Details of the trial protocols are published elsewhere (21
). All studies were conducted according to CCTRN guidelines with approval of Institutional Review Boards of each participating institution, and each patient provided written informed consent.
Collection of bone marrow and peripheral blood for storage and analysis
All patients undergo a standardized bone marrow (BM) aspiration and venipuncture on the day of treatment. Details of cell handling and processing are published elsewhere (25
). For patients who provide informed additional consent, after preparation of the treatment product (cell or cell-free) the remaining mononuclear cells and a peripheral blood (PB) specimen are sent to the Biorepository for storage and phenotypic analysis (University of Minnesota) and for functional analysis (University of Florida). The day of BM harvest and study product delivery is designated Day 0. PB is collected at predetermined time points before (Day 0), and after study product delivery ().
Blood collection across time points in CCTRN TIME, Late-TIME, and FOCUS protocols.
Phenotypic analysis by fluorescence activated cell sorting (FACS)
At each time point (), 20mL of PB is obtained in two vacutainers (BD Biosciences, Bedford, MA) containing EDTA following venipuncture; tubes are mixed well, and both BM and PB stored at 4°C until packaged shipping. Prior to shipping, tubes are wrapped in absorbent material and bubble wrap, placed in a Biohazard bag, and shipped with two refrigerated (4°C) packs and appropriate forms for next morning delivery. Upon arrival, PB samples are treated with ammonium chloride potassium (ACK) standard red blood cell lysis buffer, centrifuged at 50×g for five minutes at 4°C, and the supernatant aspirated. The pellet is resuspended in ACK lysis buffer, centrifuged at 50×g for five minutes at 4°C, the supernatant aspirated, and cells washed with PBS twice, before resuspension in FACS buffer containing PBS + 2.5% Fetal Bovine Serum. The number and viability of resulting nucleated cells is determined in an automated cell counter using the Guava ViaCount assay (Millipore, Billerica, MA). One to five million cells are stained using antibodies raised against human CD45, CD34, CD31, CD133, VEGFR2, CXCR4, CD3, CD11, CD14, CD19, and their corresponding isotype control. After incubation, cells are washed twice with, and resuspended in FACS buffer for FACS (LSRII cytometer, BD Biosciences, Bedford, MA). Data are analyzed using FlowJo software (Tree Star Inc, Ashland, OR) with standardized guidelines for gating.
To ascertain maximal cell viability during shipping, a shipping study was undertaken. Overall PB cell and subpopulation viabilities were quantified after shipping of samples with either refrigerated (4°C) packs or frozen packs. As a control, samples were not shipped but stored at 4°C overnight or left at room temperature. Room temperature storage resulted in with widely disparate sample viabilities (57.7-92.5%) and was not considered optimal for these samples, and thus was not used as a control. Optimal time-to-processing of PB was determined by evaluating viabilities by FACS-sorted PB at 0, 24, 48, or 72 hrs after harvest. Once optimal shipping conditions were determined, SOPs were generated and distributed to cell processing facilities. Standardized sample shipping containers, cold packs and wrapping materials were also provided. After distribution of materials, optimal sample shipping conditions were addressed by phone calls with network investigators and research coordinators. If BM or PB sample viability from a given center drops below 80%m for a single sample, the center is immediately contacted and shipping procedures reviewed.
Bone Marrow and Peripheral Blood Functional Analysis
On Day 0, 30mL of blood is collected in vacutainers containing EDTA following venipuncture as described above. BM and PB mononuclear cells are analyzed for viability using trypan blue staining () (Cellometer AutoT4, Nexcelom Bioscience, Lawrence, MA). Cells are reserved for endothelial and mesenchymal colony assays. When available, additional BM cells are processed for enrichment of the CD34+ stem and progenitor cell subset by immunomagnetic selection (Miltenyi, Auburn, CA). These CD34+ cells are then tested using assays for migration to stromal cell derived factor 1 (SDF-1). The CD34+ cell population is used as a surrogate for stem and progenitor cell activity in the entire BM-MNC fraction because use of the distinctly heterogeneous population of BM-MNCs generates highly variable migration data that would be difficult to interpret in the context of the CCTRN trials, which seek to recruit less than 100 subjects per trial.
Cell Function Analyses of Bone Marrow and Peripheral Blood Cells From Subjects Enrolled in CCTRN Clinical Trials
For endothelial colony assays, mononuclear cells are first plated in endothelial growth media-2 (EGM-2, Lonza, Walkersville, MD), plated on fibronectin-coated dishes (BD Biosciences, Bedford, MA) and incubated at 37°C, 5% CO2
, > 95% humidity for 48 hrs. Non-adherent cells are removed for colony forming unit Hill (CFU-Hill) assay and adherent cells are trypsinized for endothelial colony forming cell (ECFC) assay, as previously described (26
). Media is exchanged every other day and number of colony forming units, types of colonies and confluence are recorded on days 7, 14, 21 and 28 (). CFU-Hill colonies are defined as a central core of round cells with radiating elongated spindle-like cells at the periphery. ECFC colonies are defined as circumscribed monolayers of cobblestone-appearing cells.
For mesenchymal stem cell colony assay, also known as the CFU-F assay, three concentrations of BM-MNCs and PB-MNCs are plated in Mesencult serum and cytokine containing media (STEMELL Technologies, Vancouver, CA) according to manufacturer's instructions. Number of colony forming units, type of colonies and confluence are recorded on days 7, 14, 21, and 28 (). CFU-F colonies are defined as circumscribed clusters of spindle-like cells typically 1 to 8 mm in diameter.
To evaluate hematopoietic stem and progenitor cell migration to SDF-1, CD34+
cells are isolated from BM cells using immunomagnetic microbeads conjugated with anti-CD34 antibodies (Miltenyi Biotec Inc., Auburn, CA) and automated magnetic selection (AutoMACS, Miltenyi Biotec Inc.). These CD34+ cells are tested for migration to SDF-1 in a modified Boyden chamber, as previously described (28
Peripheral blood collection for cytokine or DNA/RNA analysis
At each time point, 10mL of blood are collected in vacutainers containing Heparin Sodium and mixed properly. Tubes are centrifuged within 30 min at 190×g for 10 minutes at 4°C, the top plasma layer is carefully removed for cytokine analysis, without disturbing the middle leukocyte layer. Plasma is aliquoted into cryovials kept at 4°C. For future DNA/RNA analysis, the leukocyte layer is transferred to a separate cryovial avoiding significant erythrocyte contamination. Cryovials are placed in a -80°C freezer within 30 minutes for storage until shipment on dry ice to Biorepository Cell Phenotyping and Storage Core Laboratory (UMN) for centralized storage at -86°C. All samples are handled and disposed of following institutional guidelines, with universal precautions at all times.
Cryopreservation for long-term storage
In addition to plasma and leukocytes, BM and PB from CCTRN subjects are shipped to the Biorepository Cell Phenotyping and Storage Core Laboratory for FACS profiling and long term storage. After BM and PB samples are analyzed by FACS, remaining cells are frozen in 10, 25 and 50 million cell aliquots (50 million cells per mL) in FBS and 10% DMSO according to CCTRN SOPs. Cells are slowly frozen to -80°C in a isopropanol bath apparatus for 24-48 hrs, and transferred to liquid nitrogen tanks for long-term storage (-175°C). Cells are stored for at least ten years so that if any safety questions arise, the initially harvested samples can be made available for analysis. No samples received at the Biorepository Core Laboratory are returned to the sites for clinical application. Remaining sample above those needed for safety studies are available for ancillary studies once they are approved by the CCTRN.