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J. Funct. Biomater. 2015, 6, 917-945; doi:10.3390/jfb6030917
Journal of
Functional
Biomaterials
ISSN 2079-4983
www.mdpi.com/journal/jfb
Review
Substrates for Expansion of Corneal Endothelial Cells towards
Bioengineering of Human Corneal Endothelium
Jesintha Navaratnam 1,*, Tor P. Utheim 2,3, Vinagolu K. Rajasekhar 4
and Aboulghassem Shahdadfar 1
1 Department of Ophthalmology, Oslo University Hospital, Postbox 4950 Nydalen, Oslo 0424,
Norway; E-Mail: aboulghassem.shahdadfar@medisin.uio.no
2 Department of Medical Biochemistry, Oslo University Hospital, Postbox 4950 Nydalen, Oslo 0424,
Norway; E-Mail: utheim2@gmail.com
3 Department of Oral Biology, Faculty of Dentistry, University of Oslo, Postbox 1052, Blindern,
Oslo 0316, Norway
4 Memorial Sloan Kettering Cancer Center, Rockefeller Research Building, Room 1163, 430 East
67th Street/1275 York Avenue, New York, NY 10065, USA; E-Mail: Vinagolr@mskcc.org
* Author to whom correspondence should be addressed;
E-Mail: jesintha.navaratnam@medisin.uio.no; Tel.: +47-221-17-662; Fax: +47-221-19-989.
Academic Editor: Dimitrios Karamichos
Received: 13 July 2015 / Accepted: 2 September 2015 / Published: 11 September 2015
Abstract: Corneal endothelium is a single layer of specialized cells that lines the posterior
surface of cornea and maintains corneal hydration and corneal transparency essential for
vision. Currently, transplantation is the only therapeutic option for diseases affecting the
corneal endothelium. Transplantation of corneal endothelium, called endothelial keratoplasty,
is widely used for corneal endothelial diseases. However, corneal transplantation is limited
by global donor shortage. Therefore, there is a need to overcome the deficiency of sufficient
donor corneal tissue. New approaches are being explored to engineer corneal tissues such
that sufficient amount of corneal endothelium becomes available to offset the present shortage
of functional cornea. Although human corneal endothelial cells have limited proliferative
capacity in vivo, several laboratories have been successful in in vitro expansion of human
corneal endothelial cells. Here we provide a comprehensive analysis of different substrates
employed for in vitro cultivation of human corneal endothelial cells. Advances and emerging
OPEN ACCESS
J. Funct. Biomater. 2015, 6 918
challenges with ex vivo cultured corneal endothelial layer for the ultimate goal of therapeutic
replacement of dysfunctional corneal endothelium in humans with functional corneal
endothelium are also presented.
Keywords: cell sources; corneal endothelium; human corneal endothelial cells; substrates;
tissue engineering
1. Introduction
The cornea is the transparent anterior part of the eye that transmits and focuses light onto the retina.
From anterior to posterior (Figure 1), the cornea is composed of the corneal epithelium (50 μm thick),
the Bowman’s membrane (12 μm), the stroma (480–500 μm), the Descemet’s membrane (8–10 μm), and
the endothelium (5 μm) [1]. Recently, a new layer of the cornea, Dua’s layer, was also described [2].
Figure 1. Anatomy of the cornea. (a) Section of the anterior part of the eye; (b) Section of
the cornea illustrating six layers; (c) In vivo confocal microscopy image of the corneal
endothelium. Courtesy of Geir A. Qvale.
The human cornea has a thickness of 0.5–0.6 mm centrally and 0.6–0.8 mm peripherally [3]. The
horizontal diameter of an average adult human cornea is 11.7 mm and the vertical diameter is
approximately 1 mm less than the horizontal diameter. The cornea is one of the few avascular tissues in
the body. The cornea is also one of the most heavily innervated and sensitive tissues in the body, with a
density of nerve endings about 300–400 times greater than the skin [1,4], thus diseases of the cornea
may be extremely painful. It has several functions that are essential for clear vision: The integrity and
J. Funct. Biomater. 2015, 6 919
functionality of the epithelium [5] and endothelium [6], corneal shape [1], and transparency [1]. The corneal
endothelium maintains corneal transparency by regulating water content of corneal stroma. The cornea
provides approximately two-thirds of the total refractive power of the eye (Figure 2) [6]; thus, even a
small change in corneal contour may result in refractive errors.
Figure 2. The refraction of light. The cornea provides more than two-thirds of the total
refractive power of the eye. Courtesy of Geir A. Qvale.
According to the World Health Organization’s global estimation of blindness and visual impairment
in 2010, 285 million people were reported to be visually impaired [7]. Corneal diseases are the fourth
leading cause of blindness worldwide [7]. Causes of corneal endothelial disease (CED) include
endothelial dystrophies, iridocorneal endothelial syndrome, and endothelial dysfunction following
cataract surgery and corneal transplantation. Corneal endothelial disease usually presents with a gradual
onset of decreased vision. Advanced CED can cause recurrent corneal epithelial erosions, resulting in
episodes of severe pain. The corneal endothelium is derived from embryonic neural crest cells [8].
Human corneal endothelial cells (HCECs) have limited proliferative capacity in vivo and are suggested
to be arrested in the G1-phase of cell cycle [9]. In addition, age-related decrease in corneal endothelial
cell density is reported. The mean corneal endothelial cell density decreases from 3600 cells per square
millimeter (cells/mm2) at age 5 years to 2700 cells/mm2 at age 15 years [10]. Further reduction of
the central corneal endothelial density in adults is reported at the rate of 0.6% yearly with gradual change
in cell shape and size [11]. Corneal endothelial cell density below critical level of approximately
500 cells/mm2 results in corneal edema and thereby decreased visual acuity. Significant HCEC loss and
inadequate replacement of corneal endothelial cells in vivo suggest there is a lack of or inefficient cell
division. In corneal endothelial wound healing in humans, the endothelial cells adjacent to the wound
enlarge as they elongate and slide to the wound area [12]. At present, transplantation is the only available
treatment for diseases affecting the corneal endothelium. There are two main types of corneal
transplantation for CED: Penetrating keratoplasty and endothelial keratoplasty. Penetrating keratoplasty
refers to the replacement of all corneal layers of the recipient’s cornea with a donor cornea. Selective
replacement of the diseased posterior layer of the cornea is called endothelial keratoplasty [13]. The
above surgical advancements are, however, hindered by the worldwide scarcity of available healthy
donor corneas.
J. Funct. Biomater. 2015, 6 920
A considerable research effort has been put into developing alternative methods for treatment of CED.
The remaining HCECs may be stimulated to proliferate or enhance their function with topical eye
drops [14] or cell suspension injection into the anterior chamber [15,16]. Magnetic field-guided in vitro
cultivated HCEC delivery is thought to attract the cells towards Descemet’s membrane [15,17,18].
However, the possible complications of injection of cell suspension into the anterior chamber, such as
an increase in intracellular pressure due to clogging of the trabecular meshwork, should be further
investigated before human trials are initiated. Although growth factors may promote corneal endothelial
wound healing [19], it does not induce HCEC proliferation [20]. Thus, there is a clinical interest for
engineering corneal endothelium for transplantation purposes. With increasing advances in regenerative
medicine, several research groups have investigated on expansion of corneal endothelial cells and
transplantation of tissue engineered corneal endothelium in experimental animal models [21–36].
The present review focuses on emerging substrates for improved culturing of HCECs. To provide a
background for the current use of substrates, cell sources for tissue engineering of corneal endothelium
are also described.
2. Cell Sources for Tissue Engineering of Corneal Endothelium
Various efforts have been made to increase the availability of human HCEC lines. These
include immortalization of retroviral transduction by simian virus 40 (SV40) T antigen [37,38],
Cdk4R24c/CyclinD1 [39], and/or human papilloma virus 16 E6/E7 [40]. Immortalized cells increase the
risk of tumor formation, aneuploidy [41], and structural rearrangements [42]. Recently, the establishment
of untransfected HCEC line [43] and immortalization of HCECs with human telomerase reverse
transcriptase have been explored [44]. However, the limitation of methods not using transfection is
immortalization of only a subpopulation of the primary culture.
Although the HCECs have limited proliferative capacity in vivo, these cells have the ability to
proliferate under in vitro culture conditions [18,25,29–32,36,45–119]. Primary HCECs, human
HCEC lines, and stem cells have been utilized for tissue engineering of corneal endothelium.
Donor corneoscleral rims, which remain after corneal trephination for corneal transplantation, and
human cadaver corneas that are unsuitable for corneal transplantation provide sources for primary
HCECs. The age of donors used in tissue engineering of corneal endothelium varies substantially
in the literature. Primary HCEC cultures have been established from corneas from 8-week-old human
embryos [120] up to age 80 [49,62]. The proliferative response of HCECs tends to decrease in older
donors [59,64,69,75,121–123]. Interestingly, Gao et al., in 2010, were not able to demonstrate a high
proliferative rate in human fetal corneal endothelial cells [124]. Regardless of age, human corneal
endothelial cells from peripheral areas of the cornea are reported to exhibit a higher replication
competency compared to the central area [60,73,121–124]. The lower proliferative capacity of HCECs
from the central area may be due to senescence-like characteristics of central HCECs, including
stress-induced premature senescence [122]. In addition, it remains interesting to investigate if there are
potential stem like progenitors of corneal endothelium that may have more proliferative capacity to
produce more corneal endothelial cells than their progency with limited proliferative capacity in center
areas of the growing colonies in vitro. Isolation of sphere forming HCECs has in fact been reported
vividly and has been considered as a potential source of progenitor cells [72,80,108,125–127]. It is
J. Funct. Biomater. 2015, 6 921
possible that such progenitor cells in the central region of the colonies in culture may acquire altered
epigenetic modifications which could in turn inhibit their further proliferation or result in their terminal
differentiation followed by senescence similar to that was reported with many instances of embryonic
stem cell colonies in cell cultures [128].
Stem cells are a potential source for engineering of many organs including corneal endothelium.
Organ specific adult stem cells, as well as directed differentiation competent embryonic stem cells, and
induced pluripotent stem cells (iPS cells) form such sources. Adult stem cells are suggested to reside in
the junction between the peripheral corneal endothelium and anterior part of the trabecular meshwork [129].
Embryonic stem cells [130] have the major advantages due to their characteristics of pluripotency and
an unlimited proliferation capacity. However, ethical concerns, immune rejection, and risk of teratoma
formation have limited the application of embryonic stem cells in clinical trials. The use of iPS cells in
clinical trials is also limited because of bio-safety concerns, epigenetic memory from somatic cells,
unintended genomic alterations, and related oncogenesis exacerbated by the use of retroviral or lentiviral
transducing vectors. The above said sphere forming HCECs [72,80,108,125–127] and also human
corneal stromal precursors may represent a potential source for corneal endothelial cells [131].
Other sources of human corneal endothelial-like cells for tissue engineering of corneal endothelium
include umbilical cord blood mesenchymal stem cells [132], adipose-derived stem cells [133], and bone
marrow-derived endothelial progenitor cells [134]. Functional corneal endothelium tissue engineered
from corneal stromal derived stem cells of neural crest origin in humans and mice [131], and corneal
endothelial like cells from neural crest origin in rats [135], are also reported. However, there are no
specific bio-markers for identification of corneal endothelial cells. Although sodium-potassium
adenosine triphosphatase (Na+K+ATPase) and zonula occludens-1 (ZO-1) are located on the corneal
endothelial cell membrane, both are also present in other type of cells. Therefore, the isolation of HCECs
from donor corneas has been widely followed.
In 1965, Mannagh et al. reported successful expansion of HCECs [45]. Following this report,
different isolation techniques and culture media have been introduced to harvest and expand HCECs.
At present time, isolation of HCECs technique consists of two steps. At first the Descemet’s membrane
is peeled with HCECs, thereafter the peeled membranes undergo enzymatic treatment to dissociate
the HCECs. Human corneal endothelial cells have largely been a challenging task to culture and expand.
So far, there is no superior culture medium for consistent expanding of HCECs.
3. Substrates for Cultivation of Human Corneal Endothelial Cells
In vitro expansion of HCECs is challenging, and the cells require native-like favorable growth
conditions. The cultivated corneal endothelium is fragile and difficult to handle. Therefore, the use of
substrates provides mechanical support during transplantation of ex vivo engineered human corneal
endothelial sheets. In addition, they may create a favorable microenvironment needed for cellular
activity. Ideally, the substrate should mimic Descemet’s membrane in its biological, mechanical,
chemical, and physiological characteristics. A spectrum of substrates is used in in vitro expansion of
HCECs and in reconstruction of human corneal endothelial layer. These include biological, synthetic,
and biosynthetic materials (Table 1).
J. Funct. Biomater. 2015, 6 922
For bioengineering of corneal endothelium the substrate materials should preferably fulfil the
following criteria: (i) provide favorable microenvironment for corneal endothelial cellular activity;
(ii) provide mechanical support; (iii) promote cell layer-carrier interactions, cell adhesion, and
extracellular matrix deposition; (iv) be non-toxic; (v) allow transport of gases, nutrients, and molecules;
(vi) be easy to handle during cell layer transport or surgery (endothelial keratoplasty); (vii) be
transparent; (viii) be easily reproducible (i.e., (v)–(vii) are applicable for transplantation for tissue
engineered corneal endothelial grafts).
The substrate should preferably create desired microenvironment for HCEC viability, cell proliferation,
and signaling pathways. The corneal endothelium displays high pump capacity and barrier function
in vivo in order to maintain the cornea in its relatively dehydrated physiological state. The substrate
materials must enable support of these principle functions of HCECs and corneal endothelium.
Following transplantation of tissue engineered corneal endothelial graft; the substrate should allow
sufficient transport of gases, nutrients and molecules between corneal endothelium and stroma.
The Descemet’s membrane is a specialized basement membrane. After birth, the corneal endothelium
secretes Descemet’s membrane consisting of non-banded collagen in physiological conditions [136].
In tissue engineering, it is difficult to reconstruct a substrate that totally mimics complex composition,
dynamic nature and multiple function of a native basement membrane. Therefore, it might be beneficial
if the substrates are able to stimulate collagen secretion.
The substrates should be easy to reproduce, and either degradable or non-degradable substrates may
be used in transplantation. If biodegradable, the substrate dissolution rate must be at a preset value that
does not give adverse effect on rest of the eye. As microsurgery and minimal incision operations are
increasingly used, the tissue engineered corneal endothelium on substrate should be easy to handle and
fold/unfold under the surgery.
There are various substrates applied for cultivated HCECs in experimental models (Table 1). In this
review, the substrates are classified for convenience into biological, synthetic, and biosynthetic groups
of substrates.
J. Funct. Biomater. 2015, 6 923
Table 1. Cultivation of primary human corneal endothelial cells on different types of substrate.
Main Groups
of Substrate
Specific Substrates Author(s)/Year
Cell Suspension/
Sheet on Substrate
Cell Density */
Suspension at Time of
Seeding on Substrate
Final Cell Density
on the Substrate * Morphology Phenotype
Biological Substrates
Amniotic
membrane
Denuded human AM
Ishino et al.,
2004 [68]
Cell suspension
(trypsinized)
3285 ± 62 2410 ± 31
Polygonal, uniformly
sized cells with cell-cell
and cell-AM contact
ZO-1
Decellularized/
devitalized
corneal
materials
Culture flasks + ** human
cornea denuded of endothelium
Insler and Lopez,
1986 [29]
Cell suspension
(trypsinized)
100 µL of
7.5 × 105 cells
560–1650 – –
Culture flasks + human
cornea denuded of endothelium
Insler and Lopez,
1991 [30]
Cell suspension
(trypsinized)
– – – –
Culture flasks + human
cornea denuded of endothelium
Insler and Lopez,
1991 [31]
Cell suspension
(trypsinized)
2000–2200 1000–1600 – –
Culture plates + human
cornea denuded of endothelium
Chen et al.,
2001 [62]
Cell suspension
(trypsinized)
1503–2159 1895 ± 178
Polygonal with cell-cell
adhesion complexes and
gap junction
ZO-1
Bovine ECM coated culture dishes + human cornea
denuded of endothelium
Amano,
2003 [67]
Cell suspension
(trypsinized)
Cell suspension
2 × 105 in 2 mL
2380 ± 264 Uniform in size and shape –
Bovine ECM coated culture dishes + rat cornea
denuded of endothelium and coated with fibronectin
Mimura et al.,
2004 [27]
Cell suspension
(trypsinized)
300 µL of
1 × 106 cells
2744 ± 337 Polygonal –
Bovine ECM coated culture dishes + human cornea
denuded of endothelium
Amano et al.,
2005 [71]
Cell suspension
(trypsinized)
2 mL of
2 × 105 cells
2380 ± 264
In vivo morphology with
cell-cell contact
–
Decellularized human corneal stroma
Choi et al.,
2010 [90]
Cell suspension
(trypsinized)
130–3000 – Compact cells
ZO-1, Na+K+ATPase,
connexin 43
J. Funct. Biomater. 2015, 6 924
Table 1. Cont.
Main Groups
of Substrate
Specific Substrates Author(s)/Year
Cell Suspension/
Sheet on Substrate
Cell Density */
Suspension at Time
of Seeding on
Substrate
Final Cell Density
on the Substrate * Morphology Phenotype
Decellularized/
devitalized
corneal
materials
Culture plates + decellularized posterior lamellae of
bovine cornea
Bayyoud et al.,
2012 [137]
Cell suspension
(trypsinized)
5 × 104 cells/well 2380 ± 179 Polygonal
ZO-1, Na+K+ATPase,
Na+HCO3−,
connexin 43
Culture plates + decellularized porcine cornea
Yoeruek et al.,
2012 [138]
Cell suspension
(trypsinized)
– – – –
Lens capsule
Deepithelialized human anterior lens capsule
Yoeruek et al.,
2009 [88]
Cell suspension
(trypsinized)
5 × 104 cells/well 3012 ± 109 Polygonal
ZO-1, Na+K+ATPase,
connexin 43
Culture plates + deepithelialized human anterior
lens capsule
Kopsachilis et al.,
2012 [99]
Cell suspension
(trypsinized)
5 × 104 cells/well 2455 ± 284 Hexagonal ZO-1, Na+K+ATPase
Natural
polymers
Collagen-coated, dextran-based microcarrier beads
Insler and Lopez,
1990 [56]
Cell suspension
(isolated cells)
– – Cobbelstone –
Collagen membranes
Kopsachilis et al.,
2012 [99]
Cell suspension
(trypsinized)
5 × 104 cells/well 2072 ± 325 Hexagonal –
Atelocollagen coated culture dishes + collagen
vitrigel
Yoshida et al.,
2014 [139]
Cell suspension
(trypsinized)
1.3 × 106 cells/well 2650 ± 100 – –
Type I collagen sponges
Orwin and Hubel,
2000 [65]
Cell suspension
(trypsinized)
– – Cobbelstone –
Bovine ECM coated culture dishes + type I
collagen sheet
Mimura et al.,
2004 [28]
Cell suspension
(trypsinized)
300 µL of
1 × 106 cells
– Also fibroblast like cells –
Type I collagen coated culture dishes
Choi et al.,
2013 [97]
Cell suspension
(trypsinized)
– – – ZO-1, Na+K+ATPase
Type I collagen coated culture plates
Numata et al.,
2014 [110]
Cell suspension
(trypsinized)
– – Hexagonal ZO-1, Na+K+ATPase
Type IV collagen coated culture dishes
Choi et al.,
2010 [90]
Cell suspension
(trypsinized)
– – Compact –
J. Funct. Biomater. 2015, 6 925
Table 1. Cont.
Main Groups
of Substrate
Specific Substrates Author(s)/Year
Cell Suspension/
Sheet on Substrate
Cell Density */
Suspension at Time
of Seeding on
Substrate
Final Cell Density
on the Substrate * Morphology Phenotype
Natural
polymers
Type IV collagen coated culture dishes
Yamaguchi et al.,
2011 [93]
Cell suspension
(trypsinized)
6000 – – –
Type IV collagen coated culture dishes
Choi et al.,
2013 [97]
Cell suspension
(trypsinized)
– – – ZO-1, Na+K+ATPase
Type IV collagen coated culture plates
Numata et al.,
2014 [110]
Cell suspension
(trypsinized)
– – Hexagonal ZO-1, Na+K+ATPase
Bovine ECM coated culture plates
Blake et al.,
1997 [59]
Cell suspension
(trypsinized)
– – Hexagonal –
Bovine ECM coated culture plates
Yamaguchi et al.,
2011 [93]
Cell suspension
(trypsinized)
6000 – – –
Fibronectin coated culture plates
Blake et al.,
1997 [59]
Cell suspension
(trypsinized)
– – Hexagonal –
Fibronectin coated culture plates
Choi et al.,
2010 [90]
Cell suspension
(trypsinized)
– – Compact –
Fibronectin coated culture dishes
Yamaguchi et al.,
2011 [93]
Cell suspension
(trypsinized)
6000 – – –
Fibronectin coated culture plates
Choi et al.,
2013 [97]
Cell suspension
(trypsinized)
– – – ZO-1, Na+K+ATPase
Fibronectin coated culture plates
Numata et al.,
2014 [110]
Cell suspension
(trypsinized)
– – Hexagonal ZO-1, Na+K+ATPase
FNC coating mix coated culture plates
Choi et al.,
2013 [97]
Cell suspension
(trypsinized)
– – – ZO-1, Na+K+ATPase
Gelatin coated culture flasks
Nayak and
Binder,
1984 [50]
Cell suspension
(trypsinized)
– – Flattened and polygonal –
J. Funct. Biomater. 2015, 6 926
Table 1. Cont.
Main Groups
of Substrate
Specific Substrates Author(s)/Year
Cell Suspension/
Sheet on Substrate
Cell Density */
Suspension at Time
of Seeding on
Substrate
Final Cell Density
on the Substrate * Morphology Phenotype
Natural
polymers
A mixture of laminin and chondroitin sulfate coated
culture plates
Engelmann et al.,
1988 [51]
Cell suspension
(trypsinized)
– – Mosaic pattern –
Thermoresponsive PIPAAm–grafted surfaces +
gelatin discs
Hsiue et al.,
2006 [76]
Cell sheet – – Polygonal ZO-1
Thermoresponsive PIPAAm–grafted surfaces +
gelatin discs
Lai et al.,
2007 [32]
Cell sheet 4 × 104 cells 2587 ± 272
Polygonal with
cell-cell contact
ZO-1, Na+K+ATPase
Type IV collagen coated culture dishes + gelatin
hydrogel sheets
Watanabe et al.,
2011 [94]
Cell suspension
(trypsinized)
3–5 × 103 –
Mosaic pattern with
ruffled borders
ZO-1, Na+K+ATPase,
N-cadherin
Laminin-5 coated culture dishes
Yamaguchi et al.,
2011 [93]
Cell suspension
(trypsinized)
6000 – – –
Laminin coated culture plates
Choi et al.,
2013 [97]
Cell suspension
(trypsinized)
– – – ZO-1, Na+K+ATPase
Synthetic Substrates
Rose chamber
Mannagh and
Irving,
1965 [45]
Cell suspension
(isolated cells)
– –
Elongated with
cell-cell contact
–
Tissue culture dishes or flasks
Newsome et al.,
1974 [46]
Endothelium-
Descemet’s
membrane explant
– – Flat and polygonal –
Culture flasks or Petri culture dishes
Baum et al.,
1979 [47]
Endothelium-
Descemet’s
membrane explant
– –
Small and uniform in young
donors. Large and
pleomorphic in older donors
–
J. Funct. Biomater. 2015, 6 927
Table 1. Cont.
Main Groups
of Substrate
Specific Substrates Author(s)/Year
Cell
Suspension/Sheet on
Substrate
Cell Density */
Suspension at Time
of Seeding on
Substrate
Final Cell Density
on the Substrate * Morphology Phenotype
Coverglass of disposable tissue culture chamber
Tripathi and
Tripathi,
1982 [49]
Isolation of cells by
scraping and
Descemet’s membrane
explant
– –
Flattened and hexagonal
or polygonal
–
Culture plates
Blake et al.,
1997 [59]
Cell suspension
(trypsinized)
– – Hexagonal –
Collagen type IV coated culture dishes +
thermoresponsive PIPAAm–grafted surfaces
Sumide et al.,
2006 [74]
Cell sheet 3 × 106 cells/dish 3000
Hexagonal with cilia
and microvilli
–
Thermoresponsive PIPAAm-grafted culture dishes
Ide et al.,
2006 [140]
Cell sheet – –
Polygonal with cilia
and microvilli
–
Thermoresponsive PIPAAm-grafted culture dishes
Lai et al.,
2006 [141]
Cell sheet 4 × 104 cells 2500 Hexagonal ZO-1, Na+K+ATPase
Bovine ECM coated culture dishes + culture plates
and culture inserts
Hitani et al.,
2008 [25]
Cell sheet
600 µL of
4 × 106 cells
2425 ± 83 Uniformly sized cells ZO-1, Na+K+ATPase
Culture plates
Choi et al.,
2010 [90]
Cell suspension
(trypsinized)
– – Compact –
Culture plates
Yamaguchi et al.,
2011 [93]
Cell suspension
(trypsinized)
6000 – – –
Culture plates
Kopsachilis et al.,
2012 [99]
Cell suspension
(trypsinized)
5 × 104 cells/well 2507 ± 303 Hexagonal –
Culture plates
Choi et al.,
2013 [97]
Cell suspension
(trypsinized)
– – – ZO-1, Na+K+ATPase
J. Funct. Biomater. 2015, 6 928
Table 1. Cont.
Main Groups
of Substrate
Specific Substrates Author(s)/Year
Cell
Suspension/Sheet on
Substrate
Cell Density */
Suspension at Time
of Seeding on
Substrate
Final Cell Density
on the Substrate * Morphology Phenotype
Biosynthetic Substrate
FNC coating mix coated culture dishes + FNC
coated RAFT + collagen gel (compressed plastic
and type I collagen)
Levis et al.,
2012 [101]
Cell suspension
(trypsinized)
2000 1941 Polygonal ZO-1, Na+K+ATPase
Notes: AM: Amniotic membrane; DM: Descemet’s membrane; ECM: Extracellular matrix; FNC Coating Mix®: A commercial available coating mixture consisting of
fibronectin, collagen and albumin; Na+HCO3−: Sodium bicarbonate; Na+K+ATPase: Sodium-potassium adenosine triphosphatase; PIPAAm: Poly (N-isopropylacrylamide);
Pos: Positive; RAFT: Real Architecture For 3D Tissues; ZO-1: Zona occludens. * cell density in cells/mm2 if otherwise not stated; ** change of substrate for cultivation
of cells.
J. Funct. Biomater. 2015, 6 929
3.1. Biological Substrates
3.1.1. Amniotic Membrane
Amniotic membrane (AM) is a membrane composed of collagen type IV similar to basement
membrane of conjunctiva but not cornea [142]. The anti-inflammatory [143] and non-immunogenic [144]
properties of AM are believed to be important factors that make it a suitable substrate. The AM is used
in treatment of different ocular surface diseases, and it is applied as substrate for limbal transplantation
in patients with limbal stem cell deficiency [5]. Ishino et al. used denuded AM as a substrate for
cultivated HCECs and transplanted onto rabbit corneas denuded of corneal endothelium and Descemet’s
membrane [68]. The authors demonstrated that the corneal endothelial cell density and function of
reconstructed corneal endothelial graft were similar to normal corneas. However, the tissue-engineered
grafts consisting of HCECs sheet on AM had some edema. In another study, the basement membrane of
AM was used as a carrier for transplantation of cultivated cat corneal endothelial cells on cat cornea
denuded of Descemet’s membrane and endothelium [145]. The cultivated cells predominantly displayed
hexagonal shape, and the reconstructed corneal endothelial layer maintained corneal graft thickness and
remained transparent for six weeks.
Although AM provides good biocompatibility, dependency on donor tissue is a limitation. However,
AM displays several challenges for clinical use, and thus efforts to identify alternative culture substrates
should be encouraged. First, it is semi-opaque; second, preparation is time-consuming; third, there is
possible transfer of pathogens from AM; and fourth, inter-donor and intra-donor variations and rate of
biodegradability may influence the outcome of its clinical use [146].
3.1.2. Decellularized/Devitalized Corneal Materials
The feasibility of using devitalized corneas or corneas denuded of endothelium as substrate for
HCECs is studied extensively [27,29–31,62,67,71,97,137,138]. They are applicable without substantial
redesign as they provide the desired shape, mechanical support, and transparency. Reconstructed human
corneal endothelial graft with in vitro cultivated HCECs seeded on decellularized human corneal stroma
expressed ZO-1, Na+K+ATPase and connexin 43. Proulx et al. studied the function of tissue engineered
corneal endothelium [33]. In experimental animal models they transplanted tissue engineered corneal
endothelial grafts consisting of cultivated feline corneal endothelial cells on devitalized human cornea
denuded of endothelial cells. The follow-up time after transplantation was only 7 days. In this study,
9 of 11 reconstructed corneal endothelial grafts were clear at the end of the follow-up time. The pump
function of the reconstructed corneal endothelial graft must have remained functional in order to
maintain the cornea transparent. In addition, the reconstructed corneal endothelial layers expressed
proteins related to function such as ZO-1 and Na+K+ATPase and sodium bicarbonate (Na+HCO3−)
transporter [33]. The same research group performed ultrastructural and immunohistochemical studies
of cultivated feline corneal endothelial layer on devitalized cornea [147]. Scanning and transmission
electron microscopy demonstrated a monolayer of corneal endothelium, and the tissue engineered
endothelium expressed function related proteins including ZO-1 and Na+K+ATPase and Na+HCO3−
transporter.
J. Funct. Biomater. 2015, 6 930
Bayyoud et al. seeded in vitro expanded HCECs on devitalized posterior corneal stromal lamellae.
The reconstructed corneal endothelial graft had intact barrier and expressed positive staining for
sodium-potassium pump (Na+K+ATPase), membrane transporter (Na+HCO3−), tight junction (ZO-1),
gap junction (connexin 43), and extracellular matrix protein (collagen VIII) [137].
Current methods to decellularize or devitalize cornea include scraping off corneal endothelium
mechanically [67,71], use of chemicals [62,137], or freeze/thaw method [33,147]. High-hydrostatic
pressurization is an alternative technique to decellularize cornea [148]. However, the following are some
inherent technicalities to be aware of using this approach. First, resident viable keratocytes may potentially
give raise to fibroblastic contamination. Second, biological tissues may transfer infections. Third, stroma
from donor corneas does not reduce the dependency of donor tissues.
3.1.3. Lens Capsule
The human anterior lens capsule has been evaluated as potential substrate for tissue engineered
corneal endothelium. Yoeruek et al. received human anterior lens capsule from patients who had undergone
cataract surgery [88]. They seeded HCECs on de-epithelialized anterior lens capsule and demonstrated
that the HCECs grew to confluency. The in vitro bioengineered corneal endothelium strongly expressed
staining for ZO-1, Na+K+ATPase, and connexin 43. Kopsachilis et al. compared three different
substrates; these included de-epithelialized human anterior lens capsule, collagen membrane, and
polystyrene culture plates [99]. They obtained human anterior lens capsule of a mean diameter of 10 mm
from cornea donors. The cultivated cells displayed hexagonal morphology in all groups, and the cells
formed a monolayer of corneal endothelium at two weeks. They reported higher cell density on anterior
lens capsule and culture plates in comparison to collagen membrane (Table 1). However, no statistically
significant difference in cell density was shown among all three groups. Although the de-epithelialized
human anterior lens capsule is a biocompatible substrate, it does not reduce donor dependency.
The diameter of anterior lens capsule following capsulorhexis in cataract surgery is approximately half
the size needed for a carrier for cultivated corneal endothelium for endothelial keratoplasty.
3.1.4. Natural Polymers
Extracellular protein coatings are composed of single proteins (e.g., collagen, gelatin) or combination
of different proteins (e.g., FNC coating mix®). Although the exact components and composition of the
coatings are known, the biological activity of HCECs on these coatings varies. The coating proteins influence
HCEC adhesion, proliferation, morphology, and function of HCECs. There are many different types of
coating materials available for expansion of HCECs. These include collagen [28,56,65,74,90,93,97,99,110,139],
fibronectin [59,90,93,97,110], gelatin [32,50,76,94], laminin [93,97], extracellular matrix (ECM) from
cultured bovine corneal endothelial cells [25,27,59,67,71,93], a mixture of laminin and chondroitin
sulfate [51], and a mixture of fibronectin, collagen, and albumin (FNC Coating Mix®) [97,101].
Choi et al. evaluated adhesion, proliferation, and phenotypic maintenance of HCECs on ECM coated
culture plates [97]. They studied collagen type I, collagen type IV, fibronectin, laminin, and FNC coating
mix. The HCECs expressed a number of integrin genes (integrin α1, α2, α3, αv, β1 and β5), but not
integrin gene β3. High expression of integrin genes supports HCEC binding to ECM. Although cells on
collagen type IV and fibronectin showed the highest expression and cells on collagen type I exhibited
J. Funct. Biomater. 2015, 6 931
the least expression, there were no statistically significant differences. Compared to uncoated control
plates, HCECs adhered more tightly to culture plates coated with coating proteins such as collagen I,
collagen IV, fibronectin, and FNC coating mix. The authors also investigated the cell adhesion strength,
and showed that all the coating proteins increased the adhesion strength compared to uncoated controls,
except for laminin. They were able to demonstrate that HCECs could grow into a confluent layer in a
week on all ECM tested, including uncoated culture plates. Gene expression of ZO-1 and Na+K+ATPase
was found in all conditions, but Na+K+ATPase expression was significantly higher in collagen type I,
fibronectin and laminin coated culture plates [97]. In a previous study of Choi et al., it was demonstrated
that proliferation of HCECs on fibronectin coated culture plates was significantly higher on day 2
after seeding compared to collagen type IV coated culture plates and uncoated culture plates [90].
On day 4 after seeding, however, there was no significant difference in the growth rate in any of the
experimental groups.
Yamaguchi et al. studied HCEC adhesion and proliferation in the presence of recombinant
laminin-5 [93]. Their results showed significantly higher adhesion of HCECs on recombinant laminin-5
coated dishes compared to uncoated control culture dishes. Furthermore, HCECs did not proliferate on
collagen type IV coated culture dishes, and the number of adherent HCECs on laminin-5 coated culture
dishes increased 1.5 times after 7 days of cell culture.
In few studies gelatin as substrate for HCECs was evaluated [50,76,94]. Hsiue et al. were able to
demonstrate that gelatin discs dissolved and the HCEC sheet was adherent to posterior part of corneal
stroma two weeks after transplantation of HCEC sheet [76]. Silkworm fibroin can be prepared as
a transparent membrane and used as carrier for cultivated corneal endothelial cells [149]. However,
higher cell density of B4G12 cell line was achieved on uncoated tissue culture compared to on fibroin.
Human corneal endothelial cells grew to confluency with polygonal morphology only on collagen type
IV coated fibroin [149].
Extracellular matrices from cultured bovine corneal endothelial cells are used as coating material for
in vitro cultivation HCECs [25,27,59,67,71,93]. In a study the HCECs were cultured initially on bovine
ECM coated culture dishes following seeding of the cells on type I collagen sheet [28]. The HCEC sheet
was reported to also have cells with fibroblastic-like morphology. Extracellular matrices produced
by bovine corneal endothelial cells may be reservoir for progelatinase A, a matrix metalloproteinase,
which is important for turnover of ECM and is involved in inflammation, wound healing, angiogenesis,
and metastasis [150].
Studies were carried out by using collagen type I and IV as a substrate for HCECs [74,90,93,97,110].
Cultivated monkey corneal endothelial cells were further cultured on collagen type I carrier for 4 weeks
and transplanted into monkeys. The cultivated corneal endothelial layer produced confluent monolayer
expressing ZO-1 and Na+K+ATPase. The transplanted tissue-engineered corneal endothelial graft
remained clear and had an endothelial cell density of 1992 to 2475 cells/mm2 on examination using
in vivo specular microscopy six months after transplantation [35].
Cultured HCECs on collagen sheets composed of cross-linked collagen type I were transplanted into
rabbits. Pump function was evaluated using Ussing chamber and ouabain, a Na+K+ATPase inhibitor.
The results showed that the cultured HCECs on collagen sheets maintained 76%–95% of pump function
of human donor corneas [28].
J. Funct. Biomater. 2015, 6 932
The difference in adhesion, proliferation, and phenotype displayed by HCECs on the same type of
coating in different studies can be related to different culture techniques and media used. However,
further studies should be conducted to assess the consistency of the different types of coatings. The use
of these coatings in clinical setting remains to be rigorously verified as the coatings are derived
from animals.
3.2. Synthetic Substrates
Synthetic polymers have the advantage of high purity with known chemical composition, structure
and properties. They can be reproduced at controlled conditions with known mechanical and physical
properties. Coated hydrogel lens was used as carrier for cultivated kitten and rabbit corneal endothelial
cells, and these constructs were transplanted into adult cats and rabbits with induced corneal edema,
respectively. The transplanted corneas became clear within three days after transplantation in both cats
and rabbits, and the cornea remained clear for 50 days in cats and 40 days in rabbits [23].
In few studies the HCECs were cultured on plastic culture plates without coating [45–47,49]. These
studies do not reveal details of adhesion and proliferation profiles of HCECs. The cyclic dimers of
glycolic and lactic acids are monomers used in production of biomedical devices. Glycolic and lactic
acids are by-products of metabolic pathway in normal physiological conditions. Therefore, they
are regarded as highly biocompatible with minimal systemic toxicity. Poly(lactic acid) (PLLA) and
poly(lactic-co-glycolic acid) (PLGA) are synthetic polymers extensively studied owing to their
biocompatibility and biodegradability [151]. Hadlock et al. seeded in vitro expanded rabbit corneal
endothelial cells on PLLA and PLGA [152]. In tissue culture conditions the cells grew into confluency
on the synthetic materials and stained for ZO-1 along the lateral cell borders.
Synthetic polymers are used commonly as drug delivery devices. In few ocular diseases dexamethasone
can be delivered into the vitreous cavity as an implant. Ozurdex, consisting of dexamethasone and PLGA
with hydroxypropyl methylcellulose, is injected intravitreally in patients with e.g., macular edema
secondary to retinal vein occlusion. Poly(lactic-co-glycolic acid) polymer matrix degrades slowly to
lactic and glycolic acids meaning the final degradation products are water and carbon dioxide [153].
Another dexamethasone delivery device, Surodex, consisting of PLGA with hydroxypropyl methylcellulose
is inserted into the anterior chamber following cataract surgery to treat postoperative inflammation. In a
comparative single-masked parallel-group study, Wadood and coauthors compared the safety and
efficacy of dexamethasone eye drops and Surodex inserted into the anterior chamber in patients
following phacoemulsification cataract extraction and posterior chamber intraocular lens implantation [154].
Out of 19 patients in this study, 11 patients received Surodex. Surodex remnants were present in all eyes
at 60-day post-operative control, and in 3 patients the traces of remnants were present at 32–36 months.
However, no significant complications were reported during the follow-up time of 3 years. The authors
reported peripheral anterior synechias of less than 1 clock hour at the site of Surodex implantat in
1 patient, and they regarded this as an adverse event. One patient developed high intraocular pressure
after Surodex implantation. The authors considered the patient to be a steroid responder. The intraocular
pressure normalized without treatment during the follow-up time of 36 months. Although PLGA is
considered to be well-tolerated by patients when inserted into the anterior chamber or vitreous cavity,
the removal of device in e.g., cases of endophthalmitis remains a major concern due to residing remnants.
J. Funct. Biomater. 2015, 6 933
In the early phase of cultivation of HCECs, adherence of the cells to the substrate is of great
importance to initiate cell growth, while detachment of an intact and confluent cell layer in a later phase
is necessary for transplantation purposes. Stimuli-responsive polymers have the ability to change their
molecular structures or physicochemical properties according to the variation in the environment they
are in. The design of these polymers with associated processes is highly specialized. Major changes,
such as alteration in the shape, transparency and permeability to water, can be achieved by a small
stimulus, such as change in temperature, pH or wavelength of light.
Research groups have cultivated HCECs on culture dishes grafted with temperature-responsive
polymer poly(N-isopropylacrylamide) (PIPAAm) which reversibly alter its hydrophobicity/hydrophilicity
dependent on incubation temperature [32,74,76,140,141]. They have the advantage of providing both
initial cell adhesion and later cell layer detachment. At 37 °C the seeded cells adhere and proliferate on
hydrophobic PIPAAm-grafted surfaces. The HCEC sheet detaches from PIPAAm-grafted culture dishes
as surfaces become hydrophilic when temperature is reduced below the lower critical solution temperature
of 32 °C. A circular portion of 18 mm in diameter in the center of 35 mm of culture dishes was grafted
with temperature responsive polymer, PIPAAm [74,76]. The in vitro cultivated HCECs were seeded on
PIPAAm-grafted culture dishes, and the cells reached confluency in 1–3 weeks [32,74,76,140,141]. The
gross appearance of confluent HCEC layer on hydrophobic PIPAAm-grafted surfaces was whitish gray,
and the authors related this to accumulation of ECM [32]. Upon reduction of incubation temperature
from 37 to 20 °C, the HCEC sheets detached from culture dish surfaces within 45–60 min [32,74,140,141].
Although the HCEC sheets detached as single contiguous layers, their surfaces were reported as wrinkled
by Lai et al. [140] and as having a white paper-like texture by Hsiue et al. [76]. The monolayered cell
sheets expressed ZO-1 [32,76,140,141] and Na+K+ATPase [32,74,141] proteins. Deposit of ECM on
basal surface of HCEC sheets were observed [32,74,76,140,141], and the ECM components, collagen
type IV and fibronectin, were detected by immunostaining [140]. Scanning electron microscopy
micrographs showed polygonal cells with cellular interconnections [74,76,140,141] and microvilli and
cilia [74,140], and transmission electron microscopy micrographs revealed abundant cytoplasmic
organelles, rough endoplasmic reticulum and mitochondria [32,140]. However, Hsiue et al. demonstrated
the absence of clear cell boundaries [76].
In two studies, the harvested HCEC sheets from PIPAAm-grafted surfaces were immediately
transferred to gelatin disc carriers (7 mm in diameter and 700–800 µm in thickness) [32,76]. The
reconstructed corneal endothelium was transplanted into experimental rabbit models denuded of corneal
endothelium. The gelatin discs dissolved in two weeks, and the corneas transplanted with reconstructed
corneal endothelium were clear with near normal corneal thickness at four weeks [76]. In rabbits
transplanted with tissue engineered corneal endothelium, the corneal thickness increased to 892 µm at
post-operative day 1, and then decreased to near normal corneal thickness of approximately 500 µm at
post-operative day 168 [32]. Sumide et al. transplanted HCEC sheet attached to cornea denuded of
corneal endothelium and Descemet’s membrane into rabbit models [74]. Control rabbits underwent all
procedures except for having HCEC sheet on corneal button. Minimal corneal edema was reported in
rabbits in HCEC sheet transplant group at day 7. In contrast, the corneas were opaque in control group.
The average corneal thickness in HCEC sheet transplant group was significantly lower compared to
control group at day 7. Even though the stimuli-responsive polymers are investigated extensively, their
J. Funct. Biomater. 2015, 6 934
role in corneal endothelial layer transplantations and the effect of the temperature change on the HCEC
bioactivity remains to be investigated.
3.3. Biosynthetic Substrates
Substrates made from a mixture of natural and synthetic polymers are referred to as biosynthetic
substrates in this review. Gao et al. evaluated biocompatibility and biodegradability of substrate composed
of hydroxypropyl chitosan, gelatin, and chondroitin sulfate [155]. Scanning electron microscopy images
revealed a porous structure without fibrils, and the light transmission (wavelength ranging 400–800 nm)
measurements through the substrate showed transmittance of more than 90%; both indicating the
membrane transparency. They demonstrated comparable or better glucose permeability through the
substrate in comparison to native corneas. Cultivated rabbit corneal endothelial cells on this substrate
reached confluency on day 4, and displayed characteristic cobblestone appearance. Histocompability
and biodegradability were assessed by implanting the substrates into skeletal muscle of rats. Sign of
inflammation was seen during post-mortem examination at the interface between the host tissue and
substrate even at the end of observation period of 2 months. Degradation of substrate was observed
from day 30.
Plastic compressed collagen gels [101] and a blending of chitosan and polycaprolactone [156] may
give the necessary mechanical strength as a carrier. Synthetic polymers have the advantage of being
reproduced under controlled conditions with known mechanical and physical properties. Different ratio
of chitosan and polycaprolactone in a substrate were examined. A composition of 75% chitosan and 25%
polycaprolactone supported cultivation of bovine corneal endothelial cells and gave the necessary
mechanical strength of a substrate. The cells reached confluency on day 7 and expressed ZO-1 protein
on substrate composed of chitosan and polycaprolactone at raio of 75:25 [156].
Plastic compressed collagen type I, termed Real Architecture For 3D Tissues (RAFT), can be easily
reproduced and trephined into the size required [101]. Scanning and transmission electron microscopy
imaging revealed a confluent monolayer of corneal endothelial cells on RAFT. Human corneal
endothelial cells cultivated on RAFT stained for ZO-1 and Na+K+ATPase proteins [101].
The synthetic polymers degrade slowly, and hence potential adverse effects on the eyes over long
time course remains to be investigated. Biosynthetic substrate is reported to give raise to inflammation
in experimental animal models [155]. Therefore, histocompability studies should be performed before
use of biosynthetic substrates in humans.
4. Conclusions and Future Perspective
It is obvious to date that the development and utility of different substrates in tissue engineering of
corneal endothelium is slowly evolving. Functional realization of the bioengineered corneal endothelium
has not yet been optimal due to the current limited knowledge of molecular mechanism of proliferation
of HCECs and their associated inter- and intra-signaling pathways that maintain the corneal endothelial
tissue homeostasis. Ideal substrates for cultivation of HCECs should mimic Descemet’s membrane in
molecular, physiological and mechanical terms. Therefore, it is essential to have thorough molecular and
functional insights into the microenvironment of human corneal endothelium in vivo and engineer such
characteristics into the deriving HCEC grafts. Identification of specific marker(s) of HCEC will be
J. Funct. Biomater. 2015, 6 935
extremely advantageous in optimizing differentiation of large numbers of HCECs from a variety of
available cell sources. In addition, even perhaps the patient specific iPS cells with the eventual goal of
prospectively circumventing the need for increasingly limiting donor corneas. Finally, though the
preliminary xenotransplantation studies appear promising, focused research on the discovery and
derivation of suitable substrates, optimization of HCEC culture techniques and identification of specific
marker(s) of HCESs appears very valuable before any bioengineered human corneal endothelial graft is
used clinically.
Acknowledgment
The authors would like to thank Geir A. Qvale at the Center for Eye Research, Department of
Ophthalmology, Oslo University Hospital, for contributing with the figures. We thank many investigators
that have contributed to this subject, while regretting to be unable to include all the works of others due
to space constraint.
Conflicts of Interests
The authors declare no conflict of interest.
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