Culture and characterization of canine and feline corneal epithelial organoids: A new tool for the study and treatment of corneal diseases

Abstract

In this study, we isolated and cultured canine and feline 3D corneal organoids. Samples derived from corneal limbal epithelium from one canine and one feline patient were obtained by enucleation after euthanasia. Stem cell isolation and organoid culture were performed by culturing organoids in Matrigel. Organoids were subsequently embedded in paraffin for further characterization. The expression of key corneal epithelial and stromal cell markers in canine and feline organoids was evaluated at the mRNA level by RNA-ISH and at the protein level by immunofluorescence (IF) and immunohistochemistry (IHC), while histochemical analysis was performed on both tissues and organoids using periodic-acid Schiff (PAS), Sirius Red, Gomori's Trichrome, and Colloidal Iron stains. IF showed consistent expression of AQP1 within canine and feline organoids and tissues. P63 was present in canine tissues, canine organoids, and feline tissues, but not in feline organoids. Results from IHC staining further confirmed the primarily epithelial origin of the organoids. Canine and feline 3D corneal organoids can successfully be cultured and maintained and express epithelial and stem cell progenitor markers typical of the cornea. This novel in vitro model can be used in veterinary ophthalmology disease modeling, corneal drug testing, and regenerative medicine.

Advances in organoid technology for veterinary disease modeling

“…Several veterinary organoid models also demonstrated the powerful potential of regenerative medicine ( 86 ). For example, corneal epithelial organoids in dogs and cats have successfully be cultured and maintained with expressions of cornea-specific epithelial and stem cell progenitor markers, which could be a new tool to model veterinary ophthalmology disease and test corneal drug and even further treat corneal diseases by corneal organoid transplantation or harnessing regenerative capabilities of limbal stem cells in the conception of regenerative medicine ( 87 ). The advantages of adopting a veterinary organoid system to model diseases and then applying it in precision and regenerative medicine are continually advancing, with potential benefits for both animal and human.…”

Exploring the role of antioxidants in sepsis-associated oxidative stress: a comprehensive review

“…As organoids offer numerous advantages over conventional models and have been extensively utilized in both fundamental and clinical research ( Kopper et al., 2021 ; Minkler et al., 2021 ; Bedos et al., 2022 ; Gabriel et al, 2022a ; Gabriel et al., 2022b ; Moshksayan et al., 2023 ; Sahoo et al., 2023b ; Gabriel et al, 2024 ), there are also reports using renal and intestinal organoids in sepsis. The use of organoids pre-treated with LPS serves as an effective means to study the impact of LPS-induced intestinal injury, which closely resembles sepsis, and facilitates the investigation of immune-associated mechanisms as well as the screening of potential therapeutic agents ( Huang et al., 2022 ).…”

Oxidative stress, hormones, and effects of natural antioxidants on intestinal inflammation in inflammatory bowel disease

“…Modulation of the stress response genes, including the antioxidant enzyme GPX1, was detected after LPS stimulation of the IBD enteroids and colonoids ( 57 ). Organoids have more advantages than conventional models and have been employed in fundamental and clinical research, such as for genetic and infectious diseases, regenerative medicine, and accurate and reliable drug screening ( 162 – 168 ). Murine colitis-derived intestinal organoids stimulated by LPS show reduced mRNA expression of inflammatory mediators such as TNF-α and lipocalin-2 (LCN2) when treated with quercetin ( 96 ).…”