ABSTRACT
Cell injection therapy is emerging as an alternative to treat corneal endothelial dysfunction (CED) and to avoid corneal scarring due to bullous keratopathy. However, establishing a standardized culture procedure that provides appropriate cell yield while retaining functional features remains a challenge. Here, we describe a detailed framework obtained from in vitro culture of human corneal endothelial cells (HCECs) and comparative in vivo experimental models for CED treatment with a new cell tracking approach. Two digestion methods were compared regarding HCEC morphology and adhesion. The effect of Y-27632 (ROCKi) supplementation on final cell yield was also assessed. Cell adhesion efficacy with two cell delivery systems (superparamagnetic embedding and cell suspension) was evaluated in an ex vivo human cornea model and in an in vivo rabbit CED model. The injection of supplemented culture medium or balanced salt solution (BSS) was used for the positive and negative controls, respectively. HCEC isolation with collagenase resulted in better morphology and adhesion of cultured HCEC when compared to EDTA. Y-27632 supplementation resulted in a 2.6-fold increase in final cell yield compared to the control. Ex vivo and in vivo adhesion with both cell delivery systems was confirmed by cell tracker fluorescence detection. Corneal edema and opacity improved in both animal groups treated with cultured HCEC. The corneas in the control groups remained opaque. Both HCEC delivery systems seemed comparable as treatments for CED and for the prevention of corneal scarring.
Subject(s)
Cell Tracking , Endothelium, Corneal , Animals , Humans , Rabbits , Endothelial Cells , Cicatrix/pathology , Cells, CulturedABSTRACT
ABSTRACT Purpose: This quality and reliability study aimed to identify the mass variability of multidose eyedrops and to verify the existence of a reference pattern for the drop volume of eyedrops using standard lubricant eyedrops available on the Brazilian market. Methods: Five brands of lubricant eyedrops were evaluated. An ideal standard 20 µL drop of eyedrops from each manufacturer was captured using an adjustable micropipette. The eyedrop bottles were randomly selected, and five measurements of the samples' masses were collected using calibrated precision scales. Results: The mass of the 20 µL samples varied significantly (p<0.001) among the different manufacturers. However, among eyedrops of the same brand, mass variation was not statistically different. The global mean mass of all weighed drops was 18.24 mg, and non-uniformity was identified across all eyedrop brands. Conclusion: Significant variations in the drop masses of common lubricant eyedrops were identified using standard laboratory equipment. Heterogeneity in the drop volume of standard eyedrop medications suggest that potential dosage discrepancies exist, possibly altering treatment efficacy. A pre-established reference measure may lead to the production of more appropriately sized eyedrops for use in human eyes.
RESUMO Objetivo: Este estudo de qualidade e confiabilidade teve como objetivo identificar a variabilidade da massa do volume da gota de colírios multidose e verificar a existência de um padrão de referência para o volume da gota dos colírios usando lágrimas artificiais do mercado brasileiro. Métodos: Cinco marcas de colírios lubrificantes foram avaliadas quanto ao volume da gota. Uma gota padrão ideal de 20 µL de cada fabricante foi coletada usando uma micropipeta ajustável. Os frascos dos colírios foram selecionados aleatoriamente e cinco medidas das massas das amostras foram coletadas usando escalas de precisão calibradas. Resultados: A massa das amostras de 20 µL variou significativamente (p<0,001) entre os diferentes fabricantes. No entanto, entre os colírios da mesma marca, a variação da massa não foi estatisticamente diferente. A massa média global de todas as gotas pesadas foi de 18,24 mg e foi observada uma não-uniformidade entre todas as marcas de colírios. Conclusão: Identificou-se uma variação significativa nas massas do volume das gotas dos colírios lubrificantes, usando equipamento padrão de laboratório. A heterogeneidade no volume da gota dos colírios testados sugere a existência de discrepâncias potenciais em suas posologias, possivelmente alterando a eficácia do tratamento. Uma medida de referência pré-estabelecida pode levar à produção de colírios com gotas de tamanho mais apropriado para uso em olhos humanos.
ABSTRACT
PURPOSE: This quality and reliability study aimed to identify the mass variability of multidose eyedrops and to verify the existence of a reference pattern for the drop volume of eyedrops using standard lubricant eyedrops available on the Brazilian market. METHODS: Five brands of lubricant eyedrops were evaluated. An ideal standard 20 µL drop of eyedrops from each manufacturer was captured using an adjustable micropipette. The eyedrop bottles were randomly selected, and five measurements of the samples' masses were collected using calibrated precision scales. RESULTS: The mass of the 20 µL samples varied significantly (p<0.001) among the different manufacturers. However, among eyedrops of the same brand, mass variation was not statistically different. The global mean mass of all weighed drops was 18.24 mg, and non-uniformity was identified across all eyedrop brands. CONCLUSION: Significant variations in the drop masses of common lubricant eyedrops were identified using standard laboratory equipment. Heterogeneity in the drop volume of standard eyedrop medications suggest that potential dosage discrepancies exist, possibly altering treatment efficacy. A pre-established reference measure may lead to the production of more appropriately sized eyedrops for use in human eyes.
Subject(s)
Ophthalmic Solutions , Brazil , Humans , Reproducibility of ResultsABSTRACT
ABSTRACT Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.
RESUMO Objetivos: Avaliar a capacidade das células-tronco imaturas da polpa do dente de leite que são células-tronco mesenquimais de origem da crista neural, de se diferenciarem no epitélio corneano para fins de transplante de córnea e engenharia de tecidos quando cultivadas em membrana amnióticas desepitelizadas. Métodos: Foram comparamos so imunofenótipo (ABCG2, CK3/12 e vimentina) de células cultivadas em membranas amnióticas ou em superfícies plásticas sob condições livres de soro ou em meios de cultura contendo soro ou componentes de substituição de soro. Resultados: Células-tronco imaturas da polpa do dente de leite cultivadas sobre membrana amniótica em condições basais são capazes de manter seu estado indiferenciado. Nossos dados também sugerem que o meio de cultura utilizado no presente trabalho pode modular a expressão de marcadores de células-tronco imaturas da polpa do dente de leite, induzindo a diferenciação epitelial destas células in vitro. Conclusão: Nossos resultados sugerem que a membrana amniótica é uma boa escolha para o crescimento e transplante de células-tronco mesenquimais, particularmente as células-tronco imaturas da polpa do dente de leite, na reconstrução da superfície ocular.
Subject(s)
Humans , Epithelium, Corneal/transplantation , Dental Pulp/cytology , Mesenchymal Stem Cell Transplantation/methods , Tissue Scaffolds , Mesenchymal Stem Cells/cytology , Amnion , Time Factors , Cells, Cultured , Reproducibility of Results , Fluorescent Antibody Technique , Cell Culture Techniques/methods , Corneal Diseases/surgery , Cell ProliferationABSTRACT
PURPOSE: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. METHODS: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. RESULTS: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. CONCLUSIONS: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.
Subject(s)
Amnion , Dental Pulp/cytology , Epithelium, Corneal/transplantation , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/cytology , Tissue Scaffolds , Cell Culture Techniques/methods , Cell Proliferation , Cells, Cultured , Corneal Diseases/surgery , Fluorescent Antibody Technique , Humans , Reproducibility of Results , Time FactorsABSTRACT
Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.
ABSTRACT
Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.
ABSTRACT
AIMS: To evaluate the expression of ß-galactoside-binding proteins galectin (Gal)-1 and Gal-3 in patients with keratoconus (KC) and postcorneal collagen cross-linking (CXL) treatment in vitro. METHODS: Tear fluid, cornea samples and conjunctival impression cytology specimens from control and KC patients were used to evaluate Gal-1 and Gal-3 expressions. Primary keratocytes were isolated by collagenase digestion from surgically removed corneas of five normal or KC human corneal buttons and cultured in Dulbecco's modified eagle medium/Ham's F12 medium supplemented with 2% fetal bovine serum. These cells were evaluated under two experimental conditions: control and submitted to the application of ultraviolet A light and riboflavin 0.1% (CXL) for 30 min. RESULTS: Patients with KC displayed increased levels of Gal-1 and Gal-3 in conjunctival epithelial cells compared with control. Furthermore, KC corneas were associated with intense expression of Gal-1 in the stroma, released by keratocytes. Ultrastructural analysis of keratocytes showed a marked increase of endogenous Gal-3 levels, but not Gal-1, in KC. In vitro, CXL induced significant release of Gal-1 in keratocyte supernatants (116±18 ng/mL, P<0.05) and decreased inflammatory biomarkers as interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-2 and MMP-9. Gal-3 levels were not detected in the keratocyte supernatants. CONCLUSION: Gal-1 and Gal-3 represent new interesting KC biomarkers as revealed by their different expression patterns in KC and control corneal samples. CXL has an immunosuppressive effect on keratocytes by reducing the release of cytokines and MMPs and increased expression of anti-inflammatory protein Gal-1.
Subject(s)
Galectin 1/metabolism , Galectin 3/metabolism , Keratoconus/metabolism , Adult , Biomarkers/metabolism , Case-Control Studies , Collagen/metabolism , Conjunctiva/metabolism , Cornea/metabolism , Corneal Keratocytes/drug effects , Corneal Keratocytes/metabolism , Cross-Linking Reagents/pharmacology , Cytokines/metabolism , Female , Humans , Keratoconus/drug therapy , Male , Photosensitizing Agents/pharmacology , Prospective Studies , Riboflavin/pharmacology , Tears/metabolism , Ultraviolet RaysABSTRACT
RESUMO Objetivo: Avaliar a variação intra e interexaminadores do volume de gotas dispensados de frascos de colírios lubrificantes disponíveis no mercado. Métodos: Foram estudados cinco frascos de colírios lubrificantes e dezenove voluntários participaram deste estudo. A massa média de gotas de 20µl dos colírios foi obtida utilizando micropipeta e balança de precisão e como padrão para comparação com a massa das gotas obtidas pelos voluntários. Cinco gotas de cada frasco foram pesadas individualmente com o tubo de colírio perpendicular à balança, usando o primeiro e segundo dedos da mão direita, de forma que a pressão fosse aplicada somente no meio do frasco. Os experimentos foram realizados em uma sala climatizada a temperatura ambiente (21±1°C). Resultados: Todos os frascos de colírios apresentaram variação estatisticamente significante das massas das gotas obtidas pelos examinadores quando comparadas com a massa média padrão de 0,0182±0,0014g, com exceção da comparação entre os dados do colírio A com o colírio D, que não apresentou variação estatisticamente significante. Conclusão: O presente estudo demonstra a ausência de uniformidade das gotas dispensadas pelos frascos de colírios disponíveis no mercado e a sua inadequação à real necessidade, uma vez que as gotas dispensadas são maiores do que o indicado. Esse fato torna-se um problema quando se trata de período de tratamento prolongado, especialmente com colírios dispendiosos como os indicados para a terapêutica do glaucoma. Nesse sentido, a padronização das gotas de colírios se faz necessária.
ABSTRACT Objective: To evaluate the intra and inter variations of eye drops volume dispensed from bottles available on the market. Methods: Five bottles of lubricant eye drops were studied and nineteen volunteers participated in this study. The average mass from 20µl of eye drops was obtained using accuracy micropipette and balance, and used as standard for comparison with the mass of the drops obtained by the volunteers. Five drops of each vial were individually weighed with the tube perpendicular to the balance, using the first and second fingers of the right hand, so that the pressure was applied only in the middle of the flask. The experiments were performed in a room temperature (21±1°C). Results: All eye drops bottles showed a statistically significant variation on masses of the drops obtained by examiners when compared with the standard average weight of 0.0182±0,0014g, except when compared A with D eye drops, with no statistically significant variation. Conclusion: This study demonstrates the lack of uniformity of drops dispensed by eye drops bottles available in the market and its inadequacy to the real need, since the dispensed drops are larger than indicated.This fact becomes a problem when it comes to long treatment period, especially with expensive drops as indicated for glaucoma therapy. In this sense, the standardization of drops of eye drops is necessary.
Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Drug Packaging/standards , Lubricant Eye Drops/administration & dosage , Lubricant Eye Drops/standards , Instillation, Drug , Drug Administration Schedule , Drug Costs , Equipment Design , Lubricant Eye Drops/economicsABSTRACT
PURPOSE: To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells. METHODS: Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5'-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT-PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining. RESULTS: Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM. CONCLUSIONS: Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM.
Subject(s)
Culture Media , Epithelium, Corneal/cytology , Limbus Corneae/cytology , Stem Cells/cytology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Cell Culture Techniques/methods , Cell Differentiation , Cell Proliferation , Cell Survival , Cells, Cultured , Culture Media, Serum-Free , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelium, Corneal/metabolism , Humans , Keratin-3/genetics , Keratin-3/metabolism , Limbus Corneae/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Stem Cells/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
PURPOSE: To evaluate the efficacy and ultrastructural aspects of human limbal epithelial cells cultured on amniotic membrane (AM) with and without epithelium. METHODS: Limbal epithelial cell cultures were established from cadaveric cor neo-scleral rim explants derived from 6 different donors. The explants from each donor were placed under 3 different groups: on human preserved AM with epithelium (Group 1), AM deepithelialized with trypsin (Group 2) and control (Group 3). The epithelial cell migration was evaluated under phase contrast microscopy. After 15 days, the amniotic membrane with cells cultures were removed and submitted to scanning and transmission electron microscopy to check for epithelial migration and adhesion. RESULTS: All epithelial cell cultures from the controls grew over the botton of the culture plate wells until reaching confluence. Epithelial cultures grew over all but one denuded amniotic membrane. In the group amniotic membrane with epithelium, epithelial cell growing was observed only in 1 well. CONCLUSIONS: Using this model, denuded amniotic membrane appeared to be the best substrate for epithelial cell migration and adhesion comparing to amniotic membrane with epithelium. Removal of amniotic membrane epithelial seems to be an important step for establishing limbal epithelial cell culture on amniotic membrane.
Subject(s)
Amnion , Cell Culture Techniques/methods , Epithelial Cells/cytology , Limbus Corneae/cytology , Cell Adhesion , Cell Movement , Cell Transplantation/methods , Cells, Cultured , Humans , Microscopy, Electron, Scanning , Microscopy, Electron, TransmissionABSTRACT
OBJETIVO: Avaliar a eficácia e aspecto estrutural de células límbicas epiteliais humanas cultivadas sobre membrana amniótica (MA) com e sem epitélio. MÉTODOS: As culturas límbicas foram obtidas a partir de rima corneoescleral remanescentes de transplantes de córnea de 6 diferentes doadores. Cada explante foi cultivado em três diferentes grupos: MA desepitelizada por tripsina (Grupo 1), MA com epitélio íntegro (Grupo 2) e controle (Grupo 3). A migração epitelial foi avaliada por microscopia de contraste de fase. Após 15 dias, as células cultivadas sobre MA foram submetidas à microscopia eletrônica para avaliar migração e adesão epitelial. RESULTADOS: Todas as células do grupo controle cresceram até atingir confluência. Somente uma das culturas em membrana amniótica desepitelizada não apresentou crescimento epitelial. O crescimento de células epiteliais sobre membrana amniótica epitelizada foi observada em apenas uma cultura. CONCLUSÃO: Baseando-se nestes achados, o uso de membrana amniótica desepitelizada aparenta ser o melhor substrato para migração e adesão epitelial comparando com membrana amniótica epitelizada. Remover o epitélio da membrana amniótica demonstra ser um importante passo para estabelecer culturas de células sobre membrana amniótica.
PURPOSE: To evaluate the efficacy and ultrastructural aspects of human limbal epithelial cells cultured on amniotic membrane (AM) with and without epithelium. METHODS: Limbal epithelial cell cultures were established from cadaveric cor neo-scleral rim explants derived from 6 different donors. The explants from each donor were placed under 3 different groups: on human preserved AM with epithelium (Group 1), AM deepithelialized with trypsin (Group 2) and control (Group 3). The epithelial cell migration was evaluated under phase contrast microscopy. After 15 days, the amniotic membrane with cells cultures were removed and submitted to scanning and transmission electron microscopy to check for epithelial migration and adhesion. RESULTS: All epithelial cell cultures from the controls grew over the botton of the culture plate wells until reaching confluence. Epithelial cultures grew over all but one denuded amniotic membrane. In the group amniotic membrane with epithelium, epithelial cell growing was observed only in 1 well. CONCLUSIONS: Using this model, denuded amniotic membrane appeared to be the best substrate for epithelial cell migration and adhesion comparing to amniotic membrane with epithelium. Removal of amniotic membrane epithelial seems to be an important step for establishing limbal epithelial cell culture on amniotic membrane.
