Elcp. Eye Res. (1991)
52, 121-128
Propagation
of Human
JOHN Ophthalmology
R.SAMPLES,* Research
(Received
Cornea1 Growth
PERRY S. BINDER
Laboratory,
9 June
Endothelium Factors
1989
Sharp
Cabrillo
and accepted
SHANKAR
AND
Hospital, in revised
In Vitro
of
K. NAYAKt
San Diego, form
Effect
CA 92110,
13 June
U.S.A.
1990)
Endothelial cells were obtained from 23 human corneal-scleral rims. Donors were 0.2-3 7 yr of age. Most cultures obtained from donors under 30 yr ceased to grow after the seventh passage (3-24 weeks). Growth from 17- and 25-yr-old donors were maintained for nine passages (26 weeks) and 18 passages (100 weeks), respectively. Cultures were established from 12 rims, using growth factors in uncoated culture flasks or epidermal growth factor (EGF) in an extracellular matrix-coated (ECM) flask. EGF improved the growth potential of the cells; a combination of an ECM-coated-tissue culture flask with EGF in the medium provided an improved environment for continuous growth. Key words: human ; cornea ; endothelium : growth factors ; tissue culture.
1. Introduction Cornea1endothelium growing in primary or secondary cultures has been used in biological, biochemical and pharmacological studies of these cells (Samples et al., 1985). Previous attempts at establishing long-term in vitro growth from human cornea1 endothelium using an entire cornea have met with limited success (Mannagh and Irving, 1965; Baum et al., 1979; Tripathi and Tripathi, 1982 ; Nayak and Binder, 1984). With few exceptions, endothelium from donors older than 20-30 yr has been exceedingly diicult to grow. Nayak and Binder (1984) introduced the use of human corneal-scleral rims remaining after cornea transplantation as a source of endothelium for tissue culture. The majority of these cells exhibited a reduced growth rate with an increasing number of passages, eventually reaching a stage characterized by poor growth, formation of enlarged multinucleated cells, and loss of the typical mosaic pattern. Growth-stimulating substances have been used as mitogens to enhance the in vitro growth of mammalian cells in tissue culture. Epidermal (EGF) and fibroblast (FGF) growth factors have been the most commonly used growth-stimulating substances for enhancing the growth of cornea1 endothelial cells in tissue culture (Gospodarowich, Meshcer and Birdwell, 1977; Nayak and Binder, 1984 ; Alvarado, Liu and Wood, 1986). Studies with the human (Nayak and Binder, 1984) and primate (Nayak et al., 1986) cornea1endothelial cells in culture have demonstrated that EGF and FGF alone are minimally effective in supporting cell growth. In the present study, other
mitogenic substances such as nerve growth factor (NGF), endothelial cell growth supplement (ECGS)and a synthetic nutrient mixture (NUS) were evaluated for efficacy in supporting growth and preventing the slowing of growth of human cornea1 endothelium in tissue culture. The present study dealswith the efficacy of growth factors supporting the growth (Gospodarowicz, Meshcer and Biidwell, 1977 ; Hyldahl, Auer and Sundelin, 1982) of human cornea1endothelial cells in tissueculture. Successfultissueculture of endothelium from bovine and primate (Tripathi and Tripathi, 1982 ; Nayak et al., 1986) corneas have been repeatedly documented. Methods for initiating the primary cultures of these cell types for human corneas are well established. However, attempts to grow these human cells in long-term culture through multiple passages have met with limited success.
2. Materials and Methods Cell Culture
Endothelium obtained from corneal-scleral rims of 3 5 human donors following cornea1 transplantation were used to initiate cell growth in tissue culture (Tables I and II) using a method which was previously described (Nayak and Binder, 1984). The donor age range was 0.2-37 yr (mean 20.8 + 11.95 yr). The time of death (TOD) to time of culture (TOC) varied from 18 to 72 hr (mean 50.4+ 17.8 hr, Table I). The endothelium was dissected and established as a primary explant culture in 2 S-cm2 tissue-culture flasks. Prior to anchoring the explants, the flask surface was wetted with growth-medium only, unless otherwise
* For correspondence and reprint requests at: Department of Ophthalmology, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR, U.S.A. t Director, Hoag Cancer Center, Newport Beach. CA,U.S.A.
00144835/91/020121+08
%03.00/O
stated.
Approximately
eight
to ten explants
measuring 3-5 mm in length and about 05-l width,
obtained
mm in
from each rim, were placed on the wet
surface of the flask and allowed to adhere by 0 1991 Academic
Press Limited
122
J. R. SAMPLES
TABLE
ET AL.
1
Tissue culture of endothelium obtained from human donor corneal-scleral
rims: donor information
TOD-TOC* Average donor age k-1
Donor age grow (yr) l-20 21-30 3140
Average time 0-d
11,3+6.8 24.25f1.7 33.Ok2.5
* Time of death to time of culture. t All cultures were initiated on uncoated
No. of
48.7+ 15 55f18 63+19
flask surfaces
in standard
11 8 4
culture
TABLE
No. of
cultures-t
specimens
11
8 4
medium.
II
Relationshipof donor age, TOD-TOC* and growth potential of endotheliumfrom human corneal-scleral rims in tissue culture Age group (yr)
l-20 1 1” 3
No. of positive cultures Grown to passage no. Duration (weeks)* Age group (v-1 No. of positive cultures Grown to passage no. Duration (weeks)*
21-30 2
Age grow (yr) No. of positive cultures Grown to passage no. Duration (weeks)*
3140 2 1” 6.5
* For the same passage number showing t Time of death to time of culture.
more
1 12 8
4.5
one positive
11212 3456718 8 24
16
111 3 4 11 12
9 28
4
12 1 2 20 13.5
lo
than
1
1 24
16
100
2 1 10 culture
TABLE
the duration
is presented
as the mean
duration
of culture.
III
Endotheliumfrom human cornea-scleralrims in tissueculture: Effect of growth factors Cell culture
Growth* factors
Donor age (yr)
HCE-50 HCE-55
NUS NUS NUS ECGS ECGS ECGS EGF EGF EGF EGF EGF-ECM EGF-ECM
14 25 0.3
48 60 18
26 10 8
8 2 1
19 20 21
72
24 20
6 4 5
26 37 19 0.2
72 24 72
24 24 30 23
0.4 0.5
36 48
44 15
HCE-61 HCE-52 HCE- 54 HCE-60 HCE-51 HCE-53 HCE-59 HCE-63 HCE-64 HC-65
TOD-TOCt (hr)
70 36
72
Duration (weeks)
10
Grown to passage no.+
9
10 12 5 15 4
* See Materials and Methods for the concentrations of growth factors and the preparation of ECM-coated surface. t Time of death to time of culture. $ Except for HCE-64 and HCE-65. all other cell cultures ceased to grow at passage numbers and the appropriate duration,
incubation in a CO, incubator for 30 min at 37°C. Growth medium was then added to barely cover the explants without dislodging them and was changed
twice a week. The cells were incubated undisturbed for 24 days. Upon reaching confluency the cells were passaged and routinely maintained as previously
HUMAN
CORNEAL
ENDOTHELIUM
IN VITRO
described (Nayak and Binder, 1984). Passage is defined as the transfer of cells from one flask to another following trypsinization. The passage number is the number of times the cells were transferred. In some experiments the tissue-culture dish surface was coated with ‘putative ’ extracellular matrix (ECM), i.e. culture dishes saved following the routine trypsinization of baboon cornea1 endothelial cells (Nayak et al., 1986). The ‘putative ’ ECM material left on the surface has been shown to resemble Descemet’s membrane in staining properties and collagen content (Perlman and Baum, 1974). Following the trypsinization of a 3- to 4-week-old confluent monolayer, dish surfaces were thoroughly washed four times with 0.1 M KC1 and distilled water (Hsieh and Baum, 1985). Dish surfaces were briefly treated with 1% deoxycholate, rinsed several times with distilled water and stored at 40°C in phosphate-buffered (0.1 M, pH 7.2) saline, until used. Growth
Medium
Medium NCTC-199 (Irvine Scientific, Irvine, CA) containing 1 y0 of an antibiotic solution consisting of 100 units ml penicillin-‘. 100 pg ml streptomycin-’ and 2.5 yg ml fungizone-’ solution, supplemented with 15 Y0fetal calf serum (FCS), was used as standard growth medium. Growth
Factors
Fibroblast (FGF), epidermal (EGF) and nerve growth factors (NGF), and endothelial cell growth supplement (ECGS) were commercially obtained (Collaborative Research, Inc., Bedford, MA). A consistent formulation of a nutrient mixture N&Serum (NUS, Collaborative Research, Inc.) containing EGF (5ng ml-l), ECGS (77.5 ,ug ml-‘), insulin, transferrin, tridothyronine, progesterone, estradiol-17, testosterone, hydroo-phosphorlethanolamine, cortisone, selenium, glucose, amino acids, vitamins and 25 % newborn bovine calf serum was used for the growth of cells. Except for EGF and ECGS, concentrations of other individual components could not be obtained from the vendor. Measurement of Cell Growth After trypsinization, cell density was determined using a hemacytometer and adjusted to a concentration of O-5 x lo5 cells ml-l. One milliliter of the cell suspension was distributed into a 60 x 15-mm culture dish (Falcon Plastics, Oxnard, CA) containing 4 ml of growth medium. In some experiments, a lower initial cell density was used to facilitate analysis. Kinetics of the cell growth was monitored by counting trypsinEDTA detached cells daily from triplicate dishes (Nayak and Binder, 1984; Nayak et al., 1986). Growth medium was changed twice a week. Growth pro-
123
motion of endothelial cells by various growth factors was analysed according to our previously described method (Nayak et al., 1986). Twenty-four hours following cell seeding, growth medium was replaced with fresh concentration of individual growth factors ; FGF, EGF, ECGS and NIJS. Duplicate experiments were performed for each parameter. Dishes were incubated for 10 days with two changes of medium on the 4th and 7th days. Growth factors FGF and EGF were used at concentrations of 0, 10, 50 and 100 ng ml-l, and ECGS at 0, 50, 100, 250 and 500 ng ml-’ concentrations. NU-Serum was used at 0, 2.5, 5 and 10%. Cell counts were determined from triplicate dishes at the end of incubation. Measurements of Initiation of DNA Synthesis
DNA synthesis as the measure of growth stimulation induced by growth factors was analysed as previously described (Tripathi and Tripathi, 1982: Nayak and Binder, 1984). Aliquots of 02 ml containing 5 x lo3 cells were distributed into wells of a 96-well tissue culture microplate (Falcon Plastics) and incubated at 3 7°C in a 5 % CO, plus 95 % air and humid atmosphere for 24 hr (Tripathi and Tripathi, 1982 ; Nayak and Binder, 1984). The growth medium was replaced with one containing a single fresh (O-100 ng per well) growth factor (FGF, EGF. ECGS and NGF). Triplicate wells were used for each parameter. Cultures were incubated for 48 hr and pulsed with 0.5 ,&i of [3H]thymidine per well (specific activity 47 Ci mM-‘, Amersham, Chicago, IL) for 18 hr. The incorporation of radioactive thymidine was determined as described previously (Nayak and Binder, 1984 ; Samples et al., 1985). 3. Results Cell Culture
Two groups of human cornea1 rims were utilized for growing cell cultures. In Group 1, endothelial cell cultures were started using plain tissue culture flask surfaces (no attachment factors such as rat tail collagen or gelatin were used) and standard growth medium (Tables I and II). Some of the cell cultures derived from this series of rims were used to study the effect of growth factors on the proliferation of endothelial cells. Cornea1 rims in Group 2 were used to grow endothelial cells on plain tissue culture flask surfaces and in the continuous presence of EGF, ECGS or NUS, or on an ECM-coated tissue culture flask surfaces in the continuous presence of EGF (Table III). In Group 1 uncontaminated endothelial cell growth was obtained from all 23 specimens (Table I) without the use of growth factors in the medium or attachment factors on the flask surface. The characteristic growth properties of these cells have been previously described (Nayak and Binder, 1984). Cells began to migrate
124
J. R. SAMPLES
ET AL.
morphologic study. However, we should note that appreciable differences in cell shape and size were not noted in response to the growth factors which we tested. Relationship o/‘Donor Age, TOD-TOC und Growth Potential of Endothelium
= 0”
0
I
B
P-4
Uncontaminated endothelial cell cultures from 2 3 cornea1 rims were maintained for a minimum of three and a maximum of 100 weeks (Table II). Endothelial cell growth occurred from all donors l-20 yr old (n = 11) with an average TOD-TOC of about 48 hr. One culture from a 17-yr-old donor with a TOWTOC of 48 hr was maintained for 100 weeks and 18 passages. The remaining cultures were maintained from primary culture to the seventh passage (3-24 weeks, Table II). Tn the group of donors 21-30 yr of age (n = 8) with an average TOD-TOC of 5 5 hr, one culture from a 2 5yr-old donor with a TOD-TOC of 64 hr survived for nine passages. Remaining cultures in this group were maintained for 3-20 weeks from primary culture to the fourth passage. All four donors 3140 yr of age with an average TOD-TOC of 64 hr produced positive cultures, but none survived beyond the first passage (Table III). FCS Requirement
P-6
P-8
II I
I
I;
2
3
4
I
I
;
I
I
rJ
5
6
7
8
9
IO
Days
FIG. 1. A, Dose response of FCS supplementation in standard culture medium, on the growth of HCE-32, P-4 cells derived from the endothelium of an 1%yr-old donor cornea. Bar = mean value k S.D. of triplicate cell counts. B. Growth kinetics of HCE-40 cells established from the endothelium of a 25-yr-old donor cornea. Each curve represents one passage and each point represents a mean value f S.D. of triplicate counts. Arrows indicate changing of the media.
from the explants within 2-5 days ; growth became confluent by about 2-3 weeks, at which time it was passaged for the tist time. Passaged growth was routinely maintained as previously described (Nayak and Binder, 1984). The cell growth was initially characterized by a typical hexagonal-to-polygonal shape with a centrally located nucleus, whereas most cases of continued cell growth form a mosaic pattern with multinucleated cells (Nayak and Binder, 1984). This stage of cell growth characterized by multinucleated cells, which has sometimes been termed the ‘crisis ’ stage, occurred after six to eight passages and about 8-10 months in culture. This was not a
The level of FCS supplementation in standard growth medium was determined [Fig. 1 (A)]. Cell culture HCE-36 established from the endothelium of a 26-yr-old donor cornea1 rim in its third passage was grown in the presence of O-20% FCS supplementation. In the absence of FCS. cells ceased to grow: a supplement level of either 15 or 20% yielded maximum growth. -In subsequent experiments growth medium was supplemented with 15 % FCS. Growth
Kinetics
A culture from the endothelium of a 25yr-old donor cornea1 rim, growing in the fourth to eighth passage (HCE-40) was used for studying growth kinetics [Fig. 1 (B)]. Cells growing in passage 4 (P-4) exhibited maximum growth with a doubling time (time required to double the initial cell number) of about 48 hr. The growth rate gradually decreased with increasing passage numbers reaching a plateau in P-8. EfJect
of
Growth Factors on Cell Growth
NUS, ECGS, FGF and EGF at different concentrations were tested for their ability to enhance the growth potential of endotheliai cell cultures (Fig. 2). All of the growth factors were tested using standard growth medium containing 15 % FCS. NUS at 10 or 20% in the presence of 15 % FCS in standard growth medium
HUMAN
CORNEAL
ENDOTHELIUM
125
IN VITRO
6 A 15%
FCS+NUS
4
2-
?? x * ; : "
0
5
20
IO %
NUS
ECGS
ing
ml-‘)
G ” IO C
6 6 4 2 r
FGF (ng
ml-‘)
FIG 2. Effect of growth factors on the growth of human cornea1endothelialcellsin vitro. Differentconcentrationsof growth factors were addedto standardgrowth mediumand were exposedto (A) NUS on HCE-45,P-3 cellsin the presence(0) and absence(n) of 15% FCSin medium.B, ECGSon HCE-25. P-4 cells.C, FGFon HCE-28,P-3 cells. D, EGFon HCE-40,P-4 cells. Bar = mean value& S.D. of triplicate counts.
5 o---O O---O
Control + ECGS
200
I
o----O -
Control + EGF
100 ng ml-’
rig/ml-’ I 4
4
/
d
z/
3
2
I
1F t t I
I
I
I
I
2
4
6
8
IO
Days
FIG. 3. Growth kinetics of human cornea1endothelialcellsHCE-40, P-6 in the presenceof (A) 200 ng ml ECGS-1or (B) 100 ng ml EGF-‘. Each point representsthe meanvalue+ S.D. of triplicate counts. Arrows indicate mediachanges.
exhibited a statistically sign&ant increase (P < 0.01) in the growth of HCE-36, P-4 cells compared to standard growth medium alone [Fig. 2 (A)]. When the
same level of NUS alone was added to medium (without FCS) a significant (P < 0.005) reduction in growth was observed. The addition of 500 ng ml-l of
126
J. R. SAMPLES
ET AL.
for each using paired t-test). Concentrations higher than 10 ng per well for FGF, EGF and NGF, and 20 ng per well for ECGSdid not produce a statistically significant increase in [3H]thymidine incorporation. Role of Growth Factors on the initiation of Endothelial Cell Growth In Vitro
“0 ; 4E P : 2 : k
0 I 20
I 40
FGF
? 8
I
40
I 60
(ng per
I
80
ECGS (ng
1
I 80
I 100
I
I
I
40
60
80
EGF
(per
well)
well)
/
1
120 160 200 per
I
20
well)
I
1 20 2.5
1
r
1
60
80
S NGF (ng
per
40
I
100
’ 100 well)
FIG. 4. Effect of growth factors on the initiation of DNA synthesis of HCE-28. P-2 grown with (FGF) and HCE-29. P-2 (0) cells in vitro. Cells were cultured in the presence of medium containing different concentrations of (A) FGF. (B) EGF, (C) ECGS and (D) 2.5 S NGF. Each point represents the mean value f S.D. of triplicate cpm. Open circles represent control groups.
ECGS, produced a significant (P < 005) growth increase for HCE-25, P-3 cells [Fig. 2 (B)]. The effect of ECGS at 200 ng ml-’ concentration on the growth kinetics of HCE-40, P-6 cells was not different from the control medium alone [Fig. 3 (A)]. An increased growth of HCE-29 cells growing in P- 5 occurred when 100 ng ml-l of FGF was added to standard growth medium [Fig. 2 (C)l. EGF (50 or 100 ng ml-l), induced a dramatic increase of cell growth of HCE-40 cells growing in P-4 [Figs 2(D) and 3(B)]. At the peak of growth on day 6, 100 ng ml-’ of EGF in the medium induced about a ninefold increase in cell growth, whereas a fivefold increase was seen for cells grown only in control medium [Fig. 3 (B)]. Effect of Growth Factors on DNA Synthesis Two cell cultures HCE-28, P-2 and HCE-29, P-2 were exposed to different concentrations of growth factors (Fig. 4). Growth factors FGF {Fig. 4(A)], EGF [Fig. 4(B)], ECGS [Fig. 4(C)], and NGF [Fig. 4(D)] induced significant increases in DNA synthesis in the presence of the lowest concentration tested (P < O-05
Endothelium from a total of 12 human donor cornea1 rims were established in tissue culture in the continuous presenceof growth factors EGF, ECGSand nutrient mixture NUS. All three growth factors were included in the growth medium from the initiation stage of these cell cultures. The results are summarized in Table III. Of the six cultures grown in the presence of EGF. four were maintained in culture for 5-12 passages (2 3-30 weeks). One of these cultures (HCE-63) ceased to grow after 23 weeks (five passages).The other three cultures were discontinued after 9, 10 and 12 passages.Growth of cell cultures HCE-64 and HCE-65 were initiated and maintained on ECM-coated tissue culture flasks in the presence of EGF. HCE-64 was maintained for 44 weeks in culture (15 passages).Cell culture HCE-65 was still growing in tissue culture after 15 weeks (four passages,Table III). Growth kinetics of the cells grown with EGF [Fig. 5 (A)] were different from the kinetics observed for cells grown in the presence of either ECGS [Fig. S(B)] or NUS [Fig. 5 CC)].More growth occurred in the presence of EGF than for cells growing in standard medium alone. One of three cultures grown in the presence of NlJS was maintained for 26 weeks before the cessation of growth in the eighth passage. The other two cultures in this group survived for one and two passages (S-10 weeks duration). Almost identical observations were made for three cultures grown in the presence of ECGS.These cultures ceased to grow after four to six passages(20-24 weeks; Table III). Growth kinetics of these cells did not change when they were grown in the presenceor absenceof ECGSin the medium [Fig. 5 (B)]. Similar observations were made for the cells grown in the presenceand absence of NUS [Fig. S(C)]. 4. Discussion Growth factors such as FGF and EGF have been shown to significantly influence the proliferation of bovine cornea1 endothelial cells in tissue culture (Gospodarowicz, Meshcer and Birdwell, 1977; Rheinwald and Green, 19 7 7 ; Gospodarowicz, Meshcer and Birdwell, 19 78 : Gospodarowicz et al., 1978 : Gospodarowicz, Greenburg and Alvarado, 1979). Growth factors in vitro have many actions, including induction of proliferation, differentiation, and aging (Stocker et al., 1959; Perlman and Baum, 1974). Growth factors may delay the ultimate senescenceof
HUMAN
CORNEAL
ENDOTHELIUM
IN VITRO
127
Days
I
I
I
ro
I 20
, !
Doys
FIG. 5. Growth kinetics of human cornea1endothelial cells grown in the continuous presenceof growth factors. The experimentwasinitiated with an initial inoculum of 1 x lo4 cellsper dish.Endothelialcells(A) HCE-53,P-2 grown with (. ,.. .) ) 100 ng ml EGF-‘; (B) HCE-52,P-3 grown with (. . ...) and without (-) and without (500 ng ml ECGS-‘; (C) HCE-50, P-2 grown with (. ,...) and without () 5 % in the standardgrowth medium.Each point representsthe meanvalue f S.D. of triplicate cell counts.
the cells, thereby increasing their culture life-span (Rheinwald and Green, 19 77). In the present study we have shown that human cornea1 endothelial cells grown on uncoated surfaces in standard culture medium without any growth factors responded to the stimulatory effects of four growth factors (Figs 2 and 4). Further, we have demonstrated that the inclusion of the growth factor EGF in the medium from the initiation stage of the cell culture drastically enhanced the growth potential of endothelial cells in tissue culture (Fig. 5). We found that using a flask surface-coated with ‘putative ’ ECM and adding EGF to the medium provided the best environment for growth of human cornea1 endothelial cells derived from 0*4- and 0.5-yrold donors. In a preliminary
study, culture-dish
surfaces coated with ECM were prepared by treating a 3-week-old confluent monolayer of baboon cornea1 epithelial, cells with trypsin solution as described by
Hsieh and Baum (198 5). One of the two cell cultures has been maintained beyond the typical slowing of growth and carried in continuous culture for over 44 weeks and 15 passages (Table III). Similar observations were reported in a communi-
cation by Alvarado, Lui and Wood (1986). In their abstract they described the establishment of cell cultures from donor cornea1 rims following transplantation and from specimens obtained at trabeculectomy, using an ECM-coated culture-dish surface and continuous presence of FGF. Their success is probably due to stimulation induced by FGF/EGF and may well have a less-pronounced effect on growth
properties. With their combination they were able to obtain rapidly growing cell cultures which underwent many generations. Both their work and ours suggests that a combination of specially treated culture-dish surfaces and growth factors in the medium may provide an environment conducive to growing human
128 cornea1 endothelial cells continuously in tissue culture. The development of systems which promote the sustained growth of cells can allow us to study the effects of drugs and a variety of potential regulators on the cornea1 endothelium. In addition, the propagation of large numbers of cells allows us to study cell-surface properties. extracellular matrix properties, and the unique biochemistry of the cornea1 endothelium and may ultimately make transplantation of cornea1 endothelium possible.
Acknowledgments Supported in part by grants from the National Vision Research Institute, Research to Prevent Blindness, and EY07111 from the National Eye Institute.
References Alvarado, J., Lui, G. and Wood, I. (1986). Establishment of human cornea1 endothelial cells in tissue culture. Invest. OphthalmoI. Vis. Sci. 27. 127 (abstract). Baum, J., Niedra, R., Davis, C. and Yue, Y. (1979). Mass culture of human cornea1 endothelial cells. Arch. Ophthalmol. 97, 113640. Glassma. A.. Coles, W. and Bennett, C. (1979). Cornea1 endothelium modified method for cultivation. In Vitro 15, 873-6. Gospodarowicz. D., Greenburg, G. and Alvarado. J. (1979). Transplantation of cultured bovine cornea1 endothelial cells to rabbit cornea: Clinical implications for human studies. Proc. Natl. Acad. Sci. U.S.A. 76, 464-8. Gospodarowicz, D., Greenburg, G.. Bialecki, H. and Zetter, B. (1978). Factors involved in the modulation of cell proliferation in vivo and in vitro: the role of fibroblast and epidermal growth factors in the proliferative response of mammalian cells. In Vitro 14, 85-l 18. Gospodarowicz. D., Meshcer. A. and Birdwell. C. (1977).
J. R. SAMPLES
ET AL.
Stimulation of cornea1 endothelial cell proliferation in vitro by fibroblast and epidermal growth factors. Exp. Eye Res. 25. 75-89. Gospodarowicz. D.. Mescher. A. and Birdwell, C. (1978). Control of cellular proliferation by fibroblast and epidermal growth factors. Natl. Cancer Inst. Monogr. 48. 109-30. Hsieh, P. and Baum, J. ( 1985). Effects of fibroblastic and endothelial extracellular matrices on cornea1 endothelial cells. Invest. Ophthalmol. Vis. Sci. 26. 457-63. Hyldahl. L.. Auer, G. and Sundelin, B. (1982). A novel method to establish primary cultures on bovine cornea1 endothelial cells. Cell Biol. Inter. Reports. 6. 52 3-X. Mannagh, J. and Irving, K. (1965). Human cornea1 endothelium. Growth in tissue culture. Arch. Ophthalmol. 74. 847-9. Nayak, S. and Binder, P. (1984). The growth of endothelium from human cornea1 rims in tissue culture. invest. OphthaImol. Vis. Sci. 25, 121 3-16. Nayak. S.. Samples, J.. Deg, J. and Binder. P. (1986). Growth characteristics of primate (baboon) cornea1 endothelium in vitro. Invest. Ophthalmol. Vis. Sci. 27. 607-l 1. Perlman, M. and Baum, J. ( I 9 74). Synthesis of a collagenous basal membrane by rabbit cornea1 endothelial cells in vitro. Arch. OphthaImol. 92, 238-9. Perlman. M. and Baum, J. (19 74). The mass culture of rabbit cornea1 endothelial cells. Arch. Ophthalmol. 92, 2 3 5-7. Rheinwald. J. and Green, H. (1977). Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265. 42 1-4. Samples, J.. Nayak. S.. Gualtieri, C. and Binder, P. ( 1985). Effect of hydrocortisone on cornea1 endothelial cells in vitro. Exp. Eye Res. 51. 487-95. Stocker, F., Eiring. A., Georgiade. R. and Georgiade. N. (1959). Evaluation of viability of preserved rabbit corneas by tissue culture procedures. Am. I. Ophthalmol. 47. 772-82. Tripathi. R. and Tripathi, B. (1982). Human trabecular endothelium, cornea1 endothelium, keratocytes. and scleral fibrocytes in primary culture. A comparative study of growth characteristics. morphology, and phagocytic activity by light and scanning electron microscopy. Exp. Eye Res. 35, 611-24.
52, 121-128
Propagation
of Human
JOHN Ophthalmology
R.SAMPLES,* Research
(Received
Cornea1 Growth
PERRY S. BINDER
Laboratory,
9 June
Endothelium Factors
1989
Sharp
Cabrillo
and accepted
SHANKAR
AND
Hospital, in revised
In Vitro
of
K. NAYAKt
San Diego, form
Effect
CA 92110,
13 June
U.S.A.
1990)
Endothelial cells were obtained from 23 human corneal-scleral rims. Donors were 0.2-3 7 yr of age. Most cultures obtained from donors under 30 yr ceased to grow after the seventh passage (3-24 weeks). Growth from 17- and 25-yr-old donors were maintained for nine passages (26 weeks) and 18 passages (100 weeks), respectively. Cultures were established from 12 rims, using growth factors in uncoated culture flasks or epidermal growth factor (EGF) in an extracellular matrix-coated (ECM) flask. EGF improved the growth potential of the cells; a combination of an ECM-coated-tissue culture flask with EGF in the medium provided an improved environment for continuous growth. Key words: human ; cornea ; endothelium : growth factors ; tissue culture.
1. Introduction Cornea1endothelium growing in primary or secondary cultures has been used in biological, biochemical and pharmacological studies of these cells (Samples et al., 1985). Previous attempts at establishing long-term in vitro growth from human cornea1 endothelium using an entire cornea have met with limited success (Mannagh and Irving, 1965; Baum et al., 1979; Tripathi and Tripathi, 1982 ; Nayak and Binder, 1984). With few exceptions, endothelium from donors older than 20-30 yr has been exceedingly diicult to grow. Nayak and Binder (1984) introduced the use of human corneal-scleral rims remaining after cornea transplantation as a source of endothelium for tissue culture. The majority of these cells exhibited a reduced growth rate with an increasing number of passages, eventually reaching a stage characterized by poor growth, formation of enlarged multinucleated cells, and loss of the typical mosaic pattern. Growth-stimulating substances have been used as mitogens to enhance the in vitro growth of mammalian cells in tissue culture. Epidermal (EGF) and fibroblast (FGF) growth factors have been the most commonly used growth-stimulating substances for enhancing the growth of cornea1 endothelial cells in tissue culture (Gospodarowich, Meshcer and Birdwell, 1977; Nayak and Binder, 1984 ; Alvarado, Liu and Wood, 1986). Studies with the human (Nayak and Binder, 1984) and primate (Nayak et al., 1986) cornea1endothelial cells in culture have demonstrated that EGF and FGF alone are minimally effective in supporting cell growth. In the present study, other
mitogenic substances such as nerve growth factor (NGF), endothelial cell growth supplement (ECGS)and a synthetic nutrient mixture (NUS) were evaluated for efficacy in supporting growth and preventing the slowing of growth of human cornea1 endothelium in tissue culture. The present study dealswith the efficacy of growth factors supporting the growth (Gospodarowicz, Meshcer and Biidwell, 1977 ; Hyldahl, Auer and Sundelin, 1982) of human cornea1endothelial cells in tissueculture. Successfultissueculture of endothelium from bovine and primate (Tripathi and Tripathi, 1982 ; Nayak et al., 1986) corneas have been repeatedly documented. Methods for initiating the primary cultures of these cell types for human corneas are well established. However, attempts to grow these human cells in long-term culture through multiple passages have met with limited success.
2. Materials and Methods Cell Culture
Endothelium obtained from corneal-scleral rims of 3 5 human donors following cornea1 transplantation were used to initiate cell growth in tissue culture (Tables I and II) using a method which was previously described (Nayak and Binder, 1984). The donor age range was 0.2-37 yr (mean 20.8 + 11.95 yr). The time of death (TOD) to time of culture (TOC) varied from 18 to 72 hr (mean 50.4+ 17.8 hr, Table I). The endothelium was dissected and established as a primary explant culture in 2 S-cm2 tissue-culture flasks. Prior to anchoring the explants, the flask surface was wetted with growth-medium only, unless otherwise
* For correspondence and reprint requests at: Department of Ophthalmology, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR, U.S.A. t Director, Hoag Cancer Center, Newport Beach. CA,U.S.A.
00144835/91/020121+08
%03.00/O
stated.
Approximately
eight
to ten explants
measuring 3-5 mm in length and about 05-l width,
obtained
mm in
from each rim, were placed on the wet
surface of the flask and allowed to adhere by 0 1991 Academic
Press Limited
122
J. R. SAMPLES
TABLE
ET AL.
1
Tissue culture of endothelium obtained from human donor corneal-scleral
rims: donor information
TOD-TOC* Average donor age k-1
Donor age grow (yr) l-20 21-30 3140
Average time 0-d
11,3+6.8 24.25f1.7 33.Ok2.5
* Time of death to time of culture. t All cultures were initiated on uncoated
No. of
48.7+ 15 55f18 63+19
flask surfaces
in standard
11 8 4
culture
TABLE
No. of
cultures-t
specimens
11
8 4
medium.
II
Relationshipof donor age, TOD-TOC* and growth potential of endotheliumfrom human corneal-scleral rims in tissue culture Age group (yr)
l-20 1 1” 3
No. of positive cultures Grown to passage no. Duration (weeks)* Age group (v-1 No. of positive cultures Grown to passage no. Duration (weeks)*
21-30 2
Age grow (yr) No. of positive cultures Grown to passage no. Duration (weeks)*
3140 2 1” 6.5
* For the same passage number showing t Time of death to time of culture.
more
1 12 8
4.5
one positive
11212 3456718 8 24
16
111 3 4 11 12
9 28
4
12 1 2 20 13.5
lo
than
1
1 24
16
100
2 1 10 culture
TABLE
the duration
is presented
as the mean
duration
of culture.
III
Endotheliumfrom human cornea-scleralrims in tissueculture: Effect of growth factors Cell culture
Growth* factors
Donor age (yr)
HCE-50 HCE-55
NUS NUS NUS ECGS ECGS ECGS EGF EGF EGF EGF EGF-ECM EGF-ECM
14 25 0.3
48 60 18
26 10 8
8 2 1
19 20 21
72
24 20
6 4 5
26 37 19 0.2
72 24 72
24 24 30 23
0.4 0.5
36 48
44 15
HCE-61 HCE-52 HCE- 54 HCE-60 HCE-51 HCE-53 HCE-59 HCE-63 HCE-64 HC-65
TOD-TOCt (hr)
70 36
72
Duration (weeks)
10
Grown to passage no.+
9
10 12 5 15 4
* See Materials and Methods for the concentrations of growth factors and the preparation of ECM-coated surface. t Time of death to time of culture. $ Except for HCE-64 and HCE-65. all other cell cultures ceased to grow at passage numbers and the appropriate duration,
incubation in a CO, incubator for 30 min at 37°C. Growth medium was then added to barely cover the explants without dislodging them and was changed
twice a week. The cells were incubated undisturbed for 24 days. Upon reaching confluency the cells were passaged and routinely maintained as previously
HUMAN
CORNEAL
ENDOTHELIUM
IN VITRO
described (Nayak and Binder, 1984). Passage is defined as the transfer of cells from one flask to another following trypsinization. The passage number is the number of times the cells were transferred. In some experiments the tissue-culture dish surface was coated with ‘putative ’ extracellular matrix (ECM), i.e. culture dishes saved following the routine trypsinization of baboon cornea1 endothelial cells (Nayak et al., 1986). The ‘putative ’ ECM material left on the surface has been shown to resemble Descemet’s membrane in staining properties and collagen content (Perlman and Baum, 1974). Following the trypsinization of a 3- to 4-week-old confluent monolayer, dish surfaces were thoroughly washed four times with 0.1 M KC1 and distilled water (Hsieh and Baum, 1985). Dish surfaces were briefly treated with 1% deoxycholate, rinsed several times with distilled water and stored at 40°C in phosphate-buffered (0.1 M, pH 7.2) saline, until used. Growth
Medium
Medium NCTC-199 (Irvine Scientific, Irvine, CA) containing 1 y0 of an antibiotic solution consisting of 100 units ml penicillin-‘. 100 pg ml streptomycin-’ and 2.5 yg ml fungizone-’ solution, supplemented with 15 Y0fetal calf serum (FCS), was used as standard growth medium. Growth
Factors
Fibroblast (FGF), epidermal (EGF) and nerve growth factors (NGF), and endothelial cell growth supplement (ECGS) were commercially obtained (Collaborative Research, Inc., Bedford, MA). A consistent formulation of a nutrient mixture N&Serum (NUS, Collaborative Research, Inc.) containing EGF (5ng ml-l), ECGS (77.5 ,ug ml-‘), insulin, transferrin, tridothyronine, progesterone, estradiol-17, testosterone, hydroo-phosphorlethanolamine, cortisone, selenium, glucose, amino acids, vitamins and 25 % newborn bovine calf serum was used for the growth of cells. Except for EGF and ECGS, concentrations of other individual components could not be obtained from the vendor. Measurement of Cell Growth After trypsinization, cell density was determined using a hemacytometer and adjusted to a concentration of O-5 x lo5 cells ml-l. One milliliter of the cell suspension was distributed into a 60 x 15-mm culture dish (Falcon Plastics, Oxnard, CA) containing 4 ml of growth medium. In some experiments, a lower initial cell density was used to facilitate analysis. Kinetics of the cell growth was monitored by counting trypsinEDTA detached cells daily from triplicate dishes (Nayak and Binder, 1984; Nayak et al., 1986). Growth medium was changed twice a week. Growth pro-
123
motion of endothelial cells by various growth factors was analysed according to our previously described method (Nayak et al., 1986). Twenty-four hours following cell seeding, growth medium was replaced with fresh concentration of individual growth factors ; FGF, EGF, ECGS and NIJS. Duplicate experiments were performed for each parameter. Dishes were incubated for 10 days with two changes of medium on the 4th and 7th days. Growth factors FGF and EGF were used at concentrations of 0, 10, 50 and 100 ng ml-l, and ECGS at 0, 50, 100, 250 and 500 ng ml-’ concentrations. NU-Serum was used at 0, 2.5, 5 and 10%. Cell counts were determined from triplicate dishes at the end of incubation. Measurements of Initiation of DNA Synthesis
DNA synthesis as the measure of growth stimulation induced by growth factors was analysed as previously described (Tripathi and Tripathi, 1982: Nayak and Binder, 1984). Aliquots of 02 ml containing 5 x lo3 cells were distributed into wells of a 96-well tissue culture microplate (Falcon Plastics) and incubated at 3 7°C in a 5 % CO, plus 95 % air and humid atmosphere for 24 hr (Tripathi and Tripathi, 1982 ; Nayak and Binder, 1984). The growth medium was replaced with one containing a single fresh (O-100 ng per well) growth factor (FGF, EGF. ECGS and NGF). Triplicate wells were used for each parameter. Cultures were incubated for 48 hr and pulsed with 0.5 ,&i of [3H]thymidine per well (specific activity 47 Ci mM-‘, Amersham, Chicago, IL) for 18 hr. The incorporation of radioactive thymidine was determined as described previously (Nayak and Binder, 1984 ; Samples et al., 1985). 3. Results Cell Culture
Two groups of human cornea1 rims were utilized for growing cell cultures. In Group 1, endothelial cell cultures were started using plain tissue culture flask surfaces (no attachment factors such as rat tail collagen or gelatin were used) and standard growth medium (Tables I and II). Some of the cell cultures derived from this series of rims were used to study the effect of growth factors on the proliferation of endothelial cells. Cornea1 rims in Group 2 were used to grow endothelial cells on plain tissue culture flask surfaces and in the continuous presence of EGF, ECGS or NUS, or on an ECM-coated tissue culture flask surfaces in the continuous presence of EGF (Table III). In Group 1 uncontaminated endothelial cell growth was obtained from all 23 specimens (Table I) without the use of growth factors in the medium or attachment factors on the flask surface. The characteristic growth properties of these cells have been previously described (Nayak and Binder, 1984). Cells began to migrate
124
J. R. SAMPLES
ET AL.
morphologic study. However, we should note that appreciable differences in cell shape and size were not noted in response to the growth factors which we tested. Relationship o/‘Donor Age, TOD-TOC und Growth Potential of Endothelium
= 0”
0
I
B
P-4
Uncontaminated endothelial cell cultures from 2 3 cornea1 rims were maintained for a minimum of three and a maximum of 100 weeks (Table II). Endothelial cell growth occurred from all donors l-20 yr old (n = 11) with an average TOD-TOC of about 48 hr. One culture from a 17-yr-old donor with a TOWTOC of 48 hr was maintained for 100 weeks and 18 passages. The remaining cultures were maintained from primary culture to the seventh passage (3-24 weeks, Table II). Tn the group of donors 21-30 yr of age (n = 8) with an average TOD-TOC of 5 5 hr, one culture from a 2 5yr-old donor with a TOD-TOC of 64 hr survived for nine passages. Remaining cultures in this group were maintained for 3-20 weeks from primary culture to the fourth passage. All four donors 3140 yr of age with an average TOD-TOC of 64 hr produced positive cultures, but none survived beyond the first passage (Table III). FCS Requirement
P-6
P-8
II I
I
I;
2
3
4
I
I
;
I
I
rJ
5
6
7
8
9
IO
Days
FIG. 1. A, Dose response of FCS supplementation in standard culture medium, on the growth of HCE-32, P-4 cells derived from the endothelium of an 1%yr-old donor cornea. Bar = mean value k S.D. of triplicate cell counts. B. Growth kinetics of HCE-40 cells established from the endothelium of a 25-yr-old donor cornea. Each curve represents one passage and each point represents a mean value f S.D. of triplicate counts. Arrows indicate changing of the media.
from the explants within 2-5 days ; growth became confluent by about 2-3 weeks, at which time it was passaged for the tist time. Passaged growth was routinely maintained as previously described (Nayak and Binder, 1984). The cell growth was initially characterized by a typical hexagonal-to-polygonal shape with a centrally located nucleus, whereas most cases of continued cell growth form a mosaic pattern with multinucleated cells (Nayak and Binder, 1984). This stage of cell growth characterized by multinucleated cells, which has sometimes been termed the ‘crisis ’ stage, occurred after six to eight passages and about 8-10 months in culture. This was not a
The level of FCS supplementation in standard growth medium was determined [Fig. 1 (A)]. Cell culture HCE-36 established from the endothelium of a 26-yr-old donor cornea1 rim in its third passage was grown in the presence of O-20% FCS supplementation. In the absence of FCS. cells ceased to grow: a supplement level of either 15 or 20% yielded maximum growth. -In subsequent experiments growth medium was supplemented with 15 % FCS. Growth
Kinetics
A culture from the endothelium of a 25yr-old donor cornea1 rim, growing in the fourth to eighth passage (HCE-40) was used for studying growth kinetics [Fig. 1 (B)]. Cells growing in passage 4 (P-4) exhibited maximum growth with a doubling time (time required to double the initial cell number) of about 48 hr. The growth rate gradually decreased with increasing passage numbers reaching a plateau in P-8. EfJect
of
Growth Factors on Cell Growth
NUS, ECGS, FGF and EGF at different concentrations were tested for their ability to enhance the growth potential of endotheliai cell cultures (Fig. 2). All of the growth factors were tested using standard growth medium containing 15 % FCS. NUS at 10 or 20% in the presence of 15 % FCS in standard growth medium
HUMAN
CORNEAL
ENDOTHELIUM
125
IN VITRO
6 A 15%
FCS+NUS
4
2-
?? x * ; : "
0
5
20
IO %
NUS
ECGS
ing
ml-‘)
G ” IO C
6 6 4 2 r
FGF (ng
ml-‘)
FIG 2. Effect of growth factors on the growth of human cornea1endothelialcellsin vitro. Differentconcentrationsof growth factors were addedto standardgrowth mediumand were exposedto (A) NUS on HCE-45,P-3 cellsin the presence(0) and absence(n) of 15% FCSin medium.B, ECGSon HCE-25. P-4 cells.C, FGFon HCE-28,P-3 cells. D, EGFon HCE-40,P-4 cells. Bar = mean value& S.D. of triplicate counts.
5 o---O O---O
Control + ECGS
200
I
o----O -
Control + EGF
100 ng ml-’
rig/ml-’ I 4
4
/
d
z/
3
2
I
1F t t I
I
I
I
I
2
4
6
8
IO
Days
FIG. 3. Growth kinetics of human cornea1endothelialcellsHCE-40, P-6 in the presenceof (A) 200 ng ml ECGS-1or (B) 100 ng ml EGF-‘. Each point representsthe meanvalue+ S.D. of triplicate counts. Arrows indicate mediachanges.
exhibited a statistically sign&ant increase (P < 0.01) in the growth of HCE-36, P-4 cells compared to standard growth medium alone [Fig. 2 (A)]. When the
same level of NUS alone was added to medium (without FCS) a significant (P < 0.005) reduction in growth was observed. The addition of 500 ng ml-l of
126
J. R. SAMPLES
ET AL.
for each using paired t-test). Concentrations higher than 10 ng per well for FGF, EGF and NGF, and 20 ng per well for ECGSdid not produce a statistically significant increase in [3H]thymidine incorporation. Role of Growth Factors on the initiation of Endothelial Cell Growth In Vitro
“0 ; 4E P : 2 : k
0 I 20
I 40
FGF
? 8
I
40
I 60
(ng per
I
80
ECGS (ng
1
I 80
I 100
I
I
I
40
60
80
EGF
(per
well)
well)
/
1
120 160 200 per
I
20
well)
I
1 20 2.5
1
r
1
60
80
S NGF (ng
per
40
I
100
’ 100 well)
FIG. 4. Effect of growth factors on the initiation of DNA synthesis of HCE-28. P-2 grown with (FGF) and HCE-29. P-2 (0) cells in vitro. Cells were cultured in the presence of medium containing different concentrations of (A) FGF. (B) EGF, (C) ECGS and (D) 2.5 S NGF. Each point represents the mean value f S.D. of triplicate cpm. Open circles represent control groups.
ECGS, produced a significant (P < 005) growth increase for HCE-25, P-3 cells [Fig. 2 (B)]. The effect of ECGS at 200 ng ml-’ concentration on the growth kinetics of HCE-40, P-6 cells was not different from the control medium alone [Fig. 3 (A)]. An increased growth of HCE-29 cells growing in P- 5 occurred when 100 ng ml-l of FGF was added to standard growth medium [Fig. 2 (C)l. EGF (50 or 100 ng ml-l), induced a dramatic increase of cell growth of HCE-40 cells growing in P-4 [Figs 2(D) and 3(B)]. At the peak of growth on day 6, 100 ng ml-’ of EGF in the medium induced about a ninefold increase in cell growth, whereas a fivefold increase was seen for cells grown only in control medium [Fig. 3 (B)]. Effect of Growth Factors on DNA Synthesis Two cell cultures HCE-28, P-2 and HCE-29, P-2 were exposed to different concentrations of growth factors (Fig. 4). Growth factors FGF {Fig. 4(A)], EGF [Fig. 4(B)], ECGS [Fig. 4(C)], and NGF [Fig. 4(D)] induced significant increases in DNA synthesis in the presence of the lowest concentration tested (P < O-05
Endothelium from a total of 12 human donor cornea1 rims were established in tissue culture in the continuous presenceof growth factors EGF, ECGSand nutrient mixture NUS. All three growth factors were included in the growth medium from the initiation stage of these cell cultures. The results are summarized in Table III. Of the six cultures grown in the presence of EGF. four were maintained in culture for 5-12 passages (2 3-30 weeks). One of these cultures (HCE-63) ceased to grow after 23 weeks (five passages).The other three cultures were discontinued after 9, 10 and 12 passages.Growth of cell cultures HCE-64 and HCE-65 were initiated and maintained on ECM-coated tissue culture flasks in the presence of EGF. HCE-64 was maintained for 44 weeks in culture (15 passages).Cell culture HCE-65 was still growing in tissue culture after 15 weeks (four passages,Table III). Growth kinetics of the cells grown with EGF [Fig. 5 (A)] were different from the kinetics observed for cells grown in the presence of either ECGS [Fig. S(B)] or NUS [Fig. 5 CC)].More growth occurred in the presence of EGF than for cells growing in standard medium alone. One of three cultures grown in the presence of NlJS was maintained for 26 weeks before the cessation of growth in the eighth passage. The other two cultures in this group survived for one and two passages (S-10 weeks duration). Almost identical observations were made for three cultures grown in the presence of ECGS.These cultures ceased to grow after four to six passages(20-24 weeks; Table III). Growth kinetics of these cells did not change when they were grown in the presenceor absenceof ECGSin the medium [Fig. 5 (B)]. Similar observations were made for the cells grown in the presenceand absence of NUS [Fig. S(C)]. 4. Discussion Growth factors such as FGF and EGF have been shown to significantly influence the proliferation of bovine cornea1 endothelial cells in tissue culture (Gospodarowicz, Meshcer and Birdwell, 1977; Rheinwald and Green, 19 7 7 ; Gospodarowicz, Meshcer and Birdwell, 19 78 : Gospodarowicz et al., 1978 : Gospodarowicz, Greenburg and Alvarado, 1979). Growth factors in vitro have many actions, including induction of proliferation, differentiation, and aging (Stocker et al., 1959; Perlman and Baum, 1974). Growth factors may delay the ultimate senescenceof
HUMAN
CORNEAL
ENDOTHELIUM
IN VITRO
127
Days
I
I
I
ro
I 20
, !
Doys
FIG. 5. Growth kinetics of human cornea1endothelial cells grown in the continuous presenceof growth factors. The experimentwasinitiated with an initial inoculum of 1 x lo4 cellsper dish.Endothelialcells(A) HCE-53,P-2 grown with (. ,.. .) ) 100 ng ml EGF-‘; (B) HCE-52,P-3 grown with (. . ...) and without (-) and without (500 ng ml ECGS-‘; (C) HCE-50, P-2 grown with (. ,...) and without () 5 % in the standardgrowth medium.Each point representsthe meanvalue f S.D. of triplicate cell counts.
the cells, thereby increasing their culture life-span (Rheinwald and Green, 19 77). In the present study we have shown that human cornea1 endothelial cells grown on uncoated surfaces in standard culture medium without any growth factors responded to the stimulatory effects of four growth factors (Figs 2 and 4). Further, we have demonstrated that the inclusion of the growth factor EGF in the medium from the initiation stage of the cell culture drastically enhanced the growth potential of endothelial cells in tissue culture (Fig. 5). We found that using a flask surface-coated with ‘putative ’ ECM and adding EGF to the medium provided the best environment for growth of human cornea1 endothelial cells derived from 0*4- and 0.5-yrold donors. In a preliminary
study, culture-dish
surfaces coated with ECM were prepared by treating a 3-week-old confluent monolayer of baboon cornea1 epithelial, cells with trypsin solution as described by
Hsieh and Baum (198 5). One of the two cell cultures has been maintained beyond the typical slowing of growth and carried in continuous culture for over 44 weeks and 15 passages (Table III). Similar observations were reported in a communi-
cation by Alvarado, Lui and Wood (1986). In their abstract they described the establishment of cell cultures from donor cornea1 rims following transplantation and from specimens obtained at trabeculectomy, using an ECM-coated culture-dish surface and continuous presence of FGF. Their success is probably due to stimulation induced by FGF/EGF and may well have a less-pronounced effect on growth
properties. With their combination they were able to obtain rapidly growing cell cultures which underwent many generations. Both their work and ours suggests that a combination of specially treated culture-dish surfaces and growth factors in the medium may provide an environment conducive to growing human
128 cornea1 endothelial cells continuously in tissue culture. The development of systems which promote the sustained growth of cells can allow us to study the effects of drugs and a variety of potential regulators on the cornea1 endothelium. In addition, the propagation of large numbers of cells allows us to study cell-surface properties. extracellular matrix properties, and the unique biochemistry of the cornea1 endothelium and may ultimately make transplantation of cornea1 endothelium possible.
Acknowledgments Supported in part by grants from the National Vision Research Institute, Research to Prevent Blindness, and EY07111 from the National Eye Institute.
References Alvarado, J., Lui, G. and Wood, I. (1986). Establishment of human cornea1 endothelial cells in tissue culture. Invest. OphthalmoI. Vis. Sci. 27. 127 (abstract). Baum, J., Niedra, R., Davis, C. and Yue, Y. (1979). Mass culture of human cornea1 endothelial cells. Arch. Ophthalmol. 97, 113640. Glassma. A.. Coles, W. and Bennett, C. (1979). Cornea1 endothelium modified method for cultivation. In Vitro 15, 873-6. Gospodarowicz. D., Greenburg, G. and Alvarado. J. (1979). Transplantation of cultured bovine cornea1 endothelial cells to rabbit cornea: Clinical implications for human studies. Proc. Natl. Acad. Sci. U.S.A. 76, 464-8. Gospodarowicz, D., Greenburg, G.. Bialecki, H. and Zetter, B. (1978). Factors involved in the modulation of cell proliferation in vivo and in vitro: the role of fibroblast and epidermal growth factors in the proliferative response of mammalian cells. In Vitro 14, 85-l 18. Gospodarowicz. D., Meshcer. A. and Birdwell. C. (1977).
J. R. SAMPLES
ET AL.
Stimulation of cornea1 endothelial cell proliferation in vitro by fibroblast and epidermal growth factors. Exp. Eye Res. 25. 75-89. Gospodarowicz. D.. Mescher. A. and Birdwell, C. (1978). Control of cellular proliferation by fibroblast and epidermal growth factors. Natl. Cancer Inst. Monogr. 48. 109-30. Hsieh, P. and Baum, J. ( 1985). Effects of fibroblastic and endothelial extracellular matrices on cornea1 endothelial cells. Invest. Ophthalmol. Vis. Sci. 26. 457-63. Hyldahl. L.. Auer, G. and Sundelin, B. (1982). A novel method to establish primary cultures on bovine cornea1 endothelial cells. Cell Biol. Inter. Reports. 6. 52 3-X. Mannagh, J. and Irving, K. (1965). Human cornea1 endothelium. Growth in tissue culture. Arch. Ophthalmol. 74. 847-9. Nayak, S. and Binder, P. (1984). The growth of endothelium from human cornea1 rims in tissue culture. invest. OphthaImol. Vis. Sci. 25, 121 3-16. Nayak. S.. Samples, J.. Deg, J. and Binder. P. (1986). Growth characteristics of primate (baboon) cornea1 endothelium in vitro. Invest. Ophthalmol. Vis. Sci. 27. 607-l 1. Perlman, M. and Baum, J. ( I 9 74). Synthesis of a collagenous basal membrane by rabbit cornea1 endothelial cells in vitro. Arch. OphthaImol. 92, 238-9. Perlman. M. and Baum, J. (19 74). The mass culture of rabbit cornea1 endothelial cells. Arch. Ophthalmol. 92, 2 3 5-7. Rheinwald. J. and Green, H. (1977). Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265. 42 1-4. Samples, J.. Nayak. S.. Gualtieri, C. and Binder, P. ( 1985). Effect of hydrocortisone on cornea1 endothelial cells in vitro. Exp. Eye Res. 51. 487-95. Stocker, F., Eiring. A., Georgiade. R. and Georgiade. N. (1959). Evaluation of viability of preserved rabbit corneas by tissue culture procedures. Am. I. Ophthalmol. 47. 772-82. Tripathi. R. and Tripathi, B. (1982). Human trabecular endothelium, cornea1 endothelium, keratocytes. and scleral fibrocytes in primary culture. A comparative study of growth characteristics. morphology, and phagocytic activity by light and scanning electron microscopy. Exp. Eye Res. 35, 611-24.
