1. UC-MSCs isolation and expansion process
1.1. Umbilical Cord collection
UCs were collected by the Cell Therapy Unit, Saint Louis Hospital (AP-HP, Paris, France) from maternities affiliated to the Allogeneic Cord Blood Bank (CBB), coordinated by the French Placental Blood Network of French Biomedicine Agency (ABM). The UC collection process was approved by the French regional health agency (ARS, Ile de France). Donation, procurement, testing, processing and storage were performed in accordance with the European Directive 2004/23/EC. UCs were collected from 15 healthy donors, who signed-up a fully informed donor consent. Serological tests were performed according to the European directive on tissue procurement (Additional file 1).
1.2. UC-MSCs isolation and expansion
UC-MSCs were isolated from UC according to the explant method (14). Using a sterile surgical scalpel, the UC was dissected longitudinally, blood vessels were removed and the Wharton’s jelly scratched. The UC was cut into fragments of few centimeters named explants, then seeded in one or several 6 well-plates, in a qualified complete culture medium composed of Nutristem® MSC XF Basal Medium (Biological Industries, Ref 05-200-1A) + Nutristem® MSC XF Supplement Mix (Biological Industries, Ref 05-200-1U) + 5% irradiated platelet lysate (PL) MultiPL100’i (Macopharma, Ref BC0190032) + sodium Heparin 2IU/mL (Panpharma, Ref 5520508). The UC was maintained at 37°C, in a humidified atmosphere with 5% CO2, and culture medium was changed twice a week. During the whole process, cell confluence and morphology were assessed in situ, using a phase-contrast microscope. At day 7, the UC was removed and UC-MSCs were cultured until reaching colonies with 80% confluence (passage 0). UC-MSCs were harvested using a recombinant trypsin EDTA solution (Biological Industries, Ref 03-079-1B), then seeded for further expansions in a higher surface culture, for one or several passages, until a suitable cell quantity was reached.
2. Characterization of UC-MSCs biological properties and functions
2.1. Cell proliferation assessment
UC-MSCs were enumerated after each passage using a manual Malassez counting Chamber. Doubling Time (DT) and Population Doubling (PD) were determined after each passage according to the formulas (T × log(2))/(log Y − log X) and (Y/X)/log(2) respectively (X: number of cells originally seeded, Y: number of cells harvested at passage and T : time of culture in hours).
2.2. Priming with pro-inflammatory cytokines
In order to assess the immunomodulatory properties of UC-MSCs, we created in vitro an inflammatory environment mimicking that seen in patients. UC-MSCs were treated with the pro-inflammatory cytokines IFNγ, TNFα, IFNγ+TNFα, IL1β, IL6, GM-CSF and a Mix of all cytokines (Additional file 2), at the concentration of 10 ng/mL each, during 48h. A not-treated (NT) condition was used for basal state. After priming, cells were harvested using a recombinant trypsin EDTA solution (Biological Industries, Ref 03-079-1B), washed and suspended in the adequate medium depending on the analysis.
2.3. UC-MSCs phenotype
UC-MSCs phenotype was assessed at basal state and under pro-inflammatory challenge conditions. Cells were suspended in 100 µL PBS/albumin 1% and stained with a panel of antibodies (Additional file 3), for 15 min at 4°C, protected from light. A titration was performed to determine the optimal concentration of each antibody. The following dilutions were tested: 1/50 (recommended by the supplier), 1/100, 1/200, 1/400, 1/800 and 1/1600 for anti-HLA-ABC, anti-CD86, anti-CD31 and 1/20, 1/40, 1/80, 1/160, 1/320 and 1/640 for others. Cells were washed in 1 ml PBS/Albumin 1%, centrifuged at 1500 RPM for 5min. After the removal of supernatant, cells were suspended in 300 µL PBS/albumin 1%. Negative controls were non-stained cells or Fluorescence Minus One for CD31, CD14 and CD45 antibodies. The acquisitions were performed on an Attune NxT™ Thermofisher® Flow Cytometer and analyzes were performed using Attune NxT software.
2.4. Potency assays
To assess the immunomodulatory properties of UC-MSCs, we performed, as potency assays, the following two assays according to the International Society for Cell & Gene Therapy (ISCT®) recommendations (15).
a. Activation markers expression
Activation markers expression was evaluated on non-treated UC-MSCs (NT) and after priming by pro-inflammatory cytokines for 48 hours. For Indoleamine 2,3-dioxygenase (IDO) staining, UC-MSCs were suspended in 100 µL PBS/albumin 0.1% and stained with 2 µL of human anti-CD90 FITC antibody for 15 minutes, at room temperature. After washing in PBS/Albumin 0.1%, cells were fixed with an intracellular fixation buffer for 60 minutes, at room temperature then permeabilized twice with permeabilization buffer (eBioscience, Ref 88882400). Cells were suspended in 100 µL of permeabilization buffer and labeled with 5 µL of human anti-IDO e-Fluor-660 antibody (eBioscience, Ref 50947742) for 20 min at room temperature, washed in the permeabilization buffer and then in PBS/albumin 0.1%.
The Inter Cellular Adhesion Molecule-1 (ICAM-1/CD54), Programmed Death-Ligand 1 (PD-L1/CD274), Vascular Cell Adhesion Molecule 1 (VCAM-1/CD106), CD200, INFγ-Receptor (INFγ-R/CD119) and TNFα-Receptor II (TNF-RII/CD120b) were assessed at basal state (NT) and after pro-inflammatory treatment according to the protocol described above (Additional file 3). Acquisitions were performed with an Attune NxT™ Thermofisher® Flow Cytometer and analyses using Attune NxT™ software.
b. Mixed Lymphocyte Reaction (MLR)
MLR potency assay was performed according to Nicotra et al. (16). MLR assay was performed on UC-MSCs both in a resting state (NT) and after priming for 48 hours with IFNγ, TNFα and IFNγ+TNFα. Briefly, peripheral blood mononuclear cells (PBMC) pooled from 10 healthy donors and labeled with the CellTraceÔ Violet (CTV) Cell Proliferation Kit (Invitrogen, Ref C34557) were co-cultured with UC-MSCs at 0:1 (control), 1:10, 1:30, 1:100, 1:300 and 1:1000 UC-MSCs:PBMC ratio with a constant amount of PBMC (3x105) and a decreasing amount of UC-MSCs from 3x104 down to 3x102.
Cells were co-cultured in a culture medium composed of Roswell Park Memorial Institute (RPMI) Medium 1640, GlutaMAXÔ Supplement, HEPES (Gibco, Ref 72400-013), 10% human A/B serum (Eurobio, Ref CAEHUM010U), 1% Amphotericin B/Penicillin/Streptomycin (Gibco, Ref 15240062), and 10 UI/mL Heparin (PanPharma, Ref 5520508) at 37°C, 5% CO2 for 7 days. At day 4 ± 1, 50 mL of culture medium was added. At day 7, cells were labeled with 5 µL of human antibodies anti-CD3 PE (BD Biosciences, Ref 345765), anti-CD45 FITC (BD Biosciences, Ref 345808) and 7-AAD viability dye (Beckman Coulter, Ref A07704) for 15 min at 4°C, protected from light. Cells were washed in PBS 1X before acquisition on the Attune NxT™ Thermofisher® Flow Cytometer and analyzes using Attune NxT™ software.
3. Manufacturing process of UC-MSCs-based investigational ATMP batches
The manufacturing of each UC-MSCs batch was performed from the UC of a single donor, under GMP conditions by the MEARY Cell and Gene Therapy Center at Saint-Louis Hospital (Paris, France) and according to the process developed and validated by the Cell Therapy Unit (CTU) of Saint-Louis Hospital. From the collection of the UC up to the final batch of UC-MSCs for clinical use, the process was divided into two steps. First, and according to the European Directive 2004/23/EC, UC receipt and qualification followed by UC-MSCs isolation and derivation of the Master Cell Stock (MCS) were performed at the CTU. The MCS was then transferred to the MEARY center for GMP production of the Work Cell Stock (WCS) and the clinical trial batches, using qualified and certified raw materials and equipment according to the GMP specific to ATMPs (17). The whole manufacturing process is described in the additional file 4.
3.1. Master Cell Stock (MCS)
Upon receipt at the CTU, the collected UC was washed in Gentamicin 0.2mg/mL (Panpharma, Ref 3512031) before further processing using the explants method as described above. The full UC was seeded in a 150 cm2 culture flask (Corning, Ref 90552) in complete culture medium, until passage 0. At day 11, UC-MSCs were harvested using a recombinant trypsin EDTA solution (Biological Industries, Ref 03-079-1B), then seeded in a 175 cm2 culture flask (Corning, Ref 353112) at a density of 500 cells/cm2 and expanded until passage 1 (P1). At day 17, UC-MSCs were harvested and seeded in 40 x 672 cm2 Cellstack® (Corning, Ref CE0459) at a density of 2000±1000 cells/cm2 and expanded until P2, under the same conditions as for P1. At day 21, cells were harvested, formulated for freezing in 50% Dulbecco’s Phosphate Buffered Saline (DPBS) (Macopharma, Ref BC0120030) + 40% albumin 50 g/L (Octapharma, Ref 575080-0) + 10% Dimethylsulfoxide (DMSO) (Wak Chemie, Ref USP9A1S) and filled into 4 cryobags (CryoMacs, ref 200-074-401, Miltenyi Biotec) containing 60x106 UC-MSCs/bag, then cryopreserved in a vapor nitrogen tanker as a MCS.
3.2. Work Cell Stock (WCS)
Upon transfer to the MEARY center, each MCS cryobag was thawed and UC-MSCs were expanded in the complete culture medium in 25 x 672 cm2 Cellstack® at a density of 4000 ± 1000 cells/cm2 until P3, under the same culture conditions as for P1 and P2 described above. During the whole process, cell confluence and morphology were assessed using an in situ phase-contrast microscope. At day 6 ± 1, cells were harvested, filled into cryobags at the concentration of 100x106 UC-MSCs/bag, frozen and cryopreserved as a WCS until need for clinical use to treat immune and/or inflammatory diseases.
3.3. Manufacture and formulation of the final UC-MSCs-based-ATMP
For a clinical use, we validated the following protocol to manufacture and formulate the final investigational ATMP. The day of patient injection, a WCS bag will be thawed in a dry bath at 37°C for 2-3 minutes, then cells will be washed in 0.9% NaCl (Fresenius, 367512-9) + 0.5% albumin (Octapharma, Ref 575080-0). The final medicinal product will consist of a cell suspension in a final volume of 150 mL 0.9% NaCl + 0.5% albumin, and will contain 100x106 UC-MSCs allowing each bag to deliver a dose of 1x106 cells/kg. The ATMP will be transferred to the hospital pharmacy unit at 22 ± 2°C, before distribution to the clinical department within 4 hours.
3.4. Quality controls (QC)
In order to qualify and validate the manufacturing process and owing to the limited shelf-life of the final reconstituted ATMP, QCs were performed at the MCS and WCS steps to allow releasing the final investigational medicinal product without any delay.
a. Cell counting and viability
UC-MSCs were enumerated after each passage using two methods: a manual Malassez counting chamber and the automated NucleoCounter NC-200TM (ChemoMetec). Viability was assessed using the automated NucleoCounter NC-200TM. In addition, cells’ clonogenicity were assessed using the Colony-Forming-Unit-Fibroblastic (CFU-F) assay.
UC-MSCs phenotype was evaluated by flow cytometry (MacsQuant10, Miltenyi). The surface antigens CD105, CD73, CD90, CD45, CD34, CD11b, CD19 and HLA-DR were used (hMSC analysis kit, BD biosciences, ref 562245). Viability was assessed using eBioscience Fixable Viability Dye eFluor 780 (Invitrogen, ref 65-0865).
c. Sterility assays
All sterility assays were performed according to the European Pharmacopea 10th edition. Aerobic and anaerobic BacT/ALERT® tests (Biomérieux) were used and analyzed on the BacT/ALERT® lecturer. Mycoplasmas were quantified using the Venor® GeM qEP test (Minerva Biolabs) on the QuantStudio5 Real-Time PCR System (ThermoFisher Scientific) and endotoxins using the Chromo-LAL test (Associates of Cape Cod) on the Multiskan Sky Spectrophotometer (ThermoFisher Scientific).
Karyotype was performed according to the International Organization for Standardization ISO 15189, on a minimum of 20 metaphases. Briefly, cells were blocked in metaphase by colchicine, chromosomes were dispersed by hypotonic shock and fixed by alcohol and acetic acid. Different banding techniques were used to obtain a specific staining of each chromosome. Mitoses were captured on a software and chromosomes classified by pair.
e. Potency assay
MLR potency assay was performed as described above. T cells proliferation was calculated using the area under the curve (AUC) as previously described (16).
4. Statistical analysis
Statistical analyses were performed using GraphPad PRISM® 8.4.0 software with appropriate tests as specified in the respective Results section below. Descriptive data are expressed as mean [min – max] and all other values are expressed as mean ± standard deviation. A minimum of 95% confidence interval was established for significance. A p-value < 0.05 was considered statistically significant. Kruskal-Wallis, Bonferroni’s and Dunnett's tests were used, as appropriate.