Progress in Wound-Healing Products Based on Natural Compounds, Stem Cells, and MicroRNA-Based Biopolymers in the European, USA, and Asian Markets: Opportunities, Barriers, and Regulatory Issues.

Wounds are breaks in the continuity of the skin and underlying tissues, resulting from external causes such as cuts, blows, impacts, or surgical interventions. Countless individuals suffer minor to severe injuries, with unfortunate cases even leading to death. In today's scenario, several commercial products are available to facilitate the healing process of wounds, although chronic wounds still present more challenges than acute wounds. Nevertheless, the huge demand for wound-care products within the healthcare sector has given rise to a rapidly growing market, fostering continuous research and development endeavors for innovative wound-healing solutions. Today, there are many commercially available products including those based on natural biopolymers, stem cells, and microRNAs that promote healing from wounds. This article explores the recent breakthroughs in wound-healing products that harness the potential of natural biopolymers, stem cells, and microRNAs. A comprehensive exploration is undertaken, covering not only commercially available products but also those still in the research phase. Additionally, we provide a thorough examination of the opportunities, obstacles, and regulatory considerations influencing the potential commercialization of wound-healing products across the diverse markets of Europe, America, and Asia.

Advanced Therapy and Clinical Trials to Treat Patients with Optic Nerve Diseases.

Optic nerve diseases include a wide variety of pathogenic conditions triggering injury or dysfunction of the optic nerves that lead to visual impairment or blindness in one or both eyes. Despite their pathogenic variety, most of them proceed through common mechanisms that allow them to investigate together. Nevertheless, roles of the cells, tissues, genes, growth factors, and proteins, and all underlying pathophysiological mechanisms need to be studied fully for better management of each optic nerve disease. This review presents a collection of information regarding ongoing and completed clinical trials (CT) of advanced therapies that deliver stem cell and gene therapy treatments as drugs to patients with optic nerve diseases as well as successes and failures achieved in treating these patients in the last few years. These drugs seem safe from creating neurotoxicity. It describes outcomes of a bibliographic search for stem cell therapy, gene therapy, and neuroprotection-based CT registered in the International ClinicalTrials.gov, the European EudraCT, and the Spanish REEC database, and related papers published in the PUBMED database by applying different search terminologies. This review overall informs the patients of optic neurodiseases that advanced therapies are progressing successfully in search of effective and safe treatments for them.

Factors influencing mesenchymal stromal cells in in vitro cellular models to study retinal pigment epithelial cell rescue.

Mesenchymal stromal cells (MSC) stop or slow retinal pigment epithelium (RPE) and neuroretina (NR) degeneration by paracrine activity in oxidative stress-induced retinal degenerative diseases. However, it is mandatory to develop adequate in vitro models that allow testing new treatment strategies against oxidative stress before performing in vivo studies. The viable double- and triple-layered setups are composed of separate layers of NR, MSC, and RPE (NR-MSC-RPE, NR-RPE, MSC-RPE) partially mimic in vivo retinal conditions. In this study, the paracrine neuroprotective effect of each setup's microenvironment on hydrogen peroxide (H2O2)-stressed was compared with unstressed RPE cells. RPE cell proliferation viability was assessed on day 1, 3, and 6 using Alamar Blue® (10%), MTT (10%) and a cell viability/cytotoxicity assay kit followed by data analysis. The results showed that RPE cells, highly viable (> 90%) in mixed medium of DMEM and neurobasal A (1:1), lost 50% viability on exposure to 400 µM of H2O2 (P < 0.05). The unexposed groups differed significantly from exposed groups for RPE cell growth (RPE and [Formula: see text]RPE (P < 0.0001), NR-MSC-RPE, and NR-MSC-[Formula: see text]RPE (P < 0.05), NR-RPE and NR-[Formula: see text]RPE (P < 0.01), and MSC-RPE and MSC-[Formula: see text]RPE (P < 0.01). NR-[Formula: see text]RPE and NR-RPE supported RPE cell proliferation viability better than other setups (P < 0.01) and RPE cells proliferated 0.49-fold more in NR-MSC-[Formula: see text]RPE than NR-MSC-RPE. Thus, NR and MSC presence improved significantly each setup's microenvironment for cell rescue, nevertheless, each setup also showed limitations for its use as an in vitro study tool. Health of microenvironment of such setups depends on many factors including cell-secreted trophic factors.

Acute intraocular toxicity caused by perfluorocarbon liquids: safety control systems of medical devices.

BACKGROUND: Acute retinal toxicity has been demonstrated to be associated with the intraoperative use of perfluorocarbon liquids (PFCLs), especially perfluorooctane (PFO). Recently, several cases of PFO-associated blindness have been reported in Spain, Holland, France, Italy, the Middle East, and South America. METHODS: As a result, a new ISO guideline (ISO 16672:2020) was drafted, discussed, approved, and released in 2019. This recent ISO16672:2020 guideline recommends performing direct cytotoxicity tests as an option along with chemical analysis to measure PFCL quality (purity and safety). RESULTS: In this review paper, it has been emphasized why an appropriate biological test, specifically direct exposure of PFCL to live cells, for measuring cytotoxicity must be performed with each PFCL batch along with chemical analysis. CONCLUSIONS: The paper intends to compile all available information to discuss possible approaches for avoiding adverse clinical cases in future.

Intraocular toxicity caused by MEROCTANE perfluorocarbon liquid.

Serious intraocular toxicity cases have been reported worldwide after the use of different perfluorocarbon liquids. The current study reports for the first-time the clinical pictures of cases of acute intraocular toxicity caused by MEROCTANE, a perfluoro-octane commercialized by a Turkish company and distributed in many countries. A series of 18 cases from Chile and Spain was retrospectively analysed. To evaluate the impurity profile, a suspicious MEROCTANE sample (lot OCT.01.2013) was analysed by gas chromatography mass spectrometry and compared with a non-suspicious sample of the same commercial perfluoro-octane (lot OCT 722011). Cytotoxicity was tested following a direct-contact method, taking into consideration the high volatility and hydrophobicity of perfluoro-octane and following the ISO 10993 guideline. Cytotoxicity test showed clear cytotoxic effects of the analysed batch (less than 9% of cell viability). Moreover, chemical analysis demonstrated the presence of many contaminants, some highly toxic (acids and alcohols). Perfluorocarbon liquids are useful tools for intraocular surgery but companies and Agencies of Medical Devices must implement measures that guarantee the safety of these products based on both chemical and cytotoxicity analysis for every batch. Medical staff should be encouraged to report any suspected case to their respective National Agencies.

Mesenchymal Stem Cell Secretome Enhancement by Nicotinamide and Vasoactive Intestinal Peptide: A New Therapeutic Approach for Retinal Degenerative Diseases.

Mesenchymal stem cells (MSC) secrete neuroprotective molecules that may be useful as an alternative to cell transplantation itself. Our purpose was to develop different pharmaceutical compositions based on conditioned medium (CM) of adipose MSC (aMSC) stimulated by and/or combined with nicotinamide (NIC), vasoactive intestinal peptide (VIP), or both factors; and to evaluate in vitro their proliferative and neuroprotective potential. Nine pharmaceutical compositions were developed from 3 experimental approaches: (1) unstimulated aMSC-CM collected and combined with NIC, VIP, or both factors (NIC+VIP), referred to as the aMSC-CM combined composition; (2) aMSC-CM collected just after stimulation with the mentioned factors and containing them, referred to as the aMSC-CM stimulated-combined composition; and (3) aMSC-CM previously stimulated with the factors, referred to as the aMSC stimulated composition. The potential of the pharmaceutical compositions to increase cell proliferation under oxidative stress and neuroprotection were evaluated in vitro by using a subacute oxidative stress model of retinal pigment epithelium cells (line ARPE-19) and spontaneous degenerative neuroretina model. Results showed that oxidatively stressed ARPE-19 cells exposed to aMSC-CM stimulated and stimulated-combined with NIC or NIC+VIP tended to have better recovery from the oxidative stress status. Neuroretinal explants cultured with aMSC-CM stimulated-combined with NIC+VIP had better preservation of the neuroretinal morphology, mainly photoreceptors, and a lower degree of glial cell activation. In conclusion, aMSC-CM stimulated-combined with NIC+VIP contributed to improving the proliferative and neuroprotective properties of the aMSC secretome. Further studies are necessary to evaluate higher concentrations of the drugs and to characterize specifically the aMSC-secreted factors related to neuroprotection. However, this study supports the possibility of improving the potential of new effective pharmaceutical compositions based on the secretome of MSC plus exogenous factors or drugs without the need to inject cells into the eye, which can be very useful in retinal pathologies.

Chemical compounds causing severe acute toxicity in heavy liquids used for intraocular surgery.

Perfluorocarbon liquids (PFCLs) have been considered safe for intraocular manipulation of the retina, but since 2013 many cases of acute eye toxicity cousing blindness have been reported in various countries when using various commercial PFCLs. All these PFCLs were CE marked (Conformité Européenne), which meant they had been subjected to evaluation complying with the International Organization for Standardization (ISO) guidelines. These dramatic events raised questions about the safety of PFCLs and the validity of some cytotoxicity tests performed under ISO guidelines. Samples from toxic batches were analyzed by gas chromatography-mass spectrometry combined with Raman and infrared spectrometry. Perfluorooctanoic acid, dodecafluoro-1-heptanol, ethylbenzene and tributyltin bromide were identified and evaluated by a direct contact cytotoxicity test using ARPE-19 cell line, patented by our group (EP 3467118 A1). Perfluorooctanoic acid at a concentration of >0.06 mM and tributyltin bromide at a concentration of ≥0.016 mM were shown to be toxic, whereas the concentration found in the toxic samples reached 0.48 mM, and 0.111 mM, respectively. These finding emphasized the idea that determination of partially fluorinated compounds are not enough to guarantee the safety of these medical devices.

Mesenchymal stem cells provide paracrine neuroprotective resources that delay degeneration of co-cultured organotypic neuroretinal cultures.

Through the paracrine effects of stem cells, including the secretion of neurotrophic, immunomodulatory, and anti-apoptotic factors, cell-based therapies offer a new all-encompassing approach to treatment of neurodegenerative diseases. In this study, we used physically separated co-cultures of porcine neuroretina (NR) and human mesenchymal stem cells (MSC) to evaluate the MSC paracrine neuroprotective effects on NR degeneration. NR explants were obtained from porcine eyes and cultured alone or co-cultured with commercially available MSCs from Valladolid (MSCV; Citospin S.L.; Valladolid, Spain), currently used for several approved treatments. Cultures were maintained for 72 h. MSC surface markers were evaluated before and after co-culture with NRs. Culture supernatants were collected and the concentration of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and glial-derived neurotrophic factor (GDNF) were determined by enzyme-linked immunosorbent assays. NR sections were stained by haematoxylin/eosin or immunostained for terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), glial fibrillary acidic protein, ß-tubulin III, and neuronal nuclei marker. NR morphology, morphometry, nuclei count, apoptosis rate, retinal ganglion cells, and glial cell activation were evaluated. Treatment effects were statistically analysed by parametric or non-parametric tests. The MSCs retained stem cell surface markers after co-culture with NR. BDNF and CNTF concentrations in NR-MSCV co-cultures were higher than other experimental conditions at 72 h (p < 0.05), but no GDNF was detected. NR general morphology, total thickness, and cell counts were broadly preserved in co-cultures, and the apoptosis rate determined by TUNEL assay was lower than for NR monocultures (all p < 0.05). Co-cultures with MSCV also protected retinal ganglion cells from degenerative changes and reduced reactive gliosis (both p < 0.05). In this in vitro model of spontaneous NR degeneration, the presence of co-cultured MSCs retarded neuroglial degeneration. This effect was associated with elevated concentrations of the neurotrophic factors BDNF and CNTF. Our data suggest that the paracrine secretion of these, and possibly other molecules, are a potential resource for the treatment of several neuroretinal diseases.

Acute retinal toxicity associated with a mixture of perfluorooctane and perfluorohexyloctane: failure of another indirect cytotoxicity analysis.

AIMS: To report new information related to acute retinal toxicity of Bio Octane Plus, a mixture of 90% perfluorooctane (PFO) and 10% perfluorohexyloctane. METHODS: This retrospective, descriptive case series reports the occurrence of acute retinal toxicity after vitreoretinal surgery in which Bio Octane Plus (batch number 1605148) was used as an endotamponade. Cytotoxicity biocompatibility tests and chemical analyses by Fourier-transformed infrared (FTIR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) of the presumed toxic product were performed. RESULTS: Four patients presented with acute severe visual loss after uneventful ocular surgery assisted by Bio Octane Plus (batch number 1605148) as endotamponade. Patients experienced extensive retinal vascular occlusion leading to retinal and optic nerve atrophy. The viability of ARPE-19 cells directly exposed to the suspect batch for 30 min was 0%. The agarose overlay method used by the manufacturer according to European Union regulations and International Organization for Standardization (ISO) International Standards failed to detect toxicity. FTIR spectroscopy showed small differences between the non-toxic and toxic batches. GC-MS analysis showed the presence of bromotributyl stannane (whose toxicity was demonstrated in the dose-response curve) only in the toxic batch of Bio Octane Plus. CONCLUSION: This is the third report of retinotoxicity due to PFO in 4 years. The clinical profiles may be missed as they resemble other postsurgical complications; therefore, more cases worldwide could have gone unreported. Protocols to determine cytotoxicity of intraocular medical devices and approved by the ISO International Standards based on indirect methods have failed and should be revised to ensure safety.

Retinal Toxicity of Medical Devices Used during Vitreoretinal Surgery: A Critical Overview.

Retinal toxicity/biocompatibility of medical devices in direct contact with the retina is an important subject for clinicians and scientists. As these effects are not very frequent, there is also a relative lack of information for many clinicians. The past has taught us multiple times that there is a significant safety problem associated with severe loss of vision in affected patients. In this review, we want to classify medical products that are used in the back of the eye, describe recent examples of toxicity, critically reflect on the regulations that exist and suggest improvements that can be done to ensure patient safety without hindering innovation. METHODS: Critical review of the recent papers and personal experience of the authors in this issue. Medical devices used in the back of the eye and recent examples of toxicity are described, regulations that exist are critically reflected and improvements suggested that can ensure patient safety without hindering innovation. RESULTS: There is clear evidence of toxicity after intraocular surgery in any category. Some cytotoxic indirect methods have failed in detecting this toxicity. Some ISO rules do not seem appropriate. Postmarketing safety is missing. There is little data on this issue. CONCLUSIONS: The absence of a clear regulation of the production, purification and evaluation of the toxic effects of the medical devices supposes the possibility that products are not sufficiently safe to obtain the CE mark.

Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation.

A series of recent acute blindness cases following non-complicated retinal detachment surgery caused the release of several health alerts in Spain. The blindness was attributed to certain lots of perfluoro-octane (PFO; a volatile and transient medical device). Similar cases have been reported in other countries. This has raised questions regarding the validity of cytotoxicity test methods currently used to certify the safety of PFO lots. The tests were performed according to the International Organization for Standardization (ISO) norms, using the extract dilution method or the indirect contact method as applied to L929 cells, a line derived from mouse fibroblasts. The limitations of those methods have been resolved in this study by proposing a new cytotoxicity test method for volatile substances. The new method requires direct contact of the tested substance with cells that are similar to those exposed to the substance in the clinical setting. This approach includes a few new technical steps that are crucial for detecting cytotoxicity. Our new method detected toxic PFO lots that corresponded to the lots producing clinical blindness, which previous methods failed to detect. The study suggests applying this new method to avoid occurrence of such cases of blindness.

ACUTE RETINAL DAMAGE AFTER USING A TOXIC PERFLUORO-OCTANE FOR VITREO-RETINAL SURGERY.

PURPOSE: To describe a series of retinal acute toxicity cases with severe visual loss after intraocular use of a toxic perfluoro-octane (PFO). The clinical presentation is described, and the likely causes are analyzed. New biological methods for testing safety of intraocular medical devices are proposed. METHODS: Information regarding a series of eyes suffering acute severe events after intraocular use of a toxic PFO was analyzed. Four types of spectroscopy, nuclear magnetic resonance, and chromatography were used to identify the potential PFO contaminants. Cultures of human retinal pigment epithelial cells (ARPE-19) and porcine neuroretina were used to quantify the toxicity of the suspect PFO lots. RESULTS: Of 117 cases of intraocular toxicity, 96 were considered clearly related to the use of PFO. Fifty-three cases had no light perception, and 97 had no measurable visual acuity. Retinal necrosis (n = 38) and vascular occlusion (n = 33) were the most characteristic findings. Two hydroxyl compounds, perfluorooctanoic acid and dodecafluoro-1-heptanol, and benzene derivatives were identified as the suspected toxic agents. While existing toxicity testing failed, we proposed new tests that demonstrated clear toxicity. CONCLUSION: Protocols to determine cytotoxicity of intraocular medical devices should be revised to assure safety. Acute toxic events should be reported to health authorities and scientific media.

A novel coculture model of porcine central neuroretina explants and retinal pigment epithelium cells.

PURPOSE: To develop and standardize a novel organ culture model using porcine central neuroretina explants and RPE cells separated by a cell culture membrane. METHODS: RPE cells were isolated from porcine eyes, expanded, and seeded on the bottom of cell culture inserts. Neuroretina explants were obtained from the area centralis and cultured alone (controls) on cell culture membranes or supplemented with RPE cells in the same wells but physically separated by the culture membrane. Finally, cellular and tissue specimens were processed for phase contrast, cyto-/histological, and immunochemical evaluation. Neuroretina thickness was also determined. RESULTS: Compared to the neuroretinas cultured alone, the neuroretinas cocultured with RPE cells maintained better tissue structure and cellular organization, displayed better preservation of photoreceptors containing rhodopsin, lower levels of glial fibrillary acidic protein immunoexpression, and preservation of cellular retinaldehyde binding protein both markers of reactive gliosis. Neuroretina thickness was significantly greater in the cocultures. CONCLUSIONS: A coculture model of central porcine neuroretina and RPE cells was successfully developed and standardized. This model mimics a subretinal space and will be useful in studying interactions between the RPE and the neuroretina and to preclinically test potential therapies.

Current focus of stem cell application in retinal repair.

The relevance of retinal diseases, both in society's economy and in the quality of people's life who suffer with them, has made stem cell therapy an interesting topic for research. Embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adipose derived mesenchymal stem cells (ADMSCs) are the focus in current endeavors as a source of different retinal cells, such as photoreceptors and retinal pigment epithelial cells. The aim is to apply them for cell replacement as an option for treating retinal diseases which so far are untreatable in their advanced stage. ESCs, despite the great potential for differentiation, have the dangerous risk of teratoma formation as well as ethical issues, which must be resolved before starting a clinical trial. iPSCs, like ESCs, are able to differentiate in to several types of retinal cells. However, the process to get them for personalized cell therapy has a high cost in terms of time and money. Researchers are working to resolve this since iPSCs seem to be a realistic option for treating retinal diseases. ADMSCs have the advantage that the procedures to obtain them are easier. Despite advancements in stem cell application, there are still several challenges that need to be overcome before transferring the research results to clinical application. This paper reviews recent research achievements of the applications of these three types of stem cells as well as clinical trials currently based on them.

Triple-layered mixed co-culture model of RPE cells with neuroretina for evaluating the neuroprotective effects of adipose-MSCs.

Mesenchymal stem cell (MSC) therapy is promising for neuroprotection but there is no report of an appropriate in vitro model mimicking the situation of the in vivo retina that is able to test the effect of MSCs in suspension or encapsulated with/without a drug combination. This study aims to establish a viable mixed co-culture model having three layers: neuroretina explants (NRs), retinal pigment epithelium (RPE) cells and adipose tissue-derived MSCs (AT-MSCs) for evaluating adipose-MSC effects. AT-MSCs were grown on the lower surface of a transwell membrane and RPE cells were grown on the bottom of a culture plate as monocultures. A transwell membrane was inserted into a culture plate well. NR was placed as an organotypic culture on the upper surface of the transwell membrane. Thus, a triple-layered co-culture setup was constructed. In double-layered setups, NR were co-cultured with AT-MSCs or RPE cells. Optimum medium, experiment execution period and transwell membrane permeability (TMP) were determined. MSC effects on RPE cell proliferation and NR reactive gliosis were evaluated. Limitations were discussed. Our study shows that neurobasal A with DMEM (1:1) mixed medium was suitable for viability of all three layers. AT-MSC growth decreased TMP significantly, 30-60 % in 3- to 6-day periods. Spontaneous NR reactive gliosis limits the experiment execution period to 6 days. AT-MSCs maintained their undifferentiated nature and showed no or limited neuroprotective effects. In this study, we successfully assembled viable double- and triple-layered co-culture setups for AT-MSCs, RPE and NR, optimised conditions for their survival and explored setup Limitations.

Chitosan feasibility to retain retinal stem cell phenotype and slow proliferation for retinal transplantation.

Retinal stem cells (RSCs) are promising in cell replacement strategies for retinal diseases. RSCs can migrate, differentiate, and integrate into retina. However, RSCs transplantation needs an adequate support; chitosan membrane (ChM) could be one, which can carry RSCs with high feasibility to support their integration into retina. RSCs were isolated, evaluated for phenotype, and subsequently grown on sterilized ChM and polystyrene surface for 8 hours, 1, 4, and 11 days for analysing cell adhesion, proliferation, viability, and phenotype. Isolated RSCs expressed GFAP, PKC, isolectin, recoverin, RPE65, PAX-6, cytokeratin 8/18, and nestin proteins. They adhered (28 ± 16%, 8 hours) and proliferated (40 ± 20 cells/field, day 1 and 244 ± 100 cells/field, day 4) significantly low (P < 0.05) on ChM. However, they maintained similar viability (>95%) and phenotype (cytokeratin 8/18, PAX6, and nestin proteins expression, day 11) on both surfaces (ChM and polystyrene). RSCs did not express alpha-SMA protein on both surfaces. RSCs express proteins belonging to epithelial, glial, and neural cells, confirming that they need further stimulus to reach a final destination of differentiation that could be provided in in vivo condition. ChM does not alternate RSCs behaviour and therefore can be used as a cell carrier so that slow proliferating RSCs can migrate and integrate into retina.

Bioactive substrates for human retinal pigment epithelial cell growth from elastin-like recombinamers.

The aim of this study was to investigate the use of bioactive RGD-containing elastin-like recombinamers (ELR-RGDs) as a substrate that can maintain human retinal pigment epithelial cell (hRPE) phenotype and growth pattern. Results obtained are compared with previously published behavior of ARPE19 cells. The extension of these results to hRPE is required because ARPE19 cells cannot be used clinically to treat age-related macular degeneration. hRPE cells were isolated, cultured, seeded, and grown on surface of glass, treated polystyrene (TCP), and solvent-cast ELR-RGD and ELR-IK film with no specific sequence. Cells were analyzed to study cell adhesion, proliferation, morphology, and RPE65 protein expression by staining with diamidino-2-phenylindole, Rhodamine-Phalloidin, and anti-RPE65 antibody at 12, 24, 72, 120, 168, and 360 h. hRPE cells always grew better on ELR-RGD than on glass and ELR-IK but not on TCP. The kinetic hRPE growth curves confirmed that growth differences started to appear at 24 h for these surfaces in ascending order of cell growths, namely glass, ELR-IK, ELR-RGD, and TCP. There was a clear difference at 360 h. ELR-RGD maintained hRPE cells stable morphology and RPE65 protein expression. ELR-RGD seems to be a good substrate for growing hRPE cells with stable morphology and RPE65 protein expression. As such, this work confirms our hypothesis regarding ELR-RGD substrates viability, which can be used as a Bruch's membrane prosthesis for further studies in animals. However, these results must subsequently be extrapolated to use of hRPE cells in animals to evaluate them as a transplantation vehicle in human.

Adipose derived mesenchymal stem cells partially rescue mitomycin C treated ARPE19 cells from death in co-culture condition.

Age-related macular degeneration is a retinal disease with important damage at the RPE layer. This layer is considered a target for therapeutical approaches. Stem cell transplantation is a promising option for retinal diseases. Adipose derived mesenchymal stem cells secret growth factors which might play a significant role in RPE maintenance. This study aimed to evaluate human AD-MSCs ability to rescue mitomycin C treated dying ARPE19 cells in co-culture condition. ARPE19 cells were treated with MMC (50 µg/ml, 100 µg/ml and 200 µg/ml) for 2 hours to induce cell death. These treated cells were co-cultured with hAD-MSCs in indirect co-culture system for 3 days and 3 weeks. Then the viability, growth and proliferation of these ARPE19 cells were evaluated by a cell viability/cytotoxicity assay kit and Alamar Blue (AB) assay. Untreated ARPE19 cells and human skin fibroblasts (HSF) were used as controls. MMC blocked ARPE19 cell proliferation significantly in 3 days and cells were almost completely dead after 3 weeks. Cell toxicity of MMC increased significantly with concentration. When these cells were co-cultured with hAD-MSCs, a significant growth difference was observed in treated cells compared to untreated cells. hAD-MSCs rescue capacity was also significantly higher than HSF for treated ARPE19 cells. This study showed that hAD-MSCs rescued MMC treated ARPE19 cells from death. It probably occurred due to undefined growth factors secreted by hAD-MSCs in the medium, shared by treated ARPE19 cells in co-culture conditions. This study supports further evaluation of the effect of hAD-MSCs subretinal transplantation over the RPE degeneration process in AMD patients.

Flow cytometry assessment of the purity of human retinal pigment epithelial primary cell cultures.

Culturing of human retinal pigment epithelial cells (hRPE) is the initial step in cell therapy of some retinal diseases. To transfer these cells into clinical use, it is necessary to guarantee that they are well differentiated and contamination free. Fluorescence microscopy is the easiest method to do this, but it is associated with operator subjectivity, and the results are highly variable. The aim of this study was to demonstrate the practicality of implementing flow cytometry (FC) analysis to determine the purity of human RPE primary cell cultures. An ARPE19 cell line, human skin fibroblasts, hRPE, and human corneal epithelial cells were analysed by FC to determine the percentage of the hRPE population expressing RPE65 and epithelial and fibroblast proteins. The cell viability and DNA content also were determined. FC analysis showed that the hRPE cells were healthy, stable, and expressed RPE65 protein in the study working conditions. The density of RPE65 protein expression decreased during passages 2 to 10, which was confirmed using a Western blot technique. However, the hRPE cells did not express the 112-kDa epithelial and fibroblast proteins in the current working conditions. These findings suggested that FC facilitates a detailed analysis of human RPE primary cell cultures, a necessary step in developing new cell therapies for retinal diseases.