Natural Killer Cell Expansion with Autologous Feeder Layer and Anti-CD3 Antibody for Immune Cell Therapy of Hepatocellular Carcinoma

Document Type : Research Articles


1 Department of Tissue Engineering and Applied Cell Sciences,School of Advanced Technologies in Medicine,Tehran University of Medical Sciences, Tehran, Iran.

2 Department of Tissue Engineering, Qom University of Medical Sciences, Qom, Iran.

3 Taleghani Bone Marrow Transplantation Center,Taleghani Hospital,Shahid Beheshti University of Medical Sciences,Tehran, Iran.

4 Cell-Based Therapies Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences, Tehran, Iran.

5 Dentistry Faculty, Tehran University of Medical Sciences, Tehran, Iran.

6 Department of Clinical Nutrition, Faculty of Nutrition & Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

7 Department of Stem Cell Therapy, Tabriz Valiasr Hospital, Tabriz, Iran.


Background: one of the promising approaches for treatment of some cancers is adoptive cell therapy using natural killer (NK) cells. Various methods have been investigated for ex vivo expansion of NK cells in large-scale, but most of them involved cancer or genetically modified cells as feeder layer and also some of them have the risk of T cell contamination and graft-versus-host disease (GVHD). Method: In this study, irradiated autologous peripheral blood mononuclear cells (PBMCs) as feeder layer with an anti-CD3 monoclonal antibody (mAb) were used. For activation and expansion of NK cells, human recombinant IL2 and IL15 were used. After co-culturing of expanded NK cells (eNKC) and isolated NK cells (iNKC) with hepatocellular carcinoma (HCC) cells, the viability of eNKC in compared to iNKC were analyzed by CCK-8 assay and degranulation of NK cells after co-culturing was assayed by measuring CD107a expression. Enzyme-Linked Immunosorbent Assay (ELISA) assay was used for the ability of NK cells to secretion of IFN-γ (interferon-γ) and TNF-α (Tumor Necrosis Factor-α) after co-culture with HCC cells. Real Time PCR analysis was used for expression of human Perforin and Granzyme B genes in the NK cells exposed to target HepG2 cells. Result: This method strongly expanded highly purified NK cells with powerful cytotoxicity against HCC cells. The expanded NK cells showed high level of expression of degranulation marker and human Perforin and Granzyme B genes, and also was secreted larger amounts of TNF-α and IFN-γ compared with fresh isolated NK cells. Conclusion: we proposed an effective method for expansion of cytotoxic NK cells using irradiated autologous PBMC as feeder layer for more successful transfer of allogeneic NK cell in immuno cell therapy of HCC.