B-LCL-HROC69

B-LCL-HROC69 is an Epstein-Barr virus (EBV)-immortalized B lymphoblastoid cell line established from tumor-infiltrating B cells (TiBc) isolated from a primary colorectal carcinoma specimen designated HROC69. The parental tumor originated from an adult male patient with right-sided colorectal carcinoma of conventional sporadic type and advanced stage disease. B cells were isolated from freshly resected tumor tissue and immortalized ex vivo using supernatant from the EBV-producing B95/8 marmoset cell line in the presence of cyclosporin A to suppress T- and NK-cell outgrowth. Outgrowth of EBV-transformed B-cell clones typically occurred within several weeks, and clonality was confirmed by immunoglobulin heavy- and light-chain gene rearrangement analysis using BIOMED-2 multiplex PCR protocols.

B-LCL-HROC69 secretes immunoglobulin A (IgA), as determined by isotype-specific ELISA of long-term culture supernatants. In contrast to several IgG-producing TiBc lines established in parallel, IgA derived from HROC69 was not further characterized for tumor cell binding in the initial functional screening assays. Importantly, no spontaneous outgrowth of B-cell cultures occurred in the absence of exogenous EBV, indicating that the immortalization is an in vitro event rather than the consequence of latent EBV infection in vivo. B-LCL-HROC69 therefore represents a monoclonal, antigen-experienced tumor-infiltrating B-cell model suitable for investigating humoral immune responses within the colorectal carcinoma microenvironment and for the potential identification of tumor-associated antigens recognized by locally expanded B-cell clones.

37°C, 5% CO₂, humidified atmosphere.

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

2. 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.

3. 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.

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

5. 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.

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

7. 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.

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

Mycoplasma contamination is excluded using both PCR-based assays and luminescence-based mycoplasma detection methods.

To ensure there is no bacterial, fungal, or yeast contamination, cell cultures are subjected to daily visual inspections.

For long-term preservation, place vials in vapor-phase liquid nitrogen at about −150 to −196 °C. Storage at −80 °C is acceptable only as a short interim step before transfer to liquid nitrogen.

Cryopreserved cell lines are shipped on dry ice in validated, insulated packaging with sufficient refrigerant to maintain approximately −78 °C throughout transit. On receipt, inspect the container immediately and transfer vials without delay to appropriate storage.

At Cytion, we stand behind the quality and viability of our products. We guarantee that our cells will be viable at the start of culture for 60 days after shipment from our facilities. We assure that all materials conform to the specifications described in the Certificate of Analysis and/or Product Sheet.

For full details, please refer to our Warranty Statement.