How to Culture & Edit NCI-H358 Cells: Best Practices for KRAS G12C Mutation Research

Lung cancer researchers often utilize the NCI-H358 cell line for its KRAS?G12C mutation to test inhibitors specifically targeting this mutation, as well as explore potential combination therapy strategies. Additionally, the NCI-H358 cell line is used to study the impact of the KRAS G12C mutation on the tumor immune microenvironment and to investigate the combined therapeutic effects of immune checkpoint inhibitors with KRAS G12C inhibitors.

Introduction to NCI-H358 Cell Line

NCI-H358 is a human non-small cell lung cancer (NSCLC) cell line that is widely used in research related to KRAS?gene mutations. The KRAS G12C mutation is relatively common in NSCLC and the NCI-H358 cell line harbors this mutation, which plays a critical role in tumor cell signaling and proliferation. Tumor cells carrying the KRAS G12C mutation may exhibit sensitivity to specific KRAS inhibitors. Because of this, NCI-H358 is invaluable for drug screening, particularly for testing KRAS inhibitors like Sotorasib (AMG 510), which targets the KRAS G12C mutation by forming a covalent bond with the cysteine residue at position 12 of the KRAS mutant protein,?inhibiting?its activity and affecting downstream signaling.

NCI-H358 Cell Line Details

Cell Name: NCI-H358 (Human Non-Small Cell Lung Cancer Cell)

Catalog Number: TCHu151 (Chinese Academy of Sciences)

Growth Characteristics: Epithelial-like morphology, adherent growth

Culture Conditions: 1640 medium + 10% FBS

Culture Environment: 95% air + 5% CO?; 37°C

Applications of NCI-H358 in Lung Cancer Research

NCI-H358 cells are essential in various lung cancer research applications, including:

• Drug Screening:?NCI-H358 cells exhibit sensitivity to various anticancer drugs, making them an ideal model for evaluating the efficacy of new drugs, especially those targeting the KRAS G12C mutation.
• Gene Function Studies:?Researchers use NCI-H358 to study the roles of specific genes in lung cancer by employing gene knockout or overexpression experiments.
• Signal Pathway Analysis:?This cell line is used to investigate intracellular signaling networks, particularly those related to the KRAS pathway.
• Tumor Microenvironment Simulation:?NCI-H358 is used to study interactions between tumor cells and surrounding cells, such as immune cells and stromal cells, ?providing insights into the tumor microenvironment.

Step-by-Step Guide to Culturing NCI-H358 Cells

During the actual cultivation process, the proliferation rate of NCI-H358 cells can be accelerated by optimizing the culture conditions. When the cell density is high, it is important to regularly replace the fresh culture medium or passage the cells in a timely manner. To ensure optimal growth and research outcomes, follow these best practices for NCI-H358 cell culture:

Cell Culture Procedure

Cell Thawing

• Add 6 mL of complete culture medium to a centrifuge tube to begin the thawing process.
• Thaw the cells in a water bath until the ice block is the size of a rice grain, then stop the water bath.
• Transfer the cells to the centrifuge tube and centrifuge.
• Resuspend the cells and seed them in a suitably sized culture dish.
• After 24 hours, observe the cell adhesion and change the medium once.

Cell Passaging

• Aspirate the supernatant and rinse the cells with room temperature PBS once.
• Add trypsin, ensuring it evenly covers the bottom of the dish.
• Digest the cells until some start to detach when tapping the dish, then stop the digestion.
• Gently pipette the cells to disperse them and transfer them to a centrifuge tube for centrifugation.
• Resuspend the cells and seed them at the appropriate ratio.

Cell Cryopreservation

• Digest and centrifuge the cells to obtain a cell pellet.
• Resuspend the cells in cryopreservation solution and transfer them to cryovials.
• Place the cryovials in a pre-cooled (4°C) controlled-rate freezing container, then store them in a -80°C freezer overnight.
• Transfer the cryovials to liquid nitrogen for long-term storage the next day.

Important Considerations for NCI-H358 Cell Culture

• Serum Quality:?Use high-quality fetal bovine serum; consider increasing the concentration slightly.
• Digestion Monitoring:?Avoid over-digestion by closely monitoring the process.
• Medium Replacement: As cell density increases, replace the medium or passage cells regularly to maintain optimal growth conditions.

Gene Editing Techniques for NCI-H358 Cells:?Using Smart-CRISPR?

Cyagen offers comprehensive gene editing services for the NCI-H358 cell line using the Smart-CRISPR? system, ensuring precise and efficient genetic modifications.

Gene Modification Projects

Gene modifications in the NCI-H358 cell line include gene knockout, point mutation, knock-in (KI), overexpression, and interference. Cyagen optimizes the culture conditions and monoclonal preparation for NCI-H358, enabling the delivery of NCI-H358 gene-edited monoclonal cells. This significantly enhances the accuracy and reliability of experiments, providing a more solid foundation for tumor immunology research.

Gene Knockout

Using the Smart-CRISPR? cell gene editing system, Cyagen offers custom NCI-H358 knockout (KO) cell services, delivering monoclonal homozygous cells. We use an optimized transfection system to deliver RNP (ribonucleoprotein) directly into the cells, achieving a gRNA cutting efficiency of over 90%. Compared to plasmid and virus-mediated CRISPR/Cas methods, this approach significantly increases cutting efficiency and reduces off-target effects, allowing for more precise targeting of the DNA sequence.

Point Mutation

Using the Smart-CRISPR? cell gene editing system, we offer precise and efficient custom services for point mutation and knock-in (KI) in the NCI-H358 cell line. By transfecting the target cells with a humanized Cas protein, gRNA, and donor components through an optimized α-donor system, we achieve high-efficiency homologous recombination, with HDR (homology-directed repair) rates reaching up to 49%, enabling the delivery of monoclonal homozygous cells.

Stable Cell Line

Cyagen has optimized and upgraded the transfection vector system and, leveraging years of experience in cell biology, has established a mature and stable gene overexpression system. We offer NCI-H358 stable cell pools or monoclonal cell lines that stably express exogenous genes or RNA interference elements, achieving protein expression levels more than 10 times higher.

Gene Editing Precautions

• Ensure that the cells are in good condition before transfection, with a cell density of around 70%.
• During digestion, try to disperse the cells into single cells as much as possible.
• Use the limiting dilution method to prepare monoclonal cells, employing Cyagen's custom culture medium and adding an additional 10% serum. Once clones are observed, supplement with fresh culture medium and continue cultivation.

Conclusions

The NCI-H358 cell line is a powerful tool in lung cancer research, particularly for studying the KRAS G12C mutation. By following best practices for cell culture and gene editing, researchers can unlock new insights into cancer biology and therapeutic strategies. Cyagen’s expertise in gene editing further enhances the research potential of NCI-H358 cells, offering precise and reliable modifications that support groundbreaking studies.

Custom Cell Line Gene Editing Services by Cyagen

Cyagen offers a one-stop in vitro service platform for cell line/iPSC gene editing, featuring advanced cell reprogramming, genetic engineering, and cellular differentiation technologies. By integrating our one-stop phenotypic analysis platform, we can provide in vitro model development and testing services for cell lines across various disease applications. Our optimized CRISPR-Pro technology enables rapid gene editing, including the excision of ?large fragments,?large fragment knockin, and?point mutations?with stable expression across various cell lines.

Contact our experts?and take advantage of our optimized CRISPR-Pro gene edited cell line modeling service platform for low-cost cell line generation with rapid turnaround and stable expression.

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