Stage 1: Bone Marrow harvest
Bone marrow-derived mesenchymal stromal cell (BM-MSC) source material is taken from a consented healthy donor, pre-screened for communicable diseases and infections according to the standard protocol. An average volume of 30ml – 40ml of BM is aspirated from the donor’s posterior iliac crest in an operating theatre in Galway University Hospital under local anaesthetic and transferred into heparinised tubes. The bone marrow aspirate is shipped to CCMI under ambient temperature conditions (15-250C).
Stage 2: MNC isolation from bone marrow
At CCMI, the manufacturing process starts with tubes containing fresh bone marrow aspirate which are washed in sterile saline then centrifuged to remove unwanted red blood cells. The remaining bone marrow cells are called mononuclear cells (MNCs) and usually number 500 million or more. The MNCs are placed into tissue culture flasks in sterile growth medium. This first culture step is called “passage 0” (P0) After 5 days, the growing mesenchymal stromal cells can be seen as individual round colonies which have a spindle-like appearance under the microscope. At this point, a morphology check is performed as it is possible to see the MSCs attached to the flask. The morphology of the cells (spindle-like) and colony size is assessed. From this point onwards, the flasks are checked regularly until large non-overlapping colonies are formed. This P0 culture phase can continue for up to 17 days.
Once cells are ready for subculture, the cells are detached from the tissue culture flasks using a recombinant enzyme, TrypLE Select. The medium is removed from the flasks, cells are washed with sterile saline and the TrypLE Select is added to each flask. The flasks are incubated for approximately 5 minutes at room temperature until all cells have detached. If cells are not easily detached then a further incubation for 5 minutes is carried out. The TrypLE-select is washed using complete medium. The cell suspension is then centrifuged at 400g for 5 minutes and the cell pellet is re-suspended in fresh complete medium. A cell count is performed using trypan blue to assess viability, and a total viable cell count is calculated. A target cell number of 10 – 20 million cells is required to seed the QUANTUM cell expansion system (CES) manufactured by Terumo BCT. The target cell number and volume for loading is transferred aseptically to a cell inlet bag and the cells are ready to load onto the QUANTUM CES.
Stage 3: QUANTUM CES preparation
The QUANTUM CES is a cell expansion system based on a hollow-fibre bioreactor. The bioreactor contains about 11.5 x 103 hollow fibres, providing a 2.1 m2 cell culture surface area (which is equivalent to the surface area of 120 T175 flasks). The bioreactor can be fed with reagents, cells and medium and can be harvested, all by sterile connective tube welding and sealing. The bioreactor is supplied with gas (5% CO2 / 20% O2, balance N2) that enters the system. The bioreactor and the required connected tubing is packaged as a disposable sterile closed-cell expansion system. The QUANTUM CES provides temperature control (37°C ± 1 °C) of the bioreactor environment and controls fluid movements in the bioreactor to enable its essential function to coat and wash the bioreactor and to load, feed, wash and harvest cells into a single closed-cell harvest bag.
The QUANTUM CES is primed with sterile saline and coated for 4 to 24 hrs with a single unit of cryoprecipitate (CP). Unbound CP is removed by flushing the QUANTUM fluidics system with PBS. The system is washed and filled with culture medium to acclimate for 1-4 hrs.
Stage 4: Cell collection (P0)
The cell suspension from P0 is tested for cell number and viability. Between 10 and 20 x106 viable cells in culture medium are injected into a Cell Inlet Bag and supplemented with culture medium to approximately 100 ml for immediate further expansion in the QUANTUM CES bioreactor.
Stage 5: QUANTUM CES expansion (P2)
Following the loading onto the QUANTUM CES, the P0 cells are allowed to attach to the coated hollow fibres of bioreactor for 24 hrs. The bioreactor is fed with culture medium starting at a rate of 0.1 ml culture medium/min. During the cell culture, the concentration of the metabolite lactate is monitored in the medium daily from day 3, which guides the stepwise increase of the feeding rate. Cells are expanded and are harvested within 20 hrs from the moment daily lactate levels reach >9 mmol/day OR on Day 7, whichever comes first.
Stage 10: Cell collection (P2)
The cells are harvested from the QUANTUM CES by adding 180 ml TrypLE Select to the bioreactor. After 15 minutes, the cells are flushed out of the bioreactor with sterile saline and are collected in the cell harvest bag (final volume of approximately 400 ml). The filled cell harvest bag is visually inspected and, if there is no abnormal appearance, the cell suspension is transferred to 50 ml tubes and concentrated by centrifugation. Samples are taken from the supernatant for sterility and mycoplasma release testing. The cell pellets are resuspended, pooled and diluted with 200 ml cold (2-8°C) saline. The cell suspension is sampled for viable cell count IPC testing and kept refrigerated (2-8°C) while counting. The cell suspension is centrifuged and the cell pellet is suspended within a preservation medium containing 10% DMSO, at a concentration of 16 x 106 viable cells/ml.
Cryobags are used to store the cell therapy product. The bags are filled with 5ml or 10ml cell suspension per bag, supplemented to 40 ml with Freezing medium, mixed by repetitive inversion of the bag and sealed to produce 40 ml cell products at a final cell concentration of 2×106/ml and 4×106/ml, respectively and are labelled. Additional vials are prepared at 2×106 cells/ml for release tests consisting of flow cytometry, viability and karyotype analysis. Samples are prepared for release tests mycoplasma, sterility, endotoxin and potency tests.
The cryobags containing cell therapy product and samples for testing are frozen using a programmable controlled rate freezer. The freezing programme is at -2°C / min until -80°C is reached, at which point the bags and vials are transferred to final storage. Cryobags and vials containing the cell product are cryopreserved in a freezer (≤-150°C) for long term storage. The cell product is shipped to the clinical trial sites under controlled temperature conditions in the vapour phase of liquid nitrogen (≤-150°C).