B-LCL-HROC68

B-LCL-HROC68 is an Epstein-Barr virus (EBV)-immortalized human B lymphoblastoid cell line established from tumor-infiltrating B cells (TiBc) isolated from a primary colorectal carcinoma designated HROC68. The parental tumor was a sporadic-type colorectal carcinoma resected from an adult male patient with advanced-stage disease. Fresh tumor tissue was mechanically dissociated, and B cells were cultured in the presence of EBV-containing supernatant derived from the B95/8 marmoset cell line, together with cyclosporin A to suppress outgrowth of T and NK cells. Long-term culture resulted in monoclonal expansion of B cells, as confirmed by immunoglobulin gene rearrangement analysis using BIOMED-2 multiplex PCR protocols, demonstrating a single dominant rearrangement pattern consistent with clonal origin.

B-LCL-HROC68 secretes immunoglobulin G (IgG) as its exclusive isotype, with stable production over extended culture. In cell-based ELISA screening against allogeneic colorectal cancer cell lines (HROC24, HROC46, and HCT116), IgG derived from B-LCL-HROC68 demonstrated measurable tumor cell binding, with the strongest signal observed against HCT116 cells. However, subsequent flow cytometric validation indicated comparatively weak binding affinity relative to other TiBc-derived IgGs. These findings indicate that B-LCL-HROC68 represents a monoclonal, antigen-experienced tumor-infiltrating B-cell line capable of producing functional IgG with detectable tumor cell reactivity, providing a useful in vitro tool for investigating humoral immune responses within the colorectal carcinoma microenvironment and for potential identification of tumor-associated antigens.

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.