Glaucoma Awareness Month

January is Glaucoma Awareness Month, a time to raise awareness for this vision-threatening condition, which affects an estimated 3 million people in the United States. Because glaucoma is often asymptomatic, it is known as the “sneak thief of sight,” making early detection challenging. Caused by fluid buildup in the eye, the condition elevates intraocular pressure, which can damage the optic nerve and, if left untreated, can result in significant vision loss and even blindness. Globally, it is the second-leading cause of blindness. Age is a significant risk factor for the development of glaucoma, and African American and Latino populations face a higher risk, with rates 6 to 8 times higher than Caucasians. There is currently no cure for glaucoma, which highlights the importance of early detection through regular eye examinations. And ongoing research efforts aim to develop more advanced diagnostic tools for early detection and new therapeutic interventions to mitigate the impact of this debilitating condition.

Cell Culture Tools to Advance Ocular Research

As the global population ages and lifestyles change, the incidence of eye-related conditions is on the rise, which has prompted intensified investment to enhance our understanding of these diseases and develop improved diagnostic and therapeutic strategies to address the growing vision-related challenges. Researchers use a variety of approaches, including both in vitro cell culture of primary cells and immortalized cell lines to conduct their studies.

Primary cells, including retinal ganglion cells, scleral fibroblasts, and corneal epithelial cells, derived directly from human tissues, are considered more representative models of cellular behavior in the in vivo environment. On the other hand, genetically engineered cell lines offer convenience and reproducibility, which is useful for high-throughput screenings in drug and diagnostic development. However, the concern with cell lines is that they may diverge from the behavior cells in vivo, which can affect the reliability and relevance of experimental results. A recent publication looks at the impact of different cellular immortalization methodologies using corneal epithelial cells and their influence on cellular characteristics compared to the original wildtype primary cells.

The transcriptome of wild-type and immortalized corneal epithelial cells

In a recent Nature publication by Furuya et al , the researchers conducted a comprehensive evaluation of different cell immortalization methodologies and their impact on the biological characteristics of corneal epithelial cells. Simian virus 40 (SV40) large T antigen has a long history for cellular immortalization, but it has also been associated with genomic instability, which can lead to alterations in cellular characteristics and chromosomal abnormalities. In recent years, the combination of cyclin-dependent kinase 4 R24C-mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT) has emerged as an efficient alternative for human cell immortalization. While K4DT cells are considered more advantageous than SV40 in preserving the original nature of primary cells, a lack of functional evidence, especially in epithelial-derived cells, prompted the authors to address this gap.

The study compares the transcriptome of primary corneal epithelial cells obtained from Lifeline® Cell Technology with immortalized corneal epithelial cells generated using either the SV40 or K4DT method. To assess and compare biological characteristics, the researchers employed RNA-sequencing (RNA-seq) of poly-A tailed RNA to conduct a comprehensive analysis of gene expression in primary corneal epithelial cells and their immortalized counterparts. Additionally, the study explored the influence of serum-containing culture media on the expression profiles.

Using the expression profiles, they evaluated the differentially expressed genes (DE genes) and identified 11,925 genes involved in ribosome, ubiquitin-mediated proteolysis, endocytosis, and cell cycle pathways that showed significant differences. Focusing on the cell cycle and ubiquitin-mediated proteolysis pathways due to their association with genomic instability, a heat map showed that the distance from wild-type to K4DT was smaller than that from wild-type to SV40 within these pathways, indicating greater similarity between K4DT and wild-type cells. Specifically, K4DT cells exhibited upregulated genes around the p16-pRB pathway in the cell cycle, while SV40 cells showed upregulated genes throughout the entire cell cycle. In the ubiquitin-mediated proteolysis pathway, certain F-box-related molecules of the SCF complex were upregulated in SV40-immortalized cells.

Morphological analysis, PCR, and western blotting were used to confirm the characteristics of the immortalized cells. Cell cycle analysis results showed an expected diploid pattern in wild-type and K4DT cells, while SV40 cells exhibited a polyploid formation, indicative of chromosome instability. Giemsa staining further supported this observation, showing increased chromosome numbers and intensive abnormalities in SV40 cells compared to K4DT cells. Importantly, the presence of serum did not significantly impact the characteristics of the cells in these analyses.

These findings strongly suggest that K4DT cells exhibit a gene expression profile that closely resembles wildtype cells, outperforming SV40 cells in maintaining similarity to the original cellular characteristics. This positions K4DT as a more favorable and promising alternative for cellular immortalization, potentially offering advantages in preserving the natural features of primary cells.

Lifeline Products to Support Ocular Research

Explore the Lifeline® catalog, which offers a range of essential products to support ocular research, including the following cell types and specialized culture media:

• Normal Human Corneal Epithelial Cells
• Human Scleral Fibroblasts
• OcuLife ™ Epithelial Medium Complete Kit
• FibroLife S2 Fibroblast Medium Complete Kit
• FibroLife Fibroblast Serum Free Medium Complete Kit

These Lifeline products serve as invaluable tools for researchers in the field of ocular studies, providing reliable and standardized resources to advance scientific exploration and discovery.

Have you used our products to power your research? We’d love to hear from you!