KBM 581 – Serum-Free Medium for T-Cell Expansion

Purpose-Built for T-Cell Expansion and Adoptive Immunotherapy

KBM 581 is a high-quality, serum-free medium optimised for the activation and expansion of human T cells, including cytokine-induced killer (CIK) cells, from peripheral blood mononuclear cells (PBMCs). Part of the widely adopted KBM 500 series, KBM 581 is frequently used in clinical research in Japan for adoptive immunotherapy and is now available through Atlantis Bioscience for translational research.

Key Advantages for T-Cell Therapy Development

Prepared using high-purity, GMP-grade reagents and raw materials.
Protein-free formulation except for injection-grade human serum albumin and recombinant human insulin.
Enhanced buffering capacity ensures stable pH throughout extended culture.
Long shelf life maintained even with L-glutamine present.
Contains kanamycin sulphate as an antimicrobial agent.
Manufactured in ISO 9001 and ISO 13485 certified facilities.

Applications

Expansion of PBMC-derived T cells
CIK cell generation for preclinical and translational research
Feeder-free and serum-reduced cell therapy process development
Scale-up in flasks or closed culture bags

Cell culture example

Add IFN-γ to KBM 581 to a final concentration of 1,000 IU/mL.
Add human autologous serum to the medium prepared above to a final concentration of 10%.
Count the number of mononuclear cells isolated or thawed, seed those cells at 0.52 × 10⁶ cells/mL concentration, and incubate at 37 °C with 5% CO₂.
On Day 1, add IFN-γ (to a final concentration of 300 IU/mL) and anti-CD3 antibody (to a final concentration of 50 ng/mL) to the medium prepared above.
At certain intervals from Day 4 to the cell recovery date, add IFN-γ to added new medium in proportion as cells grow (if necessary, expand the culture scale with T-Flasks and gas-permeable culture bags.) Dilute the cell suspension above by adding fresh medium to a cell concentration of 12 × 10⁶ cells/mL. Incubate between 14 to 21 days in proportion to the target number of cells.

Reference: 2002 Blackwell Science Ltd, British Journal of Haematology 116: 78–86

Reference Publications

Li, Y., Xie, S., Chen, M., Li, H., Wang, Y., Fan, Y., An, K., Wu, Y., & Xiao, W.

Development of an antibody-ligand fusion protein scFvCD16A-sc4-1BBL in Komagataella phaffii with stimulatory activity for Natural Killer cells.

Microbial Cell Factories, 22(1). (2023).

DOI: https://doi.org/10.1186/s12934-023-02082-6

Article Snippet: “Purifed PBMCs, at a density of 1.5× 106 cells/mL, were seeded in a fask (Corning, 430,168) coated with scFvCD16A-sc4-1BBL (2 μg/ml, 24 h at 4 °C), and cultured in KBM-581 medium (Corning, 88–51-CM) containing 5% heat-inactivated autologous plasma and 1000 IU/mL rhIL-2 (Jinsili, Jiangsu, China) for 13 days at 5% CO2 and 37℃, by adding fresh medium every 2–3 days to maintain the cell density between 1.3 and 3.2× 106 cells/mL.”

Li, Y., Xie, S., Chen, M. et al.

Development of an antibody-ligand fusion protein scFvCD16A_–sc4-1BBL in Komagataella phaffii with stimulatory activity for Natural Killer cells.

Microb Cell Fact 22, 67 (2023).

DOI: https://doi.org/10.1186/s12934-023-02082-6

Article Snippet: “Purified PBMCs, at a density of 1.5 × 10⁶ cells/mL, were seeded in a flask (Corning, 430,168) coated with scFvCD16A-sc4-1BBL (2 μg/ml, 24 h at 4 °C), and cultured in KBM-581 medium (Corning, 88–51-CM) containing 5% heat-inactivated autologous plasma and 1000 IU/mL rhIL-2 (Jinsili, Jiangsu, China) for 13 days at 5% CO₂ and 37℃, by adding fresh medium every 2–3 days to maintain the cell density between 1.3 and 3.2 × 10⁶ cells/mL.”

Cao, G., Zhang, G., Liu, M., Liu, J., Wang, Q., Zhu, L., & Wan, X. (2022).

GPC3-targeted CAR-T cells secreting B7H3-targeted BiTE exhibit potent cytotoxicity activity against hepatocellular carcinoma cell in the in vitro assay.

Biochemistry and Biophysics Reports, 31, 101324.

DOI: https://doi.org/10.1016/j.bbrep.2022.101324

Article Snippet: “The activated T cells were then transduced with lentiviral supernatants containing the different CAR constructs. 8ug/ml of Polybrene (Beyotime) was used when transducing GPC3-BiTE CAR-T cells. 2 days after transducing the cells with lentivirus, fresh media (KBM581 medium) were used as replacement.”

Lin, K., Xia, B., Wang, X. et al.

Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology.

J Transl Med 22, 349 (2024).

DOI: https://doi.org/10.1186/s12967-024-05159-x

Article Snippet: “T cells were purified using negative-selected magnetic beads (BD Biosciences) and cultured in KBM581 medium.”

Zheng, Y., Lai, Z., Wang, B. et al.

Natural killer cells modified with a Gpc3 aptamer enhance adoptive immunotherapy for hepatocellular carcinoma.

Discov Onc 14, 164 (2023).

DOI: https://doi.org/10.1007/s12672-023-00780-6

Article Snippet: “Lymphocyte serum-free medium (KBM581 Medium) was purchased from Corning (Cat. No: 88-581-CM, New York, USA).”

Zeng, Y., Zhang, W., Li, Z., Zheng, Y., Wang, Y., Chen, G., Qiu, L., Ke, K., Su, X., Cai, Z., Liu, J., & Liu, X.

Personalized neoantigen-based immunotherapy for advanced collecting duct carcinoma: case report.

Journal for ImmunoTherapy of Cancer, 8(1), e000217. (2020).

DOI: https://doi.org/10.1136/jitc-2019-000217

Article Snippet: “Afterwards, the adherent cells were cultured with medium (1% autologous serum+KBM581+1000 U/mL rhGM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor)+500 U/mL interleukin (IL)-4) for 48 hours, and then loaded with 13 neoantigen peptides (25 µg/peptide) accompanied by certain cytokines (tumor necrosis factor alpha, 1000 U/mL; IL-1b, 10 ng/mL; IL-6, 1000 U/mL) in the medium to induce DC maturation overnight.”

Zhou, Z., Li, J., Hong, J., Chen, S., Chen, M., Wang, L., Lin, W., & Ye, Y.

Interleukin-15 and chemokine ligand 19 enhance cytotoxic effects of chimeric antigen receptor T cells using zebrafish xenograft model of gastric cancer.

Frontiers in Immunology, 13. (2022).

DOI: https://doi.org/10.3389/fimmu.2022.1002361

Article Snippet: “PBMCs were incubated in T-lymphocyte serum-free KBM581 medium (Corning, Inc.; NY, USA), then seeded onto 6-well plates (Corning, Inc.) at a density of 2 x 10⁶ cells/mL.”

Gao, Y., Guo, J., Bao, X., Xiong, F., Ma, Y., Tan, B., Yu, L., Zhao, Y., & Lu, J.

Adoptive transfer of autologous invariant natural killer T cells as immunotherapy for advanced hepatocellular carcinoma: a Phase I clinical trial.

The Oncologist, 26(11), e1919–e1930. (2021).

DOI: https://doi.org/10.1002/onco.13899

Article Snippet: “PBMCs were resuspended in Corning serum-free cell medium KBM581 (Corning Inc., Corning, NY) and stimulated with 100 ng/mL α-GalCer (BioVision, Milpitas, CA) and 100 U/mL animal-free recombinant human interleukin-2…”

Chen, M., Liu, X., Peng, N. et al.

Construction of CD19 targeted dual- and enhanced dual-antibodies and their efficiency in the treatment of B cell malignancy.

Exp Hematol Oncol 12, 64 (2023).

DOI: https://doi.org/10.1186/s40164-023-00423-0

Article Snippet: “Patient derived T cells were isolated from B-ALL patients and cultured in lymphocyte medium KBM581 (Corning, USA) supplemented with 10% FBS and 50 IU/mL human interleukin-2 (IL-2) (R&D systems, USA).”

Qiao, Y., Chen, J., Wang, X., Yan, S., Tan, J., Xia, B., Chen, Y., Lin, K., Zou, F., Liu, B., He, X., Zhang, Y., Zhang, X., Zhang, H., Wu, X., & Lu, L.

Enhancement of CAR‐T cell activity against cholangiocarcinoma by simultaneous knockdown of six inhibitory membrane proteins.

Cancer Communications, 43(7), 788–807. (2023).

DOI: https://doi.org/10.1002/cac2.12452

Article Snippet: “The transduced T cells were expanded in KBM581 medium supplemented with 1% penicillin-streptomycin, 2 mmol/L GlutaMAX (GIBCO), 0.1 mmol/L nonessential amino acids (GIBCO) at 37°C with 5% CO₂.”

Specifications

Attribute	Details
Product Name	KBM 581
Cat. No.	16025810
Form	Liquid
Volume	1,000 mL
Storage Conditions	2–8 °C
Shelf Life	8 months from production date
Country of Manufacture	Japan
Manufacturer	KohJin Bio