Abstract
Corneal endothelial cells (CECs) are very important for the maintenance
of corneal transparency. However, in vitro, CECs display limited
proliferation and loss of phenotype via endothelial to mesenchymal
transformation (EMT) and cellular senescence. In this study, we
demonstrate that continuous supplementary nutrition using a perfusion
culture bioreactor and three-dimensional (3D) spheroid culture can be
used to improve CEC expansion in culture and to construct a
tissue-engineered CEC layer. Compared with static culture,
perfusion-derived CECs exhibited an increased proliferative ability as
well as formed close cell-cell contact junctions and numerous surface
microvilli. We also demonstrated that the CEC spheroid culture
significantly down-regulated gene expression of the proliferation
marker Ki67 and EMT-related markers Vimentin and α-SMA, whereas the
gene expression level of the CEC marker ATP1A1 was significantly
up-regulated. Furthermore, use of the perfusion system in conjunction
with a spheroid culture on decellularized corneal scaffolds and
collagen sheets promoted the generation of CEC monolayers as well as
neo-synthesized ECM formation. This study also confirmed that a CEC
spheroid culture on a curved collagen sheet with controlled
physiological intraocular pressure could generate a CEC monolayer.
Thus, our results show that the use of a perfusion system and 3D
spheroid culture can promote CEC expansion and the construction of
tissue-engineered corneal endothelial layers in vitro.
Introduction
Corneal endothelial cells (CECs), which reside at the inner surface of
the cornea, maintain corneal transparency by regulating stromal
hydration via their barrier and pump functions^[44]1. When the cell
density of CECs falls below the critical level required to maintain
normal corneal hydration, corneal edema usually occurs and vision is
gradually impaired, thereby requiring corneal transplantation to
restore normal vision^[45]1. Recently, there has been enormous interest
in strategies involving regeneration of the corneal endothelium as an
efficient alternative to endothelial keratoplasty, in which only the
posterior host corneal layers are replaced by a suitable endothelial
graft to restore endothelial function^[46]2–[47]4. Tissue-engineered
endothelial grafts that utilize biomaterials and biomimetic scaffolds
and incorporate the patient’s own cells have become a prospective
alternative to allografts in the treatment of endothelial
defects^[48]5. Although several studies have investigated the use of
amniotic membranes and silk fibroin membranes as corneal substitutes,
there are still insufficient data to permit further clinical
trials^[49]3, [50]6, [51]7. The challenge of producing CEC layers or
cells for endothelial cell therapy by tissue engineering is the
phenotypic instability of CECs. Therefore, solving this problem will
require the use of versatile cell culture technology and materials to
produce cells that can serve as artificial endothelial grafts.
CECs retain the ability to proliferate in vitro under the proper
culture conditions^[52]8. However, after long-term subculture, they
tend to display limited proliferation, senescence or fibroblastic
transformation with morphological, physiological and functional loss
caused by dedifferentiation^[53]1, [54]9–[55]11. Hence, there is a need
to develop culture systems for CECs in vitro that not only promote
proliferation but also counter dedifferentiation events such as the
endothelial-to-mesenchymal transition (EndoMT or EMT)^[56]12. The
cellular microenvironment, as one of the primary determining factors of
cellular activity in the human body, impacts cell morphology and
physiological functions^[57]13, [58]14. Accumulating evidence has
suggested that mechanical factors play an important role in influencing
cell growth, structure, and function^[59]15, [60]16. Flow perfusion
systems provide a flexible platform for developing a controllable
biomimetic environment that can be adapted for use in various
investigations of dynamic cell culture under conditions of rheological
stress and hydrostatic pressure^[61]17–[62]19. Flow perfusion systems
such as bioreactors enable the continuous and constant supply of
nutrients and oxygen to cells and can be used to improve the
environment for in vitro tissue-engineered conditions^[63]18, [64]20.
Therefore, flow perfusion systems may be an efficient method for the
construction of CEC layers and the observation of morphological and
physiological changes in CECs. Mannis et al. studied the morphology of
CECs under in vivo anterior chamber perfusion. Clinically, they were
unable to detect any signs of gross corneal dysfunction with
hypothermic perfusion at a flow rate of 5 ml/min^[65]21. Brunette et
al. maintained clear human corneas using incubation and perfusion for 3
weeks. The endothelial cells remained viable and functional^[66]22.
Thiel et al. reported that a chamber device was a reliable tool for in
vitro drug penetration and toxicity studies in isolated perfused
corneoscleral tissue^[67]23. However, to our knowledge, there are few
or no published reports concerning perfusion culture for the
construction of tissue-engineered CEC layers in vitro. To improve the
environment for cells and developing specialized tissues, the MINUSHEET
perfusion culture system was developed in 1990^[68]24. In this system,
the pigmented epithelium and neighbouring neurons of the intact retina
maintain a perfect morphology for a culture period of at least 10 days.
The development of a gingival epithelium or co-culture of keratinocytes
and osteoblast-like cells in a perfusion container yields much better
results than those obtained under static culture conditions. The
MINUSHEET perfusion culture system thus is advantageous for use in the
engineering of connective tissue, the generation of nervous tissue, and
the development of muscle tissue^[69]18.
Another type of cell culture, spheroid (SP) culture, which mimics the
microenvironment in vivo to yield a multicellular mass mediated by
cadherin, exhibits several advantages over conventional two-dimensional
(2D) culture^[70]25, [71]26. The precursors obtained from the CEC
spheroids possess longer telomeres and higher telomerase
activity^[72]27, and CEC-derived spheroid therapy has been used in a
rabbit CEC deficiency model^[73]28. Our previous studies also found
that CEC spheroids treated with Y-27632 are injectable in vitro and
have important implications for the favourable treatment of CEC
deficiency^[74]29.
The available matrices for CEC sheet generation have progressed from
natural membranes, biological polymers, and biosynthetic materials to
completely synthetic materials^[75]30. Acellular scaffolds such as the
amniotic membrane and corneal stroma transmit light and possess
mechanical properties that may support CEC attachment for
transplantation applications in vivo ^[76]6, [77]31. However, there is
currently no reliable, standardized decellularization protocol to
remove immunoreactive material and at the same time maintain tissue
properties such as corneal curvature^[78]32, [79]33. Our previous
report showed that short-term chemical-frozen decellularization of the
bovine stromal matrix allowed keratocytes to maintain their dendritic
shape, reticular arrangement and phenotypic stability even in the
presence of 10% FBS. Collagen is the most abundant protein in the
cornea and is the primary structural component of corneal
tissue^[80]34. Recently, researchers have demonstrated that collagen
vitrigel with a spherical curve, such as porcine-derived atelocollagen
vitrigel, can be used as a spherical biomimetic scaffold for artificial
endothelial grafts^[81]33, [82]35.
In this study, we for the first time employed the MINUSHEET perfusion
system to expand bovine CECs and construct CEC layers in vitro.
Furthermore, we describe the use of a spheroid process using agarose
micromolds and provide insight into how bovine CEC functions are
improved in spheroid culture. Bovine CECs were used because that made
it possible to obtain 25–50 age-matched 1-2-year-old eyes at one time
and thereby to obtain the large number of cells required for perfusion
and spheroid studies, which was not possible using human tissue^[83]36.
Cultured bovine CECs provided us with an excellent in vitro model for
the study of the differentiated functions of the corneal
endothelium^[84]37. The investigation of cellular behaviour in the
bovine CEC model can also be revealing for human CEC biology and
applications. To demonstrate this system’s application in
tissue-engineered CEC construction, conventional decellularized bovine
corneal scaffolds and flat or spherically curved collagen sheets were
investigated. Our objective was to explore whether the use of
biomimetic platforms involving a perfusion system and three-dimensional
(3D) spheroid culture not only promote CEC proliferation and counter
EMT but also enhance the construction of tissue-engineered corneal
endothelial layers close to the native cornea.
Results
Enhancement of the oxygen supply and CEC proliferation in the perfusion
system
The measurement from the optical fibre oxygen fluorescence microsensor
showed that the relative oxygen intensity in the waste medium of the
perfusion was the strongest, followed by that in the medium of the
static culture and then that in the perfusion medium. The value in the
unused medium was the weakest (Fig. [85]1E). Figure [86]1E also shows
that the relative intensities in water (H[2]O) were maintained at
nearly the same value, indicating that the microsensor remained stable
for the duration of the measurement. The dissolved oxygen concentration
is inversely proportional to the luminescence intensity. Thus, the
dissolved oxygen concentration is the highest in the unused medium,
followed by the perfusion medium, the medium from cells grown under
static conditions and the waste medium.
Figure 1.
Figure 1
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Enhancement of the oxygen supply and CEC proliferation in the perfusion
system. (A) Photograph of the perfusion system in a 37 °C incubator
containing 5% CO[2]. (B) Photographic illustration of the perfusion
system. (C) Schematic of the sensor system. (D) Photograph of the
microsensor. (E) Intensity values of the microsensor for dissolved
oxygen. (F) Growth curves of CECs in the static and perfusion systems
after seeding at an initial cell number of 0.7 × 10^4 cells/well.
Compared with static-derived CECs, perfusion-derived CEC cultures
contained greater numbers of cells at later times (Fig. [88]1F). To
investigate the effect of the perfusion system on the proliferation of
CECs, the number of EdU-positive cells and the cell cycle were also
analysed. Cells cultured in the perfusion system displayed more
EdU-positive cells than those in the static system on day 3 (D3)
(Fig. [89]2A). The percentage of EdU-positive CEC nuclei in cultures
grown in the static system and in the perfusion system on D3 were
5.38 ± 0.59% and 11.41 ± 1.0%, respectively (Fig. [90]2B). There was a
significant increase in the cell density in the perfusion system
(721 ± 36 cells/mm^2) compared with that in the static condition
(277 ± 8 cells/mm^2) (Fig. [91]2C) after 5 days of culture. Similarly,
PI flow cytometry demonstrated that the cell cycle distribution of CECs
in the perfusion system was obviously promoted. The percentages of
cells in the G1 and S phases on D3 in the perfusion system were
66.66 ± 6.90% and 17.55 ± 1.47%, respectively, whereas those in the
static condition were 75.93 ± 6.70% and 6.98 ± 4.37%, respectively
(Fig. [92]2D). These results demonstrate that perfusion culture with
continual supplement nutrition supports greater CEC proliferation
during expansion than the static culture.
Figure 2.
Figure 2
[93]Open in a new tab
Effects of the perfusion system on the growth ability of CECs. (A) EdU
assay of CECs in the static and perfusion systems. Scale bar: 100 µm.
(B) The proliferation of CECs was evaluated based on the ratio of
EdU-positive cells to total cells. (C) Quantification of the cell
density in the static and perfusion systems. (D) Flow cytometry
analysis of the cell cycle in the static and perfusion systems. The
data are presented as the mean ± SD of three independent experiments.
Differences with *P < 0.05 were considered to be statistically
significant.
Maintenance of the CEC phenotype in the perfusion system
We further explored whether the CEC phenotypes were maintained in the
perfusion system after enhancement of the oxygen supply, and CEC
proliferation in such a dynamic culture was found. Bovine CECs cultured
in the static system or the perfusion system on days 1 (D1), 3 (D3) and
5 (D5) were imaged under an inverted contrast microscope. CECs cultured
in the static system exhibited polygonal morphology. Compared with
cells in static culture, CECs grown in the perfusion system on D5 were
characterized by polygonal, especially hexagonal, morphology, smaller
size, and higher cell density (Fig. [94]3A). Immunofluorescence
staining revealed that the protein expression levels of AQP1 and ATP1A1
in CECs in both the static and perfusion systems were positive
(Fig. [95]3B). CECs passaged on 2D culture TCPS after static or
perfusion culture expressed positive Calcein-AM live cell staining and
the CEC phenotypic markers AQP1, vimentin and N-cadherin (Fig. [96]S2).
SEM micrographs showed that CECs in static culture were arranged in
flat monolayers with microvilli randomly distributed over the smooth
cell surface, whereas the CEC monolayer in the perfusion system was
characterized by a raised and rough surface topography with more
microvilli (Fig. [97]3C). These results suggest that CEC phenotype and
surface topography are maintained under perfusion culture conditions.
Figure 3.
Figure 3
[98]Open in a new tab
Maintenance of the CEC phenotype in the perfusion system. (A)
Representative phase-contrast images of CECs in the static and
perfusion systems at D1, D3 and D5. Scale bar: 100 μm. (B)
Immunofluorescence images of AQP1 and ATP1A1 staining. Scale bar:
50 μm. (C) SEM images of CECs on glass carriers in the static and
perfusion systems.
Impediment of EMT in CECs by biomimetic platforms of CEC spheroids and
perfusion culture
We first investigated the characterization of CEC spheroids in static
culture. CECs were cultured in multiwell agarose micromolds.
Preliminary spheroids formed on day 1 after seeding. The cell viability
assay (Live/Dead assay) demonstrated that most of the cells in the CEC
spheroids were viable during seven days of culture, showing an intense
green fluorescence in the live cytoplasm from Calcein AM staining. Dead
cells were located mainly in the centres of CEC spheroids after five
days of culture; red fluorescence was apparent in dead cell nuclei from
the EthD-III staining (Fig. [99]S3). Live/Dead staining also revealed
that the spheroid diameters were approximately 140 μm (small
spheroids), 300 μm (middle spheroids) and 600 μm (large spheroids)
after 600, 3,000 and 27,000 cells per microwell were seeded and
cultured for 3 days, respectively (Fig. [100]S4). QPCR revealed
significant up-regulation of the endothelial markers ATP1A1, AQP1 and
N-cadherin, the proliferation marker Ki67 and the mesenchymal markers
Vimentin and α-SMA, particularly in small spheroids (Fig. [101]S4D).
Hence, small CEC spheroids cultured for 3 days were chosen for
subsequent experiments.
Total RNA was extracted from the CECs of traditional 2D monolayer
cultures and from CEC cultures containing small spheroids (SP). The
RT-PCR results showed that expression of a proliferation marker (Ki67),
EMT-related markers (Vimentin and α-SMA) and CEC markers (ATP1A1, AQP1,
N-cadherin and TJP1) was positive in both the 2D and the SP cultures
(Fig. [102]S5). However, the QPCR assay revealed a difference between
the two groups. The QPCR results from CEC spheroids revealed
significant down-regulation of gene expression for the proliferation
marker Ki67 on D1, D3, D5 and D7 (Fig. [103]4A). The EMT-related
markers Vimentin and α-SMA were significantly down-regulated on D3, D5
and D7 (Fig. [104]4B,C), whereas there was significant up-regulation of
the endothelial marker ATP1A1 at those times (Fig. [105]4E). There was
no consistent difference in the TJP1 and N-cadherin expression levels
in the two groups (Fig. [106]4D,F). The gene expression level of the
CEC marker AQP1 was significantly down-regulated (Fig. [107]4G).
Western blot analysis also revealed significantly up-regulated
expression of the ATP1A1 protein and significant down-regulation of the
expression of Vimentin and AQP1 in cultured CEC spheroids on D3
(Fig. [108]4H,I).
Figure 4.
Figure 4
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Impediment of EMT in CECs by biomimetic platforms of CEC spheroids.
(A–G) QPCR analysis of proliferation marker and functional marker
expression normalized to GAPDH. (H) WB of vimentin, ATP1A1, AQP1 and
GAPDH in 2D and SP cultures. (I) Quantification of protein expression
levels by Western blotting using Image J software. Differences with
*P < 0.05 were considered statistically significant. (J) Hierarchical
cluster analysis of gene expression based on log ratio RPKM data. (K)
Differences in the gene expression profiles of 2D and SP cultures.
We next investigated the global gene expression of 2D and SP cultures
on D3 using RNA-seq. The Pearson correlation heatmap revealed that SP
cultures showed a drastically different gene expression pattern than 2D
cultures (Fig. [110]S6). To identify differentially expressed genes
(DEGs), these data were subjected to hierarchical clustering using the
Pearson correlation as the distance metric (Fig. [111]4J). Compare to
2D libraries, 1447 genes were up-regulated and 1220 genes were
down-regulated in the SP libraries by edgeR analysis (Fig. [112]4K).
The GO enrichment analysis results showed that up-regulated DEGs were
significantly enriched in biological processes (BP), including
regulation of metabolic process, gene expression and biosynthetic
processes (Fig. [113]S7); the down-regulated DEGs were significantly
enriched in cell division, migration, mitotic cell cycle, organelle
fission and other functions (Fig. [114]S8). This may explain the low
level of expression of the cell proliferation marker Ki67 in SP. For
molecular function (MF), the up-regulated DEGs were enriched in ion
binding, protein kinase activity, cAMP response element binding and
AMP-activated protein kinase activity (Fig. [115]S7), and the
down-regulated DEGs were enriched in ATP binding, protein binding,
anion binding and other functions (Fig. [116]S8), indicating that SP
promotes the pump function of CECs. In addition, GO cell component (CC)
analysis indicated that the up-regulated DEGs were significantly
enriched in the cell part, organelles, and lumen (Fig. [117]S7) and
that down-regulated DEGs were enriched in genes related to cell
division, the cytoskeleton, ECM, cell-substrate junctions, and other
functions (Fig. [118]S8). To identify the biological pathways that are
active, we mapped the detected genes to reference pathways in the Kyoto
Encyclopedia of Genes and Genomes (KEGG) (Fig. [119]S9).
Based on the results described above, small spheroids cultured for 3
days were directly reseeded on the glass in the perfusion system. Many
of the cells in the spheroids migrated onto the culture glass from the
adherent CEC spheroids. The spheroids disappeared, and the monolayer
CECs completely covered the glass after D7. The representative
immunofluorescence images shown in Fig. [120]5 demonstrate that CECs in
both the static and perfusion systems were all positive for N-cadherin
and vimentin on D5. However, the CECs in the static monolayer culture
displayed an irregular, enlarged pattern, whereas CECs in the perfusion
spheroid culture were arranged in confluent monolayers and were
polygonal in shape with more compact and contact-inhibited features. It
is interesting to note that the expression of N-cadherin in the static
monolayer culture appeared as cytoplasmic diffuse staining around the
nucleus, whereas N-cadherin in the perfusion spheroid culture was
obviously located in the cytoplasmic membrane. The results demonstrated
that perfusion spheroid culture promotes CEC cell-cell contact of the
adherens junction (AJ)-related protein N-cadherin at the lateral cell
borders. Taken together, the results of our characterization of
spheroid-derived and 2D culture-derived CECs show that spheroid culture
increases specific corneal endothelial expression, resulting in better
maintenance of untransformed corneal endothelium in CEC spheroids in
static culture. The use of biomimetic platforms that combine the use of
the perfusion system and 3D spheroid culture impedes EMT in CECs,
enhances CEC cell-cell contact, and promotes the generation of a
healthy corneal endothelial layer.
Figure 5.
Figure 5
[121]Open in a new tab
Phenotypic expression of CECs in the static and perfusion systems.
Immunofluorescence staining for N-cadherin and vimentin in 2D- and
SP-derived CECs in the static and perfusion systems. Cell nuclei were
counterstained with DAPI. Scale bar: 50 µm.
The perfusion system and 3D spheroid culture promote the growth of
tissue-engineered CECs on decellularized corneal scaffolds
H&E staining showed that cells were eliminated in our decellularized
bovine cornea (Fig. [122]S10B,C), whereas normal bovine corneas contain
corneal epithelial, stromal and endothelial cells (Fig. [123]S10A). SEM
evaluation showed the smooth surfaces of Bowman’s layer
(Fig. [124]S10D) and Descemet’s membrane layer (Fig. [125]S10F) as well
as the rough surfaces of the acellular stromal lamellae
(Fig. [126]S10E). Those results show that the cells of the cornea were
removed using our short-term chemical-frozen decellularization.
Based on our characterization of CECs obtained using the perfusion
system and 3D spheroid culture, we constructed tissue-engineered
corneal endothelial layers on decellularized corneas under the same
conditions. Small spheroids cultured for 3 days were reseeded on the
Descemet’s membrane surface of decellularized corneas. After incubation
for 1 week under static conditions to allow the spheroids to adhere
firmly to the decellularized corneas, they were transferred to
perfusion culture for 1 week. SEM images revealed that the spheroids
disappeared and that a monolayer of spheroid-derived CECs completely
covered the surface of the decellularized corneas. Interestingly,
spheroid-derived CECs maintained in the static condition were arranged
in flat monolayers with rare extracellular matrix (ECM) and microvilli
distributed over the smooth surface of the cell layer (Fig. [127]6A),
while the monolayer derived from spheroids in the perfusion system was
characterized by rough surface topography with many ECM fibres and more
microvilli (Fig. [128]6B). Thus, the perfusion spheroid culture in
conjunction with decellularized corneal scaffolds promoted the
generation of CEC monolayers and neo-synthesized ECM formation.
Figure 6.
Figure 6
[129]Open in a new tab
CEC spheroids cultured on decellularized corneal scaffolds in the
static and perfusion system. SEM images of CECs on decellularized
corneal scaffolds in the static (A) and perfusion (B) systems.
The perfusion system and 3D spheroid culture promote tissue engineering of
CECs on collagen sheets
The ability to expand CECs on collagen sheets provides an opportunity
to generate suitable cells for therapeutic applications. As a first
step in evaluating this potential, we tested the properties of collagen
sheets. Collagen gels were rehydrated and converted into flat
transparent collagen sheets 200 ± 50 µm in thickness (Fig. [130]7A,B).
The stress-strain assay showed that the curves for each sample (n = 3)
were clustered and that all formulations were linearly elastic up to
40% strain (Fig. [131]7C). To evaluate potential changes in the
senescence of CECs grown on collagen sheets, we conducted a senescence
assay of the passaged cells. First, we found that CECs expanded on TCPS
formed monolayers of significantly higher density at passage 3 (P3)
(664 ± 35 cells/mm^2) and passage 5 (P5) (196 ± 32 cells/mm^2) on D6
than CECs expanded on collagen sheets at P3 (246 ± 64 cells/mm^2) and
P5 (89 ± 33 cells/mm^2) (Fig. [132]7D). However, as assayed by SA-β-gal
staining, senescence was significantly decreased in CECs at P3 and at
P5 on collagen sheets on D6 compared with those on TCPS (Fig. [133]7E).
The percentages of SA-β-Gal-positive cells at P3 on TCPS and on
collagen sheets were (6.50 ± 1.73)% and (2.58 ± 0.16)%, respectively.
Moreover, serial passage dramatically increased the percentage of
SA-β-Gal-positive cells at P5 on TCPS (24.39 ± 1.82)% and on collagen
sheets (8.57 ± 1.02)% (Fig. [134]7F).
Figure 7.
Figure 7
[135]Open in a new tab
Characterization of the flat collagen sheet and the perfusion system
for corneal tissue engineering. (A) Photograph of a flat collagen
sheet. (B) Representative section of the collagen sheet. (C) Tensile
stress-strain curve for each sample. (D) Cell density was calculated
based on the total number of cells in each square millimetre. (E) Cells
at different passages on TCPS and on collagen sheets were stained to
detect SA-β-Gal activity. Scale bar: 100 μm. (F) The senescence level
of the CECs was evaluated based on the ratio of SA-β-Gal-positive cells
to total cells. (G) SEM images of the collagen sheet prepared under
vitrification conditions and representative images of CECs on collagen
sheets in static and perfusion culture. Differences with *P < 0.05 were
considered statistically significant.
We used small spheroids cultured for 3 days and reseeded them on
collagen sheets. After incubation for 1 week in the static condition,
they were maintained under perfusion culture for another week. CEC
spheroids disappeared, and the monolayer of spheroid-derived CECs on
the surface of the collagen sheet in the perfusion culture reached 100%
confluence, while their counterparts on the collagen sheet in static
culture reached approximately 80–85% confluence. Immunofluorescent
staining showed that vimentin, N-cadherin, AQP1 and F-actin were
expressed in spheroid-derived CECs on collagen sheets both in the
static system and in the perfusion system. N-cadherin and F-actin were
arranged in polygonal and hexagonal patterns indicative of lateral cell
border staining (Fig. [136]S11). SEM images showed a relatively smooth
surface on the collagen sheet. However, images from higher
magnifications revealed that the collagen sheet displayed a rough
surface with a series of collagen fibres arrayed irregularly
(Fig. [137]7G, top panel). In addition, SEM images obtained at low
magnification showed that spheroid-derived CECs in static culture
manifested as a crack-like monolayer, whereas images from higher
magnifications showed that spheroid-derived CECs covered the collagen
sheet surface with loose cell-cell junctions (Fig. [138]7G, middle
panel). In the perfusion system, however, spheroid-derived CECs
completely covered the collagen sheet and displayed tight intercellular
junctions and numerous microvilli. Higher magnification showed that the
apical surfaces of spheroid-derived CECs on the collagen sheet were
covered by an extensive newly synthesized ECM (Fig. [139]7G, bottom
panel). These results demonstrate that our thin collagen sheet is
transparent and has a certain mechanical strength. CECs expanded on the
collagen sheet show lower percentage of senescent cells than CECs
expanded on TCPS. At the same time, the collagen sheet enhances the
monolayer growth and promotes ECM formation by spheroid-derived CECs
maintained in the perfusion system with continuous supplemental
nutrition.
Tissue-engineered CECs grown on curved collagen sheets in the perfusion
system under controlled pressure
To obtain a CEC biomimetic environment to generate engineered tissue,
we used a curved scaffold and a perfusion system with controlled
pressure. To construct the biomimetic scaffold, we developed a collagen
sheet with spherical curvature, produced with a spherically curved
mould 8 mm in diameter. The collagen sheet was transparent and
displayed spherical curvature (Fig. [140]8A). In addition, we were able
to control and measure the pressure in the perfusion system, as shown
in the schematic illustration in Fig. [141]S1A. To obtain an
environment that mimics physiological intraocular pressure, we
maintained the pressure at 15 mmHg during the perfusion culture period
(Fig. [142]S1B). DAPI staining of cryosectioned curved collagen sheets
with 2D- or SP-derived CECs in static or perfusion culture at 15 mmHg
pressure showed the presence of a curved CEC monolayer after 1 week of
culture (Fig. [143]8B). Whole-mount DAPI and anti-F-actin staining
(blue fluorescence and green fluorescence, respectively) also
demonstrated that these CECs formed a monolayer at the 1-week time
point (Fig. [144]8C). Our findings show that a spherically curved
collagen sheet and perfusion system with controlled pressure may be
used to construct biomimetic tissue-engineered corneal endothelial
layers in vitro.
Figure 8.
Figure 8
[145]Open in a new tab
Tissue-engineered CECs on curved collagen sheets in the perfusion
system under controlled pressure. (A) Photograph of a curved collagen
sheet. (B) Immunohistochemical analysis of cryosectioned spherically
curved collagen sheet by DAPI. Scale bar: 50 µm. (C) Representative
fluorescence microscopy images of CEC monolayers stained for F-actin
and with DAPI on spherically curved collagen sheets in the static and
perfusion systems.
Discussion
The expansion of CECs for tissue engineering is usually performed under
2D static conditions. However, although conventional cultured CECs have
been observed to undergo one or two population doublings in vitro, they
rapidly become senescent or undergo EMT to a fibroblastic
phenotype^[146]36. Along with the understanding that the entire context
of a cell’s microenvironment is important in tissue engineering, it is
generally realized that it is necessary to reconstruct dynamic in vivo
environments in in vitro experimental systems. Recently, the
development and use of biomimetic platforms in which the presence and
levels of regulatory molecules (oxygen and nutrients), other cells (3D
context and cell-cell contacts), ECM (topology and stiffness), and
physical factors (flow shear and compression) are controlled are
increasingly being applied to the generation of engineered
tissues^[147]38. The use of dynamic bioreactors could influence major
cellular events such as differentiation, proliferation, viability and
the cell cycle. To improve the environment in which cells and
developing tissues can be maintained under in vitro conditions, the
MINUSHEET perfusion culture system was developed. In this perfusion
system, a constant flow of culture medium provides seeding cells and
tissues with a constant supply of fresh nutrients and respiratory
gases, making it possible to produce a variety of specialized tissues
of the high cell biological quality that is urgently needed in tissue
engineering^[148]18. The use of a perfusion system benefits cells by
providing a continuous supply of nutrients and constant removal of
metabolic waste. Therefore, it is suitable for the generation and
long-term maintenance of various types of specialized tissues. Wu and
co-investigators reported that perfusion culture can be used to
reconstruct an auto-lamellar cornea with favourable morphological
characteristics and satisfactory physiological function^[149]39. Wang
et al. reported that the density of bone marrow mesenchymal stem cells
(MSCs) in the perfusion system used in their study remained at a high
level over the period of induction of hepatocytic differentiation and
was almost two-fold the density of cells in the static system at the
end of the induction period. For cells in scaffolds, perfusion
induction was more effective than static induction^[150]40. Many
published papers report that dynamic perfusion culture contributes to
better growth of various cells, including endothelial progenitor
cells^[151]41, human endometrial stromal cells^[152]42 and dermal
fibroblasts^[153]43, than static culture. Moreover, perfusion
bioreactors have been introduced and shown to enhance cellular access
to oxygen and nutrients. The use of perfusion-based bioreactor culture
can significantly improve the access of cells to oxygen, enhancing the
viability and contractility of the engineered tissues^[154]44.
The results of our study are consistent with previous reports. First,
we showed that the dissolved oxygen concentration in perfusion culture,
as measured by an optical fibre oxygen fluorescence microsensor, is
higher than that in static culture. The use of such microsensors offers
a new methodology for the measurement of dissolved oxygen based on
fluorescence quenching^[155]45–[156]47. Fluorescence microsensors offer
some advantages over the traditional Clark electrode^[157]48, such as
their small size and their suitability for remote sensing and multiplex
microsensor networking. Optical fibre oxygen fluorescence microsensors
do not consume oxygen and can be used to measure oxygen in both the gas
and liquid phases. Their potential rapid response time makes them
suitable for continuous dynamic pO2 monitoring^[158]49. Second, by
comparing the survival and proliferation of CECs in perfusion and
static cultures, we showed that the perfusion condition increased the
percentage of EdU-positive nuclei and cells entering the S phase and
resulted in a higher efficiency of CEC expansion and further
development of the tissue-engineered CECs. Therefore, the
microenvironment provided by the perfusion system was more favourable
than that provided by the static system for CEC expansion in vitro.
Third, we found that the perfusion system helps maintain the AQP1 and
ATP1A1 phenotypic marker characteristics of CECs. The perfusion
environment can support the expression of the CEC phenotypic markers
AQP1, vimentin and N-cadherin, even in CECs passaged on 2D culture TCPS
after perfusion culture. Aquaporin 1 (AQP1) is the major water channel
isoform in CECs and is essential for CEC proliferation and migration
via the ERK signalling pathway^[159]50. The ATP1A1 pump is an integral
membrane protein expressed in the basolateral membrane of CECs that is
responsible for continuously pumping Na^+ out of the cell and K^+ into
the cell, utilizing energy from ATP. As such, it plays important roles
in preserving corneal dehydration and transparency^[160]51. Vimentin is
a major component of the mesenchymal cell cytoskeleton and is also
expressed in human CECs^[161]52. Due to its neuroectodermal origin, the
corneal endothelium represents a special case with respect to the
expression of cadherin. N-cadherin, but not E-cadherin, plays a central
role as an adherent junction protein in human CECs^[162]53.
Interestingly, from SEM microscopic studies, we found that CECs in
perfusion culture exhibited rough topography with many microvilli,
unlike the smooth surface profiles exhibited by cells maintained in
static culture. Based on our results, we propose that the use of a
perfusion system that provides continual supplementary nutrition
promotes CEC proliferation and phenotypic maintenance. Additionally,
augmenting the concentration of dissolved oxygen in the perfusion
system may be involved in such an effect.
Spheroid size influences cell viability and phenotypic outcome^[163]54.
In the present study, QPCR revealed significant up-regulation of the
endothelial markers ATP1A1, AQP1 and N-cadherin, the proliferation
marker Ki67 and the mesenchymal markers vimentin and α-SMA in small
spheroids compared to spheroids with larger diameters. It is possible
that the cells in small spheroids are able to obtain more nutrients and
oxygen and that as a result they show better growth status. Huang et
al. found that dermal papilla (DP) spheroids of larger size displayed
slightly decreased viability on D5^[164]55. A large spheroid core is
often deficient in oxygen and/or nutrients and shows excessive
accumulation of catabolites and low pH^[165]56, [166]57. We found that
CECs in spheroid culture displayed enhanced expression of the corneal
endothelial marker ATP1A1. In addition, the enhanced gene expression of
ATP1A1 in CEC spheroids was coincident with the results of GO
enrichment of target genes for molecular function obtained by RNA-Seq
analysis. The results of the latter assay indicated that protein kinase
activity, cAMP response element binding and AMP-activated protein
kinase activity were significantly up-regulated. Wigham et al.
demonstrated that increased concentrations of cAMP activate the ATP1A1
activity and enhance the pump activity of CECs^[167]58.
QPCR assays of CEC spheroids revealed significant down-regulation of
gene expression for the proliferation marker Ki67. RNA-Seq analysis
also confirmed the enhanced proliferation of CEC spheroids. GO
enrichment of target genes in CEC spheroids for biological process
demonstrated that down-regulated DEGs were significantly enriched in
cell division, mitotic cell cycle, organelle fission and other
functions. In the cellular component, down-regulated DEGs were
significantly enriched in cell-division-related genes (spindle,
kinetochore, condensed chromosome and others). The pathway enrichment
analysis also demonstrated that the cell cycle was significantly
down-regulated in CECs in spheroids compared with CECs in 2D culture.
These results suggest that CECs in spheroids have a low rate of
proliferation and cell division, a finding that is consistent with
other reports. For example, Cheng et al. found that adipose-derived
stem cells (ASCs) almost cease to proliferate upon spheroid formation,
although later expansion of dissociated spheroid cells in monolayer
cultures exhibited higher proliferative activity^[168]59.
EMT in CECs is the process by which endothelial cells lose their
specific markers and acquire mesenchymal characteristics. EMT usually
leads to fusiform morphology, transformed phenotype, and abnormal
function of cultured CECs. Thus, it is clear that cultured CECs that
have undergone EMT will not be of use in regenerative medicine. For the
use of cultured CECs in regenerative medicine to be practical, EMT must
be antagonized during the culture process^[169]53. Recently, spheroid
culture has gained increased attention for its ability to improve
cellular pluripotency^[170]60, functional maintenance^[171]61 and 3D
bio-printing^[172]62, [173]63. Spheroid culture enables cells to
assemble and interact under native conditions and to form biomimetic 3D
environments that enhance high-density growth, cell-cell contact and
cell-matrix interactions^[174]64. Therefore, we used spheroid culture
as a biomimetic means to improve CEC growth and to further construct
tissue-engineered corneal endothelial layers. We found that CEC
spheroid culture can reverse the gene and protein expression levels of
the EMT-related markers vimentin and α-SMA. The results of RNA-Seq
analysis of differentially expressed genes (DEGs) of CECs in 2D and SP
culture are consistent with these characteristics. GO enrichment of
target genes from CEC spheroids demonstrated that down-regulated DEGs
in the cellular component were significantly enriched in the
cytoskeleton, ECM, cell-substrate junction, and other functions. We
propose that spheroid culture of CECs decreases the expression of genes
related to the actin cytoskeleton, ECM components and CEC-ECM adhesion,
components that are closely associated with the regulation of cellular
activities such as EMT. Previous studies reported that EMT is involved
in the formation of cellular protrusions and the reorganization of the
actin cytoskeleton. Changes in the ECM are able to induce EMT.
Increased ECM density can promote EMT by enhancing cell–matrix
adhesions and weakening cell–cell adhesion^[175]65, [176]66. In our
study, the pathway enrichment scores obtained by RNA-Seq for 2D and SP
CEC cultures showed changes in the signalling pathways involving
thyroid hormone and Hippo. Lin et al. showed that thyroid hormone
(T3)/thyroid hormone receptor (TR) signalling up-regulated the
expression of cell EMT-related proteins, MMP9, p-mTOR, p-STAT3, p-AKT
and p-ERK1/2^[177]67. The downstream transcriptional co-activators of
the Hippo pathway, YAP and TAZ, have been implicated in both
physiological EMT and pathological EMT^[178]68. From the above data and
reports, we deduce that the underlying mechanism through which EMT is
impeded in CEC spheroids involves down-regulation of the thyroid
hormone and Hippo signalling pathways.
CECs cultured as spheroids in the perfusion system appeared as
confluent monolayers with a polygonal shape, a more compact appearance
and more AJ-related N-cadherin protein compared with the lateral cell
border pattern. Our data revealed, for the first time, that in vitro
biomimetic platforms using a perfusion system and 3D spheroid culture
hinder EMT in the corneal endothelium. Moreover, CEC spheroids
displayed significant down-regulation of gene expression for the
proliferation marker Ki67, indicating a lower proliferation of the CEC
cells in spheroids than in 2D culture. However, the perfusion system
was able to promote CEC proliferation, indicating that perfusion
culture can overcome the minor drawback of somewhat slow growth
associated with spheroid culture. Therefore, use of the perfusion
system in conjunction with spheroid culture improves CEC monolayer
formation.
To explore whether spheroid culture in conjunction with the perfusion
system has potential utility in tissue engineering, we constructed CEC
layers. The use of substrates not only provided mechanical support
during the transplantation of ex vivo-engineered CEC sheets but also
created a favourable microenvironment for cellular activity^[179]6.
Perfusion bioreactors have been shown to enhance the access of cells to
oxygen and nutrients as well as the homogeneity of neo-synthesized ECM
in 3D scaffolds^[180]69, [181]70. The results of our study are
consistent with these findings. We used a natural decellularized
corneal matrix and collagen sheets as scaffolds for CEC tissue
engineering. We found that the combination of perfusion spheroid
culture and decellularized corneal scaffolds or collagen sheets
promotes the formation of neo-synthesized ECM, microvilli and CEC
monolayers. We also tested the senescence of the CECs on the scaffolds.
First, we found that it is difficult to observe SA-β-gal staining in
CECs on decellularized corneal scaffolds because hydration of the
decellularized corneal matrix during culture interferes with the clear
microscopic imaging of stained cells. In addition, there are some
difficulties associated with SA-β-gal staining and evaluation of CEC
spheroids at P3 and P5. Therefore, we only compared the senescence of
dissociated CECs on collagen sheets with that of cells cultured on
TCPS. At P3 and P5, CECs on collagen sheets exhibited less staining for
the senescence marker SA-β-gal than CECs on TCPS. Collagen is the major
protein constituent of the ECM and is a commonly used component in
tissue scaffold construction^[182]71. Our transparent collagen sheet
can also be made into a spherically curved shape to fit the corneal
curvature. Zhang et al. created a cornea-shaped scaffold using
collagen^[183]72. Yoshida et al. developed a transparent porcine
atelocollagen with a curved shape and adequate mechanical strength.
They showed that the curved shape is important for adhesion of the CEC
carrier to the posterior surface of the cornea^[184]33. Kimoto et al.
also demonstrated that a spherically curved gelatin hydrogel sheet with
CECs achieves close adhesion to the posterior corneal surface^[185]73.
In the present study, we showed that curved tissue engineering of the
corneal endothelial layer can be accomplished in the perfusion system
at 15 mmHg pressure. The normal range of intraocular pressure is
10–21 mmHg. Therefore, we can construct biomimetic tissue-engineered
corneal endothelial layers from curved collagen sheets in a perfusion
system that is maintained at physiological intraocular pressure.
In this study, we established biomimetic platforms to promote the
construction of tissue-engineered corneal endothelial layers in vitro.
First, we used a perfusion system that provides a higher dissolved
oxygen concentration in the medium and enhances CEC proliferation
compared to static culture. Second, we used a spheroid culture, which
resulted in a lower EMT change. Third, we showed that use of the
perfusion system in conjunction with a spheroid culture promotes
monolayer formation by untransformed CECs with normal cell-cell contact
junctions. Fourth, perfusion CEC spheroid cultures on decellularized
corneal scaffolds and collagen sheets were shown to promote the
generation of CEC monolayers and neo-synthesized ECM formation. Fifth,
we showed that perfusion CEC spheroid cultures maintained on curved
collagen sheets under a controlled physiological intraocular pressure
can generate CEC monolayers. In summary, our comprehensive biomimetic
platforms involving a dynamic perfusion system with or without a
controlled pressure, spheroid culture, decellularized corneal
scaffolds, and flat or curved collagen sheets provide a suitable
microenvironment for the maintenance of the normal CEC physiological
context of CECs, thereby improving corneal endothelial tissue
engineering and regeneration.
Methods
Ethics statement
Bovine eyes were acquired from a local abattoir (Shipai, Guangzhou,
China). The study was conducted according to the Association for
Research in Vision and Ophthalmology Statement on Using Animals in
Ophthalmic and Vision Research and the guidelines of the Animal
Experimental Committee of Jinan University, Guangzhou, China.
Isolation and culture of bovine CECs
Bovine CECs were cultured as previously described^[186]74. Briefly, the
corneal tissues were washed three times with phosphate-buffered saline
(PBS) containing 2% penicillin-streptomycin and 50 μg/ml gentamicin.
The Descemet’s membrane was peeled away from the posterior surface of
the tissue with a sterile surgical forceps under a dissecting
microscope. The strips were incubated in trypsin/EDTA at 37 °C for
8–10 min. The cells were centrifuged (300× g, 5 min), seeded into a
6-well tissue-culture-treated polystyrene (TCPS) plate and cultured in
low-glucose DMEM supplemented with 10% FBS and 1%
penicillin-streptomycin in a 37 °C incubator under 5% CO[2].
Culture of CECs in the perfusion system
Bovine CECs were seeded at a density of 0.7 × 10^4 cells/well on glass
slides mounted in tissue carriers and allowed to remain for 1 day. The
CECs on the glass slides in the tissue carriers were then directly
transferred into the MINUSHEET flow perfusion culture container for an
additional 5 days of culture. The MINUSHEET perfusion system is
illustrated in Fig. [187]1A,B. Continuous perfusion with the culture
medium was accomplished using a slowly rotating peristaltic pump
(ISMATEC, IPC N8, Wertheim, Germany) that was able to deliver pump
rates of 1 ml per hour. During perfusion culture, CECs on the glass
slides were always exposed to fresh medium from a storage bottle; the
waste medium was collected in a separate waste bottle and was not
re-circulated. The morphology of cells transferred out of the perfusion
culture container at various times was observed using phase-contrast
microscopy. To compare the proliferation and viability of cells
cultured in the perfusion system with those of cells cultured under
static conditions, the cells were dissociated with 0.25% trypsin/EDTA
and counted using a hemocytometer at various intervals during the 5-day
period.
Oxygen measurement
To measure the change in the dissolved oxygen concentration in the
culture medium of the perfusion system, we used an optical fibre oxygen
fluorescence microsensor at an atmospheric pressure of 101.3 kPa and a
temperature of 297.13 K. Figure [188]1C shows a schematic of the sensor
system. Figure [189]1D shows a photographic image of the microsensor.
In this system, the excitation provided by an LED with a central
wavelength of 405 nm is fed to the microsensor, and the emitted
luminescence from the microsensor is transmitted to the spectrometer
through the fibre coupler. The spectrum of the luminescence is detected
by the spectrometer (Ocean Optic; USB2000).
The fluorescence intensity is directly related to the oxygen
concentration according to the Stern–Volmer equation ([190]1)^[191]75:
[MATH:
I0I=1+K⋅
[O2] :MATH]
1
where I [0] and I represent the steady-state luminescence intensities
in the absence and presence of O[2], respectively, K is the
Stern–Volmer quenching constant, and [O[2]] is the O[2] concentration.
In liquid, the O[2] concentration is the dissolved oxygen
concentration.
On D3 of CEC culture, the fibre optic microsensor probe was positioned
in the medium of the static and dynamic cell cultures successively for
75 s. For comparison, the dissolved oxygen in the unused medium and in
the waste medium of the perfusion were measured at the same time. The
relative fluorescence intensity was used to analyse the dissolved
oxygen concentration. Prior to each measurement, the fibre optic probe
was washed with H[2]O to avoid cross-contamination. To test the
stability of the microsensor, the relative fluorescence intensity with
the microsensor positioned in H[2]O was also measured.
EdU labelling assay
Bovine CECs were seeded on glass slides at 0.7 × 10^4 cells/well and
were allowed to attach for 1 day. Next, CECs were continually cultured
in the perfusion or static system for another 3 days. The EdU labelling
assay was conducted according to the manual of the EdU
labelling/detection kit (Ribobio, Guangzhou, China). Samples were then
observed and photographed under a fluorescence microscope. The
percentage of EdU-positive cells was calculated, respectively, from
five random fields in three wells.
Flow cytometry
Flow cytometry was used to determine the cell cycle distribution in
bovine CECs. Briefly, CECs were seeded on glass slides at 5 × 10^4
cells/well and allowed to attach for 1 day. The CECs were then
continually cultured in the perfusion system or the static system for
another 3 days. The cell cycle distribution was then analysed by PI
flow cytometry (FACS Calibur, BD, USA) as previously described^[192]74.
Immunofluorescence
After fixation in 4% (wt/vol) paraformaldehyde for 15 min, the CECs
were washed three times with PBS, permeabilized with 0.1% Triton X-100
in PBS for 10 min and incubated with PBS containing 5% BSA for 30 min.
The cells were then incubated overnight at 4 °C with primary
antibodies, including rabbit polyclonal anti-ATP1A1 antibody (1:200;
Santa Cruz, USA), rabbit polyclonal anti-AQP1 antibody (1:200; Santa
Cruz, USA), anti-N-cadherin (1:200; BD, USA), and anti-vimentin (1:200;
Proteintech, USA), followed by washing three times in PBS and
incubation with secondary antibody for 1 h at room temperature. For
F-actin staining, FITC/phalloidin (Yeasen, China) was used according to
the manufacturer’s instructions. For collagen sheet staining, single
CECs or CEC spheroids on flat or spherically curved collagen sheets
were stained by DAPI for 10 min. The collagen sheet was then cut into
four quadrants. Imaging was performed using a fluorescence stereo
microscope (M165 FC) and an EL6000 external light source (Leica
Microsystems).
SA-β-Gal staining and cell viability assays
The detection of senescence-associated β-galactosidase (SA-β-Gal)
activity was performed using a commercial senescence staining kit
(Beyotime Biotechnology, China) according to the manufacturer’s
instructions. Briefly, single CECs were seeded into 12-well TCPS
prepared with and without coating with collagen sheets and cultured for
6 days. The cells were then fixed in SA-β-Gal fixing solution for
15 min, stained with working solution overnight at 37 °C, and imaged
using phase-contrast microscopy. The cell viability assay was performed
with a viability/cytotoxicity assay kit (Life Technologies, USA) for
live/dead cells according to the manufacturer’s instructions.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis and
quantitative polymerase chain reaction (qPCR)
Total RNA from CECs derived from spheroids and monolayer cultures was
extracted using TRIZOL reagent. The cDNA was synthesized using a
reverse-transcriptase reagent kit (TOYOBO, Japan) according to the
manufacturer’s instructions. Gene-specific primers were synthesized by
Sangon Biotech (China); the sequences of the primers are listed in
Table [193]S1. For qPCR experiments, gene expression was analysed by
real-time PCR (Bio-Rad CFX96TM, USA) with two or three replicates per
sample. The GAPDH gene was used as an internal control. Expression
changes in the gene transcripts for each sample were calculated using
the 2^−∆∆Ct method. The results from three independent experiments were
statistically analysed.
Scanning electron microscopy (SEM)
SEM was used to observe the surface ultrastructure of acellular corneal
scaffolds and collagen sheets with and without CEC spheroid seeding.
Briefly, the samples were fixed in 2.5% glutaraldehyde for 2 h and
washed 3 times for 15 min each time in PBS. After dehydration in
increasing concentrations of ethanol (70, 80, 90, 100, 100, and 100%)
for 10 min each time, the specimens were transferred to isoamyl acetate
for 30 min, subjected to critical point drying, coated with a
gold-palladium alloy and viewed by SEM on a JSM-T300-SEM instrument
(JEOL Technics Co. Ltd., Tokyo, Japan).
Generation of cell spheroids in 3D Petri dishes
CEC spheroids were established in agarose 3D Petri dishes as described
in our previous study^[194]29. Briefly, cell suspensions containing
precisely measured numbers of cells were carefully seeded into the
microwells of agarose dishes and were allowed to stand for 10 min to
promote cell deposition. The cells were then incubated at 37 °C in a 5%
CO[2] incubator. The medium was changed every two days. CEC spheroids
were formed, followed by cell aggregation and self-assembly; the
spheroids were subsequently collected by flushing them out of the
microwells gently with a pipette for further culture and experiments.
RNA-Seq and analysis
Total RNA was extracted from 2D and SP cultures on D3 using TRIZOL
reagent. After library construction and sequencing, we calculated reads
as the number of reads per kilobase of exon model per million mapped
reads (FPKM) to obtain normalized gene expression levels. We mapped the
original RNA-seq to the reference transcriptome sequence using FANSe2
as previously described^[195]76. The correlation coefficients between
gene expression levels were calculated and plotted as a correlation
heatmap. Gene ontology (GO) analysis was performed using TopGO software
(version 2.18.0), and comparisons between the two groups were made used
Fisher’s exact test. Pathway enrichment analysis was primarily based on
the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. KOBAS
software (kobas2.0-20150126) was used, and comparisons between the two
groups were made using the hypergeometric test.
Preparation of decellularized corneal scaffolds
Decellularized corneal scaffolds were established as previously
described with minor modifications^[196]29. Briefly, bovine corneal
samples were excised from fresh eyeballs and rinsed 3–5 times in PBS
containing gentamicin sulphate. The corneal epithelium, including the
posterior corneal stroma and the anterior corneal stroma, was stripped
away with sterile surgical forceps and cut into lamellae (approximately
0.35 mm thick and 8.0 mm in diameter) using a biopsy punch under
sterile conditions. The lamellae were immersed in a solution containing
0.5% Triton X-100 and 20 mM NH[4]OH for 30 min. After three additional
rinses with PBS, the lamellae were preserved at −80 °C for 3 days and
then transferred to 100% glycerol and stored at 4 °C. Prior to use, the
decellularized corneal scaffolds were rehydrated in PBS.
Immunoblotting
Total protein was extracted using RIPA lysis buffer containing the
protease inhibitor PMSF (Beyotime, China). The proteins in the extract
were separated by SDS-PAGE and transferred to PVDF membranes
(Millipore, USA). Next, the membranes were incubated overnight at 4 °C
with the following primary antibodies: rabbit polyclonal anti-vimentin
(1:2000; Proteintech, USA); rabbit polyclonal anti-AQP1 (1:3000; Santa
Cruz Biotechnology, USA); rabbit polyclonal anti-ATP1A1 (1:500; Santa
Cruz Biotechnology, USA); and rabbit polyclonal anti-GAPDH (1:3000;
Proteintech, USA). After washing, the membranes were incubated with
HRP-conjugated secondary antibodies at room temperature for 1 h, and
immunostained bands were visualized using enhanced chemiluminescence
detection reagents (Pierce, Rockford, IL, USA).
Preparation of collagen sheets and the stress-strain assay
Collagen sheets were prepared as previously described with
modifications^[197]77. Briefly, a ring-shaped sterilized nylon membrane
with an inner and outer diameter of 23 and 33 mm, respectively, was
inserted into a polystyrene culture dish with a diameter of 35 mm.
Equal volumes of 0.5% type-I collagen solution and culture medium
[DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin] were
mixed, and 3.0 ml of the mixture was poured into the culture dish. The
culture dish was incubated at 37 °C to complete gelation of the
collagen. The collagen gel was then aseptically dried and converted
into a flat collagen sheet. To imitate the posterior corneal curvature,
a spherically curved mould with a diameter of 8 mm was placed on the
collagen gel; this resulted in curvature of the collagen gel, which was
then further dried to form a curved collagen sheet. The nylon frame
provided support for the collagen gel, making it possible to easily
separate the collagen sheet with tweezers. A dynamic mechanical
analyser (Q800, TA instrument, USA) was used to evaluate the
stress-strain on the collagen sheet in the horizontal direction. The
results obtained from three independent specimens were statistically
analysed.
Examination of frozen sections
The CEC sheets on the spherically curved collagen sheets were fixed in
4% (wt/vol) paraformaldehyde for 15 min and washed in PBS. The
structures were incubated at 4 °C in OCT compound solution for at least
4 h and then embedded in OCT compound at −80 °C. Frozen sections were
cut at a thickness of 10 μm, placed on microscope slides and stained
with DAPI. The sections were then stained and observed under a
fluorescence microscope.
Determination of the real-time perfusion pressure
Real-time perfusion pressure values were measured using a manometer. To
create a hydrostatic pressure environment, a perfusion system was
designed as shown in Fig. [198]S1A. The system consists of a storage
bottle, a peristaltic pump, a culture container, a waste bottle and a
pressure manometer (BENETECH, GM-510). The system is completely closed,
and the pressure can be maintained at a relatively stable level by
adjusting the height of the system.
Statistical analysis
Values are expressed as the mean ± SD of values obtained from three to
six samples. Statistical analysis between two groups was carried out
using Student’s t test; comparison among three groups was determined by
one-way ANOVA (SPSS16.0). P < 0.05 was considered to be statistically
significant.
Electronic supplementary material
[199]Supplementary Information^ (3.1MB, pdf)
Acknowledgements