Wil2 s

The ADCC Reporter Bioassay Kit from Promega (Madison, WI) was used according to manufacturer instructions. CD20 positive cells (WIL2-S, ATCC CRL-8885) were incubated with different concentrations of test antibody and a specific concentration of Jurkat transformed cells expressing CD16. Then a luminescent substrate was added with further incubation of 20 min. The result of the assay was expressed as % of relative potency of Kikuzubam with respect to the reference product.

This bioassay was performed as Brezski and collaborators [22 (link)] with minor changes. Briefly, cell antiproliferation was induced by Rituximab through complement-dependent cytotoxicity (CDC) to CD20 expressing cells (WIL2-S, ATCC CRL-8885) in the presence of human serum complement (Quidel, CA, USA). Cells viability, following CDC treatment with Rituximab, was measured using Alamar Blue probe (Promega, WI, USA). Rituximab potency was expressed against a reference standard of 100% potency.

Cell lines were obtained from ATCC. Jurkat (ATCC TIB152) and WIL2-S (ATCC CRL8885) cell lines were cultured in RPMI1640 medium (Gibco11875093) supplemented with 10% fetal bovine serum (FBS) (Gibco16140063). The cultures were diluted on the third day by removing part of the cell suspension and adding fresh medium. The inoculate concentration was 1 × 10⁵ cells/mL. The maximum cell concentration before seeding did not exceed 1 × 10⁶ cells/mL. Cell viability was assessed by staining with trypan blue (Gibco 15250061). Cell cultures with a viability of at least 90% were used for cloning. Cell lines were cloned by limiting dilutions at the rate of 1 cell per 3 wells of a 96-well plate. Cloning was performed in RPMI1640 medium supplemented with 20% FBS at 100 µL/well. A week later, the growth of the culture in the wells of the plate was monitored under a microscope, and 100 μL of the medium was added to the wells containing growing clones. At this stage, the wells contain from 10 to 100 cells. A week later, cells grown to the state of a monolayer were transferred into centrifuge tubes, leaving about 1/10 of the volume of the cell suspension in the wells. About 100 µL of fresh medium was added to these wells. Grown cultures were transferred to centrifuge tubes and sedimented for 5 min at 200 g.

CD20 positive cells (WIL2-S, ATCC CRL-8885) were incubated in RPMI 1640 media with 10% of FBS with different concentrations of rituximab and complement human serum for 4 h at 37°C and 5% CO₂. Then MTS substrate was added to each well with a further incubation of 2 h at the same conditions. The result of the assay was expressed as % relative potency, which is obtained comparing to the EC₅₀ of the dose-response curve of Kikuzubam with respect of the EC₅₀ of the dose-response curve of the reference product.

The DLBCL cell lines OCI-LY10 (ABC subtype) and OCI-LY1 (GCB subtype) and the human B lymphoblast cell lines WIL2S and DAKIKI were purchased from ATCC (Shanghai, China) and cultured in RPMI1640 medium (Hyclone) containing 10% FBS (Hyclone), 4 mM L-glutamine (Sigma–Aldrich, St. Louis, MO, USA), 100 U/mL of penicillin (Hyclone), and 100 U/mL of streptomycin (Hyclone). HEK-293T cells were purchased from ATCC (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (Hyclone) supplemented with 10% FBS. All cells were cultured in a humidified chamber at 37°C with an atmosphere of 5% CO₂. TILs and NILs were isolated from DLBCL tumor tissues and normal tissues, which were cultured in RPMI 1640 medium containing 10% FBS supplemented with 4 mM L-glutamine (Sigma-Aldrich), 1 μM 2-mercaptoethanol (Sigma–Aldrich), and recombinant human IL-2 (300 IU/mL) (Hyclone).

The hereditary spherocytosis cell line Wil2-S was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Wil2-S cells were cultured in RPMI (Roswell Park Memorial Institute medium, Corning, NY, USA) media supplemented with 10% fetal bovine serum (VWR, Radnor, PA, USA). Cells were seeded in culture flasks at a density of 1×10⁵ cells/mL, incubated at 37 °C with 7% CO₂, and sub-cultured when the cell density reached approximately 1.5–2×10⁶ cells/mL.

A hereditary Spherocytosis cell line Wil2-S (ATCC^® CRL-8885) and the Burkitt’s lymphoma cell line Daudi (ATCC^® CCL213) were obtained from the American Type Culture Collection (Manassas, VA, USA). Both cell lines were maintained in Gibco^® RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL), streptomycin (100 µg/mL) and non-essential amino acids (Life Technologies, Carlsbad, CA, USA). Cells were maintained with seeding density of 1 × 10⁵ cells/mL and cultured at 37 °C with 5% CO₂. Cells were subcultured when cell density reached 2 × 10⁶/mL.