Background

The isolation of CD34+ cells from human umbilical cord blood (CB) is an important step in hematopoietic stem and progenitor cell (HSPC) research. Great care must be taken to isolate CD34+ cells with high purities and yields as the frequency of CD34+ cells in CB is low (typically 0.1 - 1% of nucleated cells), sample volumes are small, and cell quality and viability can be variable between different CB samples. This technical bulletin describes two new EasySep™ cell separation kits for the isolation of CD34+ cells from whole CB that can be used with samples containing different amounts of platelets.

Positive Selection of Cord Blood CD34⁺ Cells

CD34+ cells are isolated from whole CB using a simple, two-step procedure. First, standard density gradient centrifugation is combined with pre-enrichment of CD34+ cells using a RosetteSep™ Human Cord Blood CD34 Pre-Enrichment Cocktail containing antibodies that bind to mature blood cells. Two RosetteSep™ antibody cocktails have been developed for CD34+ cell pre-enrichment.

• Human Cord Blood CD34 Positive Selection Kit II (Catalog #17896) contains a RosetteSep™ antibody cocktail that binds to T cells, B cells, red blood cells (RBCs), myeloid cells, and platelets. This cocktail is recommended for pre-enrichment of CD34+ cells from CB samples that contain large amounts of platelets, which may affect the quality and purity of CD34+ cells if not depleted.
• Human Cord Blood CD34 Positive Selection Kit III (Catalog #17897) contains a RosetteSep™ antibody cocktail that binds to T cells, B cells, RBCs, and myeloid cells. This cocktail is recommended for pre-enrichment of CD34+ cells from CB samples containing few platelets or when platelet depletion is not desired.

RosetteSep™ antibody cocktails cross-link unwanted mature cells to RBCs present in a sample, forming dense immunorosettes. Cross-linked samples are then layered over standard density gradient medium (e.g. Lymphoprep™) and separated by centrifugation (Figure 1, steps 1 - 3). The fraction enriched for CD34+ cells is collected from the interface between the plasma and density gradient medium, while unwanted mature cells and RBCs stay behind in the pellet (Figure 1, steps 3 - 4).

Density gradient centrifugation can also be performed using a SepMate™ tube. The SepMate™ tube contains a unique insert that prevents the density gradient medium and blood sample from mixing. The density gradient medium is first added to the tube by pipetting through a central hole in the insert. The sample is then poured or rapidly pipetted on top of the insert. This eliminates the need to carefully layer the sample directly onto the density gradient medium, an otherwise time-consuming step. Only 10 minutes of centrifugation is required, and this step can be carried out with the centrifuge brake on, further reducing the total time necessary for separation. After centrifugation, the fraction enriched for CD34+ cells is simply poured into a new tube.

In the second step, the pre-enriched CD34+ cells are labeled by incubating with the EasySep™ isolation cocktail and magnetic EasySep™ Dextran RapidSpheres™ (Figure 1, steps 5 - 6). The CD34+ cells are then isolated by placing the tube containing the immunomagnetically-labeled cell suspension into an appropriate EasySep™ magnet. After a brief incubation period, unwanted CD34- cells are simply poured off, while the desired CD34+ cells remain in the tube (Figure 1, steps 7 - 8). Isolated CD34+ cells can then be used for downstream applications.

Platelet Removal

Depletion of platelets during RosetteSep™ pre-enrichment, prior to EasySep™ CD34+ selection, allows for the improved recovery of higher quality purified cells than with traditional centrifugebased methods. However, in some cases platelet depletion may not be necessary or desired.

• The RosetteSep™ antibody cocktail in Kit II (Catalog #17896) labels platelets, depleting these cells during the pre-enrichment step. This kit is recommended for samples that are > 24 hours old and that may show variable amounts of platelet activation and/or aggregation. Results of typical experiments with Kit II are shown in Figure 2 and Table 1 (page 3).
• The RosetteSep™ antibody cocktail in Kit III (Catalog #17897) does not label platelets and is recommended for very fresh CB samples (< 24 hours after delivery), samples that contain very few platelets, and samples for which platelet depletion is not desired. Results of typical experiments with Kit III are shown in Figure 3 and Table 2 (page 3).

General Protocol for Pre-Enrichment and Positive Selection of CD34+ Cells

Data

Human Cord Blood CD34 Positive Selection Kit II

Human Cord Blood CD34 Positive Selection Kit III

Product Information

*Each kit contains reagents sufficient for processing up to 1000 mL of CB.

Try using these EasySep™ kits to isolate CD34+ cells from cord blood in your own lab.

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Required Reagents*

• 50 mL conical tubes, e.g. SepMate™-50 tubes (Catalog #15450)
• 5 mL or 14 mL polystyrene round-bottom tubes
• EasySep™ Buffer (Catalog #20144), RoboSep™ Buffer (Catalog # 20104), or PBS containing 2% fetal bovine serum (FBS) and 1 mM EDTA
• Density gradient medium, e.g. Lymphoprep™ (Catalog #07801)
• EasySep™ Magnet (Catalog #18000/18001/18103) or RoboSep™ fully automated cell separator (Catalog #21000/23000)

Other Kits for the Isolation of Human CD34+ Cells

• To isolate CD34+ cells from fresh adult peripheral blood or buffy coat cells, we recommend the Complete Kit for Human Whole Blood CD34+ Cells (Catalog #15086).
• To isolate CD34+ cells from other samples, including fresh or previously frozen mobilized peripheral blood or bone marrow mononuclear cells, or from previously frozen CB mononuclear cells, we recommend using the EasySep™ Human CD34 Positive Selection Kit II (Catalog #17856).

Culture of CD34+ Cells

After isolation, CD34+ cells may be cultured using specialized cell culture media such as StemSpan™ serum-free media supplemented with cytokines and/or other supplements. StemSpan™ expansion supplements contain optimized combinations of cytokines and supplements to either promote the expansion of HSPCs or their lineage-specific differentiation to generate large numbers of mature erythroid, megakaryocyte, or myeloid cells, dependant on the type of supplement used. Purified CD34+ cells can also be analyzed in colony-forming unit (CFU) assays using methylcellulose-based MethoCult™ medium, and in LTC-IC assays using MyeloCult™ medium.

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