High throughput cell-based assay of hematopoietic progenitor differentiation.

The in vitro efficacy of drug candidates relative to hematopoietic stem cell proliferation and differentiation is currently assayed through use of the clonogenic "colony assay." The extremely low throughput of this assay precludes its use in library screening and much drug discovery work. A rapid-throughput assay of progenitor cell differentiation based on the quantification of hematopoietic lineage-specific markers has been developed.

Tissue culture surface characteristics influence the expansion of human bone marrow cells.

Human cell therapy applications in tissue engineering, such as the ex vivo production of hematopoietic cells for transplantation, have recently entered the clinic. Although considerable effort has been focused on the development of biological processes to generate therapeutic cells, little has been published on the design and manufacture of devices for implementation of these processes in a robust and reproducible fashion at a clinical scale. In this study, the effect of tissue culture surface chemistry and texture was assessed in human bone marrow (BM) mononuclear cell (MNC) and CD34-enriched cell cultures.

Alternatives to animal sera for human bone marrow cell expansion: human serum and serum-free media.

The increasing use of cultured human cells in clinical trials is highlighting the need for alternatives to media containing animal sera that are typically used to support these cultures. Perfused cultures of BM mononuclear cells (MNC) were used to evaluate animal sera alternatives with respect to the output of primitive, progenitor, and stromal cells. A serum level of 20% was optimal, and this could be provided by FBS alone or by a mixture of horse serum (HoS) and FBS, but not by HoS alone.

Quantitative long-term culture-initiating cell assays require accessory cell depletion that can be achieved by CD34-enrichment or 5-fluorouracil exposure.

Characterization of hematopoietic cells and measurement of their proliferative potential is critical in many research and clinical applications. Because in vivo assay of human cells is not possible and xenogeneic assays are not yet routine, in vitro assays such as the long-term culture-initiating cell (LTC-IC) assay have been widely adopted. This study investigated LTC-IC assay linearity and reproducibility and resulting implications with respect to quantitation of primitive cell expansion.

Clinical-scale human umbilical cord blood cell expansion in a novel automated perfusion culture system.

Use of umbilical cord blood (CB) for stem cell transplantation has a number of advantages, but a major disadvantage is the relatively low cell number available. Ex vivo cell expansion has been proposed to overcome this limitation, and this study therefore evaluated the use of perfusion culture systems for CB cell expansion. CB was cryopreserved using standard methods and the thawed unpurified cells were used to initiate small-scale cultures supplemented with PIXY321,flt-3 ligand, and erythropoietin in serum-containing medium.

Importance of parenchymal:stromal cell ratio for the ex vivo reconstitution of human hematopoiesis.

Many new developments in tissue engineering rely on the culture of primary tissues which is composed of parenchymal and mesenchymal (stromal) cell populations. Because stroma regulates parenchymal function, the parenchymal:stromal cell (P:S) ratio will likely influence culture behavior. To investigate parenchymal-stromal cell interactions, the P:S ratio was systematically varied in a human bone marrow (BM) culture system, measuring the output of mature cells, immature progenitors (colony forming units-granulocyte/macrophage [CFU-GM]), and primitive stem cells (long-term culture-initiating cells [LTC-IC]).

Donor-to-donor variability in the expansion potential of human bone marrow cells is reduced by accessory cells but not by soluble growth factors.

Clinical trials assessing the utility of cultured hematopoietic cells for the support of patients receiving high-dose chemotherapy are beginning. Although many reports have described these cultures, little is known about the donor-to-donor variability that might be expected to occur in widespread use. Therefore, this study was undertaken to assess variables which might predict and reduce the donor-to-donor variability in cell expansion potential.

Different measures of human hematopoietic cell culture performance are optimized under vastly different conditions.

Hematopoiesis, the formation of mature blood cells from stem (LTC-IC) and progenitor (CFU-GM) cells in the bone marrow, is a complex tissue-forming process that leads to many important physiological functionalities. Consequently, a functioning ex vivo hematopoietic system has a variety of basic scientific and clinical uses. The design and operation of such a system presents the tissue engineer with challenges and choices.

Long-term culture-initiating cell expansion is dependent on frequent medium exchange combined with stromal and other accessory cell effects.

Despite considerable effort, the expansion of long-term culture-initiating cells (LTC-ICs) in cultures of purified hematopoietic cells has not yet been achieved. In contrast, LTC-IC expansion has been attained in cultures of bone marrow mononuclear cells (MNC) using frequent medium exchange. The use of frequent medium exchange was, therefore, examined in cultures of CD34-enriched cells.

Bioreactor expansion of human bone marrow: comparison of unprocessed, density-separated, and CD34-enriched cells.

Scale-up of human hematopoietic cultures was previously described in continuously perfused systems with bone marrow mononuclear cells (BMMNC), yielding expansion of both progenitors and long-term culture-initiating cells (LTC-IC). We report here on the use of these systems for expansion of unprocessed whole BM cells (WBMC) and CD34-enriched cells. Density separation recovered 84% of CFU-GM and 65% of LTC-IC from WBMC.