Development of transplantable genetically modified corneal epithelial cell sheets for gene therapy

The purpose of this study was to establish a method for the fabrication of exogenous gene-transferred, transplantable corneal epithelial cell sheets. Corneo-limbal epithelial cells collected from USA eye bank eyes were transduced with an EGFP-expressing lentiviral vector at differential MOI. Multi-layered corneal epithelial cell sheets were fabricated by co-cultivation of transduced cells and mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes. These cultured epithelial cells could be harvested as intact sheets by simply lowering the temperature. The number of EGFP-positive cells was increased as the MOI raised, and at an MOI of 100, nearly 100% of the superficial cells showed strong EGFP expression. Histological analysis revealed that EGFP was expressed in all layers of the cell sheet of which cell source was transduced with the lentiviral vector at an MOI of 100. Immunofluorescence data showed that p63 was also expressed in the basal layer of the same cell sheet. These results suggest that this technique will likely be applicable to ex vivo gene therapies for various corneal disorders.     

Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature

To develop new technology for harvesting transplantable cultured epithelium without dispase treatment, human keratinocytes were plated on culture dishes grafted with a thermo-responsive polymer, poly(N-isopropylacrylamide). The grafted dish surfaces are slightly hydrophobic above 32 degrees C, but reversibly change to hydrophilic below this temperature. According to the method of Rheinwald and Green, keratinocytes proliferated and made a multilayer on the grafted surfaces at 37 degrees C, as on the nongrafted culture dishes. The multilayered keratinocyte sheets were detached from the grafted surfaces only by reducing temperature to 20 degrees C without need for dispase. No cell remnants were observed on the dishes. Such cell sheet detachment was not observed on nongrafted dishes. Immunoblotting of harvested keratinocyte sheets revealed that dispase treatment disrupted E-cadherin and laminin 5, while these molecules remained intact in the keratinocyte sheets harvested by only reducing temperature from the grafted dishes. Transmission electron microscopy revealed that desmosomes were destroyed in dispase treatment but retained in low-temperature treatment. Use of thermo-responsive dishes was examined as a new tool for tissue engineering to achieve the preparation of artificial epithelium for cell transplantation as well as for the investigation of intact multilayered keratinocyte sheets.     

Anisotropic cell sheets for constructing three-dimensional tissue with well-organized cell orientation

Normal human dermal fibroblasts were aligned on micropatterned thermoresponsive surfaces simply by one-pot cell seeding. After they proliferated with maintaining their orientation, anisotropic cell sheets were harvested by reducing temperature to 20 °C. Surprisingly, the cell sheets showed different shrinking rates between vertical and parallel sides of the cell alignment (aspect ratio: approx. 3: 1), because actin fibers in the cell sheets were oriented with the same direction. The control of cell alignment provided not only a physical anisotropy but also biological impacts to the cell sheet. Vascular endothelial growth factor (VEGF) secreted by aligned fibroblasts was increased significantly, whereas transforming growth factor-β1 (TGF-β1) expression was the same level in anisotropic cell sheets as cell sheets having random cell orientations. Furthermore, although the amount of deposited type Ⅰ collagen was different non-significantly onto between cell sheets with and without controlled cell alignment, collagen deposited onto fibroblasts sheets with cell alignment also showed anisotropy, verified by a fluorescence imaging analysis. The physical and biological anisotropies of cell sheets were potentially useful to construct biomimetic tissues that were organized by aligned cells and/or extracellular matrix (ECM) including collagen in cell sheet-based regenerative medicine. Furthermore, due to the unique thermoresponsive property, the anisotropic cell sheets were successfully manipulated using a gelatin-coated plunger and were layered with maintaining their cell alignment. The combined use of the anisotropic cell sheet and cell sheet manipulation technique promises to create complex tissue that requires the three-dimensional control of their anisotropies, as one of the next-generation cell sheet technologies.     

A novel gelatin hydrogel carrier sheet for corneal endothelial transplantation

We examined the feasibility of using gelatin hydrogels as carrier sheets for the transplantation of cultivated corneal endothelial cells. The mechanical properties, transparency, and permeability of gelatin hydrogel sheets were compared with those of atelocollagen sheets. Immunohistochemistry (ZO-1, Na(+)/K(+)-ATPase, and N-cadherin), hematoxylin and eosin staining, and scanning electron microscopy were performed to assess the integrity of corneal endothelial cells that were cultured on gelatin hydrogel sheets. The gelatin hydrogel sheets displayed greater transparency, elastic modulus, and albumin permeability compared to those of atelocollagen sheets. The corneal endothelial cells on gelatin hydrogel sheets showed normal expression levels of ZO-1, Na(+)/K(+)-ATPase, and N-cadherin. Hematoxylin and eosin staining revealed the formation of a continuous monolayer of cells attached to the gelatin hydrogel sheet. Scanning electron microscopy observations showed that the corneal endothelial cells were arranged in a regular, mosaic, and polygonal pattern with normal cilia. These results indicate that the gelatin hydrogel sheet is a promising material to transport corneal endothelial cells during transplantation.     

Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: the noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces

A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive culture dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted dishes merely by reducing the temperature. However, during the early stage of culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other culture dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.     

Reconstruction of functional tissues with cell sheet engineering

The field of tissue engineering has yielded several successes in early clinical trials of regenerative medicine using living cells seeded into biodegradable scaffolds. In contrast to methods that combine biomaterials with living cells, we have developed an approach that uses culture surfaces grafted with the temperature-responsive polymer poly(N-isoproplyacrylamide) that allows for controlled attachment and detachment of living cells via simple temperature changes. Using cultured cell sheets harvested from temperature-responsive surfaces, we have established cell sheet engineering to create functional tissue sheets to treat a wide range of diseases from corneal dysfunction to esophageal cancer, tracheal resection, and cardiac failure. Additionally, by exploiting the unique ability of cell sheets to generate three-dimensional tissues composed of only cultured cells and their deposited extracellular matrix, we have also developed methods to create thick vascularized tissues as well as, organ-like systems for the heart and liver. Cell sheet engineering therefore provides a novel alternative for regenerative medicine approaches that require the re-creation of functional tissue structures.     

The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets

Primary canine oral mucosal epithelial cells were cultured on temperature-responsive dishes and cell culture inserts to fabricate transplantable epithelial cell sheets. When 3T3 feeder layers and fetal bovine serum were eliminated from dish culture, the harvested cell sheets became significantly more fragile. In contrast, when epithelial cells were cultured on inserts having submicron-scale pores, cell sheet fragility was eliminated. Keratin expression profiles showed no differences among the harvested cell sheets, but the expression of p63, a putative stem/progenitor marker, was strongly dependent on the presence of 3T3 feeder layers and serum. These results suggest that the maintenance of stem/progenitor cells is influenced by the apical/basal supply of nutrients as well as culture supplements.     

Micro-patterned cell-sheets fabricated with stamping-force-controlled micro-contact printing

Cell-sheet-engineering based regenerative medicine is successfully applied to clinical studies, though cell sheets contain uniformly distributed cells. For the further application to complex tissues/organs, cell sheets with a multi-cellular pattern were highly demanded. Micro-contact printing is a quite useful technique for patterning proteins contained in extracellular matrix (ECM). Because ECM is a kind of cellular adherent molecules, ECM-patterned cell culture surface is capable of aligning cells on the pattern of ECM. However, a manual printing is difficult, because a stamp made from polydimethylsiloxane (PDMS) is easily deformed, and a printed pattern is also crushed. This study focused on the deformability of PDMS stamp and discussed an appropriate stamping force in micro-contact printing. Considering in availability in a medical or biological laboratory, a method for assessing the stamp deformability was developed by using stiffness measurement with a general microscope. An automated stamping system composed of a load cell and an automated actuator was prepared and allowed to improve the quality of stamped pattern by controlling an appropriate stamping force of 0.1 N. Using the system and the control of appropriate stamping force, the pattern of 8-mm-diameter 80-μm-stripe fibronectin was fabricated on the surface of temperature-responsive cell culture dish. After cell-seeding and cell culture, a co-culture system with the micro-pattern of both fibroblasts and endothelial cells was completed. Furthermore, by reducing temperature to 20 °C, the co-cultured cell sheet with the micro-pattern was successfully harvested. As a result, the method would not only provide a high-quality ECM pattern but also a breakthrough technique to fabricate multi-cellular-patterned cell sheets for the next generation of regenerative medicine and tissue engineering.     

Thermo-responsive poly(NiPAAm-co-DEGMA) substrates for gentle harvest of human corneal endothelial cell sheets

Gentle harvesting of corneal endothelial cell sheets grown in culture is of interest for the development of cornea replacement strategies. Thin films of a fast responding copolymer of N-isopropylacrylamide (NiPAAm) and diethyleneglycol methacrylate (DEGMA) with a phase transition temperature of 32 degrees C were prepared and evaluated for that purpose. The polymer layers were immobilized onto fluorocarbon substrates using low pressure argon plasma treatment. Cell culture and detachment experiments were performed with L929 mouse fibroblasts and human corneal endothelial cells (HCEC) at standard conditions. The hydrogel-coated supports were found to permit adhesion, spreading, and proliferation of both cell types. Harvesting of cell sheets was achieved upon lowering the temperature to about 30 degrees C. The formation of a closed monolayer as a crucial prerequisite for maintaining ionic pump function in HCEC was proven by ZO-1 immunostainung. Labeling of fibronectin indicated that the vast majority of the extracellular matrix is detached from the hydrogel coatings together with the cell layer. Inspired by this result, the reuse of the hydrogel-coated culture carriers was investigated confirming the suitability of the substrates for repeated cell harvesting. Altogether, the introduced thermoresponsive coating was found advantageous for the efficient generation of HCEC sheets and will be further utilized in transplantation strategies.     

Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets

We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37 degrees C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37 degrees C, trypsin digestion disrupted cell-cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20 degrees C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-beta-catenin antibody revealed that functional cell-cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20 degrees C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction.     

Temperature-Responsive surface for novel co-culture systems of hepatocytes with endothelial cells: 2-D patterned and double layered co-cultures

We have developed two novel cell co-culture system, without any on cell type combination limitation, utilizing a polymer surface which is temperature-sensitive with respect to its cell adhesion characteristics. One system involves a patterned co-culture of primary hepatocytes with endothelial cells utilizing patterned masked of the electron-beam cured, temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) by masked electron beam irradiation. Hepatocytes were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. Endothelial cells were then seeded onto the same surfaces at 37 degrees C. These subsequently seeded endothelial cells adhered only to the now-exposed PIPAAm-grafted domains and could be co-cultured with the hepatocytes initially seeded at 37 degrees C in well-ordered patterns. The other system involves a double layered co-culture obtained by overlaying endothelial cell sheets of the designed shape onto hepatocyte monolayers. The endothelial cells adhered and proliferated on the PIPAAm-grafted surface, as on polystyrene tissue culture dishes at 37 degrees C. By reducing the temperature, confluent monolayers of cells detached from the PIPAAm surfaces without trypsin. Because the recovered cells maintained intact cell-cell junctions together with deposited extracellular matrix, the harvested endothelial cell sheets, with designed shapes, were transferable and readily adhered to hepatocyte monolayers. Stable double layered cell sheets could be co-cultivated. These two co-culture methods enabled long-term co-culture of primary hepatocytes with endothelial cells. Hepatocytes so co-cultured with endothelial cells maintained their differentiated functions, such as albumin synthesis for unexpectedly long periods. These novel two co-culture systems offer promising techniques for basic biologic researches upon intercellular communications, and for the clinical applications of tissue engineered constructs.     

Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia

The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.     

Human corneal endothelial cell sheets for transplantation: Thermo-responsive cell culture carriers to meet cell-specific requirements

Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options.     

Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model

Conventional periodontal regeneration methods remain insufficient to attain complete and reliable clinical regeneration of periodontal tissues. We have developed a new method of cell transplantation using cell sheet engineering and have applied it to this problem. The purpose of this study was to investigate the characteristics of human periodontal ligament (HPDL) cell sheets retrieved from culture on unique temperature-responsive culture dishes, and to examine whether these cell sheets can regenerate periodontal tissues. The HPDL cell sheets were examined histologically and biochemically, and also were transplanted into a mesial dehiscence model in athymic rats. HPDL cells were harvested from culture dishes as a contiguous cell sheet with abundant extracellular matrix and retained intact integrins that are susceptible to trypsin-EDTA treatment. In the animal study, periodontal ligament-like tissues that include an acellular cementum-like layer and fibrils anchoring into this layer were identified in all the athymic rats transplanted with HPDL cell sheets. This fibril anchoring highly resembles native periodontal ligament fibers; such regeneration was not observed in nontransplanted controls. These results suggest that this technique, based on the concept of cell sheet engineering, can be useful for periodontal tissue regeneration.     

Transplantable urothelial cell sheets harvested noninvasively from temperature-responsive culture surfaces by reducing temperature

Augmentation cystoplasty using gastrointestinal flaps may induce severe complications such as lithiasis, urinary tract infection, and electrolyte imbalance. The use of viable, contiguous urothelial cell sheets cultured in vitro should enable us to avoid these complications. Transplantable urothelial cell sheets were obtained by utilizing a temperature-responsive cell culture method, and then examined by immunostaining and electron microscopy. Canine urothelium was produced on the surfaces of temperature-responsive culture dishes covalently bonded with the thermally sensitive polymer, poly(N-isopropylacrylamide). Stratified urothelial cell sheets were cultured and then harvested intact without enzymatic treatment from these dishes by reducing the temperature. Histological structure and cell-to-cell junctions were compared between these urothelial cell sheets and those harvested with dispase. All urothelial cell sheets were harvested from the bonded surfaces by reducing the culture temperature without the need for dispase. Electron microscopy revealed well-developed microridge, microvilli, and cell junction complexes. Conversely, these same cell features were destroyed by dispase treatment. Immunoblotting revealed that dispase fragmented occludin, whereas it remained unchanged in the intact urothelial cell sheets. Novel urothelial cell sheets obtained by culture on temperature-responsive culture surfaces were successfully harvested much less destructively than with dispase. This technology should prove useful in urinary tract tissue engineering in the near future.     

Transplantable retinal pigment epithelial cell sheets for tissue engineering

The native retinal pigment epithelium (RPE) exists as a monolayer structure and is critically involved in the maintenance of photoreceptors. Damage or destruction of the RPE due to a variety of diseases therefore often results in loss of vision. With regenerative purposes in mind, we have examined various culture conditions such as the initial cell density and the addition of various supplements in an effort to produce transplantable RPE cell sheets that can be harvested without defects. We demonstrate that the cell density in cultured RPE sheets increased linearly with the number of seeded cells and that RPE sheets were harvested without defects and limited contraction due to cytoskeletal reorganization, when TGF-beta2 was added to the growth medium. Results from histological analysis and the measurement of trans-epithelial resistance also demonstrates that these RPE cell sheets exist as monolayer structure, similar to the native RPE, with intact cell-to-cell junctions. Therefore, these methods provide significant insight into the fabrication of transplantable RPE cell sheets that can be applied to RPE regenerative therapies to restore lost vision.     

Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus

The present study was designed to establish a novel tissue engineering approach for diabetes mellitus (DM) by fabricating a tissue sheet composed of pancreatic islet cells for in vivo transplantation. Pancreatic islet cell suspensions were obtained from Lewis rats, and plated onto temperature-responsive culture dishes coated with extracellular matrix (ECM) proteins. After the cells reached confluency, islet cells cultured on laminin-5 coated dishes were successfully harvested as a uniformly spread tissue sheet by lowering the culture temperature to 20 °C for 20 min. The functional activity of the islet cell sheets was confirmed by histological examination and Insulin secretion assay prior to in vivo transplantation. Histological examination revealed that the harvested islet cell sheet was comprised of insulin- (76%) and glucagon- (19%) positive cells, respectively. In vivo functionality of the islet cell sheet was maintained even 7 days after transplantation into the subcutaneous space of Lewis rats. The present study describes an approach to generate a functional sheet of pancreatic islet cells on laminin-5 coated temperature-responsive dishes, which can be subsequently transplanted in vivo. This study serves as the foundation for the creation of a novel cell-based therapy for DM to provide patients an alternative method.     

Fabrication of transferable micropatterned-co-cultured cell sheets with microcontact printing

The purpose of the present study is to develop a novel method for the fabrication of transferable micropatterned cell sheets for tissue engineering. To achieve this development, microcontact printing of fibronectin on commercially available temperature-responsive dishes was employed. Primary rat hepatocytes were seeded on the dish surfaces printed with fibronectin. Under serum-free conditions, hepatocytes were attached onto fibronectin domains selectively. Then, a second cell type of endothelial cells was seeded in the presence of serum. Double fluorescent staining revealed that endothelial cells successfully adhered to the intervals of hepatocyte domains. Finally, all the cells were harvested as a single contiguous micropatterned cell sheet upon temperature-reduction. With a cell sheet manipulator having a gelatin layer for the support of harvested cell sheets, harvested micropatterned cell sheets were transferred to new dish surfaces. This technique would be useful for the fabrication of thick tissue constructs having a complex microarchitecture.     

纤维蛋白胶为载体构建人羊膜上皮细胞植片的实验研究

目的： 探讨利用纤维蛋白胶作为支架构建组织工程人羊膜上皮细胞(HAECs)植片重建眼表的可行性。方法：取足月剖宫产胎盘羊膜,经胶原酶和胰蛋白酶消化后,获得HAECs。在体外构建的纤维蛋白胶片上培养HAECs，细胞融合成片后，利用气液界面复层化，采用倒置显微镜、组织切片、HE 染色、细胞角蛋白免疫组织化学染色和扫描电子显微镜观察HAECs的生长情况。结果：HAECs在纤维蛋白胶表面生长良好，细胞呈圆形或多角形,长满后呈上皮细胞特有的铺路石样外观，扫描电镜观察细胞表面有丰富的微绒毛，细胞广谱角蛋白单克隆抗体染色阳性。细胞有复层生长趋势，植片较为透明。结论：以纤维蛋白胶为载体构建组织工程人 HAECs 植片，具有眼表重建的潜在运用价值。     

Micro- and nanotopography with extracellular matrix coating modulate human corneal endothelial cell behavior

The human corneal endothelium plays an important role in maintaining corneal transparency. Human corneal endothelial cells have limited regenerative capability in vivo. Consequently, endothelial dysfunction can occur following corneal endothelial trauma or inherited diseases. To restore endothelial function, corneal transplantation is needed. However, there is a worldwide shortage of donor corneas, motivating the development of a tissue-engineered graft alternative using cultivated endothelial cells. To induce in vitro cell proliferation, much effort has been made to improve culture conditions and to mimic the native extracellular microenvironment. We incorporated topographical and biochemical cues in our in vitro culture of human corneal endothelial cell line B4G12 (HCEC-B4G12) and hypothesized that manipulation of the extracellular environment can modulate cell proliferation, morphometry and phenotype. The topographies tested were nanopillars, microwells and micropillars on polydimethylsiloxane, while the biochemical factors were extracellular matrix protein coatings of fibronectin-collagen I (FC), FNC® coating mix (FNC) and laminin-chondroitin sulfate (LC). Cellular morphometry, Na⁺/K⁺-ATPase and zona occludens 1 (ZO-1) gene and protein expression were analyzed 3 days after cells had formed a confluent monolayer. The cell circularity on all patterns and coatings was above 0.78. On all coatings, cell area was the lowest on micropillars. The coefficient of variation (CV) of the cell area was the lowest on nanopillars with an LC coating. With an FC coating, micropillars induced a better cellular outcome as the cells had the greatest circularity, smallest cell area and highest Na⁺/K⁺-ATPase and ZO-1 gene and protein expression. With the LC coating, HCECs grown on nanopillars resulted in the lowest CV of the cell area and the highest ZO-1 gene expression. Thus, HCEC-B4G12 morphometry and phenotype can be improved using different topographical and biochemical cues.