Fabrication of bioreactor (BECA-D)
The main body of the bioreactor was made from virgin polystyrene. Parts were injection moulded and assembled via ultrasonic welding. Both the injection moulding and the ultrasonic welding were carried out under an ISO 13485 certified manufacturing environment. The moving plunger was made from overmoulding of polycarbonate core with silicone. The profile, size and hardness of the plunger rubber were optimized to achieve optimal sealing in the cell chamber. The membrane was made from polyethylene terephthalate with a uniform pore size of 1 μm. The membrane was tested with peripheral blood mononuclear cell (PBMC) culture and it was observed cells were unable to cross the membrane via the pores. Chemical diffusion across the membrane was checked with cell culture medium of different glucose concentrations. It was found glucose could slowly diffuse across the membrane from the side of high glucose concentration to the side of low glucose concentration while the culture was kept static. All the materials used for constructing the bioreactor were subjected to in-house biocompatibility test and had shown no adverse effects on the growth of PBMCs.
Donor recruitment
SingHealth Research Centralised institutional review board approved the proposed human study (CIRB2017/2398) and all methods were performed in accordance with the relevant guidelines and regulations. An email containing the invitation letter and contacts of Principal Investigator was sent to the potential donors (age between 21 and 60 years old, weigh at least 50 kg, generally be in good health) with a request to accept or decline the invitation of blood donation. Written informed consent was obtained from all participants on the day of the blood donation. The donors underwent blood screening to check their eligibility for the blood donation (haemoglobin level of at least 12.5 g/dL). 100 mL whole blood was extracted from eligible donors and processed to obtain Peripheral Blood Mononuclear Cells (PBMCs).
Cryopreservation of PBMCs
0.1 mL Dimethyl Sulfoxide (DMSO) (ATCC) and 0.9 mL of defibrinated or anticoagulant-treated blood pooled from the same donor was dispensed into each cryovial and mixed periodically to ensure an even cell suspension. The capped vials were transferred to a cryopreservation container in ethyl alcohol. The vials were placed in Mr Frosty Freezing Container (Thermo Fisher Scientific) at − 80 °C for 3–18 h or in a controlled rate freezer of freezing rate of 1 °C per minute prior transferring to storage in liquid nitrogen.
PBMC preparation
Whole blood was mixed with an equal volume of Phosphate Buffer Saline (PBS) (ATCC) or RPMI-1640 (ATCC) and layered over Histopaque-1077 (Sigma-Aldrich) at 400×g for 40 min at room temperature. Once the centrifugation is complete, the opaque lymphocyte band at the intermediate layer and the Histopaque-1077 layer down to within 0.3 cm of the pellet at the bottom of the tube were then transferred to a new centrifuge tube and mixed with Iscove’s Modified Dulbecco’s Medium (IMDM) (ATCC) with 20% Fetal Bovine Serum (FBS) (HyClone Laboratories) and 1% penicillin/streptomycin (ATCC) and centrifuged at 260×g for 15 min. The cell pellet was washed once with IMDM with 10% FBS and 1% penicillin/streptomycin and resuspended in IMDM with 10% FBS and 1% penicillin/streptomycin. Cells were immediately used for transformation or cryopreserved for future use.
Transformation of PBMCs into lymphoblastoid cell lines (LCLs)
Complete culture medium used was IMDM with 10% of FBS and 1% of penicillin/streptomycin solution. A vial of irradiated MRC-5 feeder layer cells (ATCC) was thawed and mixed with complete culture medium. Cell suspension of ~ 4 × 105 cells was dispensed into culture flasks. The medium in the flasks of feeder layers was removed and supplemented with IMDM with 20% FBS, cell suspension containing 1–6 × 106 PBMCs and human gammaherpesvirus 4 (HHV-4) (ATCC). The contents in the flask were gently mixed and incubated at 37 °C under a 5% CO2 atmosphere. After 7 days, cells were observed for signs of transformation and complete culture medium was added to the flasks. Every 3–4 days thereafter, cells were observed for transformation and the media was refreshed. Transformed LCL were maintained at the density of 1–3 × 105 cells per mL by expanding to additional flasks. LCLs were then collected from flask and frozen down for future use using chilled freezing medium (complete culture media with 10% DMSO) at the cell density of 3–5 × 106 cells per mL.
Expansion of EBVST culture
The complete culture medium used for donor PBMC culture was 45% RPMI 1640 (Thermo Fisher Scientific), 45% EHAA Click’s Medium (Irvine Scientific), 10% heat-inactivated FBS (Thermo Fisher Scientific) and 2 mM l-glutamine (Thermo Fisher Scientific).
The complete culture medium used for donor LCL culture was RPMI-1640, 10% heat-inactivated FBS and 2 mM l-glutamine. Cryopreserved LCLs were thawed and maintained at 4 × 105 cells/mL. On days of activation, the required volume of LCLs were removed for irradiation and the remaining cells were passaged and maintained.
Expansion of EBVST culture (24-well plate)
1st activation (Day 0): PBMCs were thawed and washed to remove DMSO. PBMCs were resuspended in fresh media and cell count was performed and viability determined by Trypan Blue staining (Gibco). PBMCs were seeded into 24-well plate at density of 1.0 × 106 cells/cm2. Irradiated LCLs were seeded into the PBMC wells at the ratio of 1:40. Total volume of media in the well was 2 mL. No media change was required until the 2nd activation.
2nd to 5th activation (Day 10, 17, 24, 31): 300 uL of media samples were removed prior to cell collection for glucose and lactate measurements. Cells from each well were pooled and centrifuged at 300×g for 10 min at room temperature. Cell pellet was resuspended with complete medium and cell count and viability was determined. Cells were seeded into new 24-well plate at density of 0.5 × 106 cells/cm2. Irradiated LCLs were seeded at the ratio of 1:4. 50 IU/mL of Interluekin-2 (IL-2) (STEMCELL Technologies) was added to the culture from 3rd to 5th activation. Total volume of media in the well was 2 mL.
Media Change and IL-2 Top-up (Day 14, 21, 27, 34): 50% media change was performed by removing 1 mL of spent media from the well and replacing it with 1 mL of fresh media. 50 IU/mL of IL-2 was added to each well.
Harvest (Day 38): 300 uL of media samples were removed prior to cell collection for glucose and lactate measurements. Cells from each well were pooled and centrifuged at 300×g for 10 min at room temperature. Cell pellet was resuspended with complete medium and cell count and viability were determined. Cells were processed for characterization assays.
Expansion of EBVST culture (BECA-D)
1st activation (day 0): 180 mL of complete media was added to BECA-D media chamber and 42 mL of complete media was added to BECA-D cell chamber—back. PBMCs were thawed and washed to remove DMSO. Cell pellet was resuspended with complete medium and cell count and viability was determined. PBMCs were seeded into BECA-D cell chamber—front (12 cm2) at density of 1.0 × 106 cells/cm2. Irradiated LCLs were seeded into the cell chamber at the ratio of 1:40. Total volume of media in the cell chamber was 12 mL. No media change was required until the 2nd activation.
2nd to 5th activation (day 10, 17, 24, 31): 300 µL of media samples were removed from the media chamber and cell chamber—front prior to cell collection for glucose and lactate measurements. Cells from cell chamber were collected, and cell chamber was washed once with complete medium. The medium was pooled with the cells and centrifuged at 300×g for 10 min at room temperature. Cell pellet was resuspended with complete medium and cell count and viability was determined. Total surface area of culture was determined using cell number and density of 0.5 × 106 cells/cm2 and total volume of culture was determined for a media height of 5 mm. Cells were diluted to appropriate culture volume and seeded into the cell chamber—front. Irradiated LCLs were seeded at the ratio of 1:4 in complete PBMC medium. The Plunger was pulled to the appropriate distance to convey the surface area required for culture. 50% media change was performed by removing 90 mL of spent media from media chamber and replacing it with 90 mL of fresh media. 50 IU/mL of IL-2 was added to the media chamber from 3rd to 5th activation.
IL-2 top-up (day 14, 21, 27, 34): 50 IU/mL of IL-2 was added to the media chamber.
Harvest (day 38): 300 µL of media samples were removed prior to cell collection for glucose and lactate measurements. Cells from cell chamber were collected and cell chamber was washed once with complete medium. The medium was pooled with the cells and centrifuged at 300×g for 10 min at room temperature. Cell pellet was resuspended with complete medium and cell count and viability was determined. Cells were processed for characterization assays.
Cell surface phenotyping
T-cells generated from different culture vessels were transferred into a 1.5 mL Eppendorf tube, centrifuged at 300×g for 10 min at room temperature. Cell pellet was resuspended in 100 µL of fluorophore solution and incubated in the dark for 20 min. The samples were washed repeatedly before resuspending in a final volume of 250 µL. MACSQuant Analyser 10 (Miltenyi Biotec) was used to acquire the data and MACSQuantify Software 2.11 (Miltenyi Biotec) was used for analyses. Antibodies used in analyses: Viobility 405/520 Fixable Dye (130-109-814), CD3-VioBlue (130-113-133), CD19-PE (130-113-646), CD56-PE-Vio770 (120-113-313), CD4-APC-Vio770 (130-113-251) and CD8-FITC (130-110-677) (Miltenyi Biotec).
Enzyme-linked immunospot (ELISpot)
Human interferon gamma (IFN-ʏ) ELISpot Plus Kit (3420-4 HST-10) (Mabtech) was used to quantify IFN-ʏ secreting T-cells in vitro. A human IFN-ʏ pre-coated strip plate was prepared according to manufacturer’s protocol. 20 µL of 6.25 μg/mL antigens or controls and 100 µL of 2.5 × 106 cells/mL T-cells were added to each well. The antigens used were LMP1 PepMix, LMP2 PepMix, EBNA1 PepMix. DMSO was used as a negative control and CD3 mAb 6 μg/mL was used as a positive control. The plate was analysed using the C.T.L ImmunoSpot Analyzer Software 5.2 (Cellular Technology Limited (CTL)).
Cytotoxicity assay
Cytotoxicity capabilities of the cultures were assessed with the DELFIA® EuTDA Cytotoxicity Reagents (PerkinElmer) using Effector:Target (E:T) ratios ranging from 40:1 to 2.5:1, with matched LCLs as the target. The cytotoxicity assay was performed in a 96-well plate accordance to the manufacturer’s protocol. The plate was assessed with a Tecan Infinite 200 PRO (Tecan). Specific killing was calculated as follows:
$$small % Specific, killing=frac{(Experimental ,release, left(countsright)-Spontaneous, release, left(countsright))}{(Maximum, release, left(countsright)-Spontaneous, release, left(countsright))},times 100%$$
where labelled LCLs incubated in medium alone or in 2% Triton x-100 (Sigma-Aldrich) were used to determine Spontaneous and Maximum release respectively.
Glucose and lactate measurements
Frozen media were thawed and analysed for glucose and lactate levels using the Cedex Bio Analyzer (Roche Diagnostics). Glucose and lactate concentrations were measured accordance to the manufacturer’s protocol.
Statistical analysis
Paired Student’s two-tailed t-test was performed to assess the statistical significance of differences between two groups across the donor sets. A p-value of < 0.05 obtained from the test indicates a significant difference. Correlation between glucose media level and cell numbers were evaluated using Pearson’s correlation coefficient.
