Current Advancements in the Development and Characterization of Full-Thickness Adult Neuroretina Organotypic Culture Systems.

Retinal degenerative diseases such as macular degeneration, glaucoma, and diabetic retinopathy constitute the leading cause of blindness in the industrialized world. There is a continuous demand in investigative ophthalmic research for the development of new treatment modalities for retinal therapy. Unfortunately, efforts to identify novel neuroprotective and neuroregenerative agents have often been hindered by an experimental model gap that exists between high-throughput methods via dissociated cells and preclinical animal models. Even though dissociated cell culture is rapid and high-throughput, it is limited in its ability to reproduce the in vivo conditions. In contrast, preclinical animal models may offer greater fidelity, albeit they lack efficiency and experimental control. Retina explant cultures provide an ideal bridge to close this gap and have been used to study an array of biological processes such as retinal development and neurodegeneration. However, it is often difficult to interpret findings from these studies due to the wide variety of experimental species and culture methods used. This review provides a comprehensive overview of current ex vivo neuroretina culture methods and assessments, with a focus on their suitability, advantages, and disadvantages, along with novel insights and perspectives on the organotypic culture model as a high-throughput platform for screening promising molecules for retinal regeneration.

Accelerated epithelialization and improved wound healing metrics in porcine full-thickness wounds transplanted with full-thickness skin micrografts.

Split-thickness skin grafting (STSG) is the current gold standard for treatment of extensive burn and traumatic skin injuries. However, STSG is limited by donor-site morbidity and availability, and often leads to scarring and wound contracture. Furthermore, these thin grafts lack dermal elements such as nerves and adnexa which are important in recapitulating normal skin function. Methods of fractional skin replacement either as minced STSGs or microscopic skin tissue columns have been proposed, though these techniques have not been fully characterized and lack evidence of regenerated adnexal structures. Here, we describe an alternative method of fractional skin replacement using full-thickness skin micrografts containing deep dermal components and intact adnexa. Full-thickness wounds measuring 3 cm in diameter and 2 cm apart were created on adult female Yorkshire swine. Full-thickness skin tissue columns (FTSTCs) 1.5 mm in diameter with intact adnexa and subcutaneous tissue were obtained using a suction-assisted device. Explant culture was initiated to demonstrate the capacity of FTSTCs to act as reservoirs of viable and proliferative epidermal and dermal cells. FTSTCs were applied directly to excisional wounds at three different expansion ratios (1:16, 1:40, 1:100) in fibrin sealant. Biopsies were collected at defined time points postwounding and processed for histology and immunohistochemistry. Wounds grafted with FTSTCs showed enhanced reepithelialization and epidermal differentiation over untreated control wounds in a dosage dependent manner. Adnexal structures such as hair follicles and sweat glands were only evident in FTSTC-treated wounds. Furthermore, whereas ungrafted wounds were marked by extensive infiltration of α-Smooth Muscle Actin+ (α-SMA+ ) myofibroblasts at POD 60, α-SMA expression was sparse and largely limited to perivascular cells in FTSTC-treated wounds. The number of Ki67+ cells was also greatly reduced in FTSTC-treated wounds. Transplantation of FTSTCs containing intact adnexa improved wound healing parameters in porcine full-thickness wounds and may have implications for the treatment of large, traumatic wounds.

In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.

Neurotrophic Factors Secreted by Induced Pluripotent Stem Cell-Derived Retinal Progenitors Promote Retinal Survival and Preservation in an Adult Porcine Neuroretina Model.

Purpose: Paracrine factors released by pluripotent stem cells have shown great potential as therapeutic agents in regenerative medicine. The purpose of this study was to characterize trophic factor secretion of retinal progenitor cells (RPCs) derived from human induced pluripotent stem cells (iPSCs) and to assess its impact on retinal survival ex vivo. Methods: RPCs were generated from human 3D1 iPSCs following previously established protocols with modifications. Conditioned medium (CM) was harvested from iPSC-derived retinal progenitors and analyzed for trophic factor composition through multiplex enzyme-linked immunosorbent assay. Retina-preserving capability of the collected CM was examined using a degenerative porcine neuroretina model. Viability of the CM-treated retina explants was evaluated using the resazurin-based PrestoBlue reagent, whereas the lactate dehydrogenase (LDH) assay was used to assess retinal cytotoxicity. Retina explants were also analyzed morphologically through immunohistochemistry for glial cell activation and apoptosis. Results: We have successfully generated and characterized iPSC-derived RPCs that secreted an array of neuroprotective factors, including osteopontin, hepatocyte growth factor, stromal cell-derived factor 1, and insulin-like growth factor-1. Retina explants cultured in CM derived from iPSC-RPCs (iPSC-RPC-CM) showed better preservation of the retinal microarchitecture and fewer terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)+ nuclei, and reduced reactive gliosis. Furthermore, we saw a reduction in extracellular LDH levels in CM-treated retina explants, which also exhibited higher metabolic activity than the untreated controls. Conclusions: iPSC-derived RPCs secrete many trophic factors that have been shown to promote neuroprotection, tissue repair, and regeneration in the retina. Overall, we have demonstrated the neuroprotective effects of iPSC-RPC-CM through a degenerative neuroretina model ex vivo.

Quantitative Assessment of Retina Explant Viability in a Porcine Ex Vivo Neuroretina Model.

PURPOSE: Given that porcine and human retinas have similar structures and characteristics, ex vivo culture of porcine neuroretina provides an attractive model for studying mechanisms of human retinal injury and degenerative disease. Here, we describe the method that was used to establish and characterize an adult porcine retina culture system as a rapid screening tool for retinal survival in real time. METHODS: Neuroretina explants 8 mm in diameter were harvested from adult swine and cultured on porous cell culture inserts with adjustable heights. Retina explant viability was evaluated at 1, 4, 7, 11, and 14 days of culture using a resazurin-based metabolic assay. The explants were analyzed morphologically through immunohistochemistry for glial activation and apoptosis. Morphometric analysis was also performed on hematoxylin and eosin-stained retina sections from each time point. RESULTS: The viability of retina explants gradually decreased over time in culture. The laminar structure of the neuroretina was well preserved during the first 7 days. However, by day 14, most explants showed significant loss of cells in each laminar layer and obvious thinning. Overall, the progressive loss of retinal lamination and thickness, and increase in apoptotic nuclei with activated hypertrophic Müller cells were well correlated with the metabolic activity of the ex vivo neuroretina explants. CONCLUSIONS: This study was the first report to describe the use of a high-throughput and quantitative method for monitoring retina explant viability in real time. Ex vivo neuroretina cultures closely mimic the functional dynamics of the organ, and can be used efficiently to screen novel therapeutics for retinal neurodegenerative disease.

Scaffold-Free, Size-Controlled Three-Dimensional Culture of Rabbit Adipose-Derived Stem Cells.

Adipose-derived stem cells are capable of self-renewal and differentiation along multiple cell lineages, and have potential applications in a wide range of therapies. ASCs are commonly cultured as monolayers on tissue culture plastic, but there are indications that they may lose their cell-specific properties with time in vitro. There has been a growing interest in culturing adherent cells using three-dimensional techniques based on the understanding that growing cells on plastic surfaces cannot truly recapitulate 3D in vivo conditions. Here we describe a novel method for generating and culturing rabbit ASCs as scaffold-free 3D cell aggregates using micropatterned wells via a forced aggregation technique.