Advanced Therapy Medicinal Product production and cryopreservation
When used as therapeutic agents, MSCs are classified as “Advanced Therapy Medicinal Products” (ATMPs) according to European regulation no. 1394/2007. Each ATMP must be produced following Good Manufacturing Practice (GMP) rules in facilities authorized by the national regulatory body. Umbilical cords (UCs) for MSC production were collected during caesarean sections. The mothers provided signed informed consent (Ethics Committee act no. 16/18 of February 3rd,2018), and their blood was screened according to European Directive 2004/23/EC, for HCV/HBV/HIV1-DNA, anti-HIV 1+2 Ab, anti-HCV Ab, HBsAg and anti-T. pallidum Ab. Briefly, after collection, UCs were immediately submerged in a decontamination solution containing a cocktail of four antibiotics (BASE 128; Alchimia; Padua, Italy). Then, in the clean room, UCs were first minced into small fragments, seeded in conventional T-flasks in complete medium containing human-derived platelet lysate (produced by the blood bank of Meyer Hospital; Florence, I) and were incubated at 37°C and 5% CO2. After 7 days, tissue fragments were removed and medium was substituted. After 13 days, adherent cells were detached by using TrypLe Select (Thermo Fischer; Waltham, MA, USA) and re-seeded in complete medium. On Day 19, adherent cells were detached and expanded in HYPERFlask Cell Culture Vessels (Corning; NY, USA) and on Day 26 cells were harvested, counted and collected in CryoMACS freezing bags (Miltenyi Biotec; Bergisch Gladbach, DE) or in cryotubes (Nalgene; Rochester, NY, USA). Cells were finally frozen to -140°C in a cryogenic freezer (Feezal, Air Liquide; Paris, FR) at a cooling rate of -1°C/min to -40°C followed by a cooling rate of -5°C/min to -140°C, and finally stored in vapour phase LN2. UC-MSCs bags were released after the quality control check that included sterility, mycoplasma, endotoxins, cell count, phenotype, karyotype, cell viability and impurities tests compliant with European Pharmacopeia methods.
In vitro experimental design
To simulate the conditions to which cells would be exposed during the alternative shipment method, cells stored in LN2 vapour (n=3) were incubated for 18 h in dry ice before thawing; as a control, cells stored in LN2 were thawed directly at 37°C. Analyses of immuno-phenotype; immuno-modulation; population doubling (PD); cell viability, apoptosis; and colony forming unit-fibroblast (CFU-F) potential were performed as described in Figure 1.
Cell viability, apoptosis and population doubling
To test viability after thawing, cells were harvested and resuspended in trypan blue (Thermo Fischer) at 1:1 ratio (v:v) in medium solution. The number of viable/dead cells was scored by using a Burker hemocytometer. For apoptosis testing, 2x105 cells were harvested after thawing and centrifuged at 400g for 6 min at 8°C. Cells were then washed with binding buffer and labelled with Annexin V/7-aminoactinomycin D (7-AAD) (Thermo Fischer). After dilution with the binding buffer (1:10 v:v), fluorescence of 2x104 cells/sample was detected by using a Cytomics FC500 cytometer and analysed by using EXPO32 software (all by Beckman Coulter; Brea, CA, USA). Cell populations were separated into four subsets: viable cells were negative to both Annexin V and 7-AAD fluorescence; cells in early phase of apoptosis were Annexin V+/7-AAD–; cells in late phase of apoptosis were Annexin V+/7-AAD+, while the necrotic cells were Annexin V-/7-AAD+. Cell viability and apoptosis were determined for both transport conditions (LN2 and dry ice) immediately after thawing (Day 0) and after one week in culture (Day 7). In the last condition, the PD was calculated after 2-5-7 days as follows: PD = (Log10 Nt – Log10 N0)/ Log10 2, where Nt= number of counted cells N0= number of plated cells (n=3).
Briefly, 1x105 cells were stained with monoclonal anti-human antibodies against CD31-FITC (Clone 5.6E), CD45-ECD (Clone J.33), CD105-PE (Clone 1G2), CD90-FITC (Clone F15-42-1-5), HLA-DR-APC (Clone IMU 357) and 7-amino actinomycin D (7-AAD) (all by Beckman Coulter) and CD34-PE (Clone 8G12), CD73-PC7 (Clone AD2) (by Beckton Dickinson; Franklin Lakes, NJ, USA). Cells were incubated for 15 minutes at room temperature together with specific antibodies (CD90/CD105/CD45/7AAD; CD31/CD34/CD45/HLA-DR/CD73). After washing, at least 10,000 events were acquired using a FC500 flow cytometer and analysed using Kaluza software (both by Beckman Coulter).
Thawed peripheral blood mononuclear cells (PBMCs) collected from a single healthy donor were suspended in RPMI 1640 (Sigma-Aldrich; St. Louis, MO, USA) supplemented with 10% FBS, 2mM L-glutamine, 100U/ml penicillin, 0.1mg/ml streptomycin (all by Sigma-Aldrich) and rested overnight at 37°C in a humidified atmosphere containing 5% CO2. UC-MSCs were seeded in 96-well flat-bottom plates (Corning) at different densities: 4×104, 2×104 and 1×104 cells. To measure cell proliferation, PBMCs were stained with 5μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (CellTrace Cell Proliferation Kit; Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. PBMCs were also stimulated with 0.5 μg/ml of anti-CD3 antibody (clone OKT3, Miltenyi Biotec) and 500UI/ml of recombinant human interleukin-2 (rh-IL-2, Miltenyi Biotec) for six days before measuring the corresponding decrease in CFSE fluorescence by flow cytometry. Stimulated and unstimulated PBMCs seeded alone were used as controls. CFSE-labeled PBMCs (2×105) were then seeded on MSCs monolayers to obtain different MSC:PBMC ratios of 1:5, 1:10 and 1:20. Anti-human CD45-ECD antibody was used to assess proliferation on gated CD45+ cells. At least 20,000 events were acquired on a Cytomics FC500 cytometer (Beckman Coulter). PBMC proliferation was quantified as the percentage of cells undergoing at least one cell division (n=3).
Colony forming unit-fibroblast assay
UC-MSC clonogenic potential was evaluated by seeding 200 cells in a 100-mm plate (Corning), in duplicate for each condition, immediately after thawing (Day 0) or after 7 days (Day 7) in culture. Cells were incubated at 37°C and 5% CO2 and the medium was changed at Day 7. After 13 days in culture, cells were washed, fixed with 10% formaldheyde and stained with 0.1% Crystal Violet for one hour at room temperature. Only colonies with a minimum of 30 cells were scored (n=3).
Validation of ATMP delivery in dry ice
Two concomitant validation runs were performed during the transport of the ATMP from the LTCA in Vicenza to the Intensive Care Unit (ICU) of Ospedale Maggiore in Verona (approximately 60 km distant, corresponding to less than one hour of driving), and from the LTCA to the Haematology Unit (both in Vicenza, approximately 500 meters distant, corresponding to ten minutes of walking). A third run (worst case) was conducted by maintaining the box container at 35°C in a hood (Heraeus; Hanau, DE) for 24 hours in order to “challenge” the system. The goal was to validate the box container and packaging instructions; the quantity of dry ice to be loaded; and the maximum allowable transport time. Packaging was done in compliance with UN3373 (Biological substance, Category B) in UN1845 Dry Ice, IATA label Class 9. The box, constituted of high-density polystyrene foam (27.5x25.0x22.5cm and 3.5cm thick), was loaded with dry ice in pellets (5kg). The dry ice parcel-sized shippers comprised the payload area surrounded by an insulation medium, with the product submerged in dry-ice pellets. The box was filled halfway with dry ice and the bag containing the cells was placed horizontally in contact with a datalogger probe (iLog, Escort Scunthorpe, UK). The box was filled to the brim and closed with the lid sealed with tape. The temperature was recorded every 5 min and the box was delivered at room temperature by using the courier of the Verona Hospital. As acceptance criteria, the change in weight of dry ice at the end of the worst case test could not exceed 10%, and the temperature of the bags could never exceed -76°C.
The ATMP was supplied as a frozen, sterile, apyrogenic product in bags containing a concentration of 1 to 2x106 cells/ml. Cells were thawed by continuous agitation in sterile conditions by submerging the bag with an overpouch in a water bath at 37°C. Once thawed, the overpouch was removed and cells were diluted 1:1 (50 ml final) in thawing solution consisting of 38% saline; 50% human albumin 20% v/v (10% final); and 12% anticoagulant Citrate Dextrose Solution (Terumo; Rome, I). The bags were connected to infusion sets, delivered immediately to the clinics, and infused intravenously to patients in 30 min. The treatment consisted of two infusions of 1.1x106 UC-MSCs/kg body weight one week apart for the COVID-19 patient, and three infusions of 1.5x106 UC-MSCs/kg body weight one week apart for the aGvHD patient.
The two-tailed Student’s t-test was used to analyse statistical differences between groups. P-values <0.05 were considered statistically significant. All experiments were performed in triplicate. Data are expressed as mean ± standard deviation (SD). All statistical analyses were performed by using GraphPad Prism software (San Diego, CA, USA).