Bone marrow harvest
All experiments were approved by the OHSU Institutional Animal Care and Use Committee (IACUC) and the Department of Defense Animal Care and Use Review Office (ACURO). All experiments also adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Six-month old female Yorkshire swine (Oak Hill Farms, Glen Ellen, CA) weighing between 40 and 50 kg were anesthetized using tiletamine (8 mg/kg IM) and glycopyrrolate (0.01 mg/kg IM). A 6f bone marrow biopsy needle was inserted into the anterior pelvis and a single collection of 50–100 ml of bone marrow was aspirated into a heparin-containing syringe. The bone marrow was then transferred into a conical tube, packaged with ice packs, and then shipped via over-night shipping to UCSF. The amount and overall quality of bone marrow aspirate was similar between the donors tested.
Isolation of MSCs from bone marrow
Bone marrow aspirate was filtered with MACS Smart Strainers (pore size 100 µm) to remove any coagulated matter. Loss by volume was typically less than 5%. Filtered bone-marrow aspirate was then diluted 1:5 with media known to promote growth of MSCs [minimal essential medium (MEM) alpha, 10% MSC-grade fetal bovine serum (FBS), Glutamax, 10 µg/ml Gentamicin]. FBS is USDA certified and has been tested and qualified to support clonal efficiency of MSC derived from bone marrow and support cell expansion and differentiation. After 2 days of incubation at 37 °C, 5% CO2, media was removed, and adherent cells underwent 5 washes with PBS. Removal of red blood cells was confirmed by microscopy. Colonies of MSCs were then allowed to grow for a further 5 days. Following 7 days of growth in the primary tissue culture flasks, cells were trypsinized and were re-plated to form a homogenous population of cells. Seven days was selected as a time point for the first trypsinization, as this is sufficient to expand a significant number of cells without the colonies touching/overlapping. We generally observed a cell confluence between 60 and 70%. Furthermore, this time point was selected to prevent any emergence of senescence with smaller colonies. We did not observe any significant/unusual growth rates in the cells derived from the 3 pigs at this time point. The generation of colonies generated after the first trypsinization was designated Passage Zero (P0). When cells in these flasks reached confluence, they were trypsinized and frozen in cryovials for expansion in quantum. These cells were designated as pre-quantum expansion passage 1 (PreQE-P1) (Fig. 1).
Generation of conditioned medium
To compare cells from all 3 donors in in vitro assays, we propagated the cells manually in tissue culture flasks for all of the following experiments. This will also avoid the bias of different growth conditions that may affect cell characteristics. Cells from all three donors were expanded manually in flasks up to passage 3 and plated for different assays at each of the passages (see Fig. 1). To generate conditioned medium, cells were seeded at a density of 250,000 cells per well in a 6 well plate and allowed to grow to confluence in complete MSC medium (3 wells/cell type/passage). Similarly, cells from Pigs 1 and 3 that were expanded on quantum (QE-2) (Fig. 2) were seeded at a density of 250,000 cells per well in a 6 well plate and allowed to grow to confluence in complete MSC medium. Once the cells reached confluence, they were serum starved for 48 h and medium was collected, centrifuged to remove dead cell debris and stored at − 80 °C until evaluated in functional assays. Serum starvation is standard and is done so that the effects of what is secreted by the cell in the culture medium is not confounded by the factors present in the serum. Secondly, serum starvation is also a stress response under which cells secrete all factors along with exosomes and microvesicles.
In vitro cell analysis
Cell metabolic assay
Mesenchymal stem cells (MSCs) at PreQE-P1, and manually expanded passages 2 and 3 from all three donors were propagated in Minimum Essential Medium (Alpha MEM, ThermoFisher) with 10% fetal bovine serum (ThermoFisher) and 0.1% gentamicin (ThermoFisher). For metabolic assay, MSCs starting at each of the individual passages were seeded at a density of 12,000 cells per well (4 wells were assayed/day/donor, for a total of 5 days assay time) in a 96-well plate (ThermoFisher) (Fig. 1). In addition, media alone served as negative no cell control for each day (4 wells per day). Metabolic activity was analyzed using the Alamar blue assay following the manufacturer’s protocol (Bio-Rad). Briefly, first assay was performed at 4 h after initial seeding, Alamar blue dye was added to wells to be analyzed at this time point along with wells that served as negative control, at 1× concentration. Rest of the plated wells were left untouched and were analyzed over the course of the next 4 days. After a 4 h incubation with dye, the absorbance was read on plate reader (Epoch, Biotek) at 570 nm and 600 nm. All resulting values were adjusted for background by comparison to no-dye control wells. Wells without cells were utilized to determine a correction factor. Percentage consumption of dye was calculated by comparing the 570 nm value to the corrected 600 nm. This assay was repeated every 24 h using different wells/day.
Cell morphology
Cells from all three donors seeded from the three passages (PreQE-P1, manually expanded passages 2 and 3) were imaged on a phase contrast microscope using a 20× objective (ECHO, revolve) (Fig. 1).
Cell proliferation assay
Manually expanded MSCs from three donors from PreQE-P1, passage 2 or passage 3 were seeded at 50,000 cells per well in 12-well plates (3 wells/day/donor) (Fig. 1). At 24 h after plating, cells from each donor were counted in triplicate using a Vi-Cell XR viability analyzer (Beckman Coulter). Parameters set were as follows, 15–30 microns diameter, with a minimum circularity of 0, a “low” decluster degree, a cell sharpness of 100, and a cell brightness of 85%. Cells were determined to be viable if their cell spot area was 10% of the total cell and if their cell spot brightness was 75%. This assay was repeated for 4 days in a 12 well plate format with different wells trypsinized daily and used for cell count and viability determination. Known number of cells were also plated on T75 flask from which cells were also harvested after 1 week of plating (Fig. 1) and doubling time and fold increase were evaluated as follows.
$${\text{Population}}\;{\text{doubling}} = \frac{1}{\log \left( 2 \right)} \times \log \left( {\frac{Cx\left( t \right)}{Cx\left( 0 \right)}} \right)$$
where Cx(t) and Cx(0) are the cell numbers at the end and start of exponential growth phase respectively; t is the time (h)
$${\text{Fold}}\;{\text{increase}} = Cx\left( f \right)/Cx\left( 0 \right)$$
where Cx(f) is the final cell number at the end of passage and Cx(0) is the initial number of cells plated.
Surface marker expression
In addition, cells at passage 3 were immunophenotyped (Fig. 1) and stained for CD44 (Stem Cell Technologies), CD90 (Stem Cell Technologies), and CD105 (Novusbio). Primary antibodies were known to cross react with pig antigen and were detected with fluorescently tagged secondary antibodies, Alexafluor 488 anti-rat IgG (Life Technologies), Alexafluor 488 anti-mouse IgG (Life Technologies), and Alexafluor 594 anti-mouse Fab fragment (Cell Signaling). Images were captured using a Nikon 80i Epifluorescence microscope (Nikon) and a RTcmos camera (SPOT Imaging).
Expanding cells on the quantum bioreactor platform
To generate therapeutic MSC doses, cells underwent two cell expansions on the quantum bioreactor platform (Fig. 2). PreQE-P1, available in limited supply from the isolation and expansion from bone-marrow aspirate were expanded to form a quantum expansion 1 (QE-1) generation which formed the ‘MSC cell bank’, from which aliquots would be used for subsequent expansions on quantum [quantum expansion 2 (QE-2)]. This generation will be used as the cellular therapeutic for in vivo model and also for cell potency characterization. Only cells from pigs 1 and 3 were expanded on quantum and tested head to head. Procedurally both the QE-1 and QE-2 expansions on the quantum bioreactor platform were the same. In brief, prior to introducing cells into the quantum bioreactor a disposable cell expansion set was loaded onto the machine. The disposable set was primed with Ca2+/Mg2+ free phosphate buffered saline (PBS; Life Technologies) to purge sterile air from the set and the set was then coated overnight with 5 mg fibronectin (Corning). Fifteen million cells were loaded onto the Quantum and allowed to attach overnight. A programmed feed rate protocol was then initiated. Base media was Minimal Essential Medium (MEM) alpha supplemented with 10% MSC grade FBS and Glutamax (ThermoFisher). Feed rates were ramped every 24 h by the computer program, starting at 0.1 ml/min, culminating at 1.2 ml/min. As cells cannot be directly observed during closed system expansion in the quantum, the progression of cell expansion was monitored through glucose and lactate levels in culture media sterile sampled daily in triplicate using hand held devices (Contour and Nova Biomedical respectively). Post-expansion QE-1 cells forming the cell bank were resuspended in Cryostor 10 (BioLife solutions) at 5 million cells/ml, transferred to 2 ml pre-chilled cryovials (NUNC) and control rate frozen to − 80 °C in CoolCell LX alcohol free controlled rate freezing units (Biocision) before transfer to LN2 for long term storage. Post-expansion QE-2 cells forming the therapeutic doses were similarly cryopreserved at a density of 10 million cells/ml in 5 ml cryovials, with a total 50 million cells/vial. Cells generated from Quantum at passage QE-2 were used to generate conditioned medium to be tested in ECIS, cell marker expression by flow cytometry and cell differentiation potential.
Flow cytometry
Cryopreserved MSCs from QE-1 and QE-2 for Pigs 1 and 3 and passage 3 cells (manually expanded) for Pig 2 were thawed at 37 °C, counted with a hemocytometer, and 1 × 106 cells were transferred to polystyrene tubes for incubation with staining buffer [DPBS supplemented with 0.2% BSA (BD Biosciences)] and the appropriate antibody cocktail for 30 min at room temperature. CD44-APC, CD90-PE (Stem Cell Technologies), and CD105-Alexa405 (Novus Biologicals) were used to identify the MSC populations. CD31-FITC, CD45-FITC, and Swine-Leukocyte-Antigen Class II-FITC (SLA-DRII) (GeneTex) were used to determine the presence of non-MSC lineage positive cells in the expanded MSCs. Prior to acquisition on the BD LSRII flow cytometer, cells were stained for 10 min using 7AAD viability dye (BD Biosciences). Data were analyzed using FlowJo software version 9.9 (FlowJo). A compensation matrix was generated using single color stains on MSCs. Doublets were excluded using FSA vs. FSH gating and debri was excluded using FSA vs. SSC gating for all samples before gating on total live cells negative for 7AAD (BD Biosciences). ‘Fluorescence Minus One (FMO)’ controls were used to identify positive populations.
Tri-lineage differentiation of MSCs
Cryopreserved MSCs from QE-2 of donor Pigs 1 and 3 were thawed at 37 °C and plated in MSC growth medium [MEM Alpha 1X (ThermoFisher)] supplemented with 10% FBS and 5 µg/ml gentamicin (ThermoFisher) and incubated overnight at 37 °C before splitting into three cultures for each lineage. Adherent cells were detached with 0.25% Trypsin (ThermoFisher). For adipocyte and osteocyte cultures, 2 × 105 MSCs were grown overnight in a 12 well plate containing MSC growth medium before switching to appropriate differentiation medium (Adipogenesis differentiation kit, Osteogenesis differentiation kit; ThermoFisher). For chondrocyte cultures, 5 µl droplets at a concentration of 1 × 107 cells/ml were placed into a 37 °C CO2 incubator for 2 h in a 96 well U-bottom plate to allow for spheroid formation before adding 100 µl of differentiation medium (Chondrogenesis differentiation kit; ThermoFisher). All cultures were fed with fresh pre-warmed differentiation media every 2–3 days. After 5–7 days, adipocytes were stained with Oil Red O (Sigma Aldrich) for 20 min. After 14–21 days, osteocytes were stained with 2% Alizarin Red S (Sigma Aldrich) for 5 min. After 14–21 days, chondrocytes were stained with 1% Alcian Blue for 30 min. Prior to staining, all cells were fixed with Image-IT fixative solution containing 4% formaldehyde (ThermoFisher). Bright field images were captured throughout culture and following each stain (EVOS digital inverted microscope, Life Technologies and Leica CTR6500, JH Technologies).
Transendothelial electrical resistance
The barrier integrity of human pulmonary microvascular endothelial cells (HPMVEC, Promocell) monolayers was measured using an electric cell-substrate impedance sensing system (ECIS 1600, Applied BioPhysics). An incline or decline in transendothelial electrical resistance (TEER) across the cell monolayers, indicated decreased or increased endothelial paracellular permeability respectively. HPMVECs were grown on l-cysteine-reduced, 96-well plates containing electrodes in each well. Cells were treated with 50% plain MSC growth media as a control, or a dose curve of conditioned media generated from the different MSC donors (passage 3 from manually expanded cells for all 3 cell donors or QE-2 cells for Pigs 1 and 3), for 1 h, and then challenged with thrombin (Sigma-Aldrich) at a concentration of 0.2 U/ml. Monolayer resistance at 4/16/64 kHz was analyzed in 8-min intervals. Data were normalized to the mean resistance of cell monolayers before the treatments. Resulting resistance values were calculated in comparison to control samples as an area under curve (AUC), or as a maximum decrease in resistance.
Statistics
Statistical analyses were conducted using GraphPad Prism 7.0 (GraphPad Software). Metabolic assay and proliferation analysis were compared between donors using two-way analysis of variance (ANOVA) for tests between subject and within subject effects, followed by Sidak’s multiple comparison test. TEER values were compared to control samples using one-way ANOVA. A p-value of less than 0.05 was considered statistically significant. All values are presented as a group mean + standard error of mean (SEM).