Experimental design
Lipoaspirates were obtained from 3 healthy female donors (age between 41 and 65 years) undergoing cosmetic surgery (Printzlau Private Hospital). Each lipoaspirate was processed to obtain stromal vascular fractions (SVF), from which ASC populations were expanded. The use of lipoaspirate has been approved by The National Committee on Health Research Ethics in Denmark, protocol no. H-3-2009-119. All donors signed an informed consent.
The experimental setup is illustrated in Fig. 1. Expansion of ASCs was performed in a two-passage process using a traditional, flask-based process (F) or the Quantum system (Q). Primary expansion of the SVF was performed with both methods to produce ASC passage 0 (P0) cells, which were further expanded in a second passage to produce ASC P1 cells. Using the two expansion methods over two passages, three production strategies were investigated: flasks to flasks (F–F), flasks to Quantum system (F–Q), and Quantum system to Quantum system (Q–Q).
Preparation of lipoaspirate and SVF isolation
Liposuction of subcutaneous abdominal fat was performed under local anaesthesia to provide approximately 100 ml lipoaspirate from each donor. Each lipoaspirate was washed twice with phosphate-buffered saline (PBS) (Gibco, Life Technologies) to remove residual blood. The adipose tissue was digested by incubation with 0.6 PZ U/ml collagenase NB4 (Serva GmbH) dissolved in Hank’s Balanced Salt Solution (10 × HBSS) (+CaCl2 +MgCl2, Gibco, Life Technologies) diluted to a concentration of 2 mM Ca2+ at 37 °C for 45 min under constant rotation. The collagenase was neutralised with complete medium containing Minimum Essential Medium, MEM Alpha (αMEM) without Ribonucleosides and Deoxyribonucleosides, (Gibco, Life Technologies), 1% Penicillin/Streptomycin (10,000 U/ml and 10.000 µg/ml, respectively) (Gibco, Life Technologies) and 10% Fetal Bovine Serum (FBS) (Gamma irradiated, Australian origin, Gibco, Life Technologies) according to a well-established protocol [15, 16] after which the suspension was filtered through a 100 µm mesh (Cell Strainer, BD Falcon), centrifuged at 1200g for 10 min at room temperature (RT), and re-suspended. The number of cells in the isolated SVF was counted using a NucleoCounter® NC-100™ (ChemoMetec).
Cell culture in flasks
Primary cell cultures of ASCs were established by seeding 4.5 × 106 SVF cells per T75-flask (Nunc, Thermo Scientific) in complete medium. The chosen seeding density of SVF in flasks has been optimized previously in our laboratory.
The cells were incubated at standard conditions at 37 °C in humid air with 5% CO2. The culture medium was changed 3 days after the cells were seeded, thus removing non-adherent cells. Subsequently, the medium was changed every 3–4 days throughout the remainder of the culture period.
Reaching a confluence level of approximately 90%, the cells were harvested. For each T75 flask, the harvest procedure included an initial wash with 15 ml PBS, the addition of 3 ml TrypLE® Select (Gibco, Life Technologies), incubation for 10 min at 37 °C, and neutralisation with 7 ml complete medium. The resulting suspension was centrifuged at 300g for 5 min at RT and re-suspended in complete medium. After counting, the cells were re-seeded at 3.5 × 105 cells/T75-flask. Cell yields for ASCs at P0 and P1 were determined with a NucleoCounter® NC-100™ and calculated as means of three T75 flasks.
Cell culture in the Quantum system
The Quantum system is an automated and functionally closed system that integrates incubation, gas provision, and fluid handling for the management of a hollow fiber bioreactor. Operation of the Quantum system includes filling bags with media and reagents (e.g., media, PBS, cells, TrypLE Select), connecting these bags to the Quantum system via a sterile connection device (TSCD-II, Terumo), and controlling the system via a touch screen interface.
The Quantum system process in the current study used media and reagents that were consistent with those referenced in the “Cell Culture in Flasks” section. One additional reagent was used for coating of the bioreactor, as described in “Coating of culture surface area’’ section. Standard conditions for ASCs culture were maintained, including an incubation temperature of 37 °C and a pre-mixed gas supply (StrandMøllen) providing 5% CO2 and 20% O2, balanced with N2.
The Quantum system was prepared according to the manufacturer’s protocol for inserting of the disposable Cell Expansion Set (including the hollow fiber bioreactor), into the Quantum system and priming it with PBS.
Coating of culture surface area
Prior to loading of cells, the culture surface area of the hollow fiber bioreactor must be coated. For this purpose, 30 ml of pooled cryoprecipitate (Blood Bank, Rigshospitalet) diluted to 100 ml with PBS is loaded into the Quantum system. Coating times of 4 and 24 h were tested, in order to test if both conditions provided sufficient coating for cell attachment. Upon completion of the coating period, PBS and residual cryoprecipitate were washed out of the system and replaced with complete medium. ASCs P2 (30 × 106) were loaded into the bioreactors for evaluation of the effect of coating time upon adhesion and growth.
Pools of cryoprecipitate were manufactured from fresh-frozen plasma from blood donors (blood types low titer A+) selected and screened according to Danish law about blood supply. Frozen plasma bags (−30 °C) were air-thawed slowly at 4 °C overnight. Plasma bags were centrifuged and cryoprecipitate separated from plasma in a CompoMat G5 blood component separator device (Fresenius KABI). The remaining cryoprecipitate was pooled from four donors and aliquoted into 30 ml portions.
Culture expansion of primary SVF in the bioreactor (P0)
For primary expansion of the SVF, the Quantum system was seeded with in average 80 × 106 (52–100 × 106) SVF suspended in 100 ml complete medium. Cells were allowed to attach for 24 h at static conditions, after which perfusion automatically began at 0.1 ml/min. After 3 days of cultivation, a washout was performed to remove any non-adherent cells.
Culture expansion of pre-cultured ASCs in the bioreactor (P1)
For expansion of pre-cultured ASCs (i.e., those resulting from the primary expansion), the Quantum system was seeded with 30 × 106 ASCs suspended in 100 ml complete medium. A 24 h attachment period was provided, as consistent with the SVF expansion.
After the attachment period, complete medium was fed continuously into the system at increasing rates from 0.1 ml/min and gradually doubled up to 0.8 ml/min, maintaining lactate levels between 6 and 10 mM throughout the expansion. Growth of the cells was estimated based on glucose consumption and lactate production, as mentioned by Lambrechts et al. [17]. Samples from the supernatant were drawn from the sample port and glucose/lactate levels were measured with a blood gas analyzer ABL 835 FLEX (Radiometer).
Harvest of ASCs from bioreactor
Harvests were performed when lactate generation rates surpassed 6-9 mM/day at feeding rate 0.8 ml/min. The harvest process included a washout of the system with PBS, the addition of 180 ml TrypLE® Select, and a 15 min incubation time, after which the cells were flushed into the harvest bag using complete medium. Cell yields at P0 and P1 were determined by a NucleoCounter® NC-100™.
Storage of cells
To be able to work with a single Quantum system cells need to be stored for up till 5 h while preparing a new Cell Expansion Set for successive expansion. To assess the impact of storage media on cell viability, we tested 3 different storage media, at 4 °C and RT for a 5 h period and measured viability with a NucleoCounter® NC-100™ every hour. 10 × 106 ASCs were re-suspended in 1 ml of: 1. Isotonic saline (9 mg/ml) (Amgros I/S) with 1% human albumin (1% HA) (20%, CSL Behring GmbH) 2. Isotonic saline with 20% human albumin (20% HA) 3. Isotonic saline with 20% autologous human serum (20% HuS).
After 5 h incubation, 1 × 106 ASCs from each condition were seeded into T75 flasks. Attachment and morphology of ASCs was verified by microscopy after 24 h.
Immunophenotyping by flow cytometry
Adipose derived stromal cells P0 and P1 culture expanded in a Quantum system or flasks were analyzed by flow cytometry (Navios, Beckman Coulter). Cells from T75 flasks were detached by incubation with TrypLE® for 5–10 min at 37° C, and washed with FACS-PBS mixture [FACS-PBS (Hospital pharmacy, Copenhagen, Denmark), 1% EDTA (Hospital pharmacy, Copenhagen)], and 10% new born calf serum (GIBCO, Life Technologies). Harvested cells from a Quantum system were washed with 3 mL FACS-PBS mixture. After centrifugation (5 min at 300g), all cell pellets were re-suspended in FACS-PBS mixture, filtered, and distributed to FACS tubes with or without antibodies. The cells were incubated for 30 min. at room temperature with the following antibodies or isotypes: Cluster of Differentiation (CD)45-phycoerythrincyanin (PC7), CD34-allophycocyanin (APC), CD90-fluorescein isothiocyanate (FITC), CD73-phycoerythrin (PE), CD13-phycoerythrin and Texas red energy coupled dye (ECD), CD29-FITC, HLA-DR-FITC, CD19-ECD, CD14-PC7, IgG2a-FITC, IgG1-ECD, IgG1-APC, MsIgG1-PC7 (all Beckman Coulter), CD105-PE (R&D Sciences), CD166-PE, CD106-FITC, CD31-FITC, CD36-FITC, IgG1-PE, IgG1-FITC (all BD Bioscience). After incubation, the cells were washed with FACS-PBS mixture, centrifuged, and re-suspended in PBS before measuring on a Navios flow cytometer (Beckman Coulter) using a six-colour protocol. The mentioned isotype controls and Fluorescence Minus One tubes were run with each analysis. Viability was determined by addition of SYTOX blue 5 min. prior to analysis (SYTOX®, Invitrogen, Life Technologies). Dead cells were excluded from the final analysis, and data was analyzed using Navios software and Kaluza (Beckman Coulter).
Differentiation potential of ASC
Adipose derived stromal cells P2 harvested from a Quantum system and flasks were tested for their ability to differentiate into osteoblasts, adipocytes and chondrocytes lineages, as described in our previous publication using StemPro differentiation kit (Gibco, Life Technology), according to the manufacturer’s protocols [18].
Microbial contamination assays
Cell culture supernatants were collected from the Quantum system and from the culture flasks immediately before harvest. Microbial controls for bacteria and fungus were performed on supernatants using aerobic and anaerobic BacT/ALERT iFN and iFA plus culture bottles (Biomerieux) and the BacT/ALERT® Microbial Detection System (Biomerieux).
Presence of mycoplasmas in cell culture supernatants were detected by PCR for mycoplasma genus at Statens Serum Institute, Copenhagen, Denmark. The content of endotoxins in end cell products were quantitatively determined by the Limulus amebocyte lysate (LAL) chromogenic method, by Statens Serum Institute.
Statistics
Population doublings (PDs) were calculated from P1 using the following formula: PD = (log N − log N0)/log 2, where N is the number of harvested cells and N0 is the number of seeded cells.
PD calculations were shown to produce normally distributed data, when using cell counts from culture in flasks or a Quantum system. Equality of variance was confirmed by Levene’s test. A one-way ANOVA with Bonferroni correction was used for comparison of PD in Quantum–Quantum, flask–flask, and flask-Quantum in P1. All statistics were performed using IBM SPSS version 19. Graphs were made using SPSS and Excel 2010 (Microsoft Inc.). Data are depicted as mean ± Standard Error Mean in figures, and mean ± standard deviation in tables.