Isolation of CD34+ Cells from Human Cord Blood
- Document # 27003
- Version 2.0.0
- Dec 2024
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 贰补蝉测厂别辫鈩 cell separation kits for the isolation of CD34+ cells from whole CB that can be used with samples containing different amounts of platelets.
Advantages
- Combine your standard density gradient centrifugation step with 搁辞蝉别迟迟别厂别辫鈩 pre-enrichment, then isolate purified CD34+ cells with 贰补蝉测厂别辫鈩.
- Perform 搁辞蝉别迟迟别厂别辫鈩 separation in SepMate鈩 tubes to minimize variability between separations and increase sample throughput.
- Use on whole CB with or without platelet depletion.
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 搁辞蝉别迟迟别厂别辫鈩 Human Cord Blood CD34 Pre-Enrichment Cocktail containing antibodies that bind to mature blood cells. Two 搁辞蝉别迟迟别厂别辫鈩 antibody cocktails have been developed for CD34+ cell pre-enrichment.
- Human Cord Blood CD34 Positive Selection Kit II (Catalog #17896) contains a 搁辞蝉别迟迟别厂别辫鈩 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 搁辞蝉别迟迟别厂别辫鈩 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.
搁辞蝉别迟迟别厂别辫鈩 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. 尝测尘辫丑辞辫谤别辫鈩) 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鈩 tube. The SepMate鈩 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 贰补蝉测厂别辫鈩 isolation cocktail and magnetic 贰补蝉测厂别辫鈩 Dextran RapidSpheres鈩 (Figure 1, steps 5 - 6). The CD34+ cells are then isolated by placing the tube containing the immunomagnetically-labeled cell suspension into an appropriate 贰补蝉测厂别辫鈩 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.
贰补蝉测厂别辫鈩 Human Cord Blood CD34 Positive Selection Kit and 贰补蝉测厂别辫鈩 magnet (magnet sold separately)
Platelet Removal
Depletion of platelets during 搁辞蝉别迟迟别厂别辫鈩 pre-enrichment, prior to 贰补蝉测厂别辫鈩 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 搁辞蝉别迟迟别厂别辫鈩 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 搁辞蝉别迟迟别厂别辫鈩 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
Figure 1. Combined 搁辞蝉别迟迟别厂别辫鈩 and 贰补蝉测厂别辫鈩 Protocol for Isolation of CD34+ Cells
CD34+ cells are isolated from whole CB using 搁辞蝉别迟迟别厂别辫鈩 (steps 1 - 4) and 贰补蝉测厂别辫鈩 (steps 5 - 8). Pre-enrichment with 搁辞蝉别迟迟别厂别辫鈩 can be performed with SepMate鈩 tubes to allow faster layering of a sample over the density gradient medium as an insert within the tube prevents layers from mixing. After centrifugation, with the brake on when using SepMate鈩, or with the brake off for standard conical tubes, pre-enriched CD34+ cells are collected into a fresh tube (step 4). The 贰补蝉测厂别辫鈩 isolation cocktail and Dextran RapidSpheres鈩 are then added to the pre-enriched cells to label the CD34+ cells (steps 5 - 6). The labeled cells are placed into a magnet to positively select for CD34+ cells. After isolation, purified cells are ready for downstream applications. For detailed procedures on manual cell separation methods using either the 贰补蝉测厂别辫鈩 purple magnet (Catalog #18000), 鈥淭he Big Easy鈥 magnet (Catalog #18001), the EasyEights鈩 magnet (Catalog #18103), or for automated cell separation using RoboSep鈩 instruments (Catalog #21000/23000), please refer to the Product Information Sheet (PIS) for each kit.
Data
Human Cord Blood CD34 Positive Selection Kit II
Figure 2. Isolation of CD34+ CB Cells Using 贰补蝉测厂别辫鈩 Human Cord Blood CD34 Positive Selection Kit II
CD45 and CD34 expression of cells before separation (鈥淪tart鈥), after 搁辞蝉别迟迟别厂别辫鈩 (鈥淧re-Enriched鈥), and after selection of CD34+ cells (鈥淚solated鈥) using 贰补蝉测厂别辫鈩 Kit II (Catalog #17896). Results of a typical experiment are shown. CD45+CD34+ HSPCs (top right quadrant) have been enriched > 15-fold (from 0.6% to 10%) after 搁辞蝉别迟迟别厂别辫鈩 pre-enrichment and > 200-fold (from 0.6% to 98%) after 贰补蝉测厂别辫鈩 CD34+ selection (鈥淚solated鈥). The flow cytometry data shown are gated on cells with intermediate to high forward light scatter (FSC), and which are negative for propidium iodide (PI) staining to exclude debris, RBCs, platelets and dead cells. The results of cell separations with 15 different CB samples are summarized in Table 1.
Table 1. Purity of CD34+ Cells Isolated from Different CB Samples Using 贰补蝉测厂别辫鈩 Human Cord Blood CD34 Positive Selection Kit II
Human Cord Blood CD34 Positive Selection Kit III
Figure 3. Isolation of CD34+ CB Cells Using 贰补蝉测厂别辫鈩 Human Cord Blood CD34 Positive Selection Kit III
CD45 and CD34 expression of cells before separation (鈥淪tart鈥), after 搁辞蝉别迟迟别厂别辫鈩 (鈥淧re-Enriched鈥), and after selection of CD34+ cells (鈥淚solated鈥) using 贰补蝉测厂别辫鈩 Kit III (Catalog #17897). Results of a typical experiment are shown, gated as described in Figure 2. The results of cell separations with 10 different CB samples are summarized in Table 2.
Table 2. Purity of CD34+ Cells Isolated from Different Fresh CB Samples Using 贰补蝉测厂别辫鈩 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 贰补蝉测厂别辫鈩 kits to isolate CD34+ cells from cord blood in your own lab.
Unsure which CD34 positive selection kit to choose? We can help. Contact us by phone or email or use the LiveChat function on this page to discuss your specific needs and applications.
Required Reagents*
- 50 mL conical tubes, e.g. SepMate鈩-50 tubes (Catalog #15450)
- 5 mL or 14 mL polystyrene round-bottom tubes
- 贰补蝉测厂别辫鈩 Buffer (Catalog #20144), RoboSep鈩 Buffer (Catalog # 20104), or PBS containing 2% fetal bovine serum (FBS) and 1 mM EDTA
- Density gradient medium, e.g. 尝测尘辫丑辞辫谤别辫鈩 (Catalog #18060)
- 贰补蝉测厂别辫鈩 Magnet (Catalog #18000/18001/18103) or RoboSep鈩 fully automated cell separator (Catalog #21000/23000)
*Not included in the kits
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 贰补蝉测厂别辫鈩 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鈩 serum-free media supplemented with cytokines and/or other supplements. StemSpan鈩 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鈩 medium, and in LTC-IC assays using MyeloCult鈩 medium.
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