E. Terry Papoutsakis

Modeling pO2 Distributions in the Bone Marrow Hematopoietic Compartment. II. Modified Kroghian Models

Hematopoietic cells of various lineages are organized in distinct cellular architectures in the bone marrow hematopoietic compartment (BMHC). The homogeneous Kroghian model, which deals only with a single cell type, may not be sufficient to accurately describe oxygen transfer in the BMHC. Thus, for cellular architectures of physiological significance, more complex biophysical-transport models were considered and compared against simulations using the homogeneous Kroghian model. The effects of the heterogeneity of model parameters on the oxygen tension (pO(2)) distribution were examined using the multilayer Kroghian model. We have also developed two-dimensional Kroghian models to simulate several cellular architectures in which a cell cluster (erythroid cluster) or an individual cell (megakaryocyte or adipocyte) is located in the BMHC predominantly occupied by mature granulocytes. pO(2) distributions in colony-type cellular arrangements (erythroblastic islets, granulopoietic loci, and lymphocytic nodules) in the BMHC were also evaluated by modifying the multilayer Kroghian model. The simulated results indicate that most hematopoietic progenitors experience low pO(2) values, which agrees with the finding that low pO(2) promotes the expansion of various hematopoietic progenitors. These results suggest that the most primitive stem cells, which are located even further away from BM sinuses, are likely located in a very low pO(2) environment.

Modeling pO(2) distributions in the bone marrow hematopoietic compartment. I. Krogh's model.

Human bone marrow (BM) is a tissue of complex architectural organization, which includes granulopoietic loci, erythroblastic islets, and lymphocytic nodules. Oxygen tension (pO(2)) is an important determinant of hematopoietic stem and progenitor cell proliferation and differentiation. Thus, understanding the impact of the BM architectural organization on pO(2) levels in extravascular hematopoietic tissue is an important biophysical problem. However, currently it is impossible to measure pO(2) levels and their spatial variations in the BM. Homogeneous Kroghian models were used to estimate pO(2) distribution in the BM hematopoietic compartment (BMHC) and to conservatively simulate pO(2)-limited cellular architectures. Based on biophysical data of hematopoietic cells and characteristics of BM physiology, we constructed a tissue cylinder solely occupied by granulocytic progenitors (the most metabolically active stage of the most abundant cell type) to provide a physiologically relevant limiting case. Although the number of possible cellular architectures is large, all simulated pO(2) profiles fall between two extreme cases: those of homogeneous tissues with adipocytes and granulocytic progenitors, respectively. This was illustrated by results obtained from a parametric criterion derived for pO(2) depletion in the extravascular tissue. Modeling results suggest that stem and progenitor cells experience a low pO(2) environment in the BMHC.

Purification and characterization of the NADH-dependent butanol dehydrogenase from Clostridium acetobutylicum (ATCC 824)

Two butanol dehydrogenases with different cofactor requirements and different pH ranges have been detected in Clostridium acetobutylicum ATCC 824. The NADH-dependent butanol dehydrogenase (NADH-BDH) was purified to near homogeneity and characterized. One striking feature of the enzyme is that Zn2+ was needed to obtain a significant recovery during purification. The enzyme was a dimer composed of two subunits with subunit molecular mass of 42 kDa and a native molecular mass of 82 +/- 2 kDa. The kinetics were studied in the direction of the reduction of butyraldehyde. Inhibition studies with S-NADH and butanol indicate that the NADH-BDH follows an ordered bibi mechanism with kinetic constants of 4.86 s-1, 0.18 mM, and 16 mM for Kcat, KNADH, and Kbutyraldehyde, respectively. Activity in the reverse direction was 50-fold lower than that in the forward direction. The NADH-BDH had higher activity with longer chained aldehydes and was inhibited by metabolites containing an adenine moiety.

Stirred culture of peripheral and cord blood hematopoietic cells offers advantages over traditional static systems for clinically relevant applications

The ability to culture hematopoietic cells in readily characterizable and scalable stirred systems, combined with the capability to utilize serum-free medium, will aid the development of clinically attractive bioreactor systems for transplantation therapies. We thus examined the proliferation and differentiation characteristics of peripheral blood (PB) mononuclear cells (MNC), cord blood (CB) MNC, and PB CD34(+) cells in spinner flasks and (control) T-flask cultures in both serum-containing and serum-free media. Hematopoietic cultures initiated from all sources examined (PB MNC, CB MNC, and PB CD34(+) cells) grew well in spinner vessels with either serum-containing or serum-free medium. Culture proliferation in spinner flasks was dependent on both agitator design and RPM as well as on the establishment of critical inoculum densities (ID) in both serum-containing (2 x 10(5) MNC/mL) and serum-free (3 x 10(5) MNC/mL) media. Spinner flask culture of PB MNC in serum-containing medium provided superior expansion of total cells and colony-forming cells (CFC) at high ID (1.2 x 10(6) cells/mL) as compared to T-flask controls. Serum-free spinner culture was comparable, if not superior, to that observed in serum-containing medium. This is the first report of stirred culture of PB or CB MNC, as well as the first report of stirred CD34(+) cell culture. Additionally, this is the first account of serum-free stirred culture of hematopoietic cells from any source.

Physiologically significant effects of pH and oxygen tension on granulopoiesis

OBJECTIVE Granulocyte differentiation in the bone marrow (BM) takes place in regions with lower pH and O(2) tension (pO(2)) than those in the BM sinuses. This suggests that granulopoiesis will be enhanced at subvascular pH and pO(2). MATERIALS AND METHODS The effects of pH AND pO2 on granulocyte proliferation, differentiation, and granulocyte colony-stimulating factor receptor (G-CSFR) expression were evaluated using mobilized peripheral blood CD34(+) cells directed down the granulocytic pathway with stem cell factor, interleukin 3, interleukin 6, and G-CSF. RESULTS Cell expansion was enhanced at subvascular pH, with twice as many total cells and CD15(bright)/CD11b(+) late neutrophil precursors (myelocytes, metamyelocytes, bands) produced at pH 7.07 to 7.21 as was produced at pH 7.38. Low pH accelerated the rate of differentiation concomitant with this increase in proliferation. Also, total, CD15(bright)/CD11b(-) (promyelocytes, early myelocytes), and CD15(bright)/CD11b(+) cell expansion was enhanced at lower pO(2), with twice as many of each cell type produced at 5% O(2) as at 20% O(2). The effects of low pH and low pO(2) were additive, such that generation of total, CD15(bright)/CD11b(-), and CD15(bright)/CD11b(+) cells was 3.5-, 2.4-, and 4.0-fold greater at pH 7.21 and 5% O(2) than at the standard hematopoietic culture conditions of pH 7.38 and 20% O(2). Low pH resulted in faster upregulation of G-CSFR surface expression, whereas pO(2) had no effect on G-CSFR expression. CONCLUSION These data provide compelling evidence that pH and pO(2) gradients within the BM play significant roles in regulating hematopoiesis. More rapid granulocytic cell proliferation and differentiation at low pH may be explained in part by more rapid G-CSFR expression. The ability to alter cell development by manipulating pH and pO(2) has important implications for optimizing ex vivo production of neutrophil precursors.

The protective effect of serum against hydrodynamic damage of hybridoma cells in agitated and surface-aerated bioreactors

The effect of serum on the growth rate and metabolism of CRL-8018 hybridoma cells in an agitated, surface-aerated bioreactor was examined. In the employed well-controlled bioreactors at high agitation rates, hybridoma cells in medium containing 1% fetal bovine serum rapidly die in the presence of a vortex with accompanying gas-bubble entrainment, whereas non-agitated control cultures grow normally in medium containing 1% serum. Serum levels greater than 5% counteract the detrimental hydrodynamic effects due to agitation and bubble entrainment. The protective effect is present after short-term (less than 1 h) exposure to 10% serum concentration, suggesting a protection mechanism which is, at least in part, of a physical nature. The apparent cell yields on glucose, lactate, and glutamine decreased with decreasing growth rate due to low serum concentrations. The results are incorporated into a simple model in which the apparent growth rate is the sum of an invariable growth rate and a changing death rate.

Variations in culture pH affect the cloning efficiency and differentiation of progenitor cells in ex vivo haemopoiesis

Haemopoietic cultures may experience pH variations of as much as 0.5 units depending on culture duration and cell density. Since pH is a potent modulator of cellular proliferation and differentiation, we examined its effects on the performance of both semisolid and liquid haemopoietic cultures. Culture pH was found to have substantial effects both on progenitor cloning efficiency (as measured in liquid cultures) and on progenitor cell differentiation (as measured in methylcellulose cultures). Liquid cultures were conducted with both peripheral blood (PB) mononuclear cells (MNCs) and cord blood (CB) MNCs using growth factor combinations that promote either erythroid expansion (IL‐3/IL‐6/SCF/Epo) or granulocyte/macrophage expansion (IL‐3/IL‐6/SCF/G‐CSF/GM‐CSF). Reduced pH was found to have either a positive or neutral effect on the expansion and cloning efficiency of progenitors in ex vivo liquid cultures. Cloning efficiencies of PB BFU‐E in the erythroid combination were 9‐fold higher at low pH (7.1) when compared to high pH (7.6). A small pH increase of 0.2 units over physiological values consistently produced significant reductions (42–85%) in cloning efficiencies for all cell types and cytokine combinations tested. Methylcellulose cultures conducted using CB MNC and PB MNC indicated that differentiation of CFU‐GM into progeny was optimal between pH 7.2 and 7.4. The differentiation of erythroid progenitors (BFU‐E) progressively increased as pH was increased from 6.95 (no colonies detected) to 7.4 (maximum colonies detected), to 7.6 (maximum haemoglobin content). Methylcellulose cultures using PB CD34+ cells exhibited similar patterns to the MNC cultures. We conclude that even small variations in pH substantially affected the performance of human haemopoietic cultures. The erythroid lineage was particularly sensitive, with its extent of differentiation increasing with increasing pH. PB progenitors are more sensitive to pH variations than CB progenitors.

Development of novel perfusion chamber to retain nonadherent cells and its use for comparison of human mobilized peripheral blood mononuclear cell cultures with and without irradiated bone marrow stroma

Perfusion and static cultures of peripheral blood (PB) mononuclear cells (MNCs), obtained from patients following stem cell mobilization, were supplemented with interleukin‐3 (IL‐3), IL‐6, granulocyte colony‐stimulating factor (G‐CSF), and stem cell factor (SCF) and compared with and without a preformed irradiated allogeneic bone marrow stromal layer. Perfusion cultures without a stromal layer effectively retained nonadherent cells through the use of a novel “grooved” perfusion chamber, which was designed with minimal mass transfer barriers in order to achieve a well‐defined culture environment. The grooved chamber allowed easy and efficient culture inoculation and cell recovery. Average maximum expansion of CFU‐GM (colony‐forming unit granulocyte‐macrophage) cells was observed on day 10 for all cultures. Perfusion cultures had a maximum CFU‐GM expansion of 17‐ and 19‐fold with and without a stromal layer, respectively. In contrast, static cultures had a maximum CFU‐GM expansion of 18‐ and 13‐fold with and without a stromal layer, respectively. Average long‐term‐culture initiating cell (LTC‐IC) numbers on day 15 were 34% and 64% of input in stroma‐containing and stroma‐free perfusion cultures and 12% and 11% of input in stroma‐containing and stroma‐free static cultures, respectively. Thus, perfusion enhanced CFU‐GM expansion and LTC‐IC maintenance more for the stroma‐free cultures than for stroma‐containing cultures. This was surprising because analysis of medium supernatants indicated that the stroma‐containing cultures were metabolically more active than the stroma‐free cultures. In view of their equivalent, if not superior, performance compared to stroma‐containing cultures, stroma‐free perfusion cultures may offer significant advantages for potential clinical applications.

Hematopoietic cell culture therapies (Part I): cell culture considerations

Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, is a rapidly growing area of tissue engineering with many potential applications in bone-marrow transplantation, gene therapy and the production of blood products. Hematopoietic cultures are considerably more complex than established animal cell culture technologies owing to the presence of many cell types at various differentiation stages, and stringent medium and growth factor requirements. Culture parameters, such as pH, oxygen tension, seeding density and feeding schedules, significantly affect the proliferation and differentiation of hematopoietic cells. A number of bioreactor systems are currently under development.

Beneficial Effects of Reduced Oxygen Tension and Perfusion in Long‐Term Hematopoietic Culturesa

Growth of hematopoietic stem and progenitor cells found in the MNC fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Reduced oxygen tension increased total cell numbers by as much as 5-fold in cord blood suspension cultures, but this effect was less pronounced in cultures containing an irradiated bone marrow stromal cell layer. However, reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in both types of LTHC studied. Under low oxygen, CFU-C progenitor cell numbers were as much as 10-fold higher. Finally, reduced oxygen tension slowed the rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of human stromal and progenitor cells in vitro. These low oxygen findings were then applied to a murine model LTHC perfusion system. In this system, irradiated 3T3 stromal layer integrity was improved under low oxygen and was substantially further improved with continuous medium perfusion. Cell counts and flow cytometry analysis indicated that the total cell production and the production of immature cells from murine bone marrow MNC on irradiated 3T3 cells were significantly enhanced under low oxygen with perfusion. After three weeks of culture, a 24-fold higher number of Thy1.2lo F4/80- MAC1- cells (indicative of murine stem and progenitor cells) was observed in the perfusion system as compared with static culture under ambient oxygen.