IOVS Papers in Press. Published on March 3, 2015 as Manuscript iovs.14-15579
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A novel method using quantum dots for testing the barrier function of
2
cultured epithelial cell sheets
3
Thomas J. Duncan1, Koichi Baba2, Yoshinori Oie1, Kohji Nishida1
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1. Department of Ophthalmology, Osaka University Graduate School of
5 6
Medicine, Suita, Japan. 2. Department of Visual Regenerative Medicine, Osaka University Graduate
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School of Medicine, Suita, Japan.
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Word count: 4156
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Keywords: tight junction, quantum dots, epithelial cells, barrier function
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Funding support: This work was supported by a fellowship for overseas
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researchers from the Japan Society for the Promotion of Science (JSPS).
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Correspondence: Kohji Nishida, M.D., Ph.D.
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Department of Ophthalmology, Osaka University Graduate School of Medicine
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Rm. E7, 2-2 Yamadaoka, Suita, Osaka 565-0871, JAPAN
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Tel: 81-6-6879-3451, FAX: 81-6-6879-3459
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E-mail: [email protected]
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Copyright 2015 by The Association for Research in Vision and Ophthalmology, Inc.
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Abstract
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Purpose: The corneal epithelium provides a barrier to protect the deeper
21
structures of the eye from any particles or pathogens. Cultured epithelial cell
22
sheets are used in transplantation surgery for corneal repair or regeneration.
23
The purpose of this study was to develop a novel method utilizing fluorescent
24
quantum dot nanoparticles for validating the quality and barrier function of
25
cultured epithelial cell sheets.
26
Method: Human epithelial cell sheets, cultured from oral mucosal or corneal
27
limbal cells, were incubated in either normal calcium containing medium or
28
medium containing no calcium with a calcium chelator. Also contained in the
29
media were suspensions of two different sizes of quantum dots. Following
30
incubation, analysis of quantum dot penetration was carried out using confocal
31
microscopy.
32
Results: In contrast to the cell sheets incubated in calcium containing medium,
33
removal of extracellular calcium resulted in the disruption of tight junctions,
34
compromising the cell sheet’s barrier function. This caused a reduction in
35
transepithelial electrical resistance and deeper, more ubiquitous penetration of
36
the quantum dots into the paracellular space and interior of the cell sheet.
2
37
Conclusions: This method provides easy to interpret qualitative and
38
quantitative data on the functionality of a cell sheet’s tight junctions, as well as
39
nano and microscale structural information on its surface and interior
40
morphology, and any localized areas of damage or abnormality. This novel
41
technique could be utilized as part of the validation system for cultured epithelial
42
cell sheets for use in transplantation.
43 44
Introduction
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Anteriorly, the ocular surface is covered by specialized corneal, limbal and
46
conjunctival epithelia. The corneal epithelium is characterized by stratified
47
squamous non-keratinized epithelial cells that are responsible for maintaining
48
the smooth refractive surface of the cornea, essential for vision, whilst also
49
acting as a protective barrier to prevent the passage of foreign bodies and
50
pathogens into the deeper ocular structures. Corneal epithelial cells are derived
51
from transient amplifying cells generated by stem cell populations residing in the
52
basal layer of the limbal epithelium1, 2. Ocular disease or trauma to this area
53
such as Stevens-Johnson syndrome, thermal or chemical burns can cause
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limbal stem cell deficiencies that result in corneal invasion by adjacent
3
55
conjunctival cells, neovascularization, chronic inflammation, and stromal
56
scarring. Consequently visual acuity is severely affected3, 4. In cases of unilateral
57
damage, corneal regeneration of the affected ocular surface can be attempted
58
by transplantation of limbal grafts5 or via the cultivation of epithelial cell sheets
59
from autologous limbal stem cells taken from the contralateral eye6-8. However,
60
this requires a large limbal graft from the donor eye, carrying with it a risk of
61
infection and inducing a limbal stem cell deficiency in the donor eye9. It is also
62
unsuitable for patients who have bilateral corneal damage10. Transplantation
63
from
64
immunosuppressants11, and carries a high risk of rejection. Post-operative rates
65
of rejection after 1 year range from 20%-60% with cadaveric donor tissue5, 12,
66
and 70%-80% with living donor tissue13, 14.
allogenic
donors
necessitates
prolonged
use
of
postoperative
67 68
In an attempt to improve post-surgical results for corneal surface reconstruction,
69
recent studies have successfully fabricated and transplanted epithelial cell
70
sheets cultured ex vivo from autologous epithelium of non-ocular surface origin15,
71
16
72
autologous oral mucosal epithelial cell derived cell sheets has been
. In rabbits, the technique for harvesting, culturing and transplanting
4
73
established17,
18
74
clinical trials in humans19,
75
significantly improved visual acuity was maintained and no complications were
76
observed, with the oral mucosal keratinocytes undergoing phenotypic changes
77
to more closely resemble that of corneal keratinocytes19. Before this emerging
78
technique can become a standard medical therapy however, it is crucial to
79
establish a validation system by which the cultured oral mucosal epithelial cell
80
sheets can be evaluated prior to transplantation. Such a validation system has
81
been outlined in a previous study by Hayashi et al21.
. More recently, the same treatment has been subjected to 20
. A 14 month postoperative evaluation showed
82 83
One such parameter for evaluation is a cell sheet’s barrier function – a measure
84
by which it can control or inhibit the passage of molecules, foreign bodies or
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pathogens across its cell layers. The tight junction is the most apically located of
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the junctional complexes, forming a continuous, belt-like structure that seals the
87
paracellular pathway by joining the lateral surfaces of the most superficial layer
88
of epithelial cells at their apical edge22,
89
necessary for establishing the paracellular connections and assembly of the
90
various proteins involved in a tight junction complex24-27. Removal of calcium,
23
. Calcium has been shown to be
5
91
through the use of a calcium chelator, disrupts tight junction structure and
92
function24, 25, 28. Evaluating the barrier function and health of the tight junctions in
93
a cultured epithelial cell sheet is necessary prior to its use in transplantation
94
surgery.
95 96
Transepithelial electrical resistance (TEER) is a technique which measures the
97
electrical resistance across the full thickness of the cell sheet using two
98
electrodes placed above and below. Primarily, this technique is used to obtain
99
information on the barrier function of a cell sheet. However, TEER values are
100
often unreliable. Numerous studies have reported that TEER does not
101
accurately represent changes in tight junction structure or function29,30.
102
Furthermore, the overall electrical resistance of a cell sheet has been shown to
103
be significantly affected by other factors including temperature31, the culture
104
substrate used32 and the degree of cell-substrate contact33.
105 106
Quantum dots are nanometer scale colloidal semiconductor nanoparticles
107
composed of a few hundred to a few thousand atoms of a semiconductor
108
material such as cadmium mixed with tellurium or selenium. This is then coated
6
109
with an additional zinc sulfide semiconductor shell to contain the cadmium and
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limit its dissolution, as well as improve the quantum dot optical properties34.
111
Bio-conjugation of this shell to proteins or other small molecules serves to further
112
enhance their biological applications. Quantum dots are extremely efficient at
113
generating fluorescence, often many times greater than that of conventional
114
organic fluorescent compounds34, 35. They have a board excitation spectra with a
115
narrow emission spectra ranging from UV to near-infrared, and superior
116
photostability with a high resistance to photo-bleaching - allowing for greater
117
observation
118
advantageous property of quantum dots is that their fluorescence is tunable. A
119
predictable relationship exists between the energy emitted by these quantum
120
dots (and consequently the wavelength of their fluorescence) and the size and
121
shape of the quantum dot core – with smaller quantum dots fluorescing at
122
shorter wavelengths of light than larger quantum dots. Applying quantum dots to
123
the surface of bovine cornea in vitro has shown that penetration is limited to only
124
the superficial epithelial layers, 20µm from the corneal surface39. In this healthy
125
cornea, the epithelium acted as a barrier to limit quantum dot penetration.
126
Quantum dots may therefore represent a suitable candidate for evaluating the
time
or
long-term
imaging
experiments36-38.
One
highly
7
127
barrier function of a cell sheet.
128 129
The purpose of this study is to utilize these nanoparticles to provide qualitative
130
and quantitative data specific to a cell sheet’s morphology and tight junction
131
functionality. We hypothesize that cell sheets with damaged or disrupted barrier
132
function will permit deeper and more ubiquitous penetration of the quantum dot
133
nanoparticles. Additionally, by utilizing different sized quantum dots, we
134
anticipate a size-dependent variation in penetration and distribution.
135 136
Methods
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Cell culture – Human oral mucosal epithelial cell sheets
138
Human oral mucosal keratinocytes (HOK) (ScienCell Research Lab, CA, United
139
States) were cultured until subconfluent. Trypsin LE express (Invitrogen,
140
Carlsbad, NM, United States) was used to detach the cells from the culture flask
141
into suspension. The cell suspension was then centrifuged and the supernatant
142
removed. The cells were then re-suspended in keratinocyte culture medium and
143
seeded at an initial cell density of 1x105cells/ml into 12-well culture inserts
144
(Falcon) with mitomycin C (MMC)-treated 3T3-J2 feeder cells in KCM, separated
8
145
by the cell culture insert permeable membrane. KCM comprises Dulbecco’s
146
modified Eagle’s medium (DMEM/F12 [3:1]) supplemented with 10% fetal bovine
147
serum (Invitrogen, Japan), 1% L-Glutamine (Invitrogen, Japan) 5μg/mL insulin
148
(novolin; Novartis Pharmaceuticals, Japan), 1% penicillin-streptomycin (Meiji
149
seika, Japan), 0.4μg/ml hydrocortisone (saxizon; Kowa, Tokyo, Japan), 2nM
150
triiodothyronine (T3; Sigma Aldrich, Japan), 1nM cholera toxin (List Biological
151
Laboratories, CA, United States), and 10ng/mL EGF (Higeta Shoyu, Japan).
152
Incubation was carried out at 37°C and 5% CO2. All procedures were carried out
153
following the formation of mature, stratified epithelial cell sheets.
154 155
Cell culture – Human limbal epithelial cell sheets
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Human corneas (donor age 34-73) were obtained from eye banks (Sightlife, WA,
157
United states) and subsequently washed in DMEM before being incubated in
158
dispase for 1 hour at 37°C 5% CO2. The human limbal keratinocytes (HLK) were
159
then scraped from the limbal region of the cornea, and the resulting suspension
160
was centrifuged and the supernatant removed. The cells then underwent
161
trypsinization before being re-suspended in KCM and seeded at an initial cell
162
density of 1x105cells/ml into 12-well culture inserts (Falcon) with MMC-treated
9
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3T3-J2 feeder cells in KCM, separated by the cell culture insert permeable
164
membrane. Incubation was carried out at 37°C and 5% CO2. All procedures
165
were carried out following the formation of mature, stratified epithelial cell
166
sheets.
167 168
Quantum dot incubation and analysis
169
Incubation media was formulated to either contain the standard 1.64mM of
170
calcium, or 0mM of calcium with 5mM of the calcium chelator ethylene glycol
171
tetraacetic (EGTA). In order to standardize the level of tight junction disruption,
172
EGTA was used to remove any tight junction associated calcium, as well as any
173
calcium remaining from the use of KCM medium during the culturing period.
174
Green (525nm) and red (655nm) quantum dots, with zinc sulfide shells
175
conjugated to carboxyl molecules, were purchased as a suspension in borate
176
buffer (Life technologies Japan). 40nM suspensions of both green and red
177
quantum dots were made by diluting the commercially available quantum dots
178
into either the calcium or EGTA containing incubation media. The pH of all
179
incubation media was then adjusted to 7.4, and then applied to the superficial
180
surface of the epithelial cell sheets. These were then incubated at 37°C and 5%
10
181
CO2 for 90 minutes. The cell sheets were washed twice in PBS to remove any
182
excess quantum dots. The samples were then immunostained with a 10%
183
Hoechst solution for 10 minutes, following which they were fixed in 4%
184
paraformaldehyde and analysed using confocal microscopy (Ziess). Confocal
185
parameters (including laser power, aperture size and image thresholds) were
186
identical for all samples.
187 188
Transepithelial electrical resistance (TEER)
189
Electrical resistance across the cell sheets was measured using an EndOhm
190
chamber (WPI Inc) and EVOM2 resistance meter (WPI Inc). An insert containing
191
only medium, with no cell culture, was used as a blank. TEER was measured at
192
various time points after addition of the incubation media. The TEER value was
193
first measured at 0mins after incubation, then every 15 minutes following for a
194
period of 90 minutes. Each test was performed in triplicate for each condition.
195
The TEER value was calculated by: [average resistance of experimental wells −
196
average resistance of blank wells] Ohms × 0.33cm2 (area of the insert culture
197
surface).
198
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199
MTT cell viability assay
200
Stock solution of MTT (Sigma, USA) was diluted to 375µg/ml in KCM. A 5mM
201
EGTA solution and/or 40nM suspensions of both green and red quantum dots
202
were then added to this MTT-KCM medium. This was then pipetted into 12 well
203
inserts containing HOK or HLK derived cell sheets. After incubation at 37°C for 2
204
hours, the resulting formazan crystals were dissolved in equal quantities of
205
acidified isopropanol. The data was obtained by subtracting the background
206
absorbance at 690nm from the absorbance at 570nm recorded using a
207
spectrophotometer (V-550, UV/VIS Spectrophotometer, JASCO, Japan) and
208
expressed as a percentage of the control cell sheets incubated without quantum
209
dots or EGTA. Each test was performed in triplicate for each condition. ANOVA
210
and Dunnett’s statistical analysis was carried out using JMP software to
211
compare each test value to the control group.
212 213
Transmission electron microscopy (TEM)
214
Following incubation with the quantum dot suspensions, samples were fixed in a
215
solution of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M sodium
216
cacodylate buffer (pH 7.2) at room temperature for 3 hours. Staining was then
12
217
carried out with 2% osmium tetraoxide for 1 hour followed by 0.5% uranyl
218
acetate for 1 hour at room temperature. The samples were then dehydrated
219
through graded alcohols and propylene oxide before being embedded in Araldite
220
resin (TAAB laboratories). Resin curing was carried out at 60ºC for 3 days. 90nm
221
thick sections were cut from the cured sample blocks on a Reichert-Nissei
222
Ultracut Microtome and floated onto copper mesh-200 EM grids (Agar scientific
223
Ltd). These were then post-stained at room temperature in 2% aqueous uranyl
224
acetate and Reynolds lead citrate for 5 minutes.
225 226
In order to measure the particle size, droplets of red and green quantum dots
227
were also applied to the surface of carbon film coated slot-grids (Agar scientific
228
Ltd). Any excess solution was wicked away using filter paper. All grids were then
229
given time to dry before TEM examination.
230 231
Analysis of all samples was carried out at 80kV with a transmission electron
232
microscope
233
charge-coupled device camera (Gatan Inc., Japan).
(Hitachi
H7650,
Japan).
Images
were
obtained
with
a
234
13
235
Quantum dot size measurements
236
Micrographs were obtained of the quantum dots on carbon film coated slot-grids.
237
Length and width values for both red and green quantum dots were calculated
238
from 15 measurements taken using Fiji (Image J) software.
239 240
Fluorescence intensity measurements
241
Fluorescence intensity readings were taken across the XY-plane of the confocal
242
micrographs at 1μm interval passing through the thickness of the cell sheet. By
243
calculating the area under the curve of the subsequent fluorescence intensity
244
graph, total fluorescence intensity was determined. Mean total fluorescence
245
intensity, peak fluorescence intensity and depth of the peak fluorescence
246
intensity within the cell sheet were calculated from reading from three sample
247
cell sheets. P values were obtained via T-test statistical analysis.
248 249
Results
250
The TEM micrographs in Figure 1 show small populations of quantum dots
251
(Figure 1A arrows) residing within the paracellular space. The area marked by
252
an asterisk can be seen at higher magnification in Figure 1B, where it is possible
14
253
to discern the small green (arrow) and larger red (arrowhead) quantum dots.
254 255
The TEM micrograph Figure 2A1 shows the green quantum dots were
256
approximately spherical in shape, with an mean width of 3.16nm (SD=0.30nm)
257
and an mean length of 3.28nm (SD=0.19nm) (Figure 2A2). The red quantum
258
dots (Figure 2B1) had a more oblong shape, with a width of 5.54nm
259
(SD=0.29nm) and a length of 14.98nm (SD=1.02nm) (Figure 2B2). For a full list
260
of size measurements see Table 1 in supplementary data.
261 262
Figure 3 demonstrates the effect of incubating human oral mucosal
263
keratinocytes (HOK) and human limbal keratinocytes (HLK) cell sheets in 0mM
264
calcium medium with an EGTA calcium chelator. In 1.64mM of calcium, the initial
265
electrical resistance of the cell sheet was approximately maintained throughout
266
the 90 minutes of incubation. However, using medium containing 0mM of
267
calcium with 5mM of EGTA had the effect of rapidly reducing the cell sheet’s
268
electrical resistance within the first 15 minutes. A slowing, but continued
269
decrease occurred for the following 75 minutes until after 90 minutes the
270
resistance was significantly lower than its initial level – corresponding to an
15
271
average 18.3Ohms·cm2 reduction in the HOK cell sheets, and a 22.8Ohms·cm2
272
reduction in the HLK cell sheets. Figure 4 shows that incubation for 2 hours with
273
EGTA and/or quantum dots had no significant effect of the viability of either the
274
HOK or HLK derived cell sheets.
275 276
The confocal micrographs in Figures 5 show a significant difference in quantum
277
dot quantity and distribution across the healthy HOK (Figure 5A1) and HLK
278
(Figure 5B1) cell sheets compared to the cell sheets containing damaged tight
279
junctions (HOK Figure 5A2 and HLK Figure 5B2). In the healthy cell sheets,
280
small amounts of quantum dots can be seen residing on the surface and in the
281
border regions between cells (Figure 5A1 and 5B1 arrowhead). The damaged
282
cell sheets show significantly larger amount of quantum dots residing in the
283
paracellular space, creating a much stronger fluorescence profile for both the
284
red and green quantum dots (Figure 5A2 and 5B2 arrows).
285 286
Figure 6 shows the Z-plane surface (cross-section) rendered views through the
287
HOK and HLK cell sheets of Figure 5. The same rendered view, with the addition
288
of cell nuclei Hoechst staining, can be seen in the supplemental data Figure 1.
16
289
The healthy cell sheets (Figure 6A and 6C) contain a thin layer of quantum dots
290
residing on the surface (arrow). Both red and green quantum dots are present in
291
this residual layer. The damaged cell sheets however, contain a superficial layer
292
of quantum dots (Figure 6B and 6D arrowheads) over two times thicker than in
293
the healthy cell sheets. In addition, numerous populations of green (and to a
294
lesser extent, red) quantum dots can also be seen within the deeper cell layers.
295
The asterisk in Figure 6B2 indicates a large cluster of green quantum dots
296
residing in a deeper region. No such deeper populations can be observed in the
297
healthy cell sheet.
298 299
Figure 7 shows the fluorescence intensity readings taken from the confocal
300
micrographs in Figure 6. In the damaged cell sheets (Figure 7A2 and 7B2), the
301
initial fluorescence peak is both stronger in intensity and continues deeper into
302
the cell sheet than in the healthy cell sheets (Figure 7A1 and 7B1). Following this
303
initial peak, green quantum dot fluorescence is still present at a lower intensity
304
level throughout the thickness of the cell sheets. The second large fluorescence
305
peak seen in Figure 7A2 represents the large cluster of green quantum dots
306
residing approximately 40µm deep into the cell sheet (Figure 6B2 asterisk). In
17
307
Table 1 the mean fluorescence intensity readings taken from multiple cell sheets
308
show a statistically significant increase in both green (HOK p=0.038, HLK
309
p=0.009) and red (HOK p=0.032, HLK p=0.013) quantum dot fluorescence
310
intensity when comparing the healthy cell sheets to the damaged cell sheets.
311
Furthermore, the peak intensity readings for the quantum dots occurred 2-3
312
times deeper into the damaged cell sheet than in the healthy cell sheets. Human oral mucosal cells
Mean total fluorescence intensity (I) Mean peak fluorescence intensity (I) Mean depth of peak intensity (µm) 313
Human limbal epithelial cells
Healthy cell
Damaged cell
Healthy cell
Damaged cell
sheets
sheets
sheets
sheets
Red
Green
Red
Green
Red
Green
Red
Green
QD
QD
QD
QD
QD
QD
QD
QD
36.99
38.09
246.74
416.15
40.28
31.82
279.57 403 .00
±9.02
±5.60
±69.20 ±134.26 ±5.54
±4.59
±49.17 ±62.53
5.65
5.33
18.2
18.34
6.40
5.38
16.91
20.43
±0.57
±0.50
±1.56
±3.01
±1.08
±0.87
±2.66
±4.13
6.00
6.33
12.33
13.00
6.00
6.33
18.33
19.00
±1.00
±1.53
±2.31
±1.00
±1.73
±2.08
±4.04
±4.00
Table 1. Mean cell sheet fluorescence intensity data.
314 315
Discussion
316
We have demonstrated that quantum dots can be utilized as an effective
317
indicator of a cell sheet’s barrier function. Our hypothesis can be confirmed as 18
318
cell sheets with disrupted barrier function demonstrated deeper and more
319
widespread penetration of the quantum dot nanoparticles, particularly of the
320
smaller green quantum dots. By applying quantum dots in this way, nano and
321
microscale morphological information on a cell sheet’s surface and interior can
322
also be ascertained - in particular, making local areas of abnormality or
323
increased permeability easy to identify - something that current evaluation
324
techniques, such as TEER, are unable to provide.
325 326
TEER did however demonstrate the dramatic effect caused by EGTA calcium
327
chelation. This was in turn mirrored by the significant increase in quantum dot
328
penetration through the open paracellular spaces - penetrating even through to
329
the deepest layers of the cell sheets. These results demonstrate that tight
330
junctions are a major factor in a cell sheet’s barrier function. However, even in
331
the damaged cell sheets, the larger red quantum dots showed a more limited
332
penetration deeper than 15µm into the cell sheet. This parallels results from
333
another quantum dot penetration studies which recorded red (655nm) quantum
334
dots showing limited penetration deeper than 15-20µm into the epithelium of an
335
intact cornea after a similar 80mins incubation in vivo39. This suggests that the
19
336
size of the quantum dot was a limiting factor for its penetration through the cell
337
sheet. A model for the observed penetration of the quantum dots can be seen in
338
Figure 8. Other junctional complexes such as adherens junctions, desmosomes
339
and gap junctions serve to anchor or connect neighbouring epithelial cells
340
together. It has previously been reported that the paracellular space adjacent to
341
adherens junctions and desmosomes is approximately 20-30nm40-43. However,
342
the presence of gap junctions reduces this space to 4nm41, 43. The red quantum
343
dots - with a recorded length of 14.98nm and a width of 5.54nm – would be too
344
large to pass through these narrow paracellular regions containing gap junctions.
345
Consequently even though the damaged cell sheet had disrupted tight junctions,
346
the gap junctions may still have served to slow the rate of passage of the red
347
quantum dots through to the deeper regions of the cell sheet.
348 349
Conversely, the green quantum dots - with a recorded length of 3.28nm and a
350
width of 3.16nm, would be small enough to pass through even the narrowest
351
regions in the paracellular space. Although the rate at which they could pass
352
through these regions would still be rate limited and time dependant. This may
353
explain why although deeper populations of green quantum dots were observed
20
354
in the damaged cell sheet, a large proportion of them remained in the more
355
superficial layers. Nevertheless, a relatively large quantity of the quantum dots
356
(labelled by an asterisk in Figure 6B2) were able to accumulated at the bottom of
357
the cell sheet in such a localized manner as to suggest particular disruption or
358
damage to that area of the cell sheet. In this way, quantum dots can be used to
359
identity localized areas of abnormality within the cell sheet.
360 361
As green (to a lesser extent, red) quantum dots were able to penetrate deeply
362
into the cell sheet, we also investigated whether penetration rates could be
363
quantifiably measured by analysing the medium collected from beneath the cell
364
sheets after incubation. The results of this experiment can be seen in the
365
supplementary data Figure 2 and Table 2.
366 367
Conventional methods for evaluating epithelial barrier function or surface
368
abnormalities include measuring TEER or using fluorescent dyes such as
369
sodium fluorescein. Whilst these techniques are useful, our novel method can
370
provide rapid nano and microscale structural information on a cell sheet’s
371
surface and interior morphology as well as the health of its barrier function -
21
372
allowing localized areas of surface abnormality or damage to be easily identified.
373
Standard fluorescent dye molecules may be small enough (sodium fluorescein
374
radius=5.5 Å44) to pass through the paracellular space rapidly and with ease.
375
Quantum dots are considerably larger than these dye molecules; accordingly
376
they may show a comparably slower rate of penetration through the cell sheet.
377
The quantum dots often aggregated into small clusters as they moved through
378
the paracellular space into the deeper cell layers, with larger aggregations
379
occurring in areas of increased permeability. Subsequently, quantum dots can
380
provide structural information on the interior morphology of a cell sheet on a
381
nano and microscale – ranging from as small as 3.16nm (the width of a single
382
green quantum dot) up to the large aggregation that often measure 5-10µm in
383
size. This is something that the smaller dye molecules are unable to provide.
384 385
However, further improvements to this technique can be made. There are
386
discrepancies in the current literature regarding the toxicity of quantum dots.
387
This is largely due to the variety of quantum dots available, and a lack of
388
extensive toxicity studies. Our own investigations align with longer term studies
389
that show no appreciable toxicity45-47. Conversely, some studies have reported
22
390
that despite the zinc sulfide shell, Cd2+ ions are able to leech out of the quantum
391
dot core, causing concerns about their toxicity and safety for clinical use48.
392
Furthermore, comparative confocal micrographs in supplemental data Figure 3
393
show that even in unfixed cell sheets, the quantum dots are not readily removed
394
from the paracellular spaces once they have penetrated the cell sheet. Even so,
395
this technique could still be applied to cell sheets in vitro, together with other
396
destructive tests such as immunohistochemistry21. A range of in vivo application
397
are currently being considered using alternatives such as carbon quantum dots49
398
or indium phosphide50, 51, that have been shown to possess significantly lower
399
levels of cytotoxicity.
400 401
In addition, utilizing a greater range of quantum dot sizes would potentially
402
produce a spectrum of color across the thickness of the cell sheet, giving more
403
information on the degree to which the paracellular pathway is open. It may be
404
possible to define the scale of tight junction disruption by observing the relative
405
penetration of different sized quantum dots. Based on our experiments, the
406
smallest quantum dots would be expected to penetrate to the deepest layers.
407
Small or intermediate sized quantum dots may penetrate the superficial layers
23
408
but at a reduced rate, whilst the larger quantum dots would remain on the
409
surface or in the most superficial layer. This data would then provide greater
410
information on a cell sheet’s barrier function as well as local areas of
411
abnormality.
412 413
For this proof-of-concept study, using EGTA to disrupt tight junction function was
414
desirable as it induced a significant effect on TEER. However, future work will
415
feature a range of disruptive techniques, in order to better represent the variety
416
of abnormalities and defects common in cultured cell sheets. One such
417
candidate for future study as an alternative and milder disruptor of tight junction
418
function is TNF-α. This was tested following published protocols52-54 – briefly, the
419
cell sheets were incubated for 24hours with 10ng/ml TNF–α, before the addition
420
of red and green quantum dots. TEER readings and comparative confocal
421
micrographs can be seen in the supplemental data Figure 4.
422 423
To conclude, our method for evaluating cultured epithelial cell sheet barrier
424
function has not been reported in the literature to date. As such, it represents a
425
novel technique to provide clear, easy to interpret data on a cell sheet’s barrier
24
426
function, as well as nano and microscale structural information of its interior and
427
surface morphology. This technique can be incorporated into the range of
428
analytical tests performed prior to approval for transplantation. Furthermore, this
429
information may serve to enhance scientific knowledge on how to fabricate
430
healthy cell sheets for transplantation.
431 432
Acknowledgements
433
This work was supported by a fellowship for overseas researchers from the
434
Japan Society for the Promotion of Science (JSPS). The authors confirm that
435
there are no known conflicts of interest associated with this publication and there
436
has been no significant financial support for this work that could have influenced
437
its outcome.
438 439
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440
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Figure legends
609
Figure 1. TEM micrographs showing oral mucosal epithelial cells and the
610
penetration of quantum dots into the paracellular space. (A) Taken at x8000
611
magnification, several clusters of quantum dots can be seen residing in the
612
paracellular space (arrows). The area marked by the asterisk can be seen at
613
20,000x magnification in Figure 1B. In (B), small spherical green quantum dots
614
are indicated by an arrow. Whilst larger, more elongated red quantum dots are
615
indicated by an arrowhead. Scale bar=200nm.
616
35
617
Figure 2. TEM micrographs showing green quantum dots (A1) and red quantum
618
dots (B1). Scale bar=10nm. A2 and B2 show frequency distribution of the
619
quantum dot length and width measurements.
620 621
Figure 3. Graph showing the effect of low calcium on cell sheet TEER. Initial
622
resistance levels are maintain when the cell sheet is incubated in normal calcium
623
levels of 1.64mM. Using a calcium chelator (EGTA) in medium containing 0mM
624
of calcium resulted in a reduction in electrical resistance. Error bars show
625
standard deviation. n=3.
626 627
Figure 4. MTT cell viability assay. Following 2 hours incubation with 5mM EGTA
628
and/or green and red quantum dots, cell viability was not significantly reduced in
629
any sample of HOK or HLK derived cell sheets. P values for HOK when
630
compared to the control: QD 0.99, EGTA 0.74, EGTA QD 0.58. P values for HLK
631
when compared to the control: QD 0.89, EGTA 0.55, EGTA QD 0.44.
632 633
Figure 5. XY-plane confocal micrographs of epithelial cell sheets incubated with
634
green and red quantum dots. The healthy cell sheets (A1 and B1) show low
36
635
levels of quantum dot fluorescence on the surface and borders between cells
636
(arrowheads). Stronger fluorescence can be seen in the cell sheets with
637
damaged tight junctions (A2 and B2). The arrows indicate areas where quantum
638
dots are residing in the paracellular spaces. Scale bars=50µm.
639 640
Figure 6. Z-plane (cross-section) surface-rendered confocal micrographs of
641
epithelial cell sheets incubated with green and red quantum dots. The healthy
642
cell sheets (A and C) show a thin layer of quantum dot fluorescence in the
643
superficial most area (arrow). The damaged cell sheets (B and D) show a thick
644
layer of strong fluorescence in the superficial area (arrowheads) and deeper
645
populations of green quantum dot fluorescence (asterisk). All scales are
646
measured in micrometres.
647 648
Figure 7. Fluorescence intensity graphs from epithelial cell sheets incubated
649
with green and red quantum dots. Fluorescence intensity in the healthy (A1 and
650
B1) and damaged (A2 and B2) cell sheets was measured using data from the
651
confocal micrographs in Figure 6. These intensity graphs show quantum dot
652
fluorescence as a function of depth through the cell sheet.
37
653 654
Table 1. Mean cell sheet fluorescence intensity data. A significant increase in
655
total quantum dot fluorescence occurs in the damaged cell sheets compared to
656
the heathy cell sheets. The peak intensity was also stronger and occurred
657
deeper in the damaged cell sheets. n=3. When comparing damaged cell sheet
658
data to healthy cell sheet data, all p values
1
A novel method using quantum dots for testing the barrier function of
2
cultured epithelial cell sheets
3
Thomas J. Duncan1, Koichi Baba2, Yoshinori Oie1, Kohji Nishida1
4
1. Department of Ophthalmology, Osaka University Graduate School of
5 6
Medicine, Suita, Japan. 2. Department of Visual Regenerative Medicine, Osaka University Graduate
7
School of Medicine, Suita, Japan.
8 9
Word count: 4156
10
Keywords: tight junction, quantum dots, epithelial cells, barrier function
11
Funding support: This work was supported by a fellowship for overseas
12
researchers from the Japan Society for the Promotion of Science (JSPS).
13 14
Correspondence: Kohji Nishida, M.D., Ph.D.
15
Department of Ophthalmology, Osaka University Graduate School of Medicine
16
Rm. E7, 2-2 Yamadaoka, Suita, Osaka 565-0871, JAPAN
17
Tel: 81-6-6879-3451, FAX: 81-6-6879-3459
18
E-mail: [email protected]
1
Copyright 2015 by The Association for Research in Vision and Ophthalmology, Inc.
19
Abstract
20
Purpose: The corneal epithelium provides a barrier to protect the deeper
21
structures of the eye from any particles or pathogens. Cultured epithelial cell
22
sheets are used in transplantation surgery for corneal repair or regeneration.
23
The purpose of this study was to develop a novel method utilizing fluorescent
24
quantum dot nanoparticles for validating the quality and barrier function of
25
cultured epithelial cell sheets.
26
Method: Human epithelial cell sheets, cultured from oral mucosal or corneal
27
limbal cells, were incubated in either normal calcium containing medium or
28
medium containing no calcium with a calcium chelator. Also contained in the
29
media were suspensions of two different sizes of quantum dots. Following
30
incubation, analysis of quantum dot penetration was carried out using confocal
31
microscopy.
32
Results: In contrast to the cell sheets incubated in calcium containing medium,
33
removal of extracellular calcium resulted in the disruption of tight junctions,
34
compromising the cell sheet’s barrier function. This caused a reduction in
35
transepithelial electrical resistance and deeper, more ubiquitous penetration of
36
the quantum dots into the paracellular space and interior of the cell sheet.
2
37
Conclusions: This method provides easy to interpret qualitative and
38
quantitative data on the functionality of a cell sheet’s tight junctions, as well as
39
nano and microscale structural information on its surface and interior
40
morphology, and any localized areas of damage or abnormality. This novel
41
technique could be utilized as part of the validation system for cultured epithelial
42
cell sheets for use in transplantation.
43 44
Introduction
45
Anteriorly, the ocular surface is covered by specialized corneal, limbal and
46
conjunctival epithelia. The corneal epithelium is characterized by stratified
47
squamous non-keratinized epithelial cells that are responsible for maintaining
48
the smooth refractive surface of the cornea, essential for vision, whilst also
49
acting as a protective barrier to prevent the passage of foreign bodies and
50
pathogens into the deeper ocular structures. Corneal epithelial cells are derived
51
from transient amplifying cells generated by stem cell populations residing in the
52
basal layer of the limbal epithelium1, 2. Ocular disease or trauma to this area
53
such as Stevens-Johnson syndrome, thermal or chemical burns can cause
54
limbal stem cell deficiencies that result in corneal invasion by adjacent
3
55
conjunctival cells, neovascularization, chronic inflammation, and stromal
56
scarring. Consequently visual acuity is severely affected3, 4. In cases of unilateral
57
damage, corneal regeneration of the affected ocular surface can be attempted
58
by transplantation of limbal grafts5 or via the cultivation of epithelial cell sheets
59
from autologous limbal stem cells taken from the contralateral eye6-8. However,
60
this requires a large limbal graft from the donor eye, carrying with it a risk of
61
infection and inducing a limbal stem cell deficiency in the donor eye9. It is also
62
unsuitable for patients who have bilateral corneal damage10. Transplantation
63
from
64
immunosuppressants11, and carries a high risk of rejection. Post-operative rates
65
of rejection after 1 year range from 20%-60% with cadaveric donor tissue5, 12,
66
and 70%-80% with living donor tissue13, 14.
allogenic
donors
necessitates
prolonged
use
of
postoperative
67 68
In an attempt to improve post-surgical results for corneal surface reconstruction,
69
recent studies have successfully fabricated and transplanted epithelial cell
70
sheets cultured ex vivo from autologous epithelium of non-ocular surface origin15,
71
16
72
autologous oral mucosal epithelial cell derived cell sheets has been
. In rabbits, the technique for harvesting, culturing and transplanting
4
73
established17,
18
74
clinical trials in humans19,
75
significantly improved visual acuity was maintained and no complications were
76
observed, with the oral mucosal keratinocytes undergoing phenotypic changes
77
to more closely resemble that of corneal keratinocytes19. Before this emerging
78
technique can become a standard medical therapy however, it is crucial to
79
establish a validation system by which the cultured oral mucosal epithelial cell
80
sheets can be evaluated prior to transplantation. Such a validation system has
81
been outlined in a previous study by Hayashi et al21.
. More recently, the same treatment has been subjected to 20
. A 14 month postoperative evaluation showed
82 83
One such parameter for evaluation is a cell sheet’s barrier function – a measure
84
by which it can control or inhibit the passage of molecules, foreign bodies or
85
pathogens across its cell layers. The tight junction is the most apically located of
86
the junctional complexes, forming a continuous, belt-like structure that seals the
87
paracellular pathway by joining the lateral surfaces of the most superficial layer
88
of epithelial cells at their apical edge22,
89
necessary for establishing the paracellular connections and assembly of the
90
various proteins involved in a tight junction complex24-27. Removal of calcium,
23
. Calcium has been shown to be
5
91
through the use of a calcium chelator, disrupts tight junction structure and
92
function24, 25, 28. Evaluating the barrier function and health of the tight junctions in
93
a cultured epithelial cell sheet is necessary prior to its use in transplantation
94
surgery.
95 96
Transepithelial electrical resistance (TEER) is a technique which measures the
97
electrical resistance across the full thickness of the cell sheet using two
98
electrodes placed above and below. Primarily, this technique is used to obtain
99
information on the barrier function of a cell sheet. However, TEER values are
100
often unreliable. Numerous studies have reported that TEER does not
101
accurately represent changes in tight junction structure or function29,30.
102
Furthermore, the overall electrical resistance of a cell sheet has been shown to
103
be significantly affected by other factors including temperature31, the culture
104
substrate used32 and the degree of cell-substrate contact33.
105 106
Quantum dots are nanometer scale colloidal semiconductor nanoparticles
107
composed of a few hundred to a few thousand atoms of a semiconductor
108
material such as cadmium mixed with tellurium or selenium. This is then coated
6
109
with an additional zinc sulfide semiconductor shell to contain the cadmium and
110
limit its dissolution, as well as improve the quantum dot optical properties34.
111
Bio-conjugation of this shell to proteins or other small molecules serves to further
112
enhance their biological applications. Quantum dots are extremely efficient at
113
generating fluorescence, often many times greater than that of conventional
114
organic fluorescent compounds34, 35. They have a board excitation spectra with a
115
narrow emission spectra ranging from UV to near-infrared, and superior
116
photostability with a high resistance to photo-bleaching - allowing for greater
117
observation
118
advantageous property of quantum dots is that their fluorescence is tunable. A
119
predictable relationship exists between the energy emitted by these quantum
120
dots (and consequently the wavelength of their fluorescence) and the size and
121
shape of the quantum dot core – with smaller quantum dots fluorescing at
122
shorter wavelengths of light than larger quantum dots. Applying quantum dots to
123
the surface of bovine cornea in vitro has shown that penetration is limited to only
124
the superficial epithelial layers, 20µm from the corneal surface39. In this healthy
125
cornea, the epithelium acted as a barrier to limit quantum dot penetration.
126
Quantum dots may therefore represent a suitable candidate for evaluating the
time
or
long-term
imaging
experiments36-38.
One
highly
7
127
barrier function of a cell sheet.
128 129
The purpose of this study is to utilize these nanoparticles to provide qualitative
130
and quantitative data specific to a cell sheet’s morphology and tight junction
131
functionality. We hypothesize that cell sheets with damaged or disrupted barrier
132
function will permit deeper and more ubiquitous penetration of the quantum dot
133
nanoparticles. Additionally, by utilizing different sized quantum dots, we
134
anticipate a size-dependent variation in penetration and distribution.
135 136
Methods
137
Cell culture – Human oral mucosal epithelial cell sheets
138
Human oral mucosal keratinocytes (HOK) (ScienCell Research Lab, CA, United
139
States) were cultured until subconfluent. Trypsin LE express (Invitrogen,
140
Carlsbad, NM, United States) was used to detach the cells from the culture flask
141
into suspension. The cell suspension was then centrifuged and the supernatant
142
removed. The cells were then re-suspended in keratinocyte culture medium and
143
seeded at an initial cell density of 1x105cells/ml into 12-well culture inserts
144
(Falcon) with mitomycin C (MMC)-treated 3T3-J2 feeder cells in KCM, separated
8
145
by the cell culture insert permeable membrane. KCM comprises Dulbecco’s
146
modified Eagle’s medium (DMEM/F12 [3:1]) supplemented with 10% fetal bovine
147
serum (Invitrogen, Japan), 1% L-Glutamine (Invitrogen, Japan) 5μg/mL insulin
148
(novolin; Novartis Pharmaceuticals, Japan), 1% penicillin-streptomycin (Meiji
149
seika, Japan), 0.4μg/ml hydrocortisone (saxizon; Kowa, Tokyo, Japan), 2nM
150
triiodothyronine (T3; Sigma Aldrich, Japan), 1nM cholera toxin (List Biological
151
Laboratories, CA, United States), and 10ng/mL EGF (Higeta Shoyu, Japan).
152
Incubation was carried out at 37°C and 5% CO2. All procedures were carried out
153
following the formation of mature, stratified epithelial cell sheets.
154 155
Cell culture – Human limbal epithelial cell sheets
156
Human corneas (donor age 34-73) were obtained from eye banks (Sightlife, WA,
157
United states) and subsequently washed in DMEM before being incubated in
158
dispase for 1 hour at 37°C 5% CO2. The human limbal keratinocytes (HLK) were
159
then scraped from the limbal region of the cornea, and the resulting suspension
160
was centrifuged and the supernatant removed. The cells then underwent
161
trypsinization before being re-suspended in KCM and seeded at an initial cell
162
density of 1x105cells/ml into 12-well culture inserts (Falcon) with MMC-treated
9
163
3T3-J2 feeder cells in KCM, separated by the cell culture insert permeable
164
membrane. Incubation was carried out at 37°C and 5% CO2. All procedures
165
were carried out following the formation of mature, stratified epithelial cell
166
sheets.
167 168
Quantum dot incubation and analysis
169
Incubation media was formulated to either contain the standard 1.64mM of
170
calcium, or 0mM of calcium with 5mM of the calcium chelator ethylene glycol
171
tetraacetic (EGTA). In order to standardize the level of tight junction disruption,
172
EGTA was used to remove any tight junction associated calcium, as well as any
173
calcium remaining from the use of KCM medium during the culturing period.
174
Green (525nm) and red (655nm) quantum dots, with zinc sulfide shells
175
conjugated to carboxyl molecules, were purchased as a suspension in borate
176
buffer (Life technologies Japan). 40nM suspensions of both green and red
177
quantum dots were made by diluting the commercially available quantum dots
178
into either the calcium or EGTA containing incubation media. The pH of all
179
incubation media was then adjusted to 7.4, and then applied to the superficial
180
surface of the epithelial cell sheets. These were then incubated at 37°C and 5%
10
181
CO2 for 90 minutes. The cell sheets were washed twice in PBS to remove any
182
excess quantum dots. The samples were then immunostained with a 10%
183
Hoechst solution for 10 minutes, following which they were fixed in 4%
184
paraformaldehyde and analysed using confocal microscopy (Ziess). Confocal
185
parameters (including laser power, aperture size and image thresholds) were
186
identical for all samples.
187 188
Transepithelial electrical resistance (TEER)
189
Electrical resistance across the cell sheets was measured using an EndOhm
190
chamber (WPI Inc) and EVOM2 resistance meter (WPI Inc). An insert containing
191
only medium, with no cell culture, was used as a blank. TEER was measured at
192
various time points after addition of the incubation media. The TEER value was
193
first measured at 0mins after incubation, then every 15 minutes following for a
194
period of 90 minutes. Each test was performed in triplicate for each condition.
195
The TEER value was calculated by: [average resistance of experimental wells −
196
average resistance of blank wells] Ohms × 0.33cm2 (area of the insert culture
197
surface).
198
11
199
MTT cell viability assay
200
Stock solution of MTT (Sigma, USA) was diluted to 375µg/ml in KCM. A 5mM
201
EGTA solution and/or 40nM suspensions of both green and red quantum dots
202
were then added to this MTT-KCM medium. This was then pipetted into 12 well
203
inserts containing HOK or HLK derived cell sheets. After incubation at 37°C for 2
204
hours, the resulting formazan crystals were dissolved in equal quantities of
205
acidified isopropanol. The data was obtained by subtracting the background
206
absorbance at 690nm from the absorbance at 570nm recorded using a
207
spectrophotometer (V-550, UV/VIS Spectrophotometer, JASCO, Japan) and
208
expressed as a percentage of the control cell sheets incubated without quantum
209
dots or EGTA. Each test was performed in triplicate for each condition. ANOVA
210
and Dunnett’s statistical analysis was carried out using JMP software to
211
compare each test value to the control group.
212 213
Transmission electron microscopy (TEM)
214
Following incubation with the quantum dot suspensions, samples were fixed in a
215
solution of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M sodium
216
cacodylate buffer (pH 7.2) at room temperature for 3 hours. Staining was then
12
217
carried out with 2% osmium tetraoxide for 1 hour followed by 0.5% uranyl
218
acetate for 1 hour at room temperature. The samples were then dehydrated
219
through graded alcohols and propylene oxide before being embedded in Araldite
220
resin (TAAB laboratories). Resin curing was carried out at 60ºC for 3 days. 90nm
221
thick sections were cut from the cured sample blocks on a Reichert-Nissei
222
Ultracut Microtome and floated onto copper mesh-200 EM grids (Agar scientific
223
Ltd). These were then post-stained at room temperature in 2% aqueous uranyl
224
acetate and Reynolds lead citrate for 5 minutes.
225 226
In order to measure the particle size, droplets of red and green quantum dots
227
were also applied to the surface of carbon film coated slot-grids (Agar scientific
228
Ltd). Any excess solution was wicked away using filter paper. All grids were then
229
given time to dry before TEM examination.
230 231
Analysis of all samples was carried out at 80kV with a transmission electron
232
microscope
233
charge-coupled device camera (Gatan Inc., Japan).
(Hitachi
H7650,
Japan).
Images
were
obtained
with
a
234
13
235
Quantum dot size measurements
236
Micrographs were obtained of the quantum dots on carbon film coated slot-grids.
237
Length and width values for both red and green quantum dots were calculated
238
from 15 measurements taken using Fiji (Image J) software.
239 240
Fluorescence intensity measurements
241
Fluorescence intensity readings were taken across the XY-plane of the confocal
242
micrographs at 1μm interval passing through the thickness of the cell sheet. By
243
calculating the area under the curve of the subsequent fluorescence intensity
244
graph, total fluorescence intensity was determined. Mean total fluorescence
245
intensity, peak fluorescence intensity and depth of the peak fluorescence
246
intensity within the cell sheet were calculated from reading from three sample
247
cell sheets. P values were obtained via T-test statistical analysis.
248 249
Results
250
The TEM micrographs in Figure 1 show small populations of quantum dots
251
(Figure 1A arrows) residing within the paracellular space. The area marked by
252
an asterisk can be seen at higher magnification in Figure 1B, where it is possible
14
253
to discern the small green (arrow) and larger red (arrowhead) quantum dots.
254 255
The TEM micrograph Figure 2A1 shows the green quantum dots were
256
approximately spherical in shape, with an mean width of 3.16nm (SD=0.30nm)
257
and an mean length of 3.28nm (SD=0.19nm) (Figure 2A2). The red quantum
258
dots (Figure 2B1) had a more oblong shape, with a width of 5.54nm
259
(SD=0.29nm) and a length of 14.98nm (SD=1.02nm) (Figure 2B2). For a full list
260
of size measurements see Table 1 in supplementary data.
261 262
Figure 3 demonstrates the effect of incubating human oral mucosal
263
keratinocytes (HOK) and human limbal keratinocytes (HLK) cell sheets in 0mM
264
calcium medium with an EGTA calcium chelator. In 1.64mM of calcium, the initial
265
electrical resistance of the cell sheet was approximately maintained throughout
266
the 90 minutes of incubation. However, using medium containing 0mM of
267
calcium with 5mM of EGTA had the effect of rapidly reducing the cell sheet’s
268
electrical resistance within the first 15 minutes. A slowing, but continued
269
decrease occurred for the following 75 minutes until after 90 minutes the
270
resistance was significantly lower than its initial level – corresponding to an
15
271
average 18.3Ohms·cm2 reduction in the HOK cell sheets, and a 22.8Ohms·cm2
272
reduction in the HLK cell sheets. Figure 4 shows that incubation for 2 hours with
273
EGTA and/or quantum dots had no significant effect of the viability of either the
274
HOK or HLK derived cell sheets.
275 276
The confocal micrographs in Figures 5 show a significant difference in quantum
277
dot quantity and distribution across the healthy HOK (Figure 5A1) and HLK
278
(Figure 5B1) cell sheets compared to the cell sheets containing damaged tight
279
junctions (HOK Figure 5A2 and HLK Figure 5B2). In the healthy cell sheets,
280
small amounts of quantum dots can be seen residing on the surface and in the
281
border regions between cells (Figure 5A1 and 5B1 arrowhead). The damaged
282
cell sheets show significantly larger amount of quantum dots residing in the
283
paracellular space, creating a much stronger fluorescence profile for both the
284
red and green quantum dots (Figure 5A2 and 5B2 arrows).
285 286
Figure 6 shows the Z-plane surface (cross-section) rendered views through the
287
HOK and HLK cell sheets of Figure 5. The same rendered view, with the addition
288
of cell nuclei Hoechst staining, can be seen in the supplemental data Figure 1.
16
289
The healthy cell sheets (Figure 6A and 6C) contain a thin layer of quantum dots
290
residing on the surface (arrow). Both red and green quantum dots are present in
291
this residual layer. The damaged cell sheets however, contain a superficial layer
292
of quantum dots (Figure 6B and 6D arrowheads) over two times thicker than in
293
the healthy cell sheets. In addition, numerous populations of green (and to a
294
lesser extent, red) quantum dots can also be seen within the deeper cell layers.
295
The asterisk in Figure 6B2 indicates a large cluster of green quantum dots
296
residing in a deeper region. No such deeper populations can be observed in the
297
healthy cell sheet.
298 299
Figure 7 shows the fluorescence intensity readings taken from the confocal
300
micrographs in Figure 6. In the damaged cell sheets (Figure 7A2 and 7B2), the
301
initial fluorescence peak is both stronger in intensity and continues deeper into
302
the cell sheet than in the healthy cell sheets (Figure 7A1 and 7B1). Following this
303
initial peak, green quantum dot fluorescence is still present at a lower intensity
304
level throughout the thickness of the cell sheets. The second large fluorescence
305
peak seen in Figure 7A2 represents the large cluster of green quantum dots
306
residing approximately 40µm deep into the cell sheet (Figure 6B2 asterisk). In
17
307
Table 1 the mean fluorescence intensity readings taken from multiple cell sheets
308
show a statistically significant increase in both green (HOK p=0.038, HLK
309
p=0.009) and red (HOK p=0.032, HLK p=0.013) quantum dot fluorescence
310
intensity when comparing the healthy cell sheets to the damaged cell sheets.
311
Furthermore, the peak intensity readings for the quantum dots occurred 2-3
312
times deeper into the damaged cell sheet than in the healthy cell sheets. Human oral mucosal cells
Mean total fluorescence intensity (I) Mean peak fluorescence intensity (I) Mean depth of peak intensity (µm) 313
Human limbal epithelial cells
Healthy cell
Damaged cell
Healthy cell
Damaged cell
sheets
sheets
sheets
sheets
Red
Green
Red
Green
Red
Green
Red
Green
QD
QD
QD
QD
QD
QD
QD
QD
36.99
38.09
246.74
416.15
40.28
31.82
279.57 403 .00
±9.02
±5.60
±69.20 ±134.26 ±5.54
±4.59
±49.17 ±62.53
5.65
5.33
18.2
18.34
6.40
5.38
16.91
20.43
±0.57
±0.50
±1.56
±3.01
±1.08
±0.87
±2.66
±4.13
6.00
6.33
12.33
13.00
6.00
6.33
18.33
19.00
±1.00
±1.53
±2.31
±1.00
±1.73
±2.08
±4.04
±4.00
Table 1. Mean cell sheet fluorescence intensity data.
314 315
Discussion
316
We have demonstrated that quantum dots can be utilized as an effective
317
indicator of a cell sheet’s barrier function. Our hypothesis can be confirmed as 18
318
cell sheets with disrupted barrier function demonstrated deeper and more
319
widespread penetration of the quantum dot nanoparticles, particularly of the
320
smaller green quantum dots. By applying quantum dots in this way, nano and
321
microscale morphological information on a cell sheet’s surface and interior can
322
also be ascertained - in particular, making local areas of abnormality or
323
increased permeability easy to identify - something that current evaluation
324
techniques, such as TEER, are unable to provide.
325 326
TEER did however demonstrate the dramatic effect caused by EGTA calcium
327
chelation. This was in turn mirrored by the significant increase in quantum dot
328
penetration through the open paracellular spaces - penetrating even through to
329
the deepest layers of the cell sheets. These results demonstrate that tight
330
junctions are a major factor in a cell sheet’s barrier function. However, even in
331
the damaged cell sheets, the larger red quantum dots showed a more limited
332
penetration deeper than 15µm into the cell sheet. This parallels results from
333
another quantum dot penetration studies which recorded red (655nm) quantum
334
dots showing limited penetration deeper than 15-20µm into the epithelium of an
335
intact cornea after a similar 80mins incubation in vivo39. This suggests that the
19
336
size of the quantum dot was a limiting factor for its penetration through the cell
337
sheet. A model for the observed penetration of the quantum dots can be seen in
338
Figure 8. Other junctional complexes such as adherens junctions, desmosomes
339
and gap junctions serve to anchor or connect neighbouring epithelial cells
340
together. It has previously been reported that the paracellular space adjacent to
341
adherens junctions and desmosomes is approximately 20-30nm40-43. However,
342
the presence of gap junctions reduces this space to 4nm41, 43. The red quantum
343
dots - with a recorded length of 14.98nm and a width of 5.54nm – would be too
344
large to pass through these narrow paracellular regions containing gap junctions.
345
Consequently even though the damaged cell sheet had disrupted tight junctions,
346
the gap junctions may still have served to slow the rate of passage of the red
347
quantum dots through to the deeper regions of the cell sheet.
348 349
Conversely, the green quantum dots - with a recorded length of 3.28nm and a
350
width of 3.16nm, would be small enough to pass through even the narrowest
351
regions in the paracellular space. Although the rate at which they could pass
352
through these regions would still be rate limited and time dependant. This may
353
explain why although deeper populations of green quantum dots were observed
20
354
in the damaged cell sheet, a large proportion of them remained in the more
355
superficial layers. Nevertheless, a relatively large quantity of the quantum dots
356
(labelled by an asterisk in Figure 6B2) were able to accumulated at the bottom of
357
the cell sheet in such a localized manner as to suggest particular disruption or
358
damage to that area of the cell sheet. In this way, quantum dots can be used to
359
identity localized areas of abnormality within the cell sheet.
360 361
As green (to a lesser extent, red) quantum dots were able to penetrate deeply
362
into the cell sheet, we also investigated whether penetration rates could be
363
quantifiably measured by analysing the medium collected from beneath the cell
364
sheets after incubation. The results of this experiment can be seen in the
365
supplementary data Figure 2 and Table 2.
366 367
Conventional methods for evaluating epithelial barrier function or surface
368
abnormalities include measuring TEER or using fluorescent dyes such as
369
sodium fluorescein. Whilst these techniques are useful, our novel method can
370
provide rapid nano and microscale structural information on a cell sheet’s
371
surface and interior morphology as well as the health of its barrier function -
21
372
allowing localized areas of surface abnormality or damage to be easily identified.
373
Standard fluorescent dye molecules may be small enough (sodium fluorescein
374
radius=5.5 Å44) to pass through the paracellular space rapidly and with ease.
375
Quantum dots are considerably larger than these dye molecules; accordingly
376
they may show a comparably slower rate of penetration through the cell sheet.
377
The quantum dots often aggregated into small clusters as they moved through
378
the paracellular space into the deeper cell layers, with larger aggregations
379
occurring in areas of increased permeability. Subsequently, quantum dots can
380
provide structural information on the interior morphology of a cell sheet on a
381
nano and microscale – ranging from as small as 3.16nm (the width of a single
382
green quantum dot) up to the large aggregation that often measure 5-10µm in
383
size. This is something that the smaller dye molecules are unable to provide.
384 385
However, further improvements to this technique can be made. There are
386
discrepancies in the current literature regarding the toxicity of quantum dots.
387
This is largely due to the variety of quantum dots available, and a lack of
388
extensive toxicity studies. Our own investigations align with longer term studies
389
that show no appreciable toxicity45-47. Conversely, some studies have reported
22
390
that despite the zinc sulfide shell, Cd2+ ions are able to leech out of the quantum
391
dot core, causing concerns about their toxicity and safety for clinical use48.
392
Furthermore, comparative confocal micrographs in supplemental data Figure 3
393
show that even in unfixed cell sheets, the quantum dots are not readily removed
394
from the paracellular spaces once they have penetrated the cell sheet. Even so,
395
this technique could still be applied to cell sheets in vitro, together with other
396
destructive tests such as immunohistochemistry21. A range of in vivo application
397
are currently being considered using alternatives such as carbon quantum dots49
398
or indium phosphide50, 51, that have been shown to possess significantly lower
399
levels of cytotoxicity.
400 401
In addition, utilizing a greater range of quantum dot sizes would potentially
402
produce a spectrum of color across the thickness of the cell sheet, giving more
403
information on the degree to which the paracellular pathway is open. It may be
404
possible to define the scale of tight junction disruption by observing the relative
405
penetration of different sized quantum dots. Based on our experiments, the
406
smallest quantum dots would be expected to penetrate to the deepest layers.
407
Small or intermediate sized quantum dots may penetrate the superficial layers
23
408
but at a reduced rate, whilst the larger quantum dots would remain on the
409
surface or in the most superficial layer. This data would then provide greater
410
information on a cell sheet’s barrier function as well as local areas of
411
abnormality.
412 413
For this proof-of-concept study, using EGTA to disrupt tight junction function was
414
desirable as it induced a significant effect on TEER. However, future work will
415
feature a range of disruptive techniques, in order to better represent the variety
416
of abnormalities and defects common in cultured cell sheets. One such
417
candidate for future study as an alternative and milder disruptor of tight junction
418
function is TNF-α. This was tested following published protocols52-54 – briefly, the
419
cell sheets were incubated for 24hours with 10ng/ml TNF–α, before the addition
420
of red and green quantum dots. TEER readings and comparative confocal
421
micrographs can be seen in the supplemental data Figure 4.
422 423
To conclude, our method for evaluating cultured epithelial cell sheet barrier
424
function has not been reported in the literature to date. As such, it represents a
425
novel technique to provide clear, easy to interpret data on a cell sheet’s barrier
24
426
function, as well as nano and microscale structural information of its interior and
427
surface morphology. This technique can be incorporated into the range of
428
analytical tests performed prior to approval for transplantation. Furthermore, this
429
information may serve to enhance scientific knowledge on how to fabricate
430
healthy cell sheets for transplantation.
431 432
Acknowledgements
433
This work was supported by a fellowship for overseas researchers from the
434
Japan Society for the Promotion of Science (JSPS). The authors confirm that
435
there are no known conflicts of interest associated with this publication and there
436
has been no significant financial support for this work that could have influenced
437
its outcome.
438 439
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Figure legends
609
Figure 1. TEM micrographs showing oral mucosal epithelial cells and the
610
penetration of quantum dots into the paracellular space. (A) Taken at x8000
611
magnification, several clusters of quantum dots can be seen residing in the
612
paracellular space (arrows). The area marked by the asterisk can be seen at
613
20,000x magnification in Figure 1B. In (B), small spherical green quantum dots
614
are indicated by an arrow. Whilst larger, more elongated red quantum dots are
615
indicated by an arrowhead. Scale bar=200nm.
616
35
617
Figure 2. TEM micrographs showing green quantum dots (A1) and red quantum
618
dots (B1). Scale bar=10nm. A2 and B2 show frequency distribution of the
619
quantum dot length and width measurements.
620 621
Figure 3. Graph showing the effect of low calcium on cell sheet TEER. Initial
622
resistance levels are maintain when the cell sheet is incubated in normal calcium
623
levels of 1.64mM. Using a calcium chelator (EGTA) in medium containing 0mM
624
of calcium resulted in a reduction in electrical resistance. Error bars show
625
standard deviation. n=3.
626 627
Figure 4. MTT cell viability assay. Following 2 hours incubation with 5mM EGTA
628
and/or green and red quantum dots, cell viability was not significantly reduced in
629
any sample of HOK or HLK derived cell sheets. P values for HOK when
630
compared to the control: QD 0.99, EGTA 0.74, EGTA QD 0.58. P values for HLK
631
when compared to the control: QD 0.89, EGTA 0.55, EGTA QD 0.44.
632 633
Figure 5. XY-plane confocal micrographs of epithelial cell sheets incubated with
634
green and red quantum dots. The healthy cell sheets (A1 and B1) show low
36
635
levels of quantum dot fluorescence on the surface and borders between cells
636
(arrowheads). Stronger fluorescence can be seen in the cell sheets with
637
damaged tight junctions (A2 and B2). The arrows indicate areas where quantum
638
dots are residing in the paracellular spaces. Scale bars=50µm.
639 640
Figure 6. Z-plane (cross-section) surface-rendered confocal micrographs of
641
epithelial cell sheets incubated with green and red quantum dots. The healthy
642
cell sheets (A and C) show a thin layer of quantum dot fluorescence in the
643
superficial most area (arrow). The damaged cell sheets (B and D) show a thick
644
layer of strong fluorescence in the superficial area (arrowheads) and deeper
645
populations of green quantum dot fluorescence (asterisk). All scales are
646
measured in micrometres.
647 648
Figure 7. Fluorescence intensity graphs from epithelial cell sheets incubated
649
with green and red quantum dots. Fluorescence intensity in the healthy (A1 and
650
B1) and damaged (A2 and B2) cell sheets was measured using data from the
651
confocal micrographs in Figure 6. These intensity graphs show quantum dot
652
fluorescence as a function of depth through the cell sheet.
37
653 654
Table 1. Mean cell sheet fluorescence intensity data. A significant increase in
655
total quantum dot fluorescence occurs in the damaged cell sheets compared to
656
the heathy cell sheets. The peak intensity was also stronger and occurred
657
deeper in the damaged cell sheets. n=3. When comparing damaged cell sheet
658
data to healthy cell sheet data, all p values
