Aim, study design and setting of the study
The aim of the study was to validate a GMP method for CIKs production as ATMP for treatment of human sarcomas. In order to replace FBS, commonly used for CIKs expansion, we decided to test Human Serum (HS) and Human Pool Plasma (HPP) as supplement medium. FBS was used as a control condition as our preclinical data were obtained culturing CIKs with RPMI + 10% FBS [5].
Each expansion run started from peripheral blood (PB) and ended with the evaluation of cellular viability, growth in terms of fold increase, identity and cytotoxicity.
As GMP guidelines recommend, blood has to be kept in quarantine in a blood bank refrigerator until the results of virology and sterility tests are provided. We first tested PB stability at 24 and 48 h from donor’s harvesting. From the comparative study, we observed no differences in terms of viability, cellular growth and identity between CIKs that expanded from fresh PB and those processed at 24 and 48 h from collection (data not shown). We could therefore exert that PB is stable up to 48 h from harvesting and this allows to wait for the virologic test results before starting the expansion process.
HS is a commercial serum while HPP is produced by the Center for Production and Validation of Hemocomponents (C.P.V.H.), City of Health and Science Hospital of Turin. From literature emerged that HPP was already used instead of FBS or HS for CIKs GMP compliant expansion and that the presence of fibrinogen in HPP did not interfere with the lymphocyte expansion [12].
HPP was also subjected to Pathogen Inactivation (PI) technique by riboflavin (45–85 μM) and UV light (265–370 nm). Riboflavin, commonly used to treat platelets or plasma (MIRASOL™ Pathogen Reduction Technology System, Caridian BCT Biotechnologies, Lakewood, Colorado, USA), is a naturally occurring vitamin (vitamin B2) used as a photosensitizer in combination with light. It provides energy to inactivate living micro-organisms [13]. This process could guarantee the safety of culture medium for CIKs expansion in a GMP setting as it might obviate virus transmission problems [14]. In order to reduce donor specific batches variability, HPP was obtained through centrifugation of whole blood of multiple donors and contains a variety of valuable organic and inorganic elements. For these reasons, HPP seemed a good alternative to FBS for CIKs expansion.
Unfortunately, we obtained contradictory results (data not shown). We demonstrated that HS was not suitable for our aim as it drastically compromised the quality of CIKs expansion in terms of cellular viability, growth and identity. On the contrary, the RPMI + 10% HPP expanded CIKs had grown up more than those in HS with a fold increase similar to those of RPMI + 10% FBS expanded CIKs. However, this trend was not maintained in the following expansion runs and the immunophenotype was not always compliant to the acceptance criteria demonstrating the presence of a great variability between HPP batches. This was a bad characteristic which compromised the choice of this supplement for the CIKs expansions. Moreover, the need to freeze and thaw HPP for its storage could bring to radical oxygen species formation [15] which are toxic causing the cells viability and growth decrement.
For all these reasons we tried to find an alternative expansion medium less variable but as efficient as RPMI + 10% FBS. We decided to test three different serum free expansion media: X-VIVO 15, largely used by other groups [16] and two GMP manufactured media: Tex Macs and Cell Genix GMP SCGM. Viability, cellular growth in terms of fold increase, immunophenotype and cytotoxicity were evaluated and compared at the end of each expansion run.
Peripheral blood collection and PBMCs isolation
The Center for Production and Validation of Hemocomponents (C.P.V.H.) of the City of Health and Science Hospital of Turin provided the whole Peripheral blood from healthy donors. For ethical reasons, we received, after informed consent, only the PB which were not conform for donation purposes and so considered a waste material to be eliminated (examples: donations interrupted for technical problems in the collection with a volume lower than the reference values for the acceptance of transfusion material).
The raw material was collected and validated by C.P.V.H., which verified the compliance of all virology tests carried out on the collected PB. Blood was stored in the blood bank refrigerator (+ 4 °C) and kept in quarantine until the results of virology tests were provided. After about 24 h from collection, the PB was processed for CIKs production. The study design is illustrated in Fig. 1.
PBMCs were separated by density gradient centrifugation using HISTOPAQUE-1077 (Sigma-Aldrich St. Louis, US) and subjected to washing cycles in Phosphate buffered saline (PBS) (EuroClone, Pero, MI, Italy) and finally re-suspended in the culture medium at the cellular concentration of 2 × 106 cells/ml.
CIKs expansion
We compared three different serum-free culture media in order to obtain the best expansion condition: X-VIVO 15 (Lonza, Allendale, NJ, US), largely used in the clinical grade lymphocytes expansion [16] and other two culture media generated in GMP conditions: Cell Genix GMP SCGM (CellGenix, Freiburg, Germany) and Tex Macs (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, 30 × 106 of cells were seeded in 15 ml of culture medium. We cultured the CIKs in T75 flasks (Corning Incorporated, NY, USA). They were not a treated plastic type and they were endowed of vented cap. They were maintained in a vertical position in order to ensure a better oxygenation of cellular suspension, to reduce the culturing medium distribution surface and to have an appropriate culturing medium volume in order to re-suspend homogeneously the cells.
At Day 0, 1000 U/ml IFN-γ was added (Boehringer Ingelheim, Vienna, Austria). At Day 1, 50 ng/ml CD3 Pure, which is IgG anti-CD3 (OKT-3) (170-076-124, Miltenyi Biotec, Bergisch Gladbach, Germany), and 300 IU/ml of Proleukin which is IL-2 cytokines (Novartis, Origgio, VA, Italy) were added [4]. Fresh medium and Proleukin (300 U/ml) were added weekly (every 3 days) during culture, and the cell concentration was maintained at 1-1.5 × 106 cells [17]. The fold increase of CIKs was calculated as follow: (%CD3+ × total cells counted at Day 20–21)/(%CD3+ × seeded cells at Day 0).
After 20–21 days, CIK cells culture was stopped and the quality controls were performed (cell count and viability, immunophenotype and sterility).
Cell count and viability evaluation
CIK cells were counted at optical microscope, using Burker chamber as indicated in the European Pharmacopoeia (Chapter 2.7.29) [18]. The best counting condition in order to obtain a measurement as reliable as possible was between 80 and 150 cells in 25 squares of Burker chamber. The cellular viability was evaluated with Trypan Blue Method (Sigma-Aldrich, St. Louis, US) [18].
Flow cytometry
Flow cytometry was performed according to European Pharmacopoeia (Chapter 2.7.24) using Beckman Coulter NAVIOS (Beckman Coulter, Brea, CA, US) [19]. At Day 0, before PBMCs seeding, basal flow cytometry was performed in order to evaluate the lymphocyte subpopulations. About 0.5–1 × 106 cells were incubated for 20 min, at 4 °C in the dark with respectively CYTO-STAT TetraCHROME CD45-FITC, CD56-RD1, CD19 ECD, CD3-PC5 (Beckman Coulter, Brea, CA, US) for B lymphocytes and CYTO-STAT TetraCHROME CD45-FITC, CD4-RD1, CD8-ECD, CD3-PC5 (Beckman Coulter, Brea, CA, US) for T lymphocytes. Cells were washed and re-suspended in 300 µl of PBS. After 20–21 days of expansion flow cytometry was performed to evaluate the CIKs identity. Briefly, 0.5–1 × 106 cells were incubated as previously described with CD3-FITC, CD56-PE, CD45 KO (Beckman Coulter, Brea, CA, US). As negative control, 0.5–1 × 106 cells were incubated without antibody. For data analysis, we designed the physical gate as [A]. From this gate, we obtained the CD3+ and the CD56+ cell population. In the dot plot CD3xCD56 we gated the CIK double positive CD3+CD56+ cell population. The tail is representative of the primitive CD3+CD56+. These cells were the cellular therapy product as it was in compliance with the identity acceptance criteria.
CIKs cytotoxicity
CIKs tumor-killing ability was assessed against target primary tumor cells obtained from surgical biopsies of gastro-intestinal stromal tumor (GIST).
CIK cells were co-cultured at progressively decreasing effector:target (E:T) ratios, 40:1, 20:1, 10:1, 5:1, 2.5:1, 1:1, 1:2, and 1:4 for 72 h in 200 μl of medium with Proleukin at a concentration of 300 U/ml at 37 °C 5% CO2. A confirmatory method was tested in parallel to determine the number of viable, metabolically active, target cells in culture, based on the quantification of ATP present (CellTiter-Glo Luminescent Cell Viability Assay, Promega Italia s.r.l.). Tumor cells, in the absence of CIK cells, were used as a control to assess spontaneous mortality. The percentage of tumor-specific lysis for each E:T ratio was calculated as experimental − spontaneous mortality/100 − spontaneous mortality × 100 [5].
Sterility
Sterility test was performed on cellular supernatants at the end of CIKs expansion. Briefly, 10 ml and 4 ml of supernatant respectively for anaerobic and aerobic microorganisms and fungal/yeasts species, were inoculated within the Bact-ALERT® FN and Bact-ALERT® PF (Biomérieux, INC. Durham, NC). The sample was then analyzed by the Laboratory of Bacteriology and Virology—Pediatric Clinical Pathology of City of Health and Science of Turin. The results were available after 7–8 days of incubation.
Statistical analysis
Cell growth, viability, immunophenotype and cytotoxicity assay data were analyzed with the GraphPad program (version Prism 5). The data were expressed as mean ± standard deviation. The statistical tests were chosen on the basis of the number of samples and the distribution of the data. The latter was evaluated by Shapiro–Wilk Test, which is considered the best for the normality evaluation for a small number of samples. Paired T test was performed when the distribution was normal, on the contrary, when there was not a Gaussian distribution, Wilcoxon Rank Test was used. Finally, Bonferroni’s multiple comparison Test was conducted to compare the mean values on each data row, which represent a condition, with the control mean data row.
