (Figs 2 and 3), the cells on FCM’s exhibit many more long slender microvilli (Figs 4 and 5). The decrease in the surface area of the cell which parallels the con-
131
traction of the matrix, presumably allows the plasma membrane to form the microvilli. On the basal surface of the cell, a definite basal lamina can be seen (Figs 4 and 6). This lamina, which closely follows the convoluted appearance of the cell membrane, appears to be a product of the PE cell, although a condensation of the surface of the FCM cannot be ruled out. Thus, although we have only recently begun to investigate the growth of PE cells on floating collagen membranes, their morphology suggests that they may provide an in oitro model which more closely approximates PE cells in ho.
DIGESTION
OF PHAGOSOMES
BY CULTURED
CELLS
Shed packages of outer segments which are phagocytized by rat PE cells in &JO, are digested within 2-4 hr (LaVail, 1976). We have studied the rate of digestion of phagocytized ROS by normal PE cells in tissue culture. Confluent monolayers of PE cells on plastic dishes were incubated with isolated ROS for 6 hr. The ROS suspension was removed, and the surfaces of the PE cell monolayers were washed to remove adhering ROS. The cultures were returned to the incubator and individual monolayers were fixed and processed for microscopy at 0, 6, 12, 24, 36, and 48 hr after removal
Fig. 7. Phagosome (ph) in a PE cell monolayer which has been treated to show the presence of acid phosphatase in the cell. The weak reaction due to the presence of the enzyme is localized within the
phagosome. No lysosomes are seen ( x 89,~~).
132
M. 0. HALL and D. S. QUOK
of the ROS. Some monolayers were processed for the cytochemical localization of acid phosphatase. Phagosomes at all stages of digestion were found within PE cells at all time points studied up to 24 hr (Fig. 2). By 36 hr after removal of external ROS, the number of undigested ROS had decreased, while at 48 hr, very few phagosomes were seen. The acid phosphatase reaction in the few lysosomes seen, and in phagosomes (secondary lysosomes) was very weak at all time points (Fig. 7). Thus it appears that PE cells in tissue culture have a greatly reduced capacity to degrade ingested ROS fragments. This may be due to a decrease in the number of lysosomes within the cultured cells. Our studies thus indicate that rat PE cells in tissue culture retain some of the properties of these cells in cico. However all of the parameters which we have measured appear to function at a greatly reduced level of efficiency. This may be due to the use of PE cells from animals which have not yet differentiated their photoreceptor cell outer segments. The properties which we have measured (phagocytosis and digestion of ROS) may not be fully expressed in the PE cell until after ROS disk shedding begins at 12 or 13 days of age. We are studying this possibility.
Ackno~ledgements~-This investigation was supported by Research Grants Number EY-00046 and EY-00331 from the National Eye Institute, National Institutes of Health, by a grant from the National Retinitis Pigmentosa Foundation, Baltimore; and by a Sammy Davis Jr. Award of Fight for Sight, inc.. New York City.
REFERENCES Edwards R. B. (1977) A method of culturing rat retinal pigment epithelium. In Mtro 13, 301-305. Emerman J. T. and Pitelka D. R. (1977) Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In I&-o 13, 316328.
Hall M. 0. (1978) Phagocytosis of light and dark-adapted rod outer segments by cultured pigment epithelium. Science 202, 5266528. Hall M. 0. (1979) Hereditary retinal dystrophy: Determination of the site of expression of the genetic defect. Inrestigrrtiw Ophfhumol. I/is. Sci. IS (Supplement), 258. LaVail M. M., Sidman R. L. and Gerhardt C. 0. (1975) Congenic strains of RCS rats with inherited retinal dystrophy. J. Heredity 66, 242-244. LaVail M. M. (1976) Rod outer segment disk shedding in T94, rat retina: relationship to cyclic lighting. Sciencr 1071. 1074. Misfeldt D. S., Hamamoto S. T. and Pitelka D. R. (1976) Transepithelial transport in cell culture. Proc. natn. nccrd. Sci. U.S.A. 73, 1212. 1216.
SYMPOSIUM
DISCUSSION
Question: Why, with all the work that you have done with the RCS rat. have you now approached the problems by utilizing tissue culture’? Reply by Dr Hall: Some years ago, when we showed the defect in the RCS rat involved phagocytosis, we were at a total loss as to how to go about it from that point on, in viaa Then Matt LaVail did his beautiful chimera work, which localized the defect to the PE. It appeared that the only way we could get at the problem, which was probably a recognition problem, was to get a pure culture of PE cells. We could then study the problem in a very logical way by looking at cell surface receptors, and at the metabolic requirements of these cells, which you just can’t do easily in rat eyecups. Also, you just have no idea about what you are working with if you try and do experiments in an eyecup preparation. Comment by Dr Flood: Our purpose for studying human pigment epithelium in culture is to eventually look at human RPE with genetic defects and we feel it is the only way of studying human RPE right now. If we can establish the basic growth parameters and possibly establish some biochemical marker for the human RPE, then we will have a baseline from which we can do further studies, Comment by Dr Potts: There is another reason, which apparently involves me more than anybody in the group. That is having a test object for toxic substances that specifically affect the RPE. When one has a pure culture of RPE. and the pure toxic substance, one has the most basic conditions for studying any particular toxic action.
VisionResearch Vol.21,pp.127to 132 Pergamon PressLtd 1981. Printedm GreatBritain
TISSUE
CULTURE OF RAT PIGMENT DIFFERENT SUPPORTING MICHAEL
0. HALL
EPITHELIUM MEDIA
ON
DEBORAH S. QUON
and
The Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. Abstract-We have used tissue culture to study the process of phagocytosis of rod outer segments (ROS) by normal and dystrophic rat pigment epithelial (PE) cells. These cells in culture retain some of their in uiuocapabilities, viz. (a) the ability to distinguish between light and dark adapted ROS (Hall, 1978); (b) PE cells from dystrophic rats have a reduced phagocytic capability in comparison with PE cells from normal rats (Hall, 1979). However, we do not know whether these functions are partially reduced in tissue culture, or to what extent other in uiuoproperties are adversely affected by removing the cells from the eye. We have thus studied the growth of PE cells on different supporting media in order to find conditions under which these cells morphologically and functionally resemble PE cells in oivo. As a first approximation, we have compared the parameters of plating efficiency, morphological appearance, and the ability to phagocytize and digest ROS, of PE cells grown on different media. Not all parameters have
been measured for each supporting medium.
GENERAL CONSIDERATIONS
We have used the general technique described by Edwards (1977) to isolate and culture PE cells from normal Long Evans rats, and from the dystrophic (rdy-p+) strain described by LaVail et nl. (1975). Cells are isolated from animals ranging in age from 6 to 10 days old. Attempts to culture cells from older animals have met with limited success. When strict adherence is paid to the isolation conditions, the yield of cells from Long Evans rats is 40,000 to 50,000 cells/ eye, while the yield from rdy-p+ rats is about 25,000 cells/eye. Cells are seeded at a density of 40,000/cm2, with a plating efficiency of 10 to 100% depending on the supporting medium used. Cells from normal and dystrophic rats behave identically on each of the supporting media studied.
GROWTH ON DIFFERENT SUPPORTING MEDIA (a) Plastic
tissue
culture dishes
When freshly isolated PE cell suspensions are seeded in plastic multiwell plates (16 mm dia.), SO-100% of the cells will attach within 24 hr. Such a culture will form a confluent monolayer in 4-7 days. Cells cover the surface of the plate both by spreading and by cell division. When cells are continuously exposed to 3H-thymidine from seeding until a confluent monolayer is formed, 62 & 8% of the cells show labeled nuclei. Some of the cells undergo no division at all, even though they attach, spread and appear to make contacts with neighboring cells. The presence of epidermal growth factor (l&30 ng/ml) does not enhance the growth of these cells. Pigment epithelial cells grown on plastic assume a flattened morphology (Fig. l), often projecting long pseudopod-like extensions to contact adjacent cells.
After a few days in culture, a disorganized extracellular matrix is secreted beneath the cells (Fig. 2). These PE cells are capable of phagocytizing isolated ROS, yet they show very few surface microvilli (Figs 2 and 3), which never approximate the number seen in uiuo. It is possible that the flat, extended.shape assumed by these cells does not easily allow for the projection of plasma membrane into microvilli and that as a consequence, the rate of phagocytosis measured in vitro is only a fraction of that occurring in uiuo. (b) Plastic disks and Milliporejlters In order to allow easier handling and manipulation of cell monolayers, particularly for microscopy, we have grown PE cells on Thermanox plastic disks (Lux Scientific Corp, Newbury Park, CA), which are compatible with the organic solvents and embedding media used in light and electron microscopy. The growth characteristics of the PE cells are identical on this support to those obtained when the cells are seeded in plastic dishes. For studies of the chemistry of the plasma membrane, it is often necessary to grow cells on a supporting medium which allows access of probes to the basal as well as to the apical surface of the cell. We have thus applied the method described by Misfeldt et al. (1976) to the growth of PE cells on Millipore filters (Millipore Corp, Bedford, MA). The number of cells seeded must be increased to 100,000/cm2 as the plating efficiency is about 50% on this support. Growth and division of the cells is slower on these filters than on plastic, but confluent monolayers are formed when cells are seeded at a high density. (c) Glass tissue culture chambers In order to visualize within PE cell monolayers, 127
fluorescent labeled ROS we have grown these cells
128
M. 0. HALL and D. S. QUON
Fig.
1, Light micrograph
of PE cells grown on plastic. The cells are flattened and extended. A phagocytized ROS is seen within a cell (arrow). n = nucleus (x 1600).
on glass supports (Lab-Tek Products, Naperville, IL). The plating efficiency is lower than on plastic (S&75%) and it is thus necessary to increase the density of cells seeded/cm2 in order to obtain a confluent monolayer within a reasonable time This support provides the best surface for subsequent light microscope examination of the monolayer, and for visualization of inclusions within the PE cells.
(d) Collagen membranes Polymerized
collagen
matrices
have been used as
supports for the growth of numerous cell types. We have examined a method described by Emerman and Pitelka (1977) for growing PE cells on a floating collagen membrane (FCM). Although we have had difficulty in routinely culturing PE cells on this type of support, the appearance of the cells most closely resembles the morphology of PE cells in oiuo, and we are thus striving to develop this as the routine method for culturing PE cells. Collagen membranes are prepared by neutralizing a solution of rat tail collagen and Hams F-10 medium
Fig. 2. Electron micrograph of PE cells grown on plastic. The cells were incubated with a ROS suspension for 6 hr, washed and returned to the incubator for a further 24 hr. Phagosomes (arrows) present within the cell have undergone digestion to,different degrees. The surface of the cell is essentially devoid of microvilli. A loose extracellular matrix (em) is present beneath the cell ( x 21.000).
Different
Fig. 3. Scanning electron sion, followed by removal
supports
for pigment
epithelium
micrograph of a monolayer of PE cells after incubation with a ROS suspenof the ROS. The cell surfaces exhibit a regular array of short microvilli, which also delineate the outlines of each cell ( x 1700).
Fig. 4. Electron micrograph of a portion of a PE cell monolayer grown on a floating collagen membrane (FCM). Numerous microvilli (mv) are seen on the apical surface of the cell. The PE cell is richly endowed with pigment granules (pg), rough endoplasmic reticulum (rer) and mitochondria (m). A discrete “basal lamina” (arrows) can be seen beneath the cell ( x 9100).
129
M. 0. HALL and D. S. QUON
Fig. 5. Higher
magnification
of the apical surfaces of two adjacent PE cells grown Numerous microvilii (mv) are present (x 24,000).
on a FCM.
Fig. 6. Higher magnification of the basal surface of a PE cell grown on a FCM. The “basal lamina” (arrows) closely follows the contours of the cell membrane. The osmiophilic granules over and adjacent to the pigment granules are artifacts ( x 20,000).
Different supports for pigment epithelium (10 x ), with sterile NaOH. The collagen polymerizes in a few minutes at room temperature. Prior to seeding, the membranes are washed with growth medium. Cells should be seeded at a density of 150,000/cm2, as the plating efficiency is always low (about 10% or less). Cell attachment is also slow, thus the plates are left undisturbed in the incubator for 3 days and the medium is not changed until 4 or 5 days after seeding. When confluent islands of cells are formed, the collagen membrane is gently released from its attachment to the plastic, and allowed to float in the growth medium. It is at this point that the cells change from the typical morphology seen when they are grown on any of the above supports, to that shown in Figs 4-6. Upon release from the plastic, the FCM progressively shrinks, presumably due to the tractional forces exerted upon it by the cells. At the same time the cells assume a rounder shape and show a more regular and polarized distribution of subcellular organelles than is seen when these cells are grown on solid supports. The most obvious changes occur at the apical and basal surfaces of the cell. Apically, the number of microvilli increases dramatically (Figs 4 and S), more closely resembling the appearance of the cell surface in uioo. Rather than the short, sparsely distributed microvilli seen when PE cells are grown on plastic (Figs 2 and 3), the cells on FCM’s exhibit many more long slender microvilli (Figs 4 and 5). The decrease in the surface area of the cell which parallels the con-
131
traction of the matrix, presumably allows the plasma membrane to form the microvilli. On the basal surface of the cell, a definite basal lamina can be seen (Figs 4 and 6). This lamina, which closely follows the convoluted appearance of the cell membrane, appears to be a product of the PE cell, although a condensation of the surface of the FCM cannot be ruled out. Thus, although we have only recently begun to investigate the growth of PE cells on floating collagen membranes, their morphology suggests that they may provide an in oitro model which more closely approximates PE cells in ho.
DIGESTION
OF PHAGOSOMES
BY CULTURED
CELLS
Shed packages of outer segments which are phagocytized by rat PE cells in &JO, are digested within 2-4 hr (LaVail, 1976). We have studied the rate of digestion of phagocytized ROS by normal PE cells in tissue culture. Confluent monolayers of PE cells on plastic dishes were incubated with isolated ROS for 6 hr. The ROS suspension was removed, and the surfaces of the PE cell monolayers were washed to remove adhering ROS. The cultures were returned to the incubator and individual monolayers were fixed and processed for microscopy at 0, 6, 12, 24, 36, and 48 hr after removal
Fig. 7. Phagosome (ph) in a PE cell monolayer which has been treated to show the presence of acid phosphatase in the cell. The weak reaction due to the presence of the enzyme is localized within the
phagosome. No lysosomes are seen ( x 89,~~).
132
M. 0. HALL and D. S. QUOK
of the ROS. Some monolayers were processed for the cytochemical localization of acid phosphatase. Phagosomes at all stages of digestion were found within PE cells at all time points studied up to 24 hr (Fig. 2). By 36 hr after removal of external ROS, the number of undigested ROS had decreased, while at 48 hr, very few phagosomes were seen. The acid phosphatase reaction in the few lysosomes seen, and in phagosomes (secondary lysosomes) was very weak at all time points (Fig. 7). Thus it appears that PE cells in tissue culture have a greatly reduced capacity to degrade ingested ROS fragments. This may be due to a decrease in the number of lysosomes within the cultured cells. Our studies thus indicate that rat PE cells in tissue culture retain some of the properties of these cells in cico. However all of the parameters which we have measured appear to function at a greatly reduced level of efficiency. This may be due to the use of PE cells from animals which have not yet differentiated their photoreceptor cell outer segments. The properties which we have measured (phagocytosis and digestion of ROS) may not be fully expressed in the PE cell until after ROS disk shedding begins at 12 or 13 days of age. We are studying this possibility.
Ackno~ledgements~-This investigation was supported by Research Grants Number EY-00046 and EY-00331 from the National Eye Institute, National Institutes of Health, by a grant from the National Retinitis Pigmentosa Foundation, Baltimore; and by a Sammy Davis Jr. Award of Fight for Sight, inc.. New York City.
REFERENCES Edwards R. B. (1977) A method of culturing rat retinal pigment epithelium. In Mtro 13, 301-305. Emerman J. T. and Pitelka D. R. (1977) Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In I&-o 13, 316328.
Hall M. 0. (1978) Phagocytosis of light and dark-adapted rod outer segments by cultured pigment epithelium. Science 202, 5266528. Hall M. 0. (1979) Hereditary retinal dystrophy: Determination of the site of expression of the genetic defect. Inrestigrrtiw Ophfhumol. I/is. Sci. IS (Supplement), 258. LaVail M. M., Sidman R. L. and Gerhardt C. 0. (1975) Congenic strains of RCS rats with inherited retinal dystrophy. J. Heredity 66, 242-244. LaVail M. M. (1976) Rod outer segment disk shedding in T94, rat retina: relationship to cyclic lighting. Sciencr 1071. 1074. Misfeldt D. S., Hamamoto S. T. and Pitelka D. R. (1976) Transepithelial transport in cell culture. Proc. natn. nccrd. Sci. U.S.A. 73, 1212. 1216.
SYMPOSIUM
DISCUSSION
Question: Why, with all the work that you have done with the RCS rat. have you now approached the problems by utilizing tissue culture’? Reply by Dr Hall: Some years ago, when we showed the defect in the RCS rat involved phagocytosis, we were at a total loss as to how to go about it from that point on, in viaa Then Matt LaVail did his beautiful chimera work, which localized the defect to the PE. It appeared that the only way we could get at the problem, which was probably a recognition problem, was to get a pure culture of PE cells. We could then study the problem in a very logical way by looking at cell surface receptors, and at the metabolic requirements of these cells, which you just can’t do easily in rat eyecups. Also, you just have no idea about what you are working with if you try and do experiments in an eyecup preparation. Comment by Dr Flood: Our purpose for studying human pigment epithelium in culture is to eventually look at human RPE with genetic defects and we feel it is the only way of studying human RPE right now. If we can establish the basic growth parameters and possibly establish some biochemical marker for the human RPE, then we will have a baseline from which we can do further studies, Comment by Dr Potts: There is another reason, which apparently involves me more than anybody in the group. That is having a test object for toxic substances that specifically affect the RPE. When one has a pure culture of RPE. and the pure toxic substance, one has the most basic conditions for studying any particular toxic action.