Donor specifications
AT was obtained from two cohorts of patients included either in the service offered by SSCF Switzerland for aesthetic purpose or in the French clinical trials listed in supplemental table 1. For the latter, AT harvesting was performed in the department of plastic surgery, La Conception, University Hospital, Marseille, France. The study was conducted in accordance with the declaration of Helsinki and all subjects provided informed consent. If necessary, studies were approved by national ethics committee review board (supplemental table 1) between January 2013 and middle 2019.
Table 1 Comparative description of SSCF an d AP-HM methods. Comparative description of each critical steps of SVF manufacturing and associated quality controls performed at the SSCF and AP-HM cell-therapy facilities. DPBS: Dulbecco’s phosphate-buffered saline. HSA: human serum albumin. RL: Ringer’s Lactate.
|
SSCF
|
AP-HM
|
Adipose Tissue harvesting
|
Manual or automatic harvesting using various canula
|
Manual harvesting using a Khouri Cannula after a strict aseptic skin preparation
|
Settling
|
10 minutes
|
Five minutes
|
Packaging in syringes
|
Directly packaging in 60-mL Luer Lock syringes.
|
Packaging in 60-mL Luer Lock syringes from a bag
|
Device preparation
|
Manual collection of AT
|
Tensioning the Celution® device, settlement of consumables, and seal check
|
Adipose Tissue transfer
|
Into three 100 ml syringes
|
AT transfer in the Celution® device
|
Settling
|
10 minutes
|
Automated
|
Visual check of discarding infiltration liquid
|
Infiltration liquid removal
|
Yes
|
Weighing
|
150 ml of AT required
|
At least 100 mL of AT required
|
Washing
|
Twice with DPBS +/+
|
From two to five washings with RL and visual check
|
Addition of the enzyme
|
Addition of Liberase®
|
Addition of Celase® in the device
|
Adipose Tissue Digestion
|
45 minutes at 37°C
|
Automated
|
20 minutes at room temperature
|
Washing
|
DPBS -/- + 1% HSA
|
Automated
|
With RL
|
Filtration
|
100 µm and 40 µm
|
NA
|
Collection of the SVF
|
SVF resuspended in 5% HSA
|
SVF resuspended in RL
|
Quality Control sampling
|
1) Cell count and viability 2) Evaluation of cell subset distribution with flow cytometry
|
1) Cell count and viability 2) Evaluation of cell subset distribution with flow cytometry 3) CFU-F assay
|
Quality Control of the end product
|
Microbiological Control
|
Microbiological Control
|
Environnemental Control in process
|
1) Passive agar (A class) (2x) 2) Contact agar bench (2x) 3) Gloves fingerprints after SVF packaging (2x) 4) Air impacting (A and B class)
|
1) Passive agar (B class) 2) Passive agar (A class) 3) Gloves fingerprints after AT transfer 4) Gloves fingerprints after enzyme reconstitution and transfer 5) Gloves fingerprints after SVF collection 6) Contact agar bench 7) Contact agar working area 8) Gloves fingerprints after SVF packaging 9) Contact agar working area 10) Air impacting (A and B class)
|
Adipose Tissue harvesting
For the SSCF cohort, AT harvesting was performed in an operating room under general anesthesia. AT was collected in three 60ml syringes filled with a maximum of 50ml each with various canula; syringes were inserted in a double secondary container (2 labeled envelopes), then delivered to SSCF in a dedicated transport container at room temperature (20±10°C). The AT sample was processed within 24 hours after collection.
For the AP-HM cohort, AT harvesting was performed in an operating room under general or local anesthesia after a standardized skin aseptic preparation using a Khouri cannula (Khouri Harvester, Koume, Lipoplasty Products, Sunrise, FL, USA), a 12-gauge 12-hole multiperforated cannula, through a closed circuit, preventing contamination of the harvested product. Then, the AT was packaged in a bag; (Easyflex+, Macopharma, Mouvaux, France). Once harvesting was complete, the bag was transported to the cell therapy unit and the AT was immediately processed.
SVF manufacturing
In the SSCF facilities, AT was washed twice with Dulbecco’s phosphate buffered saline with calcium and magnesium solution (DPBS +/+, Gibco, Life Technologies, Carlsbad, CA, USA) and enzymatically digested for 45 minutes at 37°C under constant agitation in a cell culture incubator. For the latter, Liberase® MNP-S (Roche, Basel, Switzerland), i.e., a mix of collagenase I and II, and neutral proteases, was used. Enzymatic digestion was then stopped with sterile, clinical grade 1% human serum albumin (HSA, CSL Behring, King of Prussia, Pa, USA) in DPBS without calcium and magnesium (DPBS -/-, Life Technologies); the hydrophilic phase was collected and subsequently filtered with 100 µm and 40 µm sieves. At the final step of SVF manufacturing, the hydrophilic phase was centrifuged and the resulting cell pellet, i.e., the SVF, was resuspended in 5% HSA.
In the AP-HM facilities, SVF was obtained using the Celution 800/CRS automated processing system (Lorem Cytori, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, the collected lipoaspirate was washed with Ringer’s Lactate (RL; Baxter, Deerfield, IL, USA) and enzymatically digested with Celase, a GMP cocktail of enzymes provided with consumables (Worthington Biochemical Corp., Lakewood, NJ, USA). The cells were concentrated, washed, aseptically recovered, and resuspended in RL.
Viable nucleated cells concentration and cells viability
For both cohorts, the number of VNCs and percentage of cell viability were determined using a NucleoCounter NC-100 instrument (ChemoMetec, Allerød, Denmark). Recovery yield was calculated as the number of total VNCs obtained divided by the initial volume of AT measured after removal of infiltration liquid (decantation).
Microbiological testing and Environmental monitoring
For both cohorts, a microbiological testing was performed on the end product consisting in inoculating SVF samples in Bact/Alert culture bottles (aerobic and anaerobic culture vials, each containing 40 mL of medium). The Bact/Alert method (Biomerieux, Marcy l’Etoile, France) uses a computer-controlled incubation/detection system. The media used contained proprietary factors designed to inactivate a wide variety of antibacterial and antifungal agents. Bact/Alert culture bottles were incubated at 37°C and 5% CO2 for a total of 10 days (AP-HM) or 14 days (SSCF), and automated readings were taken every 10 min (according to the European Pharmacopoeia - Chapter 2.6.27 Microbiological Examination of cell-based Preparations). Detection of organisms resulted in an audible alarm and automatic recording of time of detection.
Some “in process” environmental monitoring quality controls were also carried out in both facilities, consisting in contact agar, gloves fingerprints or air impacting. The environmental controls details and a summary of both protocols are exposed in table 1.
Flow cytometry analysis of SVF cell subsets
Immunolabelling of SVF cells
For SSCF cohort, the SVF cells were characterized by cytofluorimetric analysis using a 10 channel Navios cytometer (Beckman Coulter, Nyon, Switzerland). Briefly, 500,000 VNCs were sampled and centrifuged 5 min at 400g. The pellet was resuspended in 220 µL of DPBS without calcium and magnesium (Life Technologies) supplemented with 1% human serum off the clot (PAA Laboratories, Inc), then 100 µL of the cell suspension were distributed into two test tubes and stained with the Syto40 nuclear marker, the 7AAD viability marker and antibodies or corresponding isotype controls in matched concentrations. The monoclonal antibody mix, ready to use and lyophilized (Beckman Coulter), included antibodies directed against CD146, CD45 and CD34, conjugated with the following fluorochromes: PE; KRO and APCA750 respectively, (references listed in supplemental table 2). After 20 min of incubation, erythrocytes were lysed with 1 mL of VersaLyse (Beckman Coulter). Before acquisition, 100 µL of Perfect-Count Microspheres (Cytognos, Salamanca, Spain) were added to the test tube. Data files were analyzed using Kaluza software (Beckman Coulter).
For AP-HM cohort, characterization of the SVF cell sub-populations was performed by flow cytometry using also a 10 channel Navios instrument (Beckman Coulter, Brea, CA, USA).
Aliquots of 500,000 VNCs per tube were resuspended in 100 μL of DPBS, after centrifugation 5 min at 400g, and stained for 20 min at room temperature in the dark with the DRAQ5 nuclear marker and ready to use antibody mixes or corresponding isotype controls in matched concentrations. The monoclonal antibodies mix included antibodies against CD90, CD146, CD34 and CD45, conjugated with the following fluorochromes: FITC, PE, ECD and PC5 respectively, (references listed in supplemental table 2). Red blood cells were lysed in NH4Cl for 10 min before the cells were centrifuged and resuspended in DPBS without calcium and magnesium (Life Technologies). Then, NucBlue (Thermo Fisher Scientific), which allows discrimination of viable and dead cells, was added for 5 min prior to flow cytometry analysis. Data files were analyzed using Kaluza software (Beckman Coulter).
Inter-center validation of a common flow cytometry gating strategy
In order to be able to reliably compare the flow cytometry data generated by each center, we first verified that the difference in the antibodies panel used by the two facilities did not hamper the comparability of data when a common gating strategy is used. To that aim, 14 SVF batches manufactured in the two centers using a similar manual digestion protocol[21] were immunolabelled using the SSCF antibodies panel and the AP-HM antibodies panel. Samples were then analyzed by the same operator using the same defined gating strategy using Kaluza software as follows (figure1):
Common gating strategy
- Nucleated cells (NC) were selected according to the nuclear marker used (DRAQ5 or Syto40)
- Aggregates were removed using the forward scatter
- Viable nucleated cells (VNCs) were determined based on the death marker (NucBlue or 7-AAD)
- Among VNCs, the CD45 marker was used to discriminate the hematopoietic CD45+ cells from the non-hematopoietic regenerative cells CD45-.
- A density plot CD146 versus CD34, gated on the CD45- cells, allowed the identification of ASCs (CD45-CD34+CD146-), EC (CD45-CD34+CD146+) and an heterogeneous populations containing PRs and transitional cells (CD45-CD34-CD146+).
That strategy leads to a complete characterization of the SVF, meaning the addition of the proportions of ASCs, EC, PRs and transitional cells and hematopoietic cells equals to 100 %.
Statistical Analysis
Statistical analyses were performed using Graph Pad Prism 5 (GraphPad Software, La Jolla, CA, USA). Quantitative variables are reported as the mean ± standard deviation (SD). The nonparametric Mann–Whitney t-test was used for the cohorts comparison. Paired t-test were used for the validation of the common gating strategy. A p-value < 0.05 was considered to indicate a statistically significant difference. In addition, to establish the release acceptance criteria we have chosen the best thresholds achievable in at least 80% of cases for the parameters which are related to potency.