Umbilical CB was obtained from informed and consenting donors at the Helsinki University Central Hospital, Department of Obstetrics and Gynaecology. Permit to collect and use donated stem cells has been obtained from the ethics board of the Helsinki University Central Hospital (550/E8/02) and the ethics board of the Finnish Red Cross Blood Service (40/02).
The umbilical cord was clamped according to standard hospital procedure and CB collections were performed ex utero. CB was collected into a sterile collection bag (Cord Blood Collection system, Medsep Corporation, Covina, USA) containing 25 ml of Citrate Phosphate Dextrose solution. The collection volume varied between 45–105 ml. Some CB units were volume reduced and cryopreserved in a BioArchive system at the Finnish Red Cross Blood Service, Cord Blood Bank as previously described [16, 19] and some units were processed freshly within hours from collection. Altogether, 8 cryopreserved and 12 fresh CB units were used to optimize the protocols. In addition, 10 cryopreserved and 9 fresh CB units were used to test the optimized protocols.
Handling of CB units
Cryopreserved CB unit was taken from the BioArchive system and kept for 2 minutes in the gas phase of liquid nitrogen, 3–5 minutes at room temperature and then in 37°C water bath until completely thawed. CB was transferred from the freezing bag into 50 ml tubes containing 10 ml of freezing solution: 2.5% albumin (Finnish Red Cross, Blood Service, Helsinki, Finland), 50% NaCl (Baxter Healthcare) and 50% Gentran40 (Baxter Healthcare). Cells were pelleted by centrifugation at 600 × g for 10 minutes and the supernatant was discarded. When cell aggregation occurred, the cell pellet was resuspended in 200 μl of 1 mg/ml DNaseI (Sigma-Aldrich, Steinheim, Germany). Cells were then suspended carefully in 100 ml of phosphate-buffered saline (PBS, pH 7.4) supplemented with 0.6% ACD/A. Fresh CB was diluted 1:4 with PBS supplemented with 2 mM EDTA, to reduce the size and number of cell aggregates and to give better lymphocyte yield in density gradient centrifugation.
MNCs were isolated by density gradient using 15 ml of Ficoll-Paque reagent (Amersham Biociences, Piscataway, USA) and 35 ml of diluted CB. The two-phase system was centrifuged at 400 × g for 40 minutes. MNCs, collected from the interface of the two phases, were washed twice with PBS. MNC counting was performed by automatic cell counter Sysmex K-1000 (Sysmex Corporation, Kobe, Japan).
Separation of CD34+/CD133+ cells
CD34+ and CD133+ cells were enriched through positive selection using MiniMACS or MidiMACS separation systems (Miltenyi Biotec). For the labeling of CD34+ cells, Direct CD34 Progenitor Cell Isolation Kit was used, whereas CD133+ cells were labeled using the CD133 Cell Isolation Kit. 100 μl of FcR Blocking Reagent, to inhibit unspecific or Fc-receptor mediated binding, and 100 μl of CD34/CD133 MicroBeads for magnetic labeling of cells were added per 108 cells, as per manufacturer's recommendations.
MS or LS MACS affinity columns were used depending on the number of MNCs. MS column was used for up to 2 × 108 MNCs, and LS column was used for 2 × 108 to 2 × 109 of MNCs. Labeled cell suspension was subjected to immunomagnetic separation where magnetically labeled cells retain in the column while the unlabeled cells pass through the column. After several washes, the column was removed from the magnet and the retained CD34+ or CD133+ cells were eluted with 1–5 ml of PBS supplemented with 0.5% bovine serum albumin and 2 mM EDTA. CD34+ and CD133+ cells were subjected to one or two rounds of separation and their negative counterparts were collected for control purposes. In the two-column system, an additional labeling step between the column separations was tested, using 25 μl of both FcR Blocking Reagent and MicroBeads. The optimum purity and yield was obtained when using the additional labeling step in connection with the two-column system.
Separation of Lin- cells
To enrich progenitor cells, lineage committed cells were depleted. MNCs (8 × 107/ml) were labeled with Progenitor Enrichment Cocktail containing antibodies against CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b and Glycophorin A (StemCell Technologies, Vancouver, Canada) at room temperature for 15 minutes. Subsequently, the cell suspension was incubated with magnetic iron particles at room temperature for 15 minutes. Varying concentrations of StemSep Progenitor Enrichment Cocktail (100 μl/ml, 75 μl/ml, and 50 μl/ml) and magnetic iron particles (60 μl/m, 45 μl/ml, and 30 μl/ml) were tested. Cell suspension was loaded into MACS LD column (Miltenyi Biotec) and unlabeled cells passing through the column were collected (Lin- fraction). The column was then washed twice with 1 ml of buffer and the remaining Lin+ cells were collected for control purposes. The conditions resulting in optimal separation of Lin- cells were 100 μl of Progenitor Enrichment Cocktail and 60 μl of magnetic iron particles per 8 × 107/ml MNCs, as recommended by the manufacturer.
To determine the purity of CD34+/- and CD133+/- cell fractions, 1 × 105 cells were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (clone 2D1, Becton Dickinson, Franklin Lakes, USA) and phycoerythrin (PE)-conjugated anti-CD34 (clone 345802 Becton Dickinson) or PE-conjugated anti-CD133 (clone 293C3, Miltenyi Biotec) monoclonal antibodies at 4°C for 15 minutes. Lin-/+ cells were labeled with PE-conjugated anti-mouse immunoglobulin specific polyclonal antibody (BD Biosciences, San Jose, USA). Platelets were detected with PE-conjugated anti-CD41a monoclonal antibody (BD Biosciences, San Jose, USA). Isotype-identical monoclonal antibodies served as controls. Labeled cells were analyzed using Becton Dickinson FACSCalibur™ with a 488 nm blue argon laser. Fluorescence was measured using 530/30 nm (FITC) and 585/42 nm (PE) bandpass filters. Data were analyzed using the ProCOUNT™ software (BD Biosciences) or Windows Multiple Document Interface for Flow Cytometry, WinMDI version 2.8 . CD34+, CD133+ and Lin- cell populations were defined by first gating on forward and side scatter properties excluding platelets and debris. Subsequent gates were set to exclude >99% of control cells labeled with isotype-specific antibody.
Colony forming unit assay
MNCs (1 × 105) and enriched HSCs (2 × 103) suspended in 300 μl Iscove's Modified Dulbecco's Medium supplemented with 2% fetal bovine serum (Gibco/Invitrogen, Paisley, United Kingdom) were mixed vigorously with 3 ml of MethoCult GF H4434 containing recombinant cytokines and erythropoietin (StemCell Technologies). The cells in MethoCult medium were plated in duplicate into sterile 35 mm petri dishes, and colonies were scored according to their morphological characteristics by light microscopy after a 14-day culture. Cell culture and colony scoring were performed according to accredited methodology (Finnish Cord Blood Bank, Finnish Red Cross Blood Service, Helsinki, Finland) following international standards [24, 25]. The CFU assay was performed in triplicate for each cell type (CD34+, CD133+, Lin- and MNC).
For statistical analysis of total MNC counts between cryopreserved and fresh CB samples, the non-parametric Mann-Whitney test was used, as the MNC counts were not normally distributed. The two groups, cryopreserved and fresh CB, were considered independent as they have been produced using different protocols. The recovery of CD34+, CD133+ and Lin- cells between cryopreserved and fresh CB was compared in independent samples by the t test and equality of variances was checked by Levene's test. When the assumption of equality was not satisfied, as with Lin- cells, the t test for non-equal variances was used to compensate the lack of homoscedasticity. P values less than 0.05 were considered statistically significant. The analyses were performed by Microsoft Excel and SPSS 12.0.1.