The retinal pigment epithelium (RPE) is a monolayer of cells adjacent to the photoreceptors of the retina. It plays a crucial role in maintaining photoreceptor health and survival. Degeneration or dysfunction of the RPE can lead to photoreceptor degeneration and as a consequence to visual impairment. The most common diseased state of the RPE becomes manifest in age-related macular degeneration, an increasing cause of blindness in the elderly. RPE cells are therefore of great interest to researchers working in the field of tissue engineering and cell transplantation. In fact, studies in animal models have proven that the transplantation of RPE cells can delay the course of photoreceptor degenerative diseases. Although first attempts to transplant RPE cells into the subretinal space in human individuals suffering from age-related macular degeneration were less successful, RPE cell transplantation is still favored as a future therapeutic option, and much work is done to develop and design cell transplants. Cell banking is a prerequisite to have well-differentiated and characterized cells at hand when needed for research purposes, but also for therapeutic approaches. In this chapter the authors will describe methods to isolate, culture and preserve adult human RPE cells for the purpose of RPE cell banking.
In a previous study, we observed a deleterious effect of serum-supplemented Minimal Essential Medium (MEM) on human corneal endothelial cell survival in a cell culture model. Consequently, here we studied the effects of conventional, serum-supplemented MEM and a serum-free medium in combination with two different deswelling substances on cell survival in whole corneas in a mouse model.Murine corneas were cultured for 4 days in MEM+2% fetal calf serum (FCS) or serum-free Human Endothelial-SFM (SFM), both supplemented with either 6% dextran T500 or 7.5% hydroxyethyl starch (HES) 130/0.4. Cells were examined by differential interference contrast microscopy, H&E staining, immunocytochemistry for cleaved caspase-3, Bcl-2, haem oxygenase-1 and immunoblotting for cleaved caspase-3.In MEM, endothelial cells were almost completely lost after 4 days and the number of epithelial cells was markedly reduced. The remaining cells showed fragmented nuclei and were positive for cleaved caspase-3 and strongly positive for Bcl-2. Corneas cultured in SFM retained an almost closed layer of endothelial cells. Fewer cells were positive for Bcl-2, and only a few cells were positive for cleaved caspase-3 even under staurosporine administration. HES supplementation was well tolerated by corneal cells over 4 days, while a 4-day supplementation with dextran resulted in the loss of endothelial and epithelial cells.Serum-free medium, Human Endothelial-SFM, promoted cell survival during corneal organ culture better than MEM+2% FCS. HES 130/0.4 appeared to be tolerated better by the cells than dextran T500.
Purpose.: To evaluate retroviral vectors as a tool to transduce normal human corneal endothelial cells (HCECs) and to optimize transduction to increase gene transfer efficiency. Methods.: Enhanced green fluorescent protein (EGFP) encoding retroviral vectors based on HIV-1 or murine leukemia virus (MLV), pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or a modified foamy virus envelope protein (FV Env), and prototype foamy virus (PFV) were produced. Transduction was performed in four HCEC culture media that were previously described for specific cultivation of HCECs or organ culture of donor corneas, namely enriched HCEC growth medium F99HCEC, its unsupplemented basal medium F99, MEM + 2% fetal calf serum (FCS) (MEM), and Human Endothelial-SFM (SFM). Transduction efficiency was evaluated by marker gene transfer assay, and cytotoxic effects of virus infection were evaluated by means of resazurin conversion assay. Results.: PFV- and HIV-1–based vectors showed superior transduction efficiency compared with MLV-based vectors. Pseudotyping with a modified FV Env increased transduction efficiency compared with pseudotyping with VSV-G. In medium SFM, transduction efficiency of PFV, HIV-1–/FV Env, and MLV-based vectors was markedly reduced compared with the other culture media. When cells were cultured in F99-based media, cell viability was reduced by retroviral transduction compared with uninfected or mock infected controls, but remained unaffected when cells were cultured in SFM and was even increased when cells were cultured in MEM. Conclusions.: HIV-1–based vectors pseudotyped with FV Env can efficiently be used to transduce primary HCECs in vitro. However, transduction efficiency is dependent on culture conditions and impairs metabolic activity and viability of HCECs in vitro.
Abstract Purpose The purpose of the study was to compare the anatomical and functional results including reading ability after epiretinal membrane (ERM) surgery in patients with and without the use of autologous platelet concentrate (APC). Methods Design : Prospective, comparative non‐inferiority series. Setting : Institutional. Patients : 51 eyes of 51 patients, who underwent pars‐plana vitrectomy (PPV) for ERM surgery. 29 eyes additionally received intraoperative APC, 22 eyes underwent standard procedure without APC use. Observations : anatomical and functional outcome parameters (central retinal thickness (CRT), best corrected visual acuity (BCVA) and reading ability (RA)) were compared between the two groups at 6 weeks and 6 months postoperatively. Subjective assessment of visual acuity and reading ability was also analysed. Main outcome measures : BCVA, RA and CRT. Results Both groups showed significant CRT reduction and RA improvement, while BCVA improvement was significant only in eyes with intraoperative APC use during the follow‐up time of 6 months. There was no statistically significant difference between CRT reduction, BCVA and RA improvement between the groups. Conclusion Intraoperative APC use for ERM surgery results in similar anatomical and functional outcomes compared with standard ERM surgery without APC use.
Purpose: A malfunction of the corneal endothelium leading to corneal opacity is one of the main causes of impaired vision. Currently, keratoplasty is the one and only donor cornea–dependent treatment, and this calls for alternatives because of the worldwide lack of donor corneas. Recently, the topography of Descemet membrane (DM) has been discovered as a feasible stem cell differentiation tool. With this study, we further confirm this mechanotransductive system by using preinduced Wharton jelly–derived mesenchymal stem cells (WJ-EPCs). Methods: To measure the mechanotransductive potential of Descemet-like topography (DLT), WJ-EPCs were cultivated on collagen imprints with DLT. Changes in the gene and protein expressions of corneal endothelial cells (CECs), typical markers such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured. In addition, CEC functionality has been evaluated by measuring the relative potential differences in a 2-compartment system and by measuring corneal transparency in an ex vivo rabbit cornea model. To confirm the activity of WJ-EPCs, rabbit CECs were restless deleted by collagen digestion of a thin layer of rabbit Descemet membrane. Results: The proper CEC-typical hexagonal morphology of WJ-EPCs in combination with a significant expression of ZO-1, Na/K-ATPase, PITX2, and COL-8 could be demonstrated. In addition, the WJ-EPCs were able to build up a relative potential difference of 40 mV and to keep corneas clear and transparent. Conclusions: These data indicate that a well-characterized, functional CEC monolayer was developed by using a DLT-mediated mechanotransductive differentiation of WJ-EPCs.
