NCH612

NCH612 is a patient-derived glioma cell line known to harbor a mutation in the IDH1 gene, specifically the IDH1 R132H variant. This mutation is often found in gliomas and is associated with epigenetic alterations such as the glioma CpG island methylator phenotype (G-CIMP). These epigenetic changes can lead to the silencing of tumor suppressor genes, making IDH1-mutant gliomas a target for epigenetic therapies. The NCH612 cell line shows particular sensitivity to the DNA methyltransferase inhibitor decitabine (DAC), which has demonstrated the ability to inhibit cell proliferation and growth by reducing DNMT1 protein levels, a key epigenetic regulator. NCH612, in particular, exhibits a partial deletion of the 1p/19q chromosome arms, a characteristic often associated with certain subtypes of gliomas.
When treated with DAC, NCH612 cells show a significant reduction in cell proliferation and colony formation, as demonstrated in various assays. This sensitivity to DAC is linked to the downregulation of TERT (telomerase reverse transcriptase) and the upregulation of p21, a cyclin-dependent kinase inhibitor involved in the DNA damage response. Interestingly, NCH612 is one of several IDH1-mutant-codel glioma lines that exhibit this sensitivity, whereas other lines, such as NCH1681 (which lacks the 1p/19q codeletion), are resistant to DAC. This suggests that the therapeutic effects of DAC may be particularly pronounced in gliomas with both IDH1 mutations and 1p/19q deletions.
Further molecular studies have shown that DAC treatment in NCH612 cells induces enrichment of pathways related to DNA replication, cell cycle regulation, and lysosome function, which are key to understanding the drug's antitumor effects. The repression of TERT by DAC is mediated by p21, and this interaction plays a crucial role in determining the cell's response to the drug. Given these characteristics, NCH612 is an important model for studying the effects of DNMT inhibitors like DAC in IDH1-mutant gliomas with 1p/19q codeletion.

Spheroid culture

39 years

Caucasian

DMEM:Ham's F12, w: 3.1 g/L Glucose, w: 1.6 mM L-Glutamine, w: 15 mM HEPES, w: 1.0 mM Sodium pyruvate, w: 1.2 g/L NaHCO3 (Cytion article number 820400a)

For subculturing spheroid cultures, begin by mechanically dissociating the spheroids through pipetting up and down 5 to 10 times using an Eppendorf pipette with 1000 μl filter tips. After this, centrifuge the mixture at 300g for 5 minutes at room temperature to pellet the cells. Discard the supernatant and resuspend the cell pellet in fresh culture medium. Finally, transfer the resuspended cells into new culture vessels to promote further spheroid formation. This approach ensures efficient spheroid breakdown and readies them for continued growth in a new environment.

Slow. After thawing allow the cells to recover from the freezing process for at least 48 hours.

Confirm that the vial remains deeply frozen upon delivery, as cells are shipped on dry ice to maintain optimal temperatures during transit.

Upon receipt, either store the cryovial immediately at temperatures below -150° C to ensure the preservation of cellular integrity, or proceed to step 3 if immediate culturing is required.

For immediate culturing, swiftly thaw the vial by immersing it in a 37° C water bath with clean water and an antimicrobial agent, agitating gently for 40-60 seconds until a small ice clump remains.

Perform all subsequent steps under sterile conditions in a flow hood, disinfecting the cryovial with 70% ethanol before opening.

Carefully open the disinfected vial and transfer the cell suspension into a 15 ml centrifuge tube containing 8 ml of room-temperature culture medium, mixing gently.

Centrifuge the mixture at 300 x g for 3 minutes to separate the cells and carefully discard the supernatant containing residual freezing medium.

Gently resuspend the cell pellet in 10 ml of fresh culture medium. For adherent cells, divide the suspension between two T25 culture flasks; for suspension cultures, transfer all the medium into one T25 flask to promote effective cell interaction and growth.

Adhere to established subculture protocols for continued growth and maintenance of the cell line, ensuring reliable experimental outcomes.

When planning to store a cryovial in liquid nitrogen for future thawing, it is mandatory to adhere to stringent safety measures. Appropriate protective gloves and clothing are essential, and the use of a face mask or safety goggles is required during the transfer of frozen samples to or from the liquid nitrogen tank. This is to mitigate the risk of injury from potential cryovial explosions upon removal, which can result in the projection of sharp fragments.

We stand by the promise of delivering products with high cell viability and robust culture performance. To achieve the best results, please make sure you follow the storage and culture instructions detailed in the product information sheet closely. Your adherence to these guidelines is key to success.

A ratio of 1:2 to 1:5 is recommended