Corneal Cell Cultures Research Articles

Исследование цитотоксического воздействия квантовых точек на клеточных культурах роговицы и конъюнктивы человека в аспекте перспектив лечения инфекционного кератита

Purpose. To investigate the cytotoxic effect of quantum dots on human corneal and conjunctival cell cultures to determine their safe concentration in the treatment of infectious keratitis. Material and methods. This study was conducted on HCEC (human, corneal epithelial cells) and Clone 1-5c-4 (human, normal conjunctiva) cell cultures, which were exposed to semiconductor nanoparticles – quantum dots of three types: CdTe/Cd (cadmium telluride) MPA 710, Ag (10%) InP/ZnS (indium phosphide, doped with silver) MPA 710, InP/ZnSe/ZnS (indium phosphide) 650 MPA. Results. Quantum dot solutions have a dose-dependent cytotoxic effect, they are nontoxic in concentrations from 0.001, 0.0001, 0.00001 mcg/ml for corneal (HCEC) and conjunctival cell cultures (Clone 1-5c-4). They cause toxic effects in concentrations of 0.1–0.01 mg/ml. Conclusion. This study confirmed the biocompatibility of quantum dot solutions in certain concentrations with corneal and conjunctival cells of the human eye. Results of determined safe concentrations of quantum dots on human cell cultures make it possible to plan further research on the creation of therapeutic anti-infective drugs based on them. Key words: nanomedicine, keratitis, anti-infective drugs

αB-crystallin mini-peptides support corneal healing in vitro and in vivo in rabbit model.

To evaluate if topical use of αB-crystallin mini-peptides supports corneal healing following flap surgery. Cultured corneal cells were treated with fluorescent tagged αB-crystallin mini-peptides to assess its internalization. Cultured corneal cells pre-treated with or without the mini-peptides were exposed to H2O2 and cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Elongation of neurites of cultured trigeminal neurones was examined following treatment either with αB-crystallin mini-peptides or protein. Cultured trigeminal neurones were pre-treated either with αB-crystallin mini-peptides or crystallin protein and exposed to H2O2 and presence of beading in the dendrites and axons was assessed. Corneal flap surgery was conducted on rabbit cornea and treated topically either with αB-crystallin peptide (0.5 mg/mL thrice daily for 14d) or phosphate-buffered saline (PBS). Corneal healing was evaluated under slit-lamp biomicroscope, mRNA expression of inflammatory cytokines were assessed and the corneas were evaluated by histopathology. Internalization of αB-crystallin mini-peptides was ascertained by the detection of fluorescence within the corneal cells. The MTT assay revealed that treatment with αB-crystallin mini-peptide reduced cell death induced by H2O2 treatment. The mini-peptides did not influence the elongation of trigeminal neurites, but significantly (P<0.05) reduced beading in the neurites. In rabbit eye, the treated corneas showed reduced hyper-reflective zones (P<0.05) and suppression in the expression of inflammatory cytokines. Histopathological examination also revealed reduction of inflammatory response in treated corneas. The αB-crystallin mini-peptides restrict the damage to corneal cells and neurons and aids in corneal healing.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Topical sevoflurane corneal toxicity: in vitro study

AbstractPurpose: The purpose of the study is to investigate the cytotoxic effect of sevoflurane (SF) and povidone‐iodine (PI) on cultured rabbit corneal epithelial cells in vitro.Methods: We evaluated the cytotoxic effect of a 5 min exposition of SF, PI and balanced saline solution (BSS) using an in vitro rabbit corneal cell culture model (Short‐Time Exposure Test Method). In order to quantify cytotoxicity MTT assay was employed. Repeated exposition of SF was evaluated as well as cell viability after different recovery times (30 min, 60 min and 1 h). Statistical results were obtained with Kruskal‐Wallis test and Conover post‐hoc test.Results: The median of cell viability of SF was 85.29%, PI 0.52% and BSS 97.29%, with statistically significant differences between them (p = 0.005). After a 5 minute recovery, the median of cell viability for SF was 87.77%, 74.48% after 30 minutes and 76.66% after 60 minutes. After a double exposition it was 70.59%, with no statistically significant differences between single and double exposition (p = 0.18).Conclusions: No corneal toxicity of SF was observed, in contrast with PI. Cellular damage did not change with longer recovery time or double exposition to SF.ReferencesSevoflurane (SF) has been proved recently to have analgesic/antimicrobial effects. (1, 2) We believe of interest to study the effect of SF in the ocular surface. As there is no literature about this topic, in vitro studies are required before in vivo tests.1. Chu CC, Wu SZ, Su WL, Shieh JP, Kao CH, Ho ST, et al. Subcutaneous injection of inhaled anaesthetics produces cutaneous analgesia. Can J Anesth Can Anesth. 2008;55(5):290–4.2. Manuel Gerónimo Pardo, María Martínez Serrano, Ángel Martínez Monsalve, Juan Luis Rueda Martínez. Usos Alternativos del Sevoflurano: Efecto Analgésico Tópico. Rev Electrónica AnestesiaR [Internet]. 2012 [cited 2019 Apr 15];4(5). Available from: http://revistaanestesiar.org/index.php/rear/article/view/373

Optically Clear Silk Fibroin Films with Tunable Properties for Potential Corneal Tissue Engineering Applications: A Process-Property-Function Relationship Study.

Owing to the shortage of donor corneas and issues associated with conventional corneal transplantation, corneal tissue engineering has emerged as a promising therapeutic alternative. Biocompatibility and other attractive features make silk fibroin a biomaterial of choice for corneal tissue engineering applications. The current study presents three modes of silk fibroin film fabrication by solvent casting with popular solvents, viz. aqueous (aq), formic acid (FA), and hexafluoroisopropanol (HFIP), followed by three standard modes of postfabrication annealing with water vapor, methanol vapor, and steam, and systematic characterization studies including corneal cell culture in vitro. The results indicated that silk fibroin films made from aq, FA, and HFIP solvents had surface roughness (Rq) of 1.39, 0.32, and 0.13, contact angles of 73°, 85°, and 89°, water uptake% of 58, 29, and 27%, swelling ratios of 1.58, 1.3, and 1.28, and water vapor transmission% of 39, 26, and 22%, respectively. The degradation rate was in the order of aq > HF > FA, whereas the tensile strength was in the order of aq < HF < FA. Further, the results of the annealing process indicated notable changes in morpho-topographical, physical, degradation, and tensile properties. However, the films showed no detectable changes in chemical composition and remained optically clear with >90% transmission in the visible range, irrespective of fabrication and postfabrication processing conditions. The films were noncytotoxic against L929 cells and were cytocompatible with rabbit cornea-derived SIRC cells in vitro. The study demonstrated the potential of fine-tuning various properties of silk fibroin films by varying the fabrication and postfabrication processing conditions.

Novel Osmoprotective DOPC-DMPC Liposomes Loaded with Antihypertensive Drugs as Potential Strategy for Glaucoma Treatment.

Glaucoma is a group of chronic irreversible neuropathies that affect the retina and the optic nerve. It is considered one of the leading causes of blindness in the world. Although it can be due to various causes, the most important modifiable risk factor is the elevated intraocular pressure (IOP). In this case, the treatment of choice consists of instilling antihypertensive formulations on the ocular surface. The chronicity of the pathology, together with the low bioavailability of the drugs that are applied on the ocular surface, make it necessary to instill the formulations very frequently, which is associated, in many cases, with the appearance of dry eye disease (DED). The objective of this work is the design of topical ocular formulations capable of treating glaucoma and, at the same time, preventing DED. For this, two liposome formulations, loaded with brimonidine or with travoprost, were Tadeveloped using synthetic phospholipids and enriched by the addition of compounds with osmoprotective activity. The proposed formulations not only presented physicochemical characteristics (size, pH, osmolarity, surface tension, and viscosity) and encapsulation efficiency values (EE% of 24.78% and ≥99.01% for brimonidine and travoprost, respectively) suitable for ocular surface administration, but also showed good tolerance in human corneal and conjunctival cell cultures, as well as an in vitro osmoprotective activity. The hypotensive effect of both liposomal formulations was evaluated in normotensive albino New Zealand rabbits, showing a faster and longer lasting reduction of intraocular pressure in comparison to the corresponding commercialized products used as control. According to these results, the hypotensive liposomal formulations combined with osmoprotective agents would result in a very promising platform for the treatment of glaucoma and the simultaneous protection of the ocular surface.

Membranes Prepared from Recombinant RGD-Silk Fibroin as Substrates for Human Corneal Cells.

A recombinant formulation of silk fibroin containing the arginine–glycine–aspartic acid (RGD) cell-binding motif (RGD-fibroin) offers potential advantages for the cultivation of corneal cells. Thus, we investigated the growth of corneal stromal cells and epithelial cells on surfaces created from RGD-fibroin, in comparison to the naturally occurring Bombyx mori silk fibroin. The attachment of cells was compared in the presence or absence of serum over a 90 min period and analyzed by quantification of dsDNA content. Stratification of epithelial cells on freestanding membranes was examined by confocal fluorescence microscopy and optimized through use of low molecular weight poly(ethylene glycol) (PEG; 300 Da) as a porogen, the enzyme horseradish peroxidase (HRP) as a crosslinking agent, and stromal cells grown on the opposing membrane surface. The RGD-fibroin reduced the tendency of stromal cell cultures to form clumps and encouraged the stratification of epithelial cells. PEG used in conjunction with HRP supported the fabrication of more permeable freestanding RGD-fibroin membranes, that provide an effective scaffold for stromal–epithelial co-cultures. Our studies encourage the use of RGD-fibroin for corneal cell culture. Further studies are required to confirm if the benefits of this formulation are due to changes in the expression of integrins, components of the extracellular matrix, or other events at the transcriptional level.

Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus.

Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

Experimental study of synthetic polymeric materials as a basis for developing advanced carrier matrices for cultivating limbal stem cells

Objective: to conduct an experimental study on the properties of three different types of synthetic polyester matrices, to carry out their comparative assessment, and to identify the optimal carrier for the cultivation and transplantation of limbal stem cells while eliminating limbal insufficiency. Materials and methods. Transparency, mechanical properties (strength, relative elongation at break, and elastic modulus), biocompatibility with corneal cell cultures were assessed, and duration of matrix biodegradation in vivo were studied. Results. In the course of our study, the optical and mechanical properties of matrices, made of polylactide-glycolide (PLG), polylactide-caprolactone (PLC) and poly-E-caprolactone (PCL), were studied. It has been experimentally shown that limbal stem cells of humans and rabbits, as well as human corneal epithelial cells, adhered to the surface of all types of the studied matrices. During the cultivation process, they retained the typical structure of the actin cytoskeleton, along with the ability to proliferate and migrate. Differences in the interaction of different cell cultures with different types of carriers were revealed. The biodegradation time of 5 μm thick PLC matrices was about 30 days. Conclusion. Our results obtained implied the possibility of using 5 µm thick PLC matrices as a carrier for cultured limbal stem cells.

Lyophilized Autologous Serum Eyedrops: Experimental and Comparative Study

To analyze the biological stability of autologous serum eyedrops after lyophilization. Prospective, comparative experimental study. This was a comparative study with serum obtained from 12 healthy volunteers. The concentrations of different epitheliotropic factors (eg, transforming growth factor-β [TGF-β1], epidermal growth factor [EGF], platelet-derived growth factor AB [PDGF-AB], and albumin) were measured in fresh and lyophilized serum. The samples were studied after serum preparation (fresh serum) and immediately after saline solution reconstitution of lyophilized serum (0), 15, and 30days later. The biological effects of both serum samples were also compared on conjunctival and corneal cell cultures. The pH, osmolarity, and serum density were also determined. No significant differences were found in the concentration of growth factors between fresh serum and re-dissolved serum samples after lyophilization. The concentration of growth factors remained stable during 1month at 4°C in re-dissolved lyophilized form with saline solution. No differences were found related to osmolarity, pH, and density between fresh and lyophilized serum. In addition, no differences were found on the conjunctival and corneal cells proliferation and differentiation in cells cultures between either serum preparation. The properties of autologous serum remain after lyophilization. The lyophilized serum can be easily stored without temperature restrictions and easily reconstituted for preparation of eyedrops for standard clinical use.

Isolation and Culture of Corneal Stromal Stem Cells.

An increasing body of evidence authenticates the benefit of corneal stroma-derived stem cells (CSSCs) in tissue engineering and regeneration oriented research, and potentially in the development of clinically relevant cellular therapies. Postmortem corneal tissue obtained from otherwise discarded material after keratoplasties is oftentimes the source of the cells for ex vivo research. Relatively easy to isolate and cultivate as well as inexpensive to culture, CSSCs now represent a well-described cell type with attributes of mesenchymal stem cells (MSCs). These include differentiation- and immunosuppressive potential, as well as a favorable capacity to expand in vitro. Here, we in detail describe two straightforward methods to isolate and establish CSSC cultures ex vivo.

Extracellular Vesicles and Cell-Cell Communication in the Cornea.

ABSTRACTOne question that has intrigued cell biologists for many years is, “How do cells interact to influence one another's activity?” The discovery of extracellular vesicles (EVs) and the fact that they carry cargo, which directs cells to undergo changes in morphology and gene expression, has revolutionized this field of research. Little is known regarding the role of EVs in the cornea; however, we have demonstrated that EVs isolated from corneal epithelial cells direct corneal keratocytes to initiate fibrosis. Intriguingly, our data suggest that EVs do not penetrate epithelial basement membrane (BM), perhaps providing a mechanism explaining the importance of BM in the lack of scarring in scrape wounds. Since over 100‐million people worldwide suffer from visual impairment as a result of corneal scarring, the role of EVs may be vital to understanding the mechanisms of wound repair. Therefore, we investigated EVs in ex vivo and in vivo‐like three‐dimensional cultures of human corneal cells using transmission electron microscopy. Some of the major findings were all three major cell types (epithelial, fibroblast, and endothelial cells) appear to release EVs, EVs can be identified using TEM, and EVs appeared to be involved in cell–cell communication. Interestingly, while our previous publication suggests that EVs do not penetrate the epithelial BM, it appears that EVs penetrate the much thicker endothelial BM (Descemet's membrane). These findings indicate the huge potential of EV research in the cornea and wound healing, and suggest that during homeostasis the endothelium and stromal cells are in communication. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Deregulation of Acanthamoeba castellanii steroidogenesis is amoebicidal and protects cultured corneal cells from Ac attack

Human infections by amoeba represent rare diseases, yet they are an emerging health problem that can develop into rapid death, as in cases involving the spinal cord or amoeba that penetrate the brain. Our recent papers highlighted in J. Lipid Res, 2017 and ASBMB Today magazine, 2018 focus on development of new sterol methyltransferase (SMT) inhibitors to prevent steroidogenesis in the parasitic amoeba Acanthamoeba castellanii (Ac). Now we show, Ac cultured on unexplored sterol C24‐methyltransferase (SMT) inhibitors 25‐azalanosterol and 25‐thialanosterol iodine salt are highly potent anti‐amoeba drugs exhibiting IC50 25 nM and 15 nM, respectively and a minimum amoebicidal concentration of 1 μM. Both analogs inhibited the cloned 24‐AcSMT and 28‐AcSMT yielding Ki values of 13 nM/37nM and 9nM/16nM, respectively and blocked Ac steroidogenesis at the target enzyme. Against human epithelial kidney cell growth these drugs had no effect to 40 μM or effected cholesterol biosynthesis (selective index greater than 103); in mice at 50 mg/kg, 3H‐analog could rapidly cross the blood brain barrier with minimal effect on liver cholesterol biosynthesis. Against human corneal epithelial cultures infected with Ac, the drugs protected the eye at doses of 500 nM. These new amoeba sterol biosynthesis inhibitors are currently under investigation for their therapeutic potential.Support or Funding InformationThis research was funded through a grant from the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Examination of Kynurenine Toxicity on Corneal and Conjunctival Epithelium: In vitro and in vivo Studies

Kynurenine (KYN) is a metabolite of tryptophan, proposed for the treatment of corneal diseases. Our goal was to evaluate the effects of KYN on normal human corneal and conjunctival epithelial cells in vitro and the re-epithelization of corneal erosion in rabbits. In our study, we used corneal (10.014 pRSV-T) and conjunctival (HC0597) epithelium cell cultures. KYN was applied at a concentration range of 1–100 µM for 24 and 48 h. We examined the effects on cellular metabolism, viability, interleukin-1β (IL-1β), IL-6, IL-10 secretion, cytoskeleton organization and transwell migration ability. Following a bilateral corneal de-epithelialization, the rabbits received drops containing 1% KYN and a saline solution to the contralateral control eye, 5 times daily. Digital images were analyzed using the EPCO 2000 software. The metabolic activity of cells was slightly decreased by KYN in the corneal but not in the conjunctival epithelium. The viability of both epithelia was improved by KYN; it caused alterations in the secretion of IL-6 and IL-10 but not IL-1β. It had no impact on both epithelia morphology and the organization of the cellular cytoskeleton. KYN stimulated the formation of pseudopodia projections in both epithelia in vitro, which may be important in terms of wound healing. However, there were no differences in the re-epithelization rate in vivo. At the tested concentrations, KYN was not toxic for the corneal and the conjunctival epithelium in vitro and did not affect corneal re-epithelization in rabbits in vivo. Our results suggest that KYN may be taken into consideration for the treatment of ocular disorders.

Evaluation of the Safety of 2.0% Ganciclovir Eye Drops for Hospital Preparation

The concentration of ganciclovir eye drops for hospital preparation was changed from 0.5% to 2.0% at the Nagasaki University Hospital from March 2015. We investigated the incidence of side effects in 12 patients using 2.0% ganciclovir eye drops and evaluated the cytotoxicity of 2.0% ganciclovir eye drops using cultured rabbit corneal cells in vitro. As a side effect of 2.0% ganciclovir eye drops, three patients exhibited an early feeling of transient stimulation. The 2.0% ganciclovir eye drops did not demonstrate cell cytotoxicity for cultured corneal cells after 5 min, but did after 10 min. These findings suggested that the 2.0% ganciclovir eye drops can be used without corneal epithelium disorder in clinical settings.

Optimized human platelet lysate as novel basis for a serum-, xeno-, and additive-free corneal endothelial cell and tissue culture.

The expansion of donor-derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3-fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2weeks in 0.1-mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing.

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Effect of bone marrow-derived mesenchymal stem cells and stem cell supernatant on equine corneal wound healing in vitro

BackgroundWe aimed to determine and compare the in vitro effects of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) and mesenchymal stem cell supernatant (MSC-Sp) on the wound healing capacity of equine corneal fibroblasts using a scratch assay.MethodsBone marrow aspirates and eyes were collected from normal, euthanized horses with subsequent isolation and culture of BM-MSCs and corneal stromal cells. Corneal stromal cells were culture-expanded in the culture well of transwell plates and then treated with an autologous BM-MSC suspension (dose: 2.5 × 105/100 μL media with the BM-MSCs contained within the insert well), MSC-Sp solution, or naive culture media (control) for 72 h. A linear defect in confluent cell cultures was created (i.e., corneal scratch assay) to assess the cellular closure (“healing”) over time. Three representative areas of the scratch in each culture were photographed at each time point and the scratch area was quantitated using image analysis software (ImageJ). Media from the scratches were analyzed for various growth factors using human enzyme-linked immunosorbent assay (ELISA) kits that crossreact with the horse.ResultsThere was a significant percentage decrease in the scratch area remaining in the BM-MSC and MSC-Sp groups compared to the control group. There was also a significant percentage decrease in the scratch area remaining in the BM-MSC group compared to the MSC-Sp group at 36 h post-scratch and all time points thereafter. The concentration of transforming growth factor (TGF)-β1 in the media was significantly higher in the BM-MSC group compared to the control group.ConclusionsThe significant decrease in scratch area in equine corneal fibroblast cultures treated with autologous BM-MSCs compared to MSC-Sp or control treatments suggests that BM-MSCs may substantially improve corneal wound healing in horses. MSC-Sp may also improve corneal wound healing given the significant decrease in scratch area compared to control treatments, and would be an immediately available and cost-effective treatment option.

Cultured corneas show dendritic spread and restrict herpes simplex virus infection that is not observed with cultured corneal cells

Herpes simplex virus-1 (HSV-1) causes life-long morbidities in humans. While fever blisters are more common, occasionally the cornea is infected resulting in vision loss. A very intriguing aspect of HSV-1 corneal infection is that the virus spread is normally restricted to only a small fraction of cells on the corneal surface that connect with each other in a dendritic fashion. Here, to develop a comprehensive understanding of the susceptibility of human corneal epithelial (HCE) cells to HSV-1 infection, we infected HCE cells at three different dosages of HSV-1 and measured the outcomes in terms of viral entry, gene and protein expression, viral replication and cytokine induction. In cultured cells, infectivity and cytokine induction were observed even at the minimum viral dosage tested, while a more pronounced dose-restricted infectivity was seen in ex vivo cultures of porcine corneas. Use of fluorescent HSV-1 virions demonstrated a pattern of viral spread ex vivo that mimics clinical findings. We conclude that HCE cell cultures are highly susceptible to infection whereas the cultured corneas demonstrate a higher ability to restrict the infection even in the absence of systemic immune system. The restriction is helped in part by local interferon response and the unique cellular architecture of the cornea.

Изучение биосовместимости полимерных материалов (полиметилметакрилат и бисфенол-А-диглицидилметакрилат) на модели культуры клеток стромы роговицы

Изучение биосовместимости полимерных материалов (полиметилметакрилат и бисфенол-А-диглицидилметакрилат) на модели культуры клеток стромы роговицы