Summary: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 ؋ 10 4 per cm 2 to 6.0 ؋ 10 5 per cm 2 , the output of cells, CFU-GM, and LTC-IC increased. Cell and CFU-GM output reached a plateau at 6.0 ؋ 10 5 per cm 2 , whereas LTC-IC output continued to increase up to 1.2 ؋ 10 6 per cm 2 . Because the increase in culture output did not increase linearly with increasing inoculum density, expansion ratios were greatest at 1.5 ؋ 10 5 per cm 2 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-␤, TNF-␣, and MIP-1␣) 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 ؋ 10 7 CFU-GM (298-fold expansion) from 1.2 ؋ 10 8 thawed viable nucleated CB cells (n = 3). The ability to generate Ͼ Ͼ Ͼ10 7 CFU-GM from Ͻ15 ml of CB within this closed, automated system without the need for extensive cell manipulations

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)

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.

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.

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 ( -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.