RESUMO
Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) has been introduced as a modification of the standard DMEK technique to increase the pool of endothelial grafts. In this study, we evaluated in vitro changes in endothelial cell distribution, viability and morphology of Quarter-DMEK grafts when stored in organ-culture medium. Quarter-DMEK grafts were prepared from 5 corneas and stored in organ-culture medium for 4, 7 and 11 days. Endothelial cell re-distribution was investigated by light microscopy, cell viability by a Calcein-AM assay, and expression of endothelial and non-endothelial markers by immunohistochemistry. Three standard DMEK-grafts were used as controls. After preparation, all Quarter-DMEK grafts showed a band with no viable endothelial cells along the radial cut graft edges [average width 190 (± 20) µm]. Endothelial cell density in the central graft area decreased by 12%, 23% and 26% after 4, 7, and 11 days of storage, respectively. At the same time, empty bands along the cut edges were re-populated and some cells migrated to the stromal side of the Descemet membrane (DM). These cells showed an altered phenotype, as indicated by expression of migration marker CD73 and fibroblast marker αSMA. Majority of migration occurred within the first 4 days of storage. Our data suggest that endothelial cells on Quarter-DMEK grafts re-distribute during organ-culture storage to re-populate preparation-induced empty bands and after re-distribution, cells may show further migration to the stromal DM side during storage.
Assuntos
Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Células Endoteliais/citologia , Células Endoteliais/transplante , Idoso , Idoso de 80 Anos ou mais , Contagem de Células , Feminino , Humanos , Masculino , Técnicas de Cultura de Órgãos , Fatores de Tempo , Doadores de Tecidos , Preservação de TecidoRESUMO
AIM: Studying cell migration of corneal endothelial cells in vitro is challenging because the capacity for cell migration needs to be maintained while at the same time the tissue must remain fixed on a rigid substrate. In this study, we report a thermoresponsive culture technique designed to maintain cellular viability, and to reduce tissue handling in order to analyze in vitro endothelial cell migration from corneal grafts. MATERIALS AND METHODS: As a test tissue, fifteen Quarter-Descemet membrane endothelial keratoplasty (Q-DMEK) grafts were used that were embedded in a three-dimensional culture system using a temperature-reversible hydrogel and cultured over 2-3 weeks in a humidified atmosphere at 37°C and 5% CO2. RESULTS: All grafts could be successfully cultured inside the thermoresponsive polymer solution for periods of up to 21 days. Using this system, cell migration could be assessed by light microscopy at fixed time intervals. At the end of the culture period, the gel could be removed from all grafts and immunohistochemistry analysis showed that endothelial cells were able to maintain confluence, viability, and junctional integrity. Some problems were encountered when using the thermoresponsive cell culture system. These were mostly structural inconsistencies during the sol-to-gel transition phase that resulted in the formation of tiny bubbles in the matrix. Additionally, areas with different viscosity resulted in optical distortions showing up as folds throughout the matrix which can persist even after several cycles of culture medium exchange. These effects had impact on the imaging quality but did not affect the viability of the explant tissue. CONCLUSION: This study proves that temperature-reversible hydrogel is a very useful matrix for studying in vitro corneal endothelial cell migration from explant grafts and allows for subsequent biological investigation after gel removal.
Assuntos
Técnicas de Cultura de Células/métodos , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Endotélio Corneano/citologia , Distrofia Endotelial de Fuchs/cirurgia , Doadores de Tecidos , Acuidade Visual , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Distrofia Endotelial de Fuchs/diagnóstico , HumanosRESUMO
Corneal transplantation is currently the most effective treatment to restore corneal clarity in patients with endothelial disorders. Endothelial transplantation, either by Descemet membrane endothelial keratoplasty (DMEK) or by Descemet stripping (automated) endothelial keratoplasty (DS(A)EK), is a surgical approach that replaces diseased Descemet membrane and endothelium with tissue from a healthy donor eye. Its application, however, is limited by the availability of healthy donor tissue. To increase the pool of endothelial grafts, research has focused on developing new treatment options as alternatives to conventional corneal transplantation. These treatment options can be considered as either 'surgery-based', that is tissue-efficient modifications of the current techniques (e.g. Descemet stripping only (DSO)/Descemetorhexis without endothelial keratoplasty (DWEK) and Quarter-DMEK), or 'cell-based' approaches, which rely on in vitro expansion of human corneal endothelial cells (hCEC) (i.e. cultured corneal endothelial cell sheet transplantation and cell injection). In this review, we will focus on the most recent developments in the field of the 'cell-based' approaches. Starting with the description of aspects involved in the isolation of hCEC from donor tissue, we then describe the different natural and bioengineered carriers currently used in endothelial cell sheet transplantation, and finally, we discuss the current 'state of the art' in novel therapeutic approaches such as endothelial cell injection.
Assuntos
Doenças da Córnea/cirurgia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Endotélio Corneano/transplante , Células Cultivadas , Humanos , Doadores de TecidosRESUMO
AIM: To test the feasibility of implanting human anterior lens capsules (HALCs) with porcine corneal endothelial cells (pCEC) in vivo in Göttingen minipigs and at the same time test the suitability of Göttingen minipig as model for endothelial keratoplasty. MATERIALS AND METHODS: Cell-carrier constructs of decellularized HALC with cultured (pCEC) were created for implementation in vivo. Eight Göttingen minipigs (6 months old) underwent surgery with descemetorhexis or removal of endothelium by scraping and implementation of HALC without (animal 1-4) and with (animal 5-8) pCEC. Follow-up examinations included optical coherence tomography (OCT) imaging (1,2 and 3 months) and slit-lamp examination (<1 week as well as 1,2 and 3 months). RESULTS: Intraoperative challenges included difficulties in maintaining an anterior chamber due to soft tissue and vitreous pressure, development of corneal edema and difficulties removing Descemet's membrane because of strong adhesion to stroma. Therefore, descemetorhexis was replaced by mechanical scraping of the endothelium in animal 4-8. HALCs without pCEC were implanted in animal 1-4. Apposition to the back surface was not achieved in animal 1 and 3 because of corneal edema and poor visibility. Animal 5 was sacrificed because of a lens capsule tear. HALCs with pCEC were implanted in animal 6-8. Slit-lamp examination the first week revealed corneal edema in all animals, although mild in animals 4. One-month examination showed retrocorneal membranes with overlying corneal edema in all animals. Histology showed fibrosis in the AC and on the back surface of the cornea, compatible with the clinical diagnosis of retrocorneal membrane. CONCLUSIONS: In conclusion, the minipig is not suitable for corneal transplantation studies in vivo because of intraoperative challenges and development of retrocorneal membrane postoperatively. For in vivo testing of the surgical handling and the therapeutic potential of tissue-engineered endothelial cell-carrier constructs other animal models are required.
Assuntos
Transplante de Córnea/métodos , Modelos Animais de Doenças , Endotélio Corneano/transplante , Engenharia Tecidual/métodos , Animais , Câmara Anterior/patologia , Técnicas de Cultura de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Edema da Córnea/diagnóstico , Edema da Córnea/etiologia , Desbridamento , Dinamarca , Endotélio Corneano/citologia , Endotélio Corneano/diagnóstico por imagem , Estudos de Viabilidade , Fibrose , Cápsula do Cristalino/citologia , Microscopia com Lâmpada de Fenda , Suínos , Porco Miniatura , Tomografia de Coerência ÓpticaRESUMO
Corneal transplantation is currently the only effective treatment option for dysfunctional corneal endothelial cells (CEC). In this study, we test in vitro the surgical potential of cultivated human corneal endothelial cells (hCEC) on human anterior lens capsule (HALC), LinkCell™ bioengineered collagen sheets of 20-µm thickness (LK20), and denuded Descemet membrane (dDM) as tissue-engineered grafts for Descemet membrane (DM) endothelial keratoplasty (DMEK) to bypass the problem of donor tissue availability. Primary hCEC cultured on all carriers formed a monolayer of tightly packed cells with a high cell viability rate (96% ± 4%). hCEC on HALC and LK20 showed unremarkable expression of zonula occludens-1 (ZO-1) and Na+/K+-adenosine triphosphatase (ATPase), while Na+/K+-ATPase expression of cells seeded on dDM was mainly cytoplasmic. All hCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using a human donor cornea without DM mounted on an artificial anterior chamber (AC) and a regular DMEK-graft used as a surgical reference model. During in vitro surgery, hCEC-HALC constructs behaved most similarly to a DMEK-graft during implantation and unfolding, showing good adhesion to the bare stroma. On the other hand, hCEC-LK20 and hCEC-dDM constructs required some additional handling because of challenges related to the surgical procedure, although they were both successfully unfolded and implanted in the artificial AC. The hCEC-dDM constructs showed similar graft adherence as hCEC-HALC constructs, while adherence of hCEC-LK20 constructs was less effective. After the in vitro surgery, the estimated area populated by viable cells on the hCEC-HALC and hCEC-LK20 constructs was â¼83% and â¼67%, respectively. Overall, hCEC-HALC constructs behaved most similarly to a DMEK-graft during in vitro DMEK surgery, while graft adhesion and surgical handling, respectively, are parameters still requiring optimization for hCEC-LK20 and hCEC-dDM constructs.
Assuntos
Transplante de Córnea/métodos , Células Endoteliais/fisiologia , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Humanos , Pessoa de Meia-Idade , Engenharia Tecidual/métodosRESUMO
Endothelial cell migration plays a crucial role in achieving corneal clearance after corneal transplantation when using smaller-sized endothelial grafts to increase the donor pool. In this study we investigated how different strategies of Quarter-Descemet Membrane Endothelial Keratoplasty (Quarter-DMEK) limbal graft edge modification influence peripheral endothelial cell migration in an in vitro culture environment. For this study, 15 Quarter-DMEK grafts, prepared from 7 corneas deemed ineligible for transplantation but with intact and viable endothelial cells, were embedded in a cooled biocompatible, thermoresponsive matrix for culture. The limbal edge of ten Quarter-DMEK grafts were modified, either by using a small diameter punch or by peripheral radial cuts. All Quarter-DMEK grafts showed substantial collective endothelial cell migration from the radial cut graft edges, as observed by light microscopy at standardized time intervals. Grafts were retrieved from the polymer matrix after the two-week culture for immunohistochemistry analyses of the newly formed cell monolayers; this demonstrated the presence of tightly packed and viable cells that showed higher migratory ability at the leading edge. Peripheral endothelial cell migration, however, was not triggered by increasing cell exposure to free space through surgical modifications of the far periphery. Our data suggest that alterations in the far peripheral area of Quarter-DMEK grafts were insufficient to triggering cell migration from the limbal graft edge. This may be due to transient-amplifying cells that reside in the far periphery and which lack cytokinetic directional cues. Understanding the migration capacity of the peripheral endothelium could unlock cells' therapeutic potential which are, at present, routinely discarded from transplantation. Encouraging peripheral cell migration may also improve clinical outcomes from Quarter-DMEK, but a more effective solution is required prior to clinical implementation of modified grafts.
Assuntos
Técnicas de Cultura de Células/métodos , Córnea/citologia , Células Endoteliais/citologia , Idoso , Autopsia , Movimento Celular , Células Cultivadas , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Feminino , Humanos , Masculino , Microscopia , Pessoa de Meia-IdadeRESUMO
Purpose/Aim: Evaluating the suitability of bioengineered collagen sheets and human anterior lens capsules (HALCs) as carriers for cultivated porcine corneal endothelial cells (pCECs) and in vitro assessment of the cell-carrier sheets as tissue-engineered grafts for Descemet membrane endothelial keratoplasty (DMEK). MATERIALS AND METHODS: pCECs were isolated, cultured up to P2 and seeded onto LinkCell™ bioengineered matrices of 20 µm (LK20) or 100 µm (LK100) thickness, and on HALC. During expansion, pCEC viability and morphology were assessed by light microscopy. ZO-1 and Na+/K+-ATPase expression was investigated by immunohistochemistry. Biomechanical properties of pCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using an artificial anterior chamber (AC) and a human donor cornea without Descemet membrane (DM). RESULTS: During in vitro expansion, cultured pCECs retained their proliferative capacity, as shown by the positive staining for proliferative marker Ki67, and a high cell viability rate (96 ± 5%). pCECs seeded on all carriers formed a monolayer of hexagonal, tightly packed cells that expressed ZO-1 and Na+/K+-ATPase. During in vitro surgery, pCEC-LK20 and pCEC-LK100 constructs were handled like Descemet stripping endothelial keratoplasty (DSEK) grafts, i.e. folded like a "taco" for insertion because of challenges related to rolling and sticking of the grafts in the injector. pCEC-HALC constructs behaved similar to the DMEK reference model during implantation and unfolding in the artificial AC, showing good adhesion to the bare stroma. CONCLUSIONS: In vitro DMEK surgery showed HALC as the most suitable carrier for cultivated pCECs with good intraoperative graft handling. LK20 carrier showed good biocompatibility, but required a DSEK-adapted surgical protocol. Both carriers might be notional candidates for potential future clinical applications.
Assuntos
Materiais Biocompatíveis , Colágeno , Endotélio Corneano/citologia , Cápsula do Cristalino , Alicerces Teciduais , Animais , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Endotélio Corneano/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Antígeno Ki-67/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos , Engenharia Tecidual , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
PURPOSE: To investigate in vitro central and peripheral corneal endothelial cell (EC) migration from Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) grafts. METHODS: Quarter-DMEK grafts were obtained from 10 corneas ineligible for transplantation but with intact and viable ECs. Ten Quarter-DMEK grafts were 'sandwiched' between two glass slides and cultured over 1 week in a humidified atmosphere at 37 °C and 5% CO2 . Cell migration was evaluated by light microscopy at standardized time intervals. In addition, immunohistochemistry analyses were performed to assess the detailed structural organization of ECs in the corneal centre and far periphery. RESULTS: Endothelial cell (EC) migration occurred from the radial cut graft edges, but not from the far peripheral area. Cell migration followed three different migration patterns: (1) individual cell migration, (2) uncoordinated cell migration of cell clusters and (3) collective migration in which ECs moved as a sheet. Immunostaining showed the presence of ECs up to the far periphery but with different expression patterns of phenotypical markers ZO-1, Na+ /K+ -ATPase and vimentin compared to central ECs. CONCLUSION: In vitro EC migration from Quarter-DMEK grafts occurs along the radial cut edges with a decrease in migration activity towards the corneal far periphery. No migration occurred along the outer peripheral corneal edge possibly due to a different anatomical matrix in the far periphery. Hence, ECs from the far periphery may not contribute to corneal clearance of the adjacent bare area after Quarter-DMEK surgery, but these cells may constitute a valuable cellular reserve on the graft.
Assuntos
Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Endotélio Corneano/patologia , Distrofia Endotelial de Fuchs/cirurgia , Doadores de Tecidos , Acuidade Visual , Idoso , Idoso de 80 Anos ou mais , Contagem de Células , Movimento Celular , Feminino , Distrofia Endotelial de Fuchs/patologia , Sobrevivência de Enxerto , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-IdadeRESUMO
Fibroblasts are abundantly present in the prostate tumor microenvironment (TME), including cancer-associated fibroblasts (CAFs) which play a key role in cancer development. Androgen receptor (AR) signaling is the main driver of prostate cancer (PCa) progression, and stromal cells in the TME also express AR. High-grade tumor and poor clinical outcome are associated with low AR expression in the TME, which suggests a protective role of AR signaling in the stroma against PCa development. However, the mechanism of this relation is not clear. In this study, we isolated AR-expressing CAF-like cells. Testosterone (R1881) exposure did not affect CAF-like cell morphology, proliferation, or motility. PCa cell growth was not affected by culturing in medium from R1881-exposed CAF-like cells; however, migration of PCa cells was inhibited. AR chromatin immune precipitation sequencing (ChIP-seq) was performed and motif search suggested that AR in CAF-like cells bound the chromatin through AP-1-elements upon R1881 exposure, inducing enhancer-mediated AR chromatin interactions. The vast majority of chromatin binding sites in CAF-like cells were unique and not shared with AR sites observed in PCa cell lines or tumors. AR signaling in CAF-like cells decreased expression of multiple cytokines; most notably CCL2 and CXCL8 and both cytokines increased migration of PCa cells. These results suggest direct paracrine regulation of PCa cell migration by CAFs through AR signaling.