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Expansion and Differentiation of Human Hematopoietic Cells from Static Cultures through Small-scale Bioreactors Charles A. Sardonini’ and Ying-Jye Wut CytoMed, Inc., 840 Memorial Drive, Cambridge, Massachusetts 02139
Maintenance of the progenitor cells responsible for hematopoiesis has generally been accomplished using a feeder layer of stromal cells in stationary culture. Here, we compared the expansion of the total cell and progenitor cell populations using lowdensity mononuclear cells (LDMCs) obtained from human bone marrow in static culture (T-flasks)and in different cell culture bioreactors designed for the scale-up of mammalian cells. Static cultures were performed without the presence of a previously established stromal cell layer. Expansion of marrow in all cases was accomplished through the use of added cytokines such as IL-3, GM-CSF, and c-kit ligand. The results for the total cell expansion in static culture ranged from 4.4- to 32-fold. The cell number increase was affected by such factors as patient to patient variability, freeze-thawing, and the combination of cytokines used. Due to widespread use and the small amount of marrow needed, static cultures were used as a basis for comparison with other expansion systems. The cell culture systems used to evaluate the scale-up of marrow cultures included suspension, microcarrier, airlift, and hollow fiber bioreactors. Using identical media, cytokines, and feed schedules, LDMCs in the suspension bioreactor expanded to a value of 1.6 compared to a normalized value of 1.0 for static cultures for the two runs investigated. Expansion results for microcarrier cultures averaged 0.75 when compared to static cultures. A cell number increase in the airlift bioreactor resulted in an expansion which was 0.70 of the control static culture. Granulocyte-macrophage and erythroid progenitor assay data were also evaluated for the suspension, microcarrier, and airlift bioreactors. A single run for the hollow fiber system demonstrated no observable expansion of hematopoietic cells when compared to control static cultures. However, this finding may be a reflection of the inefficiency in retrieving cells from the extracapillary space of the hollow fiber bioreactor. Although the expansion results observed here are of limited clinical utility, these studies provide a basis for future development for the scale-up of hematopoietic cultures.
Introduction Following the development of “Dexter culture” where hematopoietic cells were cocultured in the presence of a stromal layer (Dexter et al., 1984; Chang et al., 19891,new methods for supporting bone marrow cell growth in the absence of a preestablished stromal layer have been developed. Many of these studies involve the use of cytokines in liquid culture. Examples include evaluation of IL-1, IL-3, and IL-6 (Koller et al., 1992a; Ottmann et al., 1991;Iscove et al., 1989) and the effects of c-kit ligand with other cytokines (Brandt et al., 1992;Migliaccio et al., 1991; deVries et al., 1991). Other investigations have evaluated serum-deprived conditions (Migliaccio et al., 1990; Ottmann et al., 1991; Migliaccio et al., 19911, the effects of perfusion rates (Koller et al., 1992c;Caldwell et al., 1991;Schwartz et al., 1991),the effects of oxygen tension (Koller et al., 1992b,c; Smith and Broxmeyer, 1986),and the effects of sampling on culture longevity (Varma et al., 1992). Recent clinical studies have used bone marrow which has been cultured in vitro for transplantation (Chang et al., 1986; Chang et al., 1989). The above techniques involve culturing hematopoietic cells in static culture (T~~
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* Corresponding author.
+ Current address: Matritech, Inc., 763 Concord Avenue, Cambridge, MA 02138.
87567938/93/3009-0131$04.00/0
flasks and multiwell plates) with or without added cytokines under a variety of conditions. Compared to the scale of traditional in vitro culture methods (i.e., T-flasks),relatively large quantities of cells are needed in order to reconstitute the hematopoietic system of patients following chemotherapy. For example, a recent clinical study was performed on 11 patients transplanted with cells cultured in vitro (Chang et al., 1989). Here, an average of 1.9 X lo8 celldkg of patient were transplanted in an effort to reconstitute hematopoiesis following chemotherapy. This amounts to 1.3 X 1Olo cells for a typical 70-kg patient. Generation of this quantity of cells in vitro is cumbersome using these traditional culture methods due to the large amount of flasks required. Investigation of alternate culture methods for the expansion of these cells would prove useful in generating the amount of cells necessary for clinical use. To date, studies on the expansion of bone marrow in systems designed for large-scale production of hematopoietic cells have been limited. One study concentrated on mimicking the hematopoietic microenvironment in a three-dimensional culture supported by a nylon screen (Naughtonet al., 1990). Here, stromal cells were supported on the nylon screen before inoculation with hematopoietic cells. This system was found to support the growth of several hematologic lineages concurrently and could be purged to retrieve cells. A more recent study investigated
0 1993 American Chemical Soclety and American Instltute of Chemlcal Englneers
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the use of a perfusion, flat-bed bioreactor for the expansion of murine hematopoietic cells on a layer of irradiated 3T3 cells (Koller et al., 1992~).Here, it was determined that perfusion contributed to an increase in the total number of hematopoietic cells retrieved from the system. Although the large-scale culture of hematopoietic cells has received little attention in the literature, large-scale mammalian cell culture systems have been developed and refined for a number of other applications. These include systems for both anchorage-dependent and suspension cell lines largely for the production of monoclonal antibodies and genetically engineered therapeutics (Ho and Wang, 1991;Lubiniecki, 1990). The requirement of large amounts of hematopoietic cells for clinical use provides an incentive to investigate mammalian cell expansion technologies which have been developed and refined in the last decade.
Materials and Methods Preparation of Bone Marrow. Bone marrow aspirates obtained from breast cancer patients were a gift of Dr. William Peters at the Duke University Medical Center. Aspirates were centrifuged (9OOgfor 10min), and nucleated cell fractions (buffy coats) were removed and frozen for shipment. Upon thawing, marrow was washed with Iscove's Modified Dulbecco's Medium (IMDM) plus 105% FBS, 20 units/mL heparin, and 500 units/mL DNAse. The preparation was enriched for low-densitymononuclear cells by density gradient centrifugation using Histopaque 1077 (Sigma, St. Louis, MO). Cells were resuspended in 20 mL of tissue culture medium and layered onto the middle of 50-mL centrifuge tubes containing 15 mL of Histopaque and 20 mL of tissue culture medium. Tubes were centrifuged for 30 min at 2000g in a Beckman GP centrifuge. Retrieved low-density mononuclear cells were washed three times with the tissue culture medium described above. Static Cultures. LDMCs were seeded into 6-welltissue culture plates containing 3.0 mL at a concentration of 5.0 X 105 viable cells/mL. The tissue culture medium used for all experiments was IMDM with 105% FBS and added cytokines. Cytokines included IL-3 (0.5ng/mL), GM-CSF (0.25 ng/mL), and c-kit ligand (100 ng/mL). These cytokines and concentrations were chosen due to their ability to expand CD34+DR-CD15- cells in vitro (Brandt et al., 1992). Cytokines were added independent of the media changes at a rate of 3 times per week. Medium was replaced as necessary in order to keep the cell concentration under lo6cells/mL. During periods of relatively constant cell number, medium was replaced on a weekly basis by removing one-half of the volume of the culture, centrifuging, and adding back the cells with fresh medium to the original volume. During the period of rapid cell expansion, the cultures were split 1-to-2in order to reduce the cell Concentration. This was performed by removing and discarding one-half of the volume of the culture (along with cells) and replacing the volume with fresh medium. Cell counts were obtained every 2-3 days by hemacytometer counting with Trypan Blue stain (Sigma). This was performed by vigorously pipetting the cells in the well of the tissue culture plate up and down to suspend as many of the nonadherent cells as possible. Adherent cells were not included in the cell counts. Duplicate wells from eight different marrow samples were performed. Expansion values were calculated using the initial and final cell concentrations as well as the number of splits using the
formula expansion = (final cell concn/initial cell concn)2" where n is the number of 1-to-2 splits. Suspension and Microcarrier Cultures. Two runs each were performed for suspension and microcarrier cultures using different marrow samples, with each run consisting of a single spinner vessel. For each marrow sample, control 6-well plates were also cultured as described above. For both suspension and microcarrier cultures, cells were seeded into Bellco 250-mL spinner vessels (Bellco Biotechnology, Vineland, NJ) operated at 45 rpm overlaid with a slow flow rate of equilibration gas. The equilibration gas consisted of 10% CO~/balanceair. The spinner vessels were siliconized prior to use (Sigma) to prevent the attachment of anchorage-dependent cells to the glass walls of the vessel. For the microcarrier cultures, Cultisphere GL microcarriers (Hyclone, Logan, UT) were employed at a concentration of 2.5 g/L and were prepared according to the manufacturer's specifications. Dissolved oxygen, pH, pCO,, and HC03- values were monitored using a Corning Model 170 blood gas analyzer. Cell counts were obtained from the spinner vessels by direct sampling of the culture with a 1.0-mL pipet. For the microcarrier culture, the microcarriers were allowed to settle before the suspension population was sampled. The medium, seed density, and feed schedules that were followed for the spinner vessels and control plates were as described above for static cultures. For run 1, cytokines for the suspension, microcarrier, and control plate were added 3 times per week at the concentration described above. For run 2, cytokines for all three systems were added once per week. (These two cytokine addition schedules were found to result in equivalent expansions when compared in static culture as described above using identical marrow (unpublished observations).) Starting at day 0 and at weekly intervals thereafter, the suspension and microcarrier cultures were sampled for granulocyte-macrophage (CFU-GM) and erythrocyte (BFU-e) progenitor cells using methylcellulose colony assays (CytoMed, Inc., Cambridge, MA). The colony assay protocols followed were supplied by the manufacturer. Assay plates were scored for the presence of CFU-GM and BFU-e colonies at day 14. The CFU-GM and BFU-e expansion values were calculated using the initial and final progenitor cell concentrations (colonies per 105 cells) as well as the total cell expansion, as described above. Airlift Bioreactor. One run was performed in a Celllift airlift bioreactor (Ventrex, Portland, ME) employing a working volume of 575 mL. The sparge flow rate was 4-5 mL/min using 10% Codbalance air equilibration gas. This rate was found to minimize foaming while maintaining an appropriate medium circulation rate. Dissolved gasses and pH values were monitored using a blood gas analyzer as described above. The seed density was 1.1X 105viable cells/mL. Medium, cytokines, and feed schedules were followed as described above for the static cultures. No antifoams were employed. CFU-GM and BFU-e methylcellulose colony assays were performed according to the protocols described above for the suspension and microcarrier cultures. Control cultures in 6-well plates were performed using the same marrow and feeding conditions. Expansion values were calculated as described above. Hollow Fiber Culture. In order to evaluate the ability of a hollow fiber system to expand LDMCs, a system was assembled using the medium and cytokines described above. Medium from a 250-mL reservoir was passed through a peristaltic pump (Cole-Parmer, Chicago, IL)
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Figure 1. Typical expansion curves for human low-density mononuclear cells in static culture: 0 , duplicate expansions of the same marrow sample with cytokines; W, similar expansions without cytokines.
into the hollow fiber cartridge at a flow rate of approximately 50 mL/min. The bioreactor employed was a Cell Pharm Model BRllO (UniSyn Fibertec, San Diego, CA) with a 1.0 sq f t fiber surface area and a 10 000 molecular weight cutoff with an extracapillary space (ECS) volume of 12 mL. Medium was then passed through a hollow fiber oxygenator (Cell Pharm Model OXY-1)and returned to the reservoir. The equilibration gas used was 10% COz/ balance air. All tubing used for medium and gas flows was norprene (Cole-Parmer, Chicago, IL). The oxygen and CO1concentrations entering and exiting the bioreactor were monitored by sampling the fluid stream and injecting into a blood gas analyzer. Cytokines were added both to the extracapillary space (cell space) of the hollow fiber bioreactor as well as to the medium reservoir a t the concentrations employed for static cultures at a rate of 3 times per week. Low-density mononuclear cells (4.4 X 10') obtained as described above were seeded into the extracapillary space of the hollow fiber cartridge. The cell number in the ECS was periodically monitored by flushing the cells out of the ECS by vigorous washing followed by hemacytometer counting. After counting, the cells were returned to the ECS with fresh medium and added cytokines. The oxygen consumption rate of the cartridge was also monitored using the entering and exiting oxygen concentrations and the medium flow rate.
Results An example of an expansion curve for static culture operation is shown in Figure 1. Duplicate wells using the same marrow preparation are shown by the solid circles. After initial seeding, no increase in the total cell number was observed for the first 7 days. A stromal layer formed after 1-2 weeks in culture. This attached population of cells could not be removed from the plate by vigorous pipetting and was not included in the cell expansion curves. Following the lag period, an expansion phase lasting about 2 weeks was observed where the cell number increased significantly. Following the expansion phase, the culture entered a death phase where the total cell number decreased with time. The squares represent two wells which were cultured in the absence of cytokines. Under these conditions, no increase in the total cell number was observed. Expansion values in 6-well plates from marrow obtained from eight different patients are listed in Table I. When
Table I. Expansion of Low-Density Mononuclear Cells Obtained from Human Marrow from Several Different Patients in Static Culture
patient
run 1 30 5.3 14 9.6 4.4 19 26 15
run 2 32 6.1 17 9.0 5.4 22 31 22
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std devtmean. % 4.6 9.9 13 4.6 14 10 11 24
duplicate expansions on the marrow from individual patients were performed, the results were generally in good agreement, with standard deviations ranging from 5 to 25% of the mean expansion value. However, significant patient to patient variability was observed when marrow was expanded from different patients. Patient 1exhibited increases of 30- and 32-fold,while the marrow from patient 5 increased only 4.4- and 5.4-fold. Examples of expansion curves for suspension, microcarrier, and airlift operation in 250-mL spinner vessels are shown in Figure 2. The growth curve in each bioreactor run (denoted by solid squares) is compared to the equivalent curve performed in duplicate using static culture with the same marrow sample (denoted by solid circles). Cell growth profiles in these bioreactors were similar to the profiles observed for the static cultures with respect to stationary, exponential, and death phases. Total expansion results are presented in Table I1 and represent the results of two runs for the suspension and microcarrier spinner vessels and a single run for the airlift bioreactor. In each case, the suspension cultures expanded to a greater extent than did the control static runs, while the microcarrier and airlift cultures expanded to an equal or lesser degree than the corresponding controls. While the suspension and microcarrier cultures exhibited no foaming, a foam layer 2-4 cm thick was present a t the medium/ surface interface of the airlift vessel throughout the duration of the experiment. Expansion data for granulocytemacrophage and erythroid progenitor cells are presented in Figures 3-5 for suspension, microcarrier, and airlift cultures, respectively. For the suspension and microcarrier cultures, an increase in these progenitor cell populations was observed on day 7 before declining. The maximum increase in the CFU-
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in the "other" category at the start of the experiment may represent cells in the LDMC preparation which were adversely affected by the freeze-thaw process. During the life span of the culture, the fraction of blasts steadily decreased from 47 % to 2 % by day 28. The population of granulocytes increased to 31 % by day 14before declining. The dominant population by the end of the experiment was cells in the macrophage/monocyte lineage comprising 89% of the culture. No cells in the erythroid lineage were observed during the experiment. Total cell expansion results for the hollow fiber bioreactor system demonstrated no observable expansion of cells throughout the life span of the culture as measured by the amount of cells removed from the ECS. The control plate using identical starting marrow exhibited an expansion of 31-fold. In addition, no observable oxygen consumption rate was measured using the inlet and outlet oxygen concentrations of the bioreactor employing a minimum detection limit of 0.007 mmol of Oz/h.
20 SUSPENSION BIOREACTOR
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Discussion
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Figure 2. Expansion curves for suspension, microcarrier, and airlift cultures. In each figure, represents the expansion in the bioreactor while 0 represents the expansion of the control static culture in 6-well plates using the same marrow sample. Table 11. Expansion Values for Human Bone Marrow in Stationary, Suspension, Microcarrier, Hollow Fiber, and Airlift Cultures micro- hollow suspension carrier fiber airlift run 1 total expansion 15 9.0 0.0 12 9.0 9.0 31 17 control plate expansion 1.6 1.0 0.0 0.7 relative expansion run 2 total expansion 27 8.1 NIA NIA control plate expansion 17 17 NIA NIA relative expansion 1.6 0.5 NIA NIA
GM population was 150% over the starting value and was observed in the suspension culture. For the airlift bioreactor, no significant increase in either progenitor cell population was observed. Results of the morphological analysis using Wright Giemsa stain which was performed on the suspension culture are presented in Table 111. These data demonstrated that a large fraction of the cells in the culture were blasts at the time of seeding. The large percentage of cells
Prior to the development of growth-promotingcytokines, the maintenance of cultures of bone marrow cells required the use of a stromal layer to provide the necessary growth factors for cell proliferation. The discovery of these growth factors and the ability of these factors to act synergistically have allowed cells retrieved from bone marrow to expand in the absence of a preestablished stromal layer (Brandt et al., 1992). When highly enriched progenitor/stem cell populations were cultured, an expansion in excess of 10 000-fold was found to occur in the liquid phase (Brandt et al., 1992). During this cell number increase, no significant stromal layer was established. The gignificance of these advances is that cells retrieved from patients requiring bone marrow transplantation can be expanded in vitro and returned to the patient following chemol radiation therapy. In this study, we have evaluated the most commonly used mammalian cell culture scale-up techniques for the expansion low-densitymononuclear cells retrieved from bone marrow. This study was designed to provide an initial screening of the types of expansion systems which are currently available in order to provide a basis for future development. Expansion of low-density mononuclear cells obtained from human marrow in vitro using tissue culture medium containing IL-3, GM-CSF, and c-kit ligand resulted in a 5-31-fold increase in the total number of cells when cultured in 6-well plate static culture. An association between the established stromal layer and selected populations of nonadherent cells may have affected the expansion results. Using this culture method, a lag period as long as 20 days was observed before the cell number increased. One possible explanation for the presence of a lag phase is that it represents the sum of the growth and death rates of the different populations present in the culture. The growth rates of these populations are strongly affected by the presence of exogenously added growth factors, as demonstrated by the lack of growth when culturing LDMCs without cytokines. In one scenario, populations such as the terminally differentiated cell types are in a period of decline while other populations such as progenitors are in a period of expansion, with the total growth of the culture being the sum of the changes of the individual cell types. The presence of the exponential growth phase may represent the outgrowth of certain populations of cells which arise after the maturation of progenitor cells. Although the expansions of identical marrow samples were found to be in good agreement when performed in
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Figure 3. Expansion of granulocyte-macrophage (CFU-GM)and erythroid (BFU-e)progenitor cells in suspension bioreactor culture:
H, CFU-GM; 0,BFU-e.
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Figure 4. Expansion of granulocyte-macrophage (CFU-GM)and CFU-GM; O , BFU-e. duplicate using static culture, a significant amount of variability was observed when the total cell number increase using marrow from different patients was compared. Patient to patient variability could result from variability in the physiological state of the donor as well as differences in marrow collection and processing. In order to provide a basis for the scale-up of hematopoietic cells, suspension, microcarrier, airlift, and hollow fiber cultures were investigated for the ability to expand low-density bone marrow cells. The large amount of marrow needed in order to complete these experiments necessitated the use of marrow from different patients. In order to allow a comparison of the different expansion systems, all bioreactor expansions were referenced to results obtained in static culture using identical marrow in 6-well plates. By normalizing the expansion values of static cultures to 1.0, the greatest amount of relative expansion was found to occur in suspension culture, which demonstrated an increase in the total cell yield of 60 5% for the two runs investigated. This was followed by airlift and microcarrier cultures, which expanded the cells to a
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erythroid (BFU-e) progenitor cells in microcarrier culture: m, similar or lesser degree than static culture. Hollow fiber culture exhibited no increase in the ability to expand these cells, although this finding may only be reflecting the difficulties in retrieving cells from the extracapillary space of the bioreactor. A morphological analysis demonstrated that the dominant lineage in suspension culture was found to be cells of the macrophage/monocyte lineage. These results agree with Brandt et al. (1992),who found the monocyte lineage to dominate when sorted CD34+DR-CD15- cells were cultured with an IL-3, GM-CSF fusion protein in the presence of c-kit ligand. In this study, the dominant cell populations resulting from in vitro culture of these cells were found to vary with the type of cytokine added to the cultures. The environment to which cells are exposed can have a significant impact on growth. When cells were grown in static culture, expansions were found to be a function of the feed schedules employed (Schwartz et al., 1991).In our studies, attempts were made to provide similar growth conditions in all of the systems investigated as much as
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TIME (days) Figure 5. Expansion of granulocyte-macrophage (CFU-GM) and erythroid (BFU-e) progenitor cells in airlift culture: 0,BFU-e.
Table 111. Morphological Analysis of the Cell Expansion Observed in Suspension Culture Determined Using Wright Giemsa Stains day blasts gran mono eryth other 0 2 0 51 0 47 30 39 0 3 7 28 31 57 0 0 14 12 21 72 0 2 21 5 8 89 0 1 28 2
Abbreviations are as follows: gran, granulocytes;mono, monocyte or macrophage; eryth, erythroid.
possible. As well as using identical medium and cytokines, temperature, oxygen, pH, pCO2, and HC03-were all kept to within similar ranges to the extent possible in each system investigated. However, environmental differences inherent to a given bioreactor may have played a role in the expansion process. For example, mechanical agitation in the suspension cultures could have affected growth patterns. Fluid shear due to bubble formation and breakage may have negatively affected the cells in the airlift system. Although no growth was detected in the hollow fiber system, high localized cell concentrations and intimate cell to cell contact in the extracapillary space may have influenced cell proliferation. In addition, the degree of adherence of the stromal cells to the walls of the hollow fiber is unknown. Cells adhering to each other as well as the fiber wall may have affected the ability to retrieve cells. It is important to note that the degree of optimization in a given expansion process can have a significant influence on the performance of that process. Each technology investigated has its own inherent parameters which could improve performance if optimized. In the stirred tank vessels, agitation rates and the resulting fluid shear could be improved. For the airlift system, sparge rates and antifoam additions could be optimized to minimize the effects of foaming. In the hollow fiber system, parameters such as fiber type, ICs and ECS flow rates, and improved methods for cell retrieval could be optimized for increased cell yields. The studies performed in these experiments were designed to provide an initial screen of the currently available technologiesfor cell expansion in order to provide a basis for further optimization. The expansion results
.,
CFU-GM;
obtained in this study have limited clinical application. In order to improve the clinicalrelevance of in vitro expansion, a significant increase in the expansion of progenitor and stem cell populations has to be achieved. Since cytokines play an important role in the expansion of hematopoietic cells, increased proliferation of these cells may be realized by alternate combinations and/or concentrations of these growth factors. As suggested by the expansion results obtained by Brandt et al. (19921,preselection of the marrow by such techniques as cell sorting may also increase the total overall yield of useful hematopoietic cells. An alternate type of bioreactor which closely simulates the in vivo environment may also serve to improve in vitro hematopoiesis.
Literature Cited Brandt, J.;Briddell, R. A.; Srour, E. F.; Leemhius, T. B.; Hoffman, R. Role of c-kit ligand in the expansion of human hematopoietic progenitor cells. Blood 1992, 79, 634-641. Caldwell, J.; Palsson, B. 0.; Locey, B.; Emerson, S. G. Culture perfusion schedules influence the metabolic activity and granulocyte-macrophage colony stimulating factor production rates of human bone marrow stromal cells. J. Cell. Physiol. 1991,147, 344-353. Chang, J.; Coutinho, L. H.; Morgenstern, G. R.; Scarffe, J. H.; Deakin, D. P.; Harrison, C.; Testa, N. G.; Dexter, T. M. Reconstitution of haemopoietic system with autologous marrow taken during relapse of acute myeloblastic leukaemia and grown in long-term culture. Lancet 1986, 294-296. Chang, J.; Morgenstern, G. R.; Coutinho, L. H.; Scarffe, J. H.; Carr, T.; Deakin, D. P.; Testa, N. G.; Dexter, T. M. The use of bone marrow cells grown in long-term culture for autologous bone marrow transplantation in acute myeloid leukaemia: an update. Bone Marrow Transplant. 1989, 4 , 5-9. deVries, P.; Brasel, K. A.; Eisenman, J. R.; Alpert, A. R.; Williams, D. E. The effect of recombinant mast cell growth factor on purified murine hematopoietic stem cells. J.Exp. Med. 1991, 173, 1205-1211. Dexter, T. M.; Spooncer, E.; Simmons, P.; Allen, T. D. Long Term bone marrow culture: an overview of techniques and experience. In Long Term Bone Marrow Culture; Wright, D. G . ,Greenberger, J. S., Eds.; Alan R. Liss: New York, 1984; pp 57-96. Ho, C. S., Wang, D. I. C., Eds. Animal Cell Bioreactors; Butterworth-Heinemann: Boston, 1991.
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Iscove, N. N.; Shaw, A. R.; Keller, G. Net increase of pluripotential hematopoietic precursors in suspension culture in response to IL-1 and IL-3. J. Immunol. 1989,142,2332-2337. Koller, M. R.; Bender, J. G.; Papoutsakis, E. T.; Miller, W. M. Effects of synergistic cytokine concentrations,low oxygen, and irradiated stroma on the expansion of human cord blood progenitors. Blood 1992a,80, 403-411. Koller, M. R.; Bender, J. G.; Miller, W. M.; Papoutsakis, E. T. Reduced oxygen tension increases hematopoiesis in long-term culture of human stem and progenitor cells from cord blood and bone marrow. E x p . Hematol. 1992b,20, 264-270. Koller, M. R.; Bender, J. G.; Papoutsakis, E. T.; Miller, W. M. Beneficial effects of reduced oxygen tension and perfusion in long-term hematopoietic cultures. Ann. N. Y. Acad. Sci. 1992c, 665, 105-116. Lubiniecki, A. S., Ed. Large-Scale Mammalian Cell Technology; Marcel Dekker: New York, 1990. Migliaccio, G.; Migliaccio, A. R.; Adamson, J. W. The biology of hematopoietic growth factors: studies in vitro under serum deprived conditions. Exp. Hematol. 1990,18, 1049-1055. Migliaccio, G.;Migliaccio, A. R.; Valinsky, J.; Langley, K.; Zsebo, K.; Visser, J. W. M.; Adamson, J. W. Stem cell factor induces proliferation and differentiation of highly enriched murine hematopoietic cells. Proc. Natl. Acad. Sci. U.S.A. 1991,88,
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7420-7424. Naughton, B. A.; Jacob, L.; Naughton, G. K. A three-dimensional system for the growth of hematopoietic cells. In Bone Marrow Purging and Processing; Gross, S., Gee, A. P., Worthington-White,D. A., Eds.; Alan R. Liss: New York, 1990; pp 435-445. Ottman, 0. G.; Stella, C. C.; Eder, M.; Xeutzel, P.; Strocker, S.; Hoelzer, D.; Ganser, A. Regulation of early hematopoiesis in serum-deprived cultures of mafosfamide-treated and untreated CD34-enriched bone marrow cells. Exp. Hernatol. 1991, 19, 3812-3817. Schwartz, R. M.; Palsson, B. 0.;Emerson, S. G. Rapid medium perfusion rate significantly increases the productivity and longevity of human bone marrow cultures. Proc. Natl. Acad. Sci. U.S.A. 1991,88, 6760-6764. Smith, S.; Broxmeyer, H. E. The influence of oxygen tension on the long-term growth in vitro of haematopoietic progenitor cells from human cord blood. Br. J. Haematol. 1986,63,2434. Varma, A.; El-Awar, F. Y.; Palsson, B. 0.;Emerson, S. G.; Clarke, M. F. Can Dexter cultures support stem cell proliferation. Exp. Hematol. 1992,20, 87-91. Accepted December 7, 1992.
