February 2007
Volume 48, Issue 2
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Cornea  |   February 2007
Proliferation and Differentiation of Transplantable Rabbit Epithelial Sheets Engineered with or without an Amniotic Membrane Carrier
Author Affiliations
  • Kazunari Higa
    From the Department of Ophthalmology, Tokyo Dental College, Chiba, Japan; and the
  • Shigeto Shimmura
    From the Department of Ophthalmology, Tokyo Dental College, Chiba, Japan; and the
    Departments of Ophthalmology and
  • Naoko Kato
    Departments of Ophthalmology and
  • Tetsuya Kawakita
    From the Department of Ophthalmology, Tokyo Dental College, Chiba, Japan; and the
  • Hideyuki Miyashita
    Departments of Ophthalmology and
  • Yuji Itabashi
    Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo, Japan.
  • Keiichi Fukuda
    Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo, Japan.
  • Jun Shimazaki
    From the Department of Ophthalmology, Tokyo Dental College, Chiba, Japan; and the
    Departments of Ophthalmology and
  • Kazuo Tsubota
    From the Department of Ophthalmology, Tokyo Dental College, Chiba, Japan; and the
    Departments of Ophthalmology and
Investigative Ophthalmology & Visual Science February 2007, Vol.48, 597-604. doi:10.1167/iovs.06-0664
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      Kazunari Higa, Shigeto Shimmura, Naoko Kato, Tetsuya Kawakita, Hideyuki Miyashita, Yuji Itabashi, Keiichi Fukuda, Jun Shimazaki, Kazuo Tsubota; Proliferation and Differentiation of Transplantable Rabbit Epithelial Sheets Engineered with or without an Amniotic Membrane Carrier. Invest. Ophthalmol. Vis. Sci. 2007;48(2):597-604. doi: 10.1167/iovs.06-0664.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To report a novel method of engineering transplantable, carrier-free corneal epithelial sheets by using a biodegradable fibrin sealant and to compare its characteristics with epithelial sheets cultivated on denuded amniotic membrane carriers.

methods. Stratified corneal epithelial sheets were prepared in culture dishes coated with biodegradable fibrin glue. Amniotic membrane (AM) carriers served as the control. The quality of cultivated sheets was compared by immunohistochemistry for cytokeratin (K)3, K12, K14, p63, occludin, and integrin β1; electron microscopy; and colony-forming assays. K3 protein expression was compared by Western blot analysis. In a limbal-deficient rabbit transplantation model, postoperative adaptation and proliferation of BrdU-labeled cell sheets were examined by histology and anti-Ki67 staining.

results. Epithelial sheets were successfully engineered by using a biodegradable fibrin sealant. Cell sheets in both groups were multilayered, expressed K3, K12, and K14, and had functioning occludin+ apical tight junctions as well as p63 and integrin β1 staining in basal cells. The carrier-free sheets appeared to be more differentiated than the AM sheets, which was also demonstrated by the higher levels of K3 in the Western blots. The colony-forming efficiency of dissociated cells was similar in both groups, although larger colonies were observed on the AM sheets. AM sheets retained higher levels of BrdU-labeled cells and fewer Ki67+ cells compared with carrier-free sheets after transplantation.

conclusions. Tissue engineering with a commercially available fibrin sealant was an effective means of creating a carrier-free, transplantable corneal epithelial sheet. Carrier-free sheets were more differentiated compared with AM sheets, while retaining similar levels of colony-forming progenitor cells.

The use of allogenic or autologous cell sources for regenerative surgery is already common practice in the reconstruction of the ocular surface in patients with stem cell deficiency. 1 2 After the success of limbal transplantation, a second generation of regenerative corneal surgery has emerged in the form of cell sheet transplantation by tissue-engineering techniques. 3 Cell sheet transplants currently in clinical use are prepared by using biological carriers such as fibrin 4 or amniotic membrane (AM) 5 6 7 or as carrier-free cell sheets. 8 9 Although there is still debate as to whether the cultivated sheets include progenitor or stem cells, both carrier and carrier-free techniques have restored a clear ocular surface for at least 1 year, the empiric goal for successful stem cell surgery. 7 9  
One of the major benefits of cell sheet transplants, is that it can avoid the problem of donor availability. In vitro expansion provides a stratified cell sheet suitable for transplantation from a millimeter-scale tissue source procured from the healthy eye of the same patient or from a living relative in the case of bilateral disease. Ectopic cell sources such as the buccal membrane can also be modified in vitro to form a stratified epithelial sheet for ocular surface reconstruction with autologous tissue. 9 10 11 Yet, the number of clinical cases has not met the needs of patients because of ethical and technical constraints. Using AM as a carrier is one possibility as a standardized technique to produce transplantable epithelial sheets; however, AM tissue may not be readily available. 
The development of a carrier-free method to produce corneal epithelial sheets was first reported by Nishida et al., 8 who used a novel temperature-responsive polymer that changes molecular conformation and hydrophobicity at 20°C to release intact sheets. Clinical cases in which this technique has been used have shown that a carrier-free strategy is feasible and that transplantation can be performed without the use of sutures. In the present study, we developed a different technique by using commercially available fibrin sealants to produce carrier-free sheets. Our method is different from the fibrin carrier sheets described by Rama et al., 4 as we allowed the fibrin to be degraded by intrinsic proteases before transplantation. 
Materials and Methods
Antibodies
Mouse monoclonal antibodies (mAbs) for cytokeratin (K)3, K14, laminin, p63, integrin β1, and Ki67 were purchased from Progen (AE5; Heidelberg, German), Abcam (B429; Cambridgeshire, UK), Laboratory Vision (4C7; Fremont, CA), Calbiochem (4A4; Merck KGaA, Darmstadt, Germany), Chemicon International Inc. (LM534; Temecula, CA), and DakoCytomation (MIB-1; Glostrup, Denmark), respectively. Mouse IgM antibody for fibrin was purchased from Monosan (Uden, The Netherlands). Rabbit polyclonal antibody for K12, goat polyclonal antibody for type IV collagen and rat mAb for BrdU (ICR1) were purchased from TransGenic, Inc. (Kumamoto, Japan), Southern Biotechnology Associates, Inc. (Birmingham, AL) and Abcam. Isotype goat IgG, mouse IgG1, mouse IgM, rabbit IgG and rat IgG as control were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Dako Cytomation, and Jackson ImmunoResearch Laboratories (West Grove, PA), respectively. FITC-, rhodamine-, and Cy3-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories and Chemicon International Inc. 
Preparation of Epithelial Cells Sheets
All experimental procedures and protocols were approved by the Animal Care and Use Committee of Tokyo Dental College and conformed to the National Instituted of Health Guide for the Care and Use of Laboratory Animals. Fibrin sealant was purchased from Fujisawa (Bolheal; Osaka, Japan), and its constitution was performed as reported previously. 12 In brief, a solution containing 40 mg of human fibrinogen and 0.18 U of thrombin was diluted with 7.5 mL saline, and 0.3 mL was spread rapidly onto the upper chambers of a six-well plate with culture inserts (Transwell; Costar Corning, Corning, NY). Two hours later, the polymerized fibrin-coated top chambers were obtained and stored at 4°C. AMs were donated by mothers who were seronegative for human immunodeficiency virus and hepatitis B and C virus at the time of cesarean section, after written informed consent was obtained, in accordance with the Declaration of Helsinki. AM was stored with 15% dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO) with PBS at −80°C until use. Denuded AM was prepared as previously described. 7 Membranes were rinsed in PBS, spread onto the upper chambers of a six-well insert, frozen at −80°C, and air-dried at room temperature. 
Primary cultures of limbal epithelial cells were prepared from eyes of 2.5- to 3.0-kg female Japanese white rabbits (Japan CLEA, Tokyo, Japan) with anesthesia induced by intravenous injection of 4 mL pentobarbital sodium (50 mg/mL). Limbal rims of corneoscleral tissue were prepared by careful removal of excess sclera, iris, corneal endothelium, and central cornea. Epithelial sheets were isolated as described previously. 13 Dispersed epithelial sheets were treated with trypsin-ethylenediaminetetracetic acid (EDTA) for 10 minutes, to suspend cells, which were seeded onto fibrin- or AM-coated wells (2 × 105 cells/mL) with supplemented hormonal epithelial medium (SHEM) 7 containing 666 KIU/mL aprotinin (Wako, Osaka, Japan) and cocultured with mitomycin C (MMC)-treated 3T3 fibroblasts (Fig. 1A) . The cultures were submerged in medium until confluence, cultured in air-liquid interface for 1 week, and finally incubated without aprotinin for 4 days. To evaluate the proliferation of transplanted epithelium and to identify cells of donor origin, cell sheets were labeled with 10 μM BrdU for 48 hours before surgery. After labeling with BrdU, the epithelial cell sheets were washed with fresh medium and then used for surgery. 
Transmission Electron Microscopy
Epithelial cell sheets were processed for transmission electron microscopy. Epithelial cell sheets from both groups were fixed in 2.5% glutaraldehyde solution in 60 mM HEPES buffer solution for 4 hours. After washing, samples were postfixed in 1% osmium tetroxide, dehydrated in a series of ethanol and propylene oxide, and embedded in epoxy resin. Semithin sections (1-μm) were stained with toluidine blue. Then, ultrathin specimens were sectioned with a microtome (LKB, Gaithersburg, MD). Sections in the range of gray to silver were collected on 150-mesh grid, stained with uranyl acetate and lead citrate, and examined under an electron microscope (mode1 1200 EXII; JEOL, Tokyo, Japan). 
Colony-Forming Efficiency
To evaluate the proliferative potential of cells in the cultured sheets, MMC-treated 3T3 fibroblasts were used in a colony-forming efficiency (CFE) assay, as previously described. 14 15 16 NIH 3T3 fibroblasts in DMEM containing 10% FCS were treated with MMC (4 μg/mL) for 2 hours at 37°C and then treated with trypsin-EDTA and plated at a density of 3 × 106 cells in 100-mm culture dishes. Single cells were prepared from both treated epithelial cell sheets (Acutase; Innovative Cell Technologies, Inc., San Diego, CA) for 60 minutes at 37°C. Each dish was seeded at 1 × 103 cells/dish. CFE was calculated by the percentage of colonies at day 14 generated by the number of epithelial cells plated in the dish. Quantification of size (in square millimeters) and number of colonies obtained from AM or fibrin sheets (n = 5) was performed by NIH Image (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Growth capacity was evaluated on day 14 when cultured cells were stained with rhodamine B (Wako) for 30 minutes. 
Epithelial Sheet Transplantation
All animals were handled in full accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and institutional guidelines. Rabbits were anesthetized with intramuscular injection of xylazine hydrochloride (2.5 mg/mL) and ketamine hydrochloride (37.5 mg/mL). The left eye in each rabbit was rendered totally limbal stem cell deficient by 1-n-heptanol (Sigma-Aldrich) mechanical debridement of the corneal epithelium, and surgical removal of the limbal and conjunctival epithelium was performed up to 2 mm from the limbus. Carrier-free sheets were gently detached from the mesh with a cell scraper, 12 transferred by microforceps and then expanded on the bare corneal stroma with a surgical sponge or forceps. Cell sheets were allowed to attach for 5 minutes without sutures. AM carrier sheets were sutured to the corneal surface with 10-0 nylon sutures. Rabbits with denuded corneas without sheet transplants served as the control. After surgery, all rabbits were fitted with a bandage contact lens and topical antibiotic (levofloxacin), and steroids (betamethasone) were applied twice daily. 
The percentage of the cornea covered by epithelium at 1 week after surgery was calculated by measuring the area of the epithelial defects. The defect area was analyzed by tracing fluorescein images and calculated using the NIH Image program. Rabbits were then killed to observe BrdU-labeled cells as a means to confirm the donor origin of epithelium. The proliferation of transplanted epithelial cells was examined by calculating the percentage of BrdU+ and Ki67+ nuclei by immunohistochemistry. 
Immunohistochemistry
Paraffin sections (K3, K14, p63, BrdU, and Ki67) were deparaffinized in xylene and rehydrated. Frozen sections (type IV collagen and laminin) were fixed for 10 minutes in cold acetone before blocking. Frozen sections (integrin β1 and K12) were fixed for 10 minutes in 2% paraformaldehyde (Wako). Sections were blocked by incubation with 10% normal donkey serum (Chemicon International Inc., Temecula, CA) and 1% bovine serum albumin (Sigma-Aldrich) for 1 hour at room temperature (RT). Antibodies to K3 (1:50), K12 (1:100), K14 (1:100), p63 (1:50), BrdU (1:100), Ki67 (1:50), type IV collagen (1:50), laminin (1:50), and integrin β1 (1:100) were applied and incubated for 90 minutes at RT, followed by incubation with rhodamine- or Cy3-conjugated secondary antibody. After three washes with TBST, the sections were incubated with 1 mg/mL 4′,6-diamidino-2-phenylindole (DAPI; Dojindo Laboratories, Tokyo, Japan) at RT for 5 minutes. Finally, the sections were washed three times in TBST and coverslipped after mounting with an antifade medium (50 mM Tris buffer saline, 90% glycerin; Wako), 10% 1,4-diazabicyclo-2,2,2-octane (Wako). 
Western Blot Analysis
Epithelial sheets were dissociated with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Nonidet P-40; Calbiochem, Darmstadt, Germany) and homogenized. Each epithelial cell sheet was incubated for 40 minutes at 4°C, and then centrifuged at 15,000 rpm for 30 minutes at 4°C. Protein concentration of the supernatant was determined by a protein assay (DC assay; Bio-Rad Laboratory, Hercules, CA). All samples were then diluted in 2× sample buffer (100 mM Tris-HCl [pH 6.8]), 4% SDS (Invitrogen, Carlsbad, CA), 20% glycerol (Wako), 12% 2-mercaptoethanol (Wako), and boiled. Ten micrograms of each sample were loaded on a 10% Bis-Tris gel (Novex NuPAGE; Invitrogen) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked with 5% skim milk (Difco Laboratories, Detroit, MI), 1.5% normal goat serum, and PBS for 60 minutes at RT. The membranes were reacted with K3 (AE5) and β-actin (mabcam8226; Abcam) for 60 minutes at RT. After the membranes were washed three times in TBST, donkey biotinylated anti-mouse IgG (Jackson ImmunoResearch Laboratories) was added for 30 minutes at room temperature. Protein bands were visualized (Vectastain ABC Elite Kit; Vector Laboratories, Burlingame, CA) with DAB (Vector Laboratories) as the substrate. The plot profile of the bands was analyzed with the NIH image 1.63 software with band density of AM sheets in each group standardized at 1.0. 
Statistical Analysis
Statistical comparisons of Western blot band intensity, CFE, epithelialization, and BrdU and Ki67 staining were performed with the nonpaired Student’s t-test (Excel; Microsoft, Redmond, WA). 
Results
Generation of Carrier-Free Epithelial Cell Sheets
Rabbit corneal epithelial cells were cultured with 3T3 feeder cells for 1 to 2 weeks, followed by airlift cultures to produce stratified epithelium on plastic coated with fibrin polymer (Fig. 1A) . Fibrin remained at the bottom of the cell sheet when cultured with aprotinin (Figs. 1B 1D)and was dissolved after removal of aprotinin, presumably due to intrinsic proteolytic activity (Figs. 1C 1E)
In Vitro Characteristics of Cultivated Sheets
We performed a comparative study of carrier-free corneal epithelial sheets with epithelial sheet cultivated on AM carriers. Stratified epithelium was engineered on both AM (Fig. 2A)and plastic coated with degradable fibrin polymer (Fig. 2B) . The use of aprotinin did not affect cell growth or stratification on the AM carriers. 
Immunohistochemistry using anti-K3 and K12 antibodies showed that carrier-free cultures produced uniform layers of cells expressing both differentiation markers (Figs. 2C 2D 2E 2F) . Sporadic cells in the basal layer were K3 negative, which is characteristic of immature limbal basal cells in vivo. Both AM and carrier-free sheets expressed K14 (Figs. 2G 2H)and p63 (Figs. 2I 2J) . The epithelium on AM carriers appeared to express higher levels of K14 and p63, and less K3, K12, suggesting that the AM maintains epithelial cells in a less differentiated state. The difference in K3 expression was also demonstrated by Western blot analysis (Figs. 2K 2L) . Both AM and carrier-free sheets show an intact superficial tight junction, as shown by immunohistochemistry of occludin (Figs. 3G 3H)and the exclusion of HRP (Figs. 3I 3J) . Basement membrane components such as collagen IV and laminin were more prominent in the AM sheet in vitro (Figs. 3C 3E) . These proteins were not as evident in the carrier-free sheets before transplantation (Figs. 3D 3F) . However, the adhesion molecule integrin β1 was expressed in both sheets (Figs. 3A 3B)
Transmission electron microscopy revealed that the ultrastructure of the epithelium was similar between the AM and carrier-free sheets, consisting of five to six layers of stratified epithelial cells with typical columnar basal cells and superficial cells with microvilli (Figs. 4A 4B) . Both cells sheets showed tight junction formation in apical cells (Figs. 4C 4D)and desmosome formation (Figs. 4E 4F) . Although a basement membrane structure was observed in AM sheets (Fig. 4G) , the carrier-free sheets showed residual material attached to the basal epithelial membrane (Fig. 4H)which may represent components of the basement membrane structure such as integrin β1, collagen IV, and laminin observed by immunohistochemistry (Figs. 3B 3D 3F)
Colony-Forming Efficiency
Isolated epithelial cells from both carrier-free (6.6% ± 1.2%) and AM carrier sheets (8.6% ± 2.7%) maintained the ability to form colonies in a 3T3 feeder layer (Figs. 5A 5B) . Although the number of large colonies (100 mm2) was higher in AM sheets (AM: 2.8 ± 1.3 colonies, fibrin: 0.6 ± 0.8 colonies, P = 0.014, n = 5), the difference in the total number of colonies was not statistically significant (Fig. 5C)
Cultivated Sheet Transplantation
BrdU labeling was performed on the AM (Fig. 6A)and carrier-free (Fig. 6B)sheets before surgery, showing that most of the cells in both groups are viable with proliferative potential. Rabbits without cell sheet transplants characteristically had epithelial defects at 1 week after surgery, as shown by the positive staining with fluorescein dye in Figure 6H(dotted line). An intact epithelial layer excluded the dye in the AM (Fig. 6F)and carrier-free (Fig. 6G)sheet transplants. The irregular staining in the AM sheet corresponds to folds in the transplant. Optical clarity was higher in the carrier-free group (Fig. 6D)than in the group with AM sheet transplants (Fig. 6C) . Rabbits in the AM sheet group also had inflammation of the conjunctiva due to the presence of sutures. The area of intact epithelium at 1 week after surgery was significantly higher in both AM (79.4% ± 20.4%) and carrier-free (90.7% ± 17.4%) epithelial sheet groups compared with the sham-surgery control (40.2% ± 18.3%, P < 0.05; Fig. 6I ). 
Immunohistochemistry of postoperative corneas showed normal K3 expression in the transplant sheets (Figs. 7C 7D) . Sham-surgery eyes exhibited partial epithelialization by K3-negative epithelium of conjunctival origin. Basement membrane components such as collagen IV were more prominent in the AM sheet group (Figs. 7E 7F) . Proliferation of transplanted cells was observed by the distribution of BrdU staining, which was uniformly present in cells before surgery. BrdU-positive cells were observed in both groups, indicating that these cells were of donor origin and were slow cycling during the 1-week period after surgery (Figs. 7G 7H) . The number of BrdU-positive cells in the AM group was significantly higher than that in the fibrin group (AM: 18.4% ± 4.2%, fibrin: 1.1% ± 1.1%, P = 0.0002, n = 4; Fig. 7K ). Staining of the proliferation marker Ki67 was also observed in the basal layers of both groups (Figs. 7I 7J) , with significantly less Ki67-positive cells in the AM group than in the fibrin group (AM: 6.3% ± 1.2%, fibrin: 12.6% ± 3.1%, P = 0.0087, n = 4; Fig. 7L ). BrdU- and Ki67-positive cells were uniformly distributed throughout the epithelial sheet in both groups, and there was no tendency of higher localization of label retaining cells in the limbus under the conditions of the study. 
Discussion
The homeostasis of cells undergoing constant turnover depends on the healthy supply of regenerating cells, as well as an intact interaction between surrounding tissues. In the case of the corneal epithelium, stem cells in the basal limbus supply transient amplifying (TA) cells to the corneal basal layer, which proliferate and slough off the ocular surface after approximately 2 weeks. The proliferation and differentiation of epithelial cells is regulated by stromal-epithelial interaction with keratocytes, the major mesenchymal cell in the corneal stroma. Chemokines and growth factors secreted by keratocytes are involved in the proliferation and differentiation of the overlying epithelium. 17 We found that although both AM sheets and carrier-free sheets were viable in transplant-recipient eyes, carrier-free transplants demonstrated a more robust layer of fully differentiated cells. 
We observed more BrdU labeled cells and fewer Ki67-labeled cells in AM sheets compared with carrier-free sheets after transplantation. Previous studies have shown that cell-cycle kinetics and cell phenotype characteristic of limbal epithelial progenitor cells are preserved during ex vivo expansion on AM. 18 19 The difference in cell-cycle kinetics may be due to the presence of the AM basement membrane, which may modulate epithelial cell adhesion, proliferation, and differentiation. 18 20 21 In contrast, epithelial cells in carrier-free sheets seem to become integrated into the host tissue earlier, suggesting that the AM may be interfering with interactions between the epithelium and stromal cells. The absence of a carrier will restore epithelium-stromal interactions immediately after surgery, may have several advantages in maintaining a healthy epithelium, and may also allow the regeneration of a normal subepithelial nerve plexus. It can be argued that a larger yield of undifferentiated cells may be preferable in the treatment of stem cell-depleted cases. However, a mature corneal epithelium is also required for the ocular surface to act as a barrier against invading organisms, as well as to provide a smooth surface for visual clarity. The clinical data available to date show that both AM sheets and carrier-free sheets can restore the epithelium for more than 1 year, 7 9 which would not be possible without the restoration of progenitor cells. 
Another major benefit of carrier-free cell sheets is the surgical technique, which does not require the use of sutures for donor fixation. The mechanisms involved may be multiple, however, Nishida et al. 8 show that intact basement membrane substrates and adhesion molecules may play a major role. We have confirmed the presence of β1 integrin in the carrier-free group, which may have aided the carrier-free sheets in remaining on the ocular surface without sloughing off. In contrast, AM sheets require sutures for transplantation, and ingrowth of cells was observed under the AM carrier in several cases. These results show that attachment of cell sheets to the underlying stroma is stronger with carrier-free sheets during the early postoperative stage. Furthermore, the method we describe for engineering carrier-free sheets is different from previous approaches involving temperature-responsive dishes and does not require any specialized equipment or high levels of technical expertise. 
The design of our study made use of rabbits with denuded epithelium, including the limbal area. We did not take into account any damage to the underlying stromal tissue, which is sometimes observed in clinical cases after severe chemical and thermal burns. The conclusions drawn from our study therefore should be interpreted as being based on epithelial sheet transplantation in situations with relatively intact stromal tissue. The AM is rich in basement membrane components since the amnion itself supports epithelial cells in the uterus. The use of an AM carrier may therefore have benefits in cases with extensive damage and inflammation in the underlying stroma. 
There are still several issues to be resolved before the generalization of epithelial sheet surgery. The manufacture of stratified epithelial sheets requires the use of 3T3 feeder cells and culture-grade serum. Although adverse effects have not been reported, xeno-free techniques should be pursued. Similarly, the choice of whether to use carriers or not requires elucidation. Our data clearly show that cell sheets engineered without carriers reconstruct host tissue nearly to its original state as early as 1 week after surgery. Further refinements in surgical technique and quality control of cultured sheets should expand the therapeutic indications for tissue-engineered cell sheet transplantation. 
 
Figure 1.
 
Cultivation of carrier-free epithelial sheets. Limbal epithelial cells were collected and seeded on fibrin- or AM-coated chambers (A). After 1 to 2 weeks in submerged culture with MMC-treated 3T3 feeder fibroblasts, the cells were allowed to stratify at the air-liquid interface for 1 week. HE staining (B, C) and immunohistochemistry against fibrin (green) and K12 (red) (D, E) showed that fibrin acted as a scaffold during cultivation with the protease inhibitor aprotinin (B, D) and was allowed to dissolve by removing the aprotinin before transplantation (C, E).
Figure 1.
 
Cultivation of carrier-free epithelial sheets. Limbal epithelial cells were collected and seeded on fibrin- or AM-coated chambers (A). After 1 to 2 weeks in submerged culture with MMC-treated 3T3 feeder fibroblasts, the cells were allowed to stratify at the air-liquid interface for 1 week. HE staining (B, C) and immunohistochemistry against fibrin (green) and K12 (red) (D, E) showed that fibrin acted as a scaffold during cultivation with the protease inhibitor aprotinin (B, D) and was allowed to dissolve by removing the aprotinin before transplantation (C, E).
Figure 2.
 
Differentiation markers in epithelial sheets. Hematoxylin and eosin staining of AM (A) and carrier-free (B) epithelial cell sheets. (CJ) Immunohistochemistry of K3, K12, K14, and p63 in epithelial sheets. Carrier-free sheets showed stronger K3/K12 staining and weaker K14/p63 staining than did AM sheets. Nuclei of cells were stained with DAPI. Scale bar, 50 μm. The difference in K3 expression was confirmed by Western blot (K), which showed significantly higher levels of K3 in carrier-free sheets than in AM sheets, when compared semiquantitatively (L, n = 6, *P = 0.002).
Figure 2.
 
Differentiation markers in epithelial sheets. Hematoxylin and eosin staining of AM (A) and carrier-free (B) epithelial cell sheets. (CJ) Immunohistochemistry of K3, K12, K14, and p63 in epithelial sheets. Carrier-free sheets showed stronger K3/K12 staining and weaker K14/p63 staining than did AM sheets. Nuclei of cells were stained with DAPI. Scale bar, 50 μm. The difference in K3 expression was confirmed by Western blot (K), which showed significantly higher levels of K3 in carrier-free sheets than in AM sheets, when compared semiquantitatively (L, n = 6, *P = 0.002).
Figure 3.
 
Basement membrane components and barrier function in epithelial sheets. Immunohistochemistry of integrin β1, collagen type IV, laminin, and occludin in AM (A, C, E, G) and carrier-free (B, D, F, H) epithelial sheets. Nuclei of cells were stained with DAPI. (I, J) Barrier function (HRP permeability) of the epithelial sheets. Scale bar, 50 μm.
Figure 3.
 
Basement membrane components and barrier function in epithelial sheets. Immunohistochemistry of integrin β1, collagen type IV, laminin, and occludin in AM (A, C, E, G) and carrier-free (B, D, F, H) epithelial sheets. Nuclei of cells were stained with DAPI. (I, J) Barrier function (HRP permeability) of the epithelial sheets. Scale bar, 50 μm.
Figure 4.
 
Transmission electron micrographs of AM and carrier-free sheets. Both AM (A, C, E, G) and carrier-free (B, D, F, H) sheets formed five to six layers of well-stratified epithelial cells, with columnar basal epithelial cells. High-magnification views show tight junction formation in apical cells (C, D, white arrowheads), and desmosome formation in the intermediate layers (E, F, black arrowheads). Basal cells formed an intact basement membrane in the AM sheets (G, arrows), whereas carrier-free sheets had residual material attached to the basal cell membrane (H, white arrows).
Figure 4.
 
Transmission electron micrographs of AM and carrier-free sheets. Both AM (A, C, E, G) and carrier-free (B, D, F, H) sheets formed five to six layers of well-stratified epithelial cells, with columnar basal epithelial cells. High-magnification views show tight junction formation in apical cells (C, D, white arrowheads), and desmosome formation in the intermediate layers (E, F, black arrowheads). Basal cells formed an intact basement membrane in the AM sheets (G, arrows), whereas carrier-free sheets had residual material attached to the basal cell membrane (H, white arrows).
Figure 5.
 
Colony formation by disassembled cells. Colony formation by epithelial cells dissociated from AM (A) and carrier-free (B) sheets. Colonies were stained with rhodamine B after 2 weeks. (C) Quantification of size and number of colonies obtained from epithelial sheets (n = 5, mean ± SD). There was no significant difference in total colony formation. When cultures were compared by the area of each colony, a significant difference was observed only in the largest colony size (*P = 0.014; Student’s t-test, n = 5).
Figure 5.
 
Colony formation by disassembled cells. Colony formation by epithelial cells dissociated from AM (A) and carrier-free (B) sheets. Colonies were stained with rhodamine B after 2 weeks. (C) Quantification of size and number of colonies obtained from epithelial sheets (n = 5, mean ± SD). There was no significant difference in total colony formation. When cultures were compared by the area of each colony, a significant difference was observed only in the largest colony size (*P = 0.014; Student’s t-test, n = 5).
Figure 6.
 
Epithelial sheet transplantation in rabbits. Immunohistochemistry of BrdU (A, B) in epithelial sheets before transplantation. Slit lamp photographs (CE) and fluorescein staining (FH) of rabbit eyes 1 week after epithelial sheet transplantation. (C, F) AM, (D, G) carrier-free, (E, H) sham. (I) Area of intact epithelium was larger in rabbit eyes after epithelial sheet transplantation compared with sham (*P < 0.05). Carrier-free sheets had a smoother epithelial surface with minimal inflammation. Scale bar, 50 μm.
Figure 6.
 
Epithelial sheet transplantation in rabbits. Immunohistochemistry of BrdU (A, B) in epithelial sheets before transplantation. Slit lamp photographs (CE) and fluorescein staining (FH) of rabbit eyes 1 week after epithelial sheet transplantation. (C, F) AM, (D, G) carrier-free, (E, H) sham. (I) Area of intact epithelium was larger in rabbit eyes after epithelial sheet transplantation compared with sham (*P < 0.05). Carrier-free sheets had a smoother epithelial surface with minimal inflammation. Scale bar, 50 μm.
Figure 7.
 
Postoperative histology of epithelial sheet transplantation. Light micrograph of hematoxylin and eosin-stained sections of AM (A) and carrier-free (B) epithelial sheets. Immunohistochemistry of K3 (C, D), Collagen type IV (E, F), BrdU (G, H), and Ki67 (I, J). AM sheets retained significantly higher levels of BrdU (K) and expressed lower levels of Ki67 (L) than did carrier-free sheets (*P < 0.05, Student’s t-test). Scale bar, 50 μm.
Figure 7.
 
Postoperative histology of epithelial sheet transplantation. Light micrograph of hematoxylin and eosin-stained sections of AM (A) and carrier-free (B) epithelial sheets. Immunohistochemistry of K3 (C, D), Collagen type IV (E, F), BrdU (G, H), and Ki67 (I, J). AM sheets retained significantly higher levels of BrdU (K) and expressed lower levels of Ki67 (L) than did carrier-free sheets (*P < 0.05, Student’s t-test). Scale bar, 50 μm.
The authors thank Mifuyu Oshima and Tomomi Sekiguchi for technical assistance and the staff of the Cornea Center Eye Bank for administrative support. 
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Figure 1.
 
Cultivation of carrier-free epithelial sheets. Limbal epithelial cells were collected and seeded on fibrin- or AM-coated chambers (A). After 1 to 2 weeks in submerged culture with MMC-treated 3T3 feeder fibroblasts, the cells were allowed to stratify at the air-liquid interface for 1 week. HE staining (B, C) and immunohistochemistry against fibrin (green) and K12 (red) (D, E) showed that fibrin acted as a scaffold during cultivation with the protease inhibitor aprotinin (B, D) and was allowed to dissolve by removing the aprotinin before transplantation (C, E).
Figure 1.
 
Cultivation of carrier-free epithelial sheets. Limbal epithelial cells were collected and seeded on fibrin- or AM-coated chambers (A). After 1 to 2 weeks in submerged culture with MMC-treated 3T3 feeder fibroblasts, the cells were allowed to stratify at the air-liquid interface for 1 week. HE staining (B, C) and immunohistochemistry against fibrin (green) and K12 (red) (D, E) showed that fibrin acted as a scaffold during cultivation with the protease inhibitor aprotinin (B, D) and was allowed to dissolve by removing the aprotinin before transplantation (C, E).
Figure 2.
 
Differentiation markers in epithelial sheets. Hematoxylin and eosin staining of AM (A) and carrier-free (B) epithelial cell sheets. (CJ) Immunohistochemistry of K3, K12, K14, and p63 in epithelial sheets. Carrier-free sheets showed stronger K3/K12 staining and weaker K14/p63 staining than did AM sheets. Nuclei of cells were stained with DAPI. Scale bar, 50 μm. The difference in K3 expression was confirmed by Western blot (K), which showed significantly higher levels of K3 in carrier-free sheets than in AM sheets, when compared semiquantitatively (L, n = 6, *P = 0.002).
Figure 2.
 
Differentiation markers in epithelial sheets. Hematoxylin and eosin staining of AM (A) and carrier-free (B) epithelial cell sheets. (CJ) Immunohistochemistry of K3, K12, K14, and p63 in epithelial sheets. Carrier-free sheets showed stronger K3/K12 staining and weaker K14/p63 staining than did AM sheets. Nuclei of cells were stained with DAPI. Scale bar, 50 μm. The difference in K3 expression was confirmed by Western blot (K), which showed significantly higher levels of K3 in carrier-free sheets than in AM sheets, when compared semiquantitatively (L, n = 6, *P = 0.002).
Figure 3.
 
Basement membrane components and barrier function in epithelial sheets. Immunohistochemistry of integrin β1, collagen type IV, laminin, and occludin in AM (A, C, E, G) and carrier-free (B, D, F, H) epithelial sheets. Nuclei of cells were stained with DAPI. (I, J) Barrier function (HRP permeability) of the epithelial sheets. Scale bar, 50 μm.
Figure 3.
 
Basement membrane components and barrier function in epithelial sheets. Immunohistochemistry of integrin β1, collagen type IV, laminin, and occludin in AM (A, C, E, G) and carrier-free (B, D, F, H) epithelial sheets. Nuclei of cells were stained with DAPI. (I, J) Barrier function (HRP permeability) of the epithelial sheets. Scale bar, 50 μm.
Figure 4.
 
Transmission electron micrographs of AM and carrier-free sheets. Both AM (A, C, E, G) and carrier-free (B, D, F, H) sheets formed five to six layers of well-stratified epithelial cells, with columnar basal epithelial cells. High-magnification views show tight junction formation in apical cells (C, D, white arrowheads), and desmosome formation in the intermediate layers (E, F, black arrowheads). Basal cells formed an intact basement membrane in the AM sheets (G, arrows), whereas carrier-free sheets had residual material attached to the basal cell membrane (H, white arrows).
Figure 4.
 
Transmission electron micrographs of AM and carrier-free sheets. Both AM (A, C, E, G) and carrier-free (B, D, F, H) sheets formed five to six layers of well-stratified epithelial cells, with columnar basal epithelial cells. High-magnification views show tight junction formation in apical cells (C, D, white arrowheads), and desmosome formation in the intermediate layers (E, F, black arrowheads). Basal cells formed an intact basement membrane in the AM sheets (G, arrows), whereas carrier-free sheets had residual material attached to the basal cell membrane (H, white arrows).
Figure 5.
 
Colony formation by disassembled cells. Colony formation by epithelial cells dissociated from AM (A) and carrier-free (B) sheets. Colonies were stained with rhodamine B after 2 weeks. (C) Quantification of size and number of colonies obtained from epithelial sheets (n = 5, mean ± SD). There was no significant difference in total colony formation. When cultures were compared by the area of each colony, a significant difference was observed only in the largest colony size (*P = 0.014; Student’s t-test, n = 5).
Figure 5.
 
Colony formation by disassembled cells. Colony formation by epithelial cells dissociated from AM (A) and carrier-free (B) sheets. Colonies were stained with rhodamine B after 2 weeks. (C) Quantification of size and number of colonies obtained from epithelial sheets (n = 5, mean ± SD). There was no significant difference in total colony formation. When cultures were compared by the area of each colony, a significant difference was observed only in the largest colony size (*P = 0.014; Student’s t-test, n = 5).
Figure 6.
 
Epithelial sheet transplantation in rabbits. Immunohistochemistry of BrdU (A, B) in epithelial sheets before transplantation. Slit lamp photographs (CE) and fluorescein staining (FH) of rabbit eyes 1 week after epithelial sheet transplantation. (C, F) AM, (D, G) carrier-free, (E, H) sham. (I) Area of intact epithelium was larger in rabbit eyes after epithelial sheet transplantation compared with sham (*P < 0.05). Carrier-free sheets had a smoother epithelial surface with minimal inflammation. Scale bar, 50 μm.
Figure 6.
 
Epithelial sheet transplantation in rabbits. Immunohistochemistry of BrdU (A, B) in epithelial sheets before transplantation. Slit lamp photographs (CE) and fluorescein staining (FH) of rabbit eyes 1 week after epithelial sheet transplantation. (C, F) AM, (D, G) carrier-free, (E, H) sham. (I) Area of intact epithelium was larger in rabbit eyes after epithelial sheet transplantation compared with sham (*P < 0.05). Carrier-free sheets had a smoother epithelial surface with minimal inflammation. Scale bar, 50 μm.
Figure 7.
 
Postoperative histology of epithelial sheet transplantation. Light micrograph of hematoxylin and eosin-stained sections of AM (A) and carrier-free (B) epithelial sheets. Immunohistochemistry of K3 (C, D), Collagen type IV (E, F), BrdU (G, H), and Ki67 (I, J). AM sheets retained significantly higher levels of BrdU (K) and expressed lower levels of Ki67 (L) than did carrier-free sheets (*P < 0.05, Student’s t-test). Scale bar, 50 μm.
Figure 7.
 
Postoperative histology of epithelial sheet transplantation. Light micrograph of hematoxylin and eosin-stained sections of AM (A) and carrier-free (B) epithelial sheets. Immunohistochemistry of K3 (C, D), Collagen type IV (E, F), BrdU (G, H), and Ki67 (I, J). AM sheets retained significantly higher levels of BrdU (K) and expressed lower levels of Ki67 (L) than did carrier-free sheets (*P < 0.05, Student’s t-test). Scale bar, 50 μm.
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