Optimization of Culture Media for Ex vivo T-Cell Expansion for Adoptive T-Cell Therapy.

Background: Adoptive T-cell therapy is a promising treatment strategy for cancer immunotherapy. The ability of immunotherapy based on the adoptive cell transfer of genetically modified T cells to generate powerful clinical responses has been highlighted by recent clinical success. Techniques which are used to expand large numbers of T cells from different sources are critical in adoptive cell therapy. In this study, we evaluated the expansion, proliferation, activation of T lymphocytes, in the presence of various concentrations of interleukin-2, phytohemagglutinin (PHA), and insulin. Materials and Methods: The effect of different supplemented culture media on T cell expansion was evaluated using MTT assay. The expression level of the Ki-67 proliferation marker was evaluated by real-time polymerase chain reaction. In addition, flow cytometry analysis was performed to access T cell subpopulations. Results: Our results showed that supplemented culture media with an optimized concentration of PHA and interleukin-2 increased total fold expansion of T cells up to 500-fold with approximately 90% cell viability over 7 days. The quantitative assessment of Ki-67 in expanded T cells showed a significant elevation of this proliferation marker. Flow cytometry was also used to assess the proportion of CD4+ and CD8+ cells, and the main expanded population was CD3+ CD8+ cells. Conclusions: Based on these findings, we introduced a low-cost and rapid method to support the efficient expansion of T cells for adoptive cell therapy and other in vivo experiments.

Expression and purification of biologically active recombinant rabbit monocyte chemoattractant protein1 in Escherichia coli.

Monocyte chemoattractant protein 1 (MCP1) with recruiting monocytes is an important factor at the beginning of inflammatory disorders such as atherosclerosis which seems its blocking preclude this process and help improvement of related diseases. To perform clinical research in this field, MCP1 protein is required but firstly, animal studies should be done. As the rabbit is a suitable model for many inflammatory disorders, and Escherichia coli BL21(DE3) (BL21) cell is a high-efficiency host for protein expression, we decided to produce recombinant rabbit MCP1 (rRMCP1) in BL21/pET28a system. After codon usage, a construct containing RMCP1 sequence was synthesized, cloned into the pET28a plasmid, and overexpressed in BL21 cells. Followed that, with changing expression condition such as cell concentration before the induction, time period, temperature, shaking rate and inducer concentration (IPTG), rRMCP1 expression was optimized, and purified by Ni-NTA. The biological activity of the expressed protein was verified using monocyte migration assay. Using this expression system, nearly 28 mg/mL rRMCP1 was produced at 26°C/180 rpm for 24 h in LB broth medium with 1 mM IPTG. Therefore, we were succeeded to express the intermediate level of rRMCP1 with this method. This amount of protein is sufficient for biological researches in the laboratory.

Use of integrase-minus lentiviral vector for transient expression.

OBJECTIVE: Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector. MATERIALS AND METHODS: In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein (GFP) gene expression by a fluorescence microscopy. RESULTS: Recombinant and wild lentiviruses titer was about 5~8×10(6) transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed. CONCLUSION: This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.