Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line

Morita, Maresuke; Fujita, Naoki; Abe, Momoko Koyama; Hayashimoto, Koji; Nakagawa, Takayuki; Nishimura, Ryohei; Tsuzuki, Keiko

doi:10.1016/j.exer.2018.03.003

Cited by 5 publications

(6 citation statements)

References 47 publications

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“…Studies have successfully isolated canine corneal epithelial cells and cultured them on amniotic membrane or feeder layer cells [ 1 , 21 ], which is favorable for transplantation but not suitable for extensive in vitro experiments. In another report, a corneal epithelial cell line was obtained by continuous passage of limbal epithelial cells [ 22 ], which could be continuously passaged in vitro independently of the amniotic membrane or feeder cells. In our study, epithelial cells filled with the cell flask could be obtained in approximately one week.…”

Section: Discussionmentioning

confidence: 99%

Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells

Guo

Wang

et al. 2022

BMC Vet Res

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Background Primary canine corneal epithelial cells (CCECs) easily become senescent, and cell proliferation is limited. Therefore, sampling for experimentation requires a large number of animals, which is problematic in terms of animal welfare and fails to maintain the stability of the cells for in vitro analyses. Results In this study, CCECs were separated and purified by trypsin and dispase II enzymatic analysis. Next, the cells were immortalized by transfection with a lentiviral vector expressing Simian vacuolating virus 40 large T (SV40T). The immortalized canine corneal epithelial cell line (CCEC-SV40T) was established by serial passages and monoclonal selection. The biological characteristics of CCEC-SV40T cells were evaluated based on the cell proliferation rate, cell cycle pattern, serum dependence, karyotype, and cytokeratin 12 immunofluorescence detection. In addition, we infected CCEC-SV40T cells with Staphylococcus pseudintermedius (S. pseudintermedius) and detected the inflammatory response of the cells. After the CCEC-SV40T cells were passaged continuously for 40 generations, the cells grew in a cobblestone pattern, which was similar to CCECs. The SV40T gene and cytokeratin 12 can be detected in each generation. CCEC-SV40T cells were observed to have a stronger proliferation capacity than CCECs. CCEC-SV40T cells maintained the same diploid karyotype and serum-dependent ability as CCECs. After CCEC-SV40T cells were infected with S. pseudintermedius, the mRNA expression levels of NLRP3, Caspase-1 and proinflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-α, were upregulated, and the protein levels of MyD88, NLRP3 and the phosphorylation of Iκbα and p65 were upregulated. Conclusions In conclusion, the CCEC-SV40T line was successfully established and can be used for in vitro studies, such as research on corneal diseases or drug screening.

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Section: Discussionmentioning

confidence: 99%

Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells

Guo

Wang

et al. 2022

BMC Vet Res

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“…To overcome some of these limitations a so-called 2.5D organoid culture system was recently developed in which 3D organoids are cultured on top of (rather than as separate organoids) within a thick Matrigel layer (74). A further improvement in the tumor organoid field was the creation of these 2.5D structures without the need of 3D pre-culture (76). Applying this system, it seems possible to create tumor organoids and to study veterinary cancer biology, yet it is still dependent on Matrigel.…”

Section: Thyroid Cancer Organoidsmentioning

confidence: 99%

Companion animal organoid technology to advance veterinary regenerative medicine

Penning

Boom

2023

Front. Vet. Sci.

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First year medical and veterinary students are made very aware that drugs can have very different effects in various species or even in breeds of one specific species. On the other hand, the “One Medicine” concept implies that therapeutic and technical approaches are exchangeable between man and animals. These opposing views on the (dis)similarities between human and veterinary medicine are magnified in regenerative medicine. Regenerative medicine promises to stimulate the body's own regenerative capacity via activation of stem cells and/or the application of instructive biomaterials. Although the potential is enormous, so are the hurdles that need to be overcome before large scale clinical implementation is realistic. It is in the advancement of regenerative medicine that veterinary regenerative medicine can play an instrumental and crucial role. This review describes the discovery of (adult) stem cells in domesticated animals, mainly cats and dogs. The promise of cell-mediated regenerative veterinary medicine is compared to the actual achievements, and this will lead to a set of unanswered questions (controversies, research gaps, potential developments in relation to fundamental, pre-clinical, and clinical research). For veterinary regenerative medicine to have impact, either for human medicine and/or for domesticated animals, answering these questions is pivotal.

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“…Since stem cells are responsible for the continuous re-population of the corneal epithelium, diseases that induce limbal stem cell deficiency (LSCD) result in corneal conjunctivalization and neovascularization, corneal scarring, and chronic inflammation (1). In human medicine, selected congenital and acquired limbal stem cell deficiencies include aniridia, various forms of keratitis, dyskeratosis congenita, epidermal dysplasia, chemical and thermal burns, ocular surgeries involving the limbal region, use of contact lenses, ocular surface inflammatory diseases, and Stevens-Johnson syndrome (7).…”

Section: Erent Morphological Types Of Feline Organoids ( X) the Image...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…The corneal epithelium is a stratified squamous nonkeratinized epithelium that comprises the outermost layer of the cornea (1). It is a critical tissue for maintaining ocular surface health by providing a barrier to microbes and toxins, anchoring the mucin layers of the tear film, and providing a smooth optical surface for light to enter the eye, among other key functions (1). Corneal epithelial cells have a high turnover rate, continuously being replenished by new epithelial cells originating from progenitors cells (limbal epithelial stem cells) in the corneoscleral junction (limbus).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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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.

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Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line

Cited by 5 publications

References 47 publications

Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells

Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells

Companion animal organoid technology to advance veterinary regenerative medicine

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

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