Discovering ATCC Hematopoietic Progenitor Cells: Model Systems to Study the Immune and Cardiovascular Systems

ATCC does not use cells from donors injected with granulocyte colony stimulating factor.

Expansion, as measured by total nucleated cells (TNC), was monitored during differentiation. Depending on the cytokine cocktail used, we observed expansion of TNC ranging from 25-fold to over 1000-fold.

Most ATCC primary cells are from single donors. However, we do have pooled normal Human Primary Umbilical Vein Endothelial Cells (ATCC PCS-100-013), which are derived from 10 individual donors, minimizing the lot-to-lot variability associated with cells derived from single donors.

Please contact ATCC Technical support at [email protected] so that we may be able to assist you further for requests for primary cells from donors with specific characteristics.

No, we have not performed any side-by-side comparisons of CD34+ hematopoietic cells with other types of stem cells.

ATCC primary hematopoietic cells are isolated directly from screened, healthy human donors according to strict AABB and FDA guidelines and immediately cryopreserved so the likelihood of mycoplasma infection is sufficiently low, therefore our primary hematopoietic cells are not tested for mycoplasma. If you wish to request a mycoplasma tested lot, or to request testing of a specific lot please contact ATCC Technical Support ([email protected]). If you wish to test for the presence of mycoplasma in your cells we offer a Universal Mycoplasma Detection Kit (ATCC 30-1012K) for purchase. We also offer ATCC’s Mycoplasma Testing Service where you may send us your cell culture samples for mycoplasma testing.

We have not assessed CD34+ cells for markers other than CD34 and CD45. The makeup of specific CD34+ subtypes contained within each vial of cells will vary due to donor and lot variability.

No, at this time ATCC does not offer CD34+ cells from diseased state donors.

We have not tested changes in marker expression in freshly isolated PBMCs or after cryopreservation. There is published literature suggesting that cryopreservation can modulate the expression of some cell surface markers and reduce apparent cell counts, particularly CD62L+ and CD4+ CD25+ FoxP3+ cells; although, some of the results are contradictory, most likely due to differences in cryopreservation methodologies. We recommend that if these markers are critical for your application to use freshly isolated PBMCs.

We have only assessed the normal Human Primary Peripheral Blood CD14+ Monocytes (ATCC PCS-800-010) for expression of CD45.

We currently have the primary cell products in stock and they are available to ship immediately.

We do not currently have plans to offer peripheral blood derived CD34+ cells. We do have the normal Human Primary Bone Marrow CD34+ Cells (ATCC PCS-800-012) and normal Human Primary Cord Blood CD34+ Cells (ATCC PCS-800-014) available.

CD133+ is another surface marker for stem-like and progenitor cells, particularly in the area of cancer stem cells. We do not currently offer cryopreserved CD133+ cells and have not tested its expression.

The literature is conflicting on the impact of a rest period on PBMCs, particularly in the context of T-cell function and immunophenotype. Published protocols vary from no rest, to a rest of several hours, to overnight. There are reports that a rest period may alter T-cell phenotype. Therefore, it is up to the end user to determine whether a rest period will or will not impact their specific application with PBMCs.

We incubate the cells in EDTA in cold PBS on ice for 15-20 minutes. Pipetting gently up and down, or using a cell scraper, will release the macrophages. We do not recommend the use of trypsin, which can damage the cells. If you are still experiencing difficulties harvesting the cells you may also try low-attachment plates.

We have not assessed CD11b expression in the mononuclear cells. Frequencies of specific cell types, as assessed by surface marker expression, will vary from donor to donor. Expressions of other surface markers (e.g., CD3, CD8) tested are reported on the Certificate of Analysis.

Negative immunomagnetic selection for these subsets of cells is not available at this time. Please contact ATCC Technical Support at [email protected] so that we may be able to assist you further with your specific requirements.

The basal medium for PBMC culture is application-specific; however, RPMI 1640 (ATCC 30-2001) supplemented with 5-10% fetal bovine serum (ATCC 30-2020) is commonly used.

The proliferation of CD34+ cells in culture may vary greatly based on the growth media that the cells are maintained in. The choice of growth media used for maintenance of the cells should be designed to selectively promote the expansion of CD34+ cells without inducing differentiation. In the context of cytokine-induced differentiation, we have observed expansion of the total cell count ranging from 25-fold to over a thousand-fold within a week.

The highest yield of total CD34+ cells from a single donor typically comes from adult bone marrow tissue. Phenotypic differences between CD34+ cord blood-derived versus bone marrow derived cells have been reported. Refer to the following literature for more information:

1. Kim D, et al. Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells. Stem Cells 17(5):286-294, 1999.
2. Kita K, et al. Cord blood-derived hematopoietic stem/progenitor cells: current challenges in engraftment, infection, and ex vivo expansion. Stem Cells Int, 2011.
3. Yang S, et al. Hematopoietic reconstitution of CD34+ cells derived from short-term cultured cord blood mononuclear cells. Biotech Bioprocess Eng 14(4):429-435, 2009.

We recommend swirling the plate vigorously to remove loosely attached cells and potentially contaminating lymphocytes and other unwanted cell types (that are not adherent). This step is critical only when using cells that are not enriched for monocytes, such as PBMCs.

Primary immune cells are screened for the following human pathogenic viruses: Hepatitis B, Hepatitis C, HIV (I/II) and HTLV(I/II). More information on ATCC’s virus testing may be found on the Certificate of Analysis for each lot of cells.

STEMCELL Technologies. More information may be found in the FAQ section on the ATCC website for the expansion/differentiation of CD34+ primary cells.