Characterization of a Novel Human Organotypic Retinal Culture Technique

Summary

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment epithelium-choroid (RPE-choroid) and sclera.

Abstract

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.

After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, Müller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.

H&E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved Müller cell integrity. The Luminex assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.

We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

Introduction

The retina is a highly specialized ocular structure responsible for transforming incoming light energy to electric signals, which are then processed by the brain for visual perception. The human retina contains a dynamic range of cell types, highly organized in a unique lamellar structure consisting of two synaptic and three nuclei layers¹ (Figure 1). Retinal homeostasis is sustained by the intricate connections between neuroretinal cells, blood vessels, nerves, connective tissues and the RPE¹. Due to the sophisticated retinal anatomy and physiology, mechanisms of many retinal diseases still....

Protocol

Human donor eye cups were obtained from the New Zealand National Eye Bank following corneal excision for transplantation and as approved by the Northern B Health and Disability Ethics Committee (NTX/06/19/CPD/AM07).

NOTE: The culture should be done in a Class II biosafety cabinet to ensure sterile tissue culture conditions. The tissues must be cultured within 24 h post-mortem to avoid significant retinal integrity loss.

Figure 2: Images showing....

Results

Retinal integrity was preserved in this HORC model. Retinal integrity was preserved in the cultured sandwich retinal explants but was lost in the retina cultured without adjacent structures. H&E was conducted to examine the structural integrity of sectioned sandwich retinal explants after 72 h in culture. The sandwich retinal explants showed preserved integrity and a distinct lamellae structure from GCL to ONL with compact nuclei in INL and ONL (F.......

Discussion

HORC is currently the most clinically translatable model in preclinical retinal research. Compared to in vitro cell culture models, HORC can better represent the anatomy of the human retina in situ, through retaining the dynamic retinal cell types and their connections with neurons, vasculatures and the extracellular environment¹⁹. Compared to animal models, HORC are more advantageous in studying the pathophysiology of and designing pharmaceutical treatments for human retinal diseases due to inter.......

Materials

Name	Company	Catalog Number	Comments
Disposable Biopsy Punches (5 mm)	Integra York PA Inc., USA	21909-142	Referred as surgical trephines in this article
Human Recombinant IL-1β	Peprotech, USA	200-01B	Working concentration: 10 ng/mL
Human Recombinant  TNF-a	Peprotech, USA	300-01A	Working concentration: 10 ng/mL
DAPI (1 µg/mL)	Sigma Aldrich, Germany	#D9542	Nuclear stain. Working dilution 1:1000
DMEM/F-12, GlutaMAX supplement	Gibco, Thermofisher, Scientific Inc., USA	10565018	Dulbecco’s Modified Eagle Medium nutrient mixture F-12 containing a 1× antibiotics and antimycotics mixture (AA, 100× stock)
Fine Scissors - Sharp-Blunt	Fine Science Tools (F.S.T)	14028-10	Tips: Sharp-Blunt, Cutting Edge: 27mm, Length: 10cm, Alloy/Material: Stainless Steel, Serrated: No, Tip Shape: Straight
Graefe Forceps	Fine Science Tools (F.S.T)	11050-10	Length:10cm, Tip shape: Straight, Tips: Serrated, Tip Width: 0.8mm, Tip Dimensions:0.8 x 0.7mm, Alloy/Materials: Stainless Steel
Mouse monoclonal GFAP-Cy3	Sigma Aldrich, Germany	#C9205	Primary antibody conjugated to Cy3. Working dilution 1:1000.
Mouse monoclonal Vimentin-Cy3	Sigma Aldrich, Germany	#C9080	Primary antibody conjugated to Cy3. Working dilution 1:50.
Rabbit polyclonal TUNEL (In Situ Cell Death Detection Kit, Fluorescein)	Sigma Aldrich, Germany	#11684795910	Primary antibody conjugated to Fluorescein-dUTP. Working dilution 1:10 with enzyme-buffer solution.