(d) After two courses of CIK cell treatment, the tumor burden in the inferior lobe of the left lung was significantly reduced (compared to the corresponding image in (a))

(d) After two courses of CIK cell treatment, the tumor burden in the inferior lobe of the left lung was significantly reduced (compared to the corresponding image in (a)). (Rocky Hill, NJ, USA). Anti-CD3 monoclonal antibody was obtained from Pharmingen (San Diego, CA, USA). Thymopentin for injection was purchased from Beijing Shuanglu Pharmaceutical Co. Ltd. (Beijing, China) and antibodies for T lymphocyte subsets were from BD (Franklin Lakes, NJ, USA). The FACS-420 flow cytometer was from Becton-Dickinson FACS Systems (Sunnyvale, CA, Bavisant dihydrochloride hydrate USA), and data analysis was performed with CellFit software (Becton-Dickinson Inc., San Jose, CA, USA). 2.4. Preparation of Cytokine-Induced Killer Cells All the professionals for CIK cell culture and quality control were healthy and received training in good manufacturing practices. Informed consent was obtained from all patients prior to the study. A total of 54?mL of venous blood was obtained in the morning under fasting conditions, and peripheral blood mononuclear cells (PBMCs) were subsequently isolated. The PBMCs were produced in serum free medium and cell density was adjusted to meet predetermined criteria; the growth medium was supplemented with rhIFN-(final concentration of 2000?U/mL). The cells were maintained in gas-permeable cell culture bags at 37C and 5% CO2. On the following day, rhIL-2 and CD3 McAb were added to a final concentration of 1000?U/mL and 50?ng/mL, respectively. On day 0 of culture, 1000?U/mL recombinant human interferon-(IFN-) (Peprotech, New Jersey, USA) and 1000?U/mL recombinant human interleukin-2 (rhIL-2; Peprotech) were added to the culture medium. The cells were cultured in a humidified 5% CO2 incubator at 37C. Fresh GT-T551 medium with 1000?U/mL rhIL-2 was added every 3 days. After about 14 days of culture, the CIK cells had to meet the following criteria prior to transfusion: the proportions of CD3+, CD8+ and CD3+/CD56+ cells were 90%, 65%, and 20%, respectively, and cell viability, detected using trypan blue staining, was 95%. Approximately 2~10 109 CIK cells were harvested per Bavisant dihydrochloride hydrate flask, with a survival rate of 95%. 2.5. Antibodies and Flow Cytometric Analysis The following antihuman antibodies were used to stain cell surface markers to establish the CIK phenotype: CD4-fluorescein isothiocyanate (FITC), CD8-phycoerythrin (PE), CD3-chlorophyll protein complex (PerCP), and CD56-allophycocyanin (APC). The antibodies and isotype-matched monoclonal antibodies were purchased from BD Biosciences (California, USA). Data acquisition was performed using a FACSCalibur flow cytometer (BD Biosciences). 2.6. Treatment Regimen of Cytokine-Induced Killer Cells The patients received thymopentin (20?mg/day) via intramuscular injection 1 week before PBMC collection for 7 consecutive days. After PBMC collection, thymopentin (20?mg) was injected intramuscularly three times per week until 1 week before the next cycle (Physique 1). After CIK cell transfusion, patients were injected subcutaneously Bavisant dihydrochloride hydrate with 1 mU rhIL-2 each day for 10 days (from day 17 to day 26). CIK cell transfusion (1~5 109 CIK cells per infusion and 2~10 109 CIK cells infusions totally) was performed and transfused back to the patients for two consecutive days intravenously during one course of treatment. Two weeks after the final transfusion, blood was collected, and CIK cells were harvested. The patients participating in this study did not receive any other treatment during CIK cell therapy. Open in a separate window Physique 1 (a) Trials and treatments of the two groups sectionalization. (b) Treatment protocol: cytokine-induced killer (CIK) cell transfusion cycle. Peripheral blood mononuclear cells (PBMCs) were cultured for 14 days in the presence of recombinant human interferon gamma (rhIFN- 0.05 was considered to be statistically significant. 3. Results 3.1. Quality Control in Cell Culture Cell cultures were Bavisant dihydrochloride hydrate routinely evaluated for the presence of bacteria, fungi, and mycoplasma by the Department of Microbiology and our laboratory. Cells testing unfavorable for all bacteria, fungi, and mycoplasma were defined as negative. All the cells used for transfusion were unfavorable for these microorganisms, which ensured the safety of treatment. 3.2. Phenotype Changes The average culture duration for peripheral blood lymphocytes was 13.39 1.6 days. The average number of mature lymphocytes was (3.6 0.77) 109 cells, and the average fold change of amplification was 463 156.86. The survival rate of these cells was 97.681 1.41%. Cells were analyzed by flow cytometry immediately after blood collection and again after 13 days of culture. Analysis of phenotypes showed a significant increase in the proportion of CD3+, CD8+, CD3+/CD8+, and CD3+/CD56+ T lymphocytes and a slight decrease in the number of CD3+/CD4+ T lymphocytes FLJ12788 (Physique 2, Table 2). Open in a separate window Physique 2 Phenotype analysis of cells from patients and detection of CIK cells and leukemic markers by FACS analysis. All cell samples for phenotype analysis were stained with FITC-conjugated antibodies against CD4, PE-conjugated antibodies against.