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. The CELISA assay employs a single incubation with a lanthanide-conjugated primary antibody and subsequent time-resolved fluorescence spectroscopy. The rapid-throughput nature of this assay is enhanced by the use of cell culture-compatible filter plates to reduce the number of manipulations as compared to currently available cell-based assays. The culture and assay are done in 96-well plates, and the quantitation process requires approximately 1 hour. The myeloid, erythroid, and megakaryocytic lineages can be objectively quantified; data from the assay correlate extremely well with data generated through use of the traditional colony assay. This assay makes possible both rapid-throughput drug discovery and toxicity screening in the area of hematopoiesis.

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. Growth and differentiation was assessed by total, progenitor (CFU-GM), stromal (CFU-F), and primitive (LTC-IC) cell output. Tissue culture treated (TCT) plastic significantly increased MNC culture output as compared with non-TCT plastic, whereas CD34-enriched cell cultures gave lower output (than MNC cultures) that was unaffected by TCT plastic. Interestingly, the level of MNC culture output was significantly different on four commercial TCT surfaces, with the best performing surface giving output that was 1.6- to 2.8-fold greater than the worst one. The surface giving the highest output was the best at supporting development of a distinct morphological feature in the adherent layer (i.e. cobblestone area) indicative of primitive cells, and X-ray photoelectron spectroscopy (XPS) was used to characterize this surface. For custom injection molding of culture devices, the use of three different resins resulted in MNC culture output that was equivalent to commercial cultureware controls, whereas CD34-enriched cell cultures were highly sensitive to resins containing additives. When the texture of molded parts was roughened by sandblasting of the tool, MNC culture output was significantly reduced and higher spikes of IL-6 and G-CSF production were observed, presumably due to macrophage activation. In conclusion, the manufacture of BM MNC culture devices for clinical applications was optimized by consideration of plastic resin, surface treatment, and texture of the culture substratum. Although CD34-enriched cells were insensitive to surface treatment, they were considerably more sensitive to biocompatibility issues related to resin selection. The development of robust systems for BM MNC expansion will enable clinical trials designed to test the safety and efficacy of cells produced in this novel tissue engineering application.

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. Allogeneic human plasma (20%) supported half the level of cell, CFU-GM, and LTC-IC output as compared with animal sera-containing control. Significant donor-to-donor variability in human plasma was observed, but this was mitigated by pooling of plasma samples. Autologous and allogeneic human plasma performed equivalently. The use of autologous or allogeneic human serum was found to be equivalent to the use of human plasma, but all were inferior to animal sera. Animal sera supported typical stroma and cobblestone formation, whereas stroma in human serum cultures was less dense. Eight commercial serum-free media were tested and found to support MNC expansion to varying degrees, but none approached the performance of the animal serum-containing control, particularly with respect to stromal (i.e., CFU-F) support. In fact, when MNC were cultured in parallel with CD34-enriched cells, output (from MNC) was higher only in control medium, apparently because serum-free media reduced accessory cell effects. Because of these results, a new serum-free medium was developed for MNC cultures. This formulation outperformed all commercial serum-free media, resulting in cell and LTC-IC output equivalent to that of control. However, CFU-GM and CFU-F output were 66% and 9% of control, respectively. Precoating the culture surface with collagen increased CFU-F (and Thy-1+ cell) output to control levels, although CFU-GM output was still lower than control. The addition of either fibronectin or PDGF had no measurable effect, nor did the use of 5-100-fold greater concentrations of growth factor supplementation. The serum-free medium also increased CD41+ and CD61+ cell output to 150%-220% of control levels. The development of this new serum-free medium has potential for use in the perfused BM MNC culture systems currently in clinical trials to test the efficacy of expanded cells after cytoablative chemotherapy.

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. Measurement of secondary colony-forming cells (2 degrees CFCs) from 5-week cultures of bone marrow (BM) mononuclear cells (MNCs) showed that 2 degrees CFC frequency varied with assay plating density in a nonlinear fashion. The measured 2 degrees CFC frequency increased from 4.6 to 63.8 (per 10(5) MNCs) as assay plating density was decreased from 5 x 10(5) to 2 x 10(4) MNCs per well (P < 10(-6), n = 37). In contrast, assay of CD34-enriched cells was linear within the range studied. Assays of cells obtained from expansion cultures initiated with either MNCs or CD34-enriched cells were also nonlinear. Consequently, calculated 2 degrees CFC expansion ratios were ambiguous and dependent on the assay plating densities used. Limiting dilution analysis (LDA) results were also nonlinear, with LTC-IC frequency increasing from 8. 2 to 22.4 per 10(5) MNCs (P < 10(-4), n = 100) as assay plating densities were decreased. Despite the nonlinearity, 2 degrees CFC and LTC-IC assay results were consistent and reproducible over time with different samples and techniques and gave a semiquantitative indication of relative primitive cell frequency. Although CD34-enriched cells gave linear assay output, purification of cells for every assay is impractical. Therefore, exposure of cells to 5-fluorouracil (5-FU) was explored for improving assay linearity. Incubation of MNCs in 250 microg/mL 5-FU for 1 to 2 hours depleted accessory cells and resulted in a cell population that gave linear 2 degrees CFC readout. The 5-FU-resistant LTC-ICs accounted for 49% of the total LTC-IC population, adding the potential benefit of restricting assay measurement to more primitive noncycling LTC-ICs. Consequently, similar linear assay results can be obtained with either the bulk 2 degrees CFC or LDA LTC-IC methods after 5-FU, but multiple plating densities are nevertheless still required in both methods due to the greater than 100-fold range in primitive cell frequency present in normal human donor BM.

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. Twelve days of culture resulted in the optimal output from most CB samples. Frequent medium exchange led to significant increases in cell (93%), CFU-GM (82%) and LTC-IC (350%) output as compared with unfed cultures. As the inoculum density was increased from 7.5 x 10(4) per cm2 to 6.0 x 10(5) per cm2, the output of cells, CFU-GM, and LTC-IC increased. Cell and CFU-GM output reached a plateau at 6.0 x 10(5) per cm2, whereas LTC-IC output continued to increase up to 1.2 x 10(6) per cm2. Because the increase in culture output did not increase linearly with increasing inoculum density, expansion ratios were greatest at 1.5 x 10(5) per cm2 for cells (6.4-fold) and CFU-GM (192-fold). Despite the lack of adherent stroma, CB cultures expressed mRNA for many growth factors (G-CSF, GM-CSF, IL-1, IL-6, LIF, KL, FL, Tpo, TGF-beta, TNF-alpha, and MIP-1alpha) that were also found in bone marrow (BM) cultures, with the exception of IL-11 (found only in BM) and IL-3 (found in neither). Culture output was remarkably consistent from 10 CB samples (coefficient of variation 0.3) indicating that the procedure is robust and reproducible. Two commercial serum-free media were evaluated and found to support only approximately 25% of the culture output as compared with serum-containing medium. Implementation of optimal conditions in the clinical scale, automated cell production system (CPS) showed that the process scaled-up well, generating 1.7 x 10(7) CFU-GM (298-fold expansion) from 1.2 x 10(8) thawed viable nucleated CB cells (n = 3). The ability to generate >10(7) CFU-GM from <15 ml of CB within this closed, automated system without the need for extensive cell manipulations will enable clinical studies to test the safety and efficacy of expanded CB cells in the transplant setting.

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]). When parenchymal CD34-enriched cells were grown without stroma, cell and CFU-GM output increased linearly as inoculum density was increased, resulting in constant cell and CFU-GM expansion ratios. On irradiated preformed stroma (IPFS), culture output was significantly higher and less dependent on CD34-enriched cell inoculum density, resulting in greater expansion ratios at lower inoculum densities. The number of IPFS cells required to support CD34-enriched cells was independent of the CD34-enriched cell number, suggesting that IPFS did not provide discrete niches, but instead acted through soluble signals. Experiments using conditioned medium (CM) from IPFS confirmed the presence of soluble signals, but CM did not completely substitute for direct contact between CD34-enriched cells and IPFS. Because of known differences between IPFS and stroma growing within BM mononuclear cell (MNC) cultures, experiments were next performed using mixtures of CD34-enriched and CD34-depleted fractions of MNC. When inoculated with a fixed CD34+lin- cell number, culture output was optimal near the P:S ratio of the unmanipulated MNC sample and declined as CD34- cell number was increased or decreased. In cultures inoculated with a fixed total cell number, CFU-GM output increased as CD34+lin- cell number was increased, whereas LTC-IC output reached a plateau. These data suggest that a limited number of LTC-IC supportive niches were present in MNC stroma, whereas IPFS lacks these niches and acts predominantly through a less potent soluble mechanism. These studies underscore the importance of parenchymal-stromal cell interactions in the ex vivo reconstitution of tissue function and offer insight into the nature of these interactions in the human BM culture system.

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. CD34-enriched cell cultures, plated to contain 3000 CD34+lin- cells per well, exhibited a wide range of cell output (0.02 to 5.07 x 10(6)) with a high coefficient of variation (CV = 0.69, n = 52). The range in CFU-GM output was even greater (12 to 9455, CV = 0.90). Addition of preformed stroma had a significant positive effect, and resulted in narrower ranges of cell (0.19 to 8.27 x 10(6), CV = 0.41) and CFU-GM (218 to 17586, CV = 0.54) output. A wide range of stromal-dependency was exhibited by CD34-enriched cells from different donors, with stroma augmenting cell output by 1.2- to 14-fold (mean 3.5), and CFU-GM output by 1.7- to 24-fold (mean 6.5). In contrast, changes in the soluble growth factor combination affected cells from different donors in a similar fashion, thereby altering the mean level of performance without reducing donor-to-donor variability. Experiments were next performed to assess the relative contribution of CD34+lin- cells and stromal cells to culture variability by culturing CD34+lin- cells from three donors on preformed stroma from three donors in parallel. Variability in culture output was attributed to the CD34+lin- cell donor, whereas stroma from different autologous or allogeneic donors gave similar performance. Therefore, both expansion potential and stromal-dependency were inherent characteristics of CD34+lin- cells from different donors. Donor characteristics (i.e., sex, age, weight, and height) and flow cytometric assays (i.e., CD34+lin- cell purity, and CD38-, Thy-1+, and c-kit+ subsets thereof) were not well correlated with expansion potential. In contrast, many of the different biological characteristics (i.e., inoculum CFU-GM, cell and CFU-GM output, and stromal-dependency) were strongly correlated with each other. Mononuclear cell (MNC) cultures, which provide an accessory cell environment (including endogenous stroma) in which CD34+lin- cells grow, were compared with CD34-enriched cell cultures. MNC cultures (containing 3000 CD34+lin- cells) were found to give the greatest and most consistent cell (2.51 to 5.20 x 10(6), CV = 0.17) and CFU-GM (2618 to 14,745, CV = 0.46) output. These results have significant implications for the design of clinical trials of cultured hematopoietic cells, as well as for the understanding of diversity in human stem cell behavior. Furthermore, the results demonstrate the importance of a large sample size in scientific studies of primary human hematopoietic cell behavior.

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. In this study, three culture variables were used to control ex vivo human hematopoiesis. Systematic variation of inoculum density (ID), medium exchange interval (MEI), and the use of preformed stroma (PFS) showed that (1) all three variables significantly influenced culture performance, (2) the three variables interacted strongly, and (3) the variables could be manipulated to achieve the optimization of different performance criteria. Donor-to-donor variability in culture performance was great at low ID but was minimized at higher ID. PFS had a large positive effect on cell and CFU-GM output at low ID, but had minimal effect at higher ID. In fact, PFS caused a decrease in LTC-IC output at high ID. The effects of PFS indicated that stromal cell elements became more limiting than proliferative cell elements as ID was reduced.In cultures without PFS, maximum cell output was obtained with high ID using a short MEI, whereas the greatest cell expansion ratio was obtained at low ID with an intermediate MEI. Maximum CFU-GM output was obtained from cultures with high ID using a short to intermediate MEI, whereas the greatest CFU-GM expansion ratio was obtained at intermediate ID with an intermediate MEI. The addition of PFS altered the locations of these maxima. In general, PFS moved the maxima to lower ID, and culture output became more sensitive to MEI. Therefore, the optimization of one performance criterion always resulted in a decline of the others. This study demonstrates that ex vivo tissue function is sensitive to many culture variables in an interactive fashion and that systematic multivariable studies are required to characterize tissue function. Once the effects of individual variables and their interactions are known, this knowledge can be used to optimize tissue performance with respect to desired criteria.

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. In stromal-free, CD34-enriched cell cultures, medium exchange intervals ranging from 2 days to no feeding for 14 days gave similar results. Six different growth factor combinations, reported by other groups to give optimal expansion of CD34-enriched cells, were tested in comparison with the control combination of IL-3/GM-CSF/Epo/SCF. None of the combinations resulted in improved colony-forming unit-granulocyte macrophage (CFU-GM) expansion or LTC-IC maintenance, although two were equivalent. All stromal-free cultures resulted in loss of LTC-IC to half of input. Because of the limited effect of medium exchange and growth factor variations on CD34-enriched cell cultures, the effect of preformed stroma was next examined. Preformed stroma increased cell (3-fold), CFU-GM (5-fold), and LTC-IC (3-fold) output, but only when the medium was exchanged every other day. Under these conditions, the number of LTC-IC was maintained near input level. The lack of LTC-IC expansion in CD34-enriched cell cultures prompted experiments to examine the effect of cell purification. Parallel cultures were performed at CD34+lin- cell purities of 20%, 40%, 70%, and 95%, with each well containing exactly 4,000 CD34+lin- cells in addition to the CD34- accessory cells required to give the desired percentage. Also, MNC from the same source (approximately 2% CD34+lin-) were cultured at a concentration to give 4,000 CD34+lin- cells per well. As CD34+lin- cell purity was decreased from 95% to 2%, the output of cells, CFU-GM, and LTC-IC increased by threefold to fivefold. The loss of culture performance with purification was likely due to the removal of important accessory cells, because the levels of endogenously produced leukemia inhibitory factor and IL-6 were found to decline significantly with increasing CD34+lin- cell purity. In summary, preformed stroma abrogated the decrease in cell and CFU-GM output from cultured CD34-enriched cells and led to LTC-IC maintenance. In contrast, MNC inocula resulting in a growing stromal layer during the culture led to LTC-IC expansion (3.2-fold).(ABSTRACT TRUNCATED AT 400 WORDS)

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. Subsequent CD34 selection recovered 17% of CFU-GM and 48% of LTC-IC from the MNC fraction. The unabsorbed (CD34-depleted) fraction contained 37% of CFU-GM and 38% of LTC-IC, accounting for most of the lost cells. WBMC, BMMNC, and CD34-depleted cells were each placed directly in bioreactors, whereas CD34-enriched cells were placed in bioreactors containing preformed irradiated stroma. After 14 days, an average of 3.82 x 10(7) (12.7-fold expansion), 3.54 x 10(7) (11.8-fold), 2.85 x 10(7) (9.5-fold), and 3.65 x 10(7) (1298-fold) total cells were obtained from bioreactors inoculated with WBMC, BMMNC, CD34-depleted, and CD34-enriched cells on stroma, respectively. These cultures yielded 1.64 x 10(5) (27.9-fold expansion), 1.69 x 10(5) (14.3-fold), 8.36 x 10(4) (13.0-fold), and 1.91 x 10(5) (41.4-fold) CFU-GM each, respectively. Cell recovery and expansion data were combined to determine the number of expanded CFU-GM obtained per ml of BM aspirate, allowing direct comparison of performance between the four culture inocula. WBMC generated 3.76 x 10(6) CFU-GM per ml BM aspirate, whereas MNC resulted in 1.42 x 10(6) CFU-GM. CD34-enriched cells (on irradiated stroma) gave 7.00 x 10(5) CFU-GM per ml BM aspirate, whereas CD34-depleted cells generated 4.97 x 10(5) CFU-GM. The high productivity from WBMC cultures was studied further and was found to be reproducible at different inoculum densities. WBMC cultures had elevated levels of endogenous EGF and PDGF production, which may have been responsible for the more extensive stromal development observed. Flow cytometric analysis showed that the final culture composition, with respect to T and B lymphocytes, monocytes, granulocytes, and erythrocytes, was not significantly affected by the inoculum composition and in all cases was comprised of multiple lineages. Therefore, each step in cell purification resulted in the loss of primitive and accessory cells, which in turn resulted in a net decrease in the number of expanded cells obtained per ml BM aspirate.