Comparative analysis of co-culture and monoculture models in simulating diabetic neurovascular dysfunction: insights into diabetic retinopathy

Qiyun Wang; Zhixin Qiao; Wenting Kang; Ling Zhu; Xinyuan Zhang

doi:10.3389/fendo.2023.1215218

Comparative analysis of co-culture and monoculture models in simulating diabetic neurovascular dysfunction: insights into diabetic retinopathy

Front Endocrinol (Lausanne). 2023 Sep 8:14:1215218. doi: 10.3389/fendo.2023.1215218. eCollection 2023.

Authors

Qiyun Wang^{1

2}, Zhixin Qiao³, Wenting Kang³, Ling Zhu⁴, Xinyuan Zhang^{1

2}

Affiliations

¹ Beijing Tongren Eye Center, Tongren Hospital, Capital Medical University, Beijing, China.
² Beijing Retinal and Choroidal Vascular Disorders Study Group, Beijing, China.
³ Clinical Research Center, Tongren Hospital, Capital Medical University, Beijing, China.
⁴ Save Sight Institute, Department of Ophthalmology, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.

Abstract

Background: Interaction between retinal vascular endothelial cells and neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR). This study aims to compare an in vitro model over a monoculture model to simulate the neurovascular coupling under the hyperglycemic microenvironment of diabetes.

Methods: Rat retinal vascular endothelial cells (RRMECs) and ganglion cells (RGCs) were seeded mono- or co-cultured in a normal (NG, 5.5 mM) and high (HG, 75 mM) glucose concentrations culture medium. Cell viability was detected by the cell counting kit-8 (CCK-8) assay. The ability of migration and lumen formation of RRMECs were determined by scratch wound, transwell migration, and lumen formation assays. The apoptosis index of cells was calculated and detected by propidium iodide (PI)/Hoechst staining. Quantitative and morphological analysis of RGCs was performed through the labeling of RGCs by brain-specific homeobox/POU domain protein 3A (BRN3A) and anti-beta-III tubulin (TUJ1). The gene and protein expression levels of occludin (OCLN) and zonula occludens-1 (ZO-1) were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay.

Results: The viability, migration, and lumen formation abilities of RRMECs in the HG group significantly increased (P<0.05) in both mono- and co-culture models. Migration and lumen formation abilities of RRMECs in the co-culture with HG were lower than that in the monoculture group (P<0.05). The viability of RGCs cells with HG significantly decreased in both mono- and co-culture models (P_mono<0.001, P_co<0.001), the apoptosis index of RGCs in the co-culture with HG was higher than that in the monoculture (P=0.010). The protein and gene expression of OCLN, and ZO-1 in RRMECs significantly decreased with HG culture medium in both culture models (P<0.05). In the HG group, the protein and gene expression level of the ZO-1 and OCLN of RRMECs significantly decreased in the co-culture model than that in the monoculture model (P<0.05).

Conclusion: Compared with mono cell culture, the established co-culture in vitro system for diabetic neurovascular dysfunction can better stimulate the micro-environment of the retinal neurovascular unit.

Keywords: co-culture; diabetic neurovascular dysfunction; ganglion cells; monoculture; retinal vascular endothelium cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Culture Techniques
Coculture Techniques
Culture Media
DNA-Binding Proteins
Diabetes Mellitus*
Diabetic Retinopathy*
Endothelial Cells
Rats
Retina

Substances

Culture Media
DNA-Binding Proteins

Grants and funding

This work was supported by the National Natural Science Foundation of China [Grant 81570850; and 82070988] and the Ministry of Science and Technology Foundation of China [Grant 2016YFC1305604].