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W M Miller

Publications and source records attributed to W M Miller.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

Antigens, CD34↗
PubMed1998

Ammonia inhibits neural cell adhesion molecule polysialylation in Chinese hamster ovary and small cell lung cancer cells.

Ammonia is a major concern in biotechnology because it often limits recombinant protein production by animal cells. Conditions, such as ammonia accumulation, in large-scale production systems can parallel those that develop within fast-growing solid tumors such as small cell lung cancer (SCLC). Ammonia's specific inhibition of the sialylation of secreted glycoproteins is well documented, but it is not known how ammonia affects membrane-bound proteins, nor what role it may have on important glycosylation determinants in cancer. We therefore examined the effects of NH4Cl on polysialic acid (PolySia) in the neural cell adhesion molecule (NCAM). By using flow cytometry combined with two NCAM antibodies, one specific for the peptide backbone and another that recognizes PolySia chains, we show that ammonia causes rapid, dose-dependent, and reversible inhibition of NCAM polysialylation in Chinese hamster ovary (CHO) and SCLC NCI-N417 cells. The decrease in PolySia was accompanied by a small increase in NCAM, suggesting that the changes were specific to the oligosaccharide. Inhibition by ammonia was greater for CHO cells, with PolySia cell surface content decreasing to 10% of control after a 4-day culture with 10 mM NH4Cl, while N417 cell PolySia was reduced by only 35%. Ammonia caused a 60% decrease in the CHO cell yield from glucose, while N417 cells were barely affected, suggesting that increased resistance to ammonia by N41 7 cells is a global rather than glycosylation-specific phenomenon. The data presented show that the tumor microenvironment may be an important factor in the regulation of PolySia expression.

Ammonia↗
PubMed1998

pH is a potent modulator of erythroid differentiation.

Physiological parameters such as pH and oxygen tension probably play significant roles in the regulation of haemopoiesis in the bone marrow microenvironment, but these roles have yet to be characterized in detail. We have found that changes in culture pH (0.2 units) can cause significant changes in the culture composition of mature cells and colony-forming cells (CFCs), especially in the presence of erythropoietin (Epo). Peripheral blood (PB) CD34+ cells cultured at different pH values (7.15-7.6) were characterized using total cell counts, colony assays, morphological analysis. haemoglobin staining, flow cytometry, immunocytochemical staining, and Western blots. Cultures performed at high (7.6) pH contained greater numbers of haemoglobin-positive and band-3-positive cells. and acquired these erythroid differentiation markers sooner than standard (7.35) and low (7.1) pH cultures. Flow cytometry using CD71 and CD45RA antigens also indicated that erythroid differentiation proceeds faster at high pH and is blocked at an intermediate stage by low pH. Morphological data confirmed that high pH cultures had been shifted towards late-stage erythroid compartments as compared to low and standard pH cultures. These findings have important implications both in elucidating the regulatory role of pH in the bone marrow microenvironment and for the design of in vitro systems to study the development of erythroid cells.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1998

Oxygen tension alters the effects of cytokines on the megakaryocyte, erythrocyte, and granulocyte lineages.

Many investigators believe that cytokines have exclusive control over proliferation and differentiation in stroma-free hematopoietic cultures. Although cytokines are indeed necessary, other culture parameters such as oxygen (O2) tension can greatly influence both hematopoietic cell proliferation and differentiation. We investigated the effects of cytokine combinations and O2 tension on the expansion of megakaryocyte (size and number of CD41a+ cells and number of colony-forming units-megakaryocyte [CFU-Mk]), granulocyte (CD15+ cells and CFU-granulocyte monocyte [CFU-GM]), and erythrocyte (hemoglobin+ cells and burst-forming units-erythrocyte [BFU-E]) lineages. Peripheral blood CD34+ cells were cultured in serum-free medium with interleukin (IL)-3, stem cell factor (SCF), and various combinations of thrombopoietin (TPO), erythropoietin (EPO), and/or granulocyte-colony stimulating factor (G-CSF). The effects of TPO, EPO, and G-CSF on Mks, erythrocytes, and granulocytes, respectively, were dependent on the O2 tension. Thrombopoietin-containing cultures under a gas phase of 20%) O2 tension produced 1.4- to 2.2-fold more Mks than those under 5% O2. The increase in Mk size with TPO was also much greater under 20% O2. Similarly, 2.1- to 2.4-fold more hemoglobin-containing cells were produced in EPO-containing cultures under 20% vs. 5% O2. In contrast, approximately twice as many CD15+ cells were produced in G-CSF-containing cultures under 5% vs. 20%) O2. The numbers of CFU-Mk in TPO-containing and CFU-GM in G-CSF-containing cultures were larger under 5% O2. Although the O2 tension had no effect on BFU-E production in EPO-containing cultures, BFU-E production under 5% O2 in cultures without EPO was equal to that in EPO-containing cultures. Our data also suggest that TPO, EPO, and G-CSF elicit stimulatory cross-lineage effects in the presence of IL-3 and SCF, and that these effects, too, are often dependent on O2 tension.

Cell Count↗
PubMed1997

Culture materials affect ex vivo expansion of hematopoietic progenitor cells.

Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment. While many of the culture bag materials gave satisfactory results, all of the tubing materials severely inhibited total cell and CFU-GM expansion. Taken together, our results show that many materials approved for blood contact or considered biocompatible are not suitable for use with hematopoietic cells cultured in serum-free medium. As hematopoietic cultures are scaled up for a variety of clinical applications, it will be essential to carefully examine the biocompatibility of all materials involved.

Antigens, CD34↗
PubMed1997

Evaluation of cytokines for expansion of the megakaryocyte and granulocyte lineages.

The goal of our study was to identify cytokine combinations that would result in simultaneous ex vivo expansion of both the megakaryocyte (Mk) and granulocyte lineages, since these cell types have the potential to reduce the periods of thrombocytopenia and neutropenia following chemotherapy. We investigated the effects of cytokine combinations on expansion of the Mk (CD41a+ cells and colony forming unit [CFU]-Mk) and granulocyte (CD15+ cells and CFU-granulocyte/monocyte [GM]) lineages. Peripheral blood CD34+ cells were cultured in serum-free medium with interleukin 3 (IL-3), stem cell factor (SCF), and various combinations of thrombopoietin (TPO), IL-6, GM-CSF, and/or G-CSF. The Mk lineage was primarily influenced by TPO in our cultures, although Mk and CFU-Mk numbers were increased when TPO was combined with IL-6. The primary stimulator of the granulocyte lineage was G-CSF, although many synergistic and additive effects were observed with addition of other factors. Expansion of CFU-GM increased upon addition of more cytokines. The cytokine combination of IL-3, SCF, TPO, IL-6, GM-CSF and G-CSF produced the greatest number of granulocytes and CFU-GM. The minimum cytokines necessary for expansion of both the Mk and granulocyte lineages included TPO and G-CSF, since no other factors examined could increase Mk and granulocyte numbers to the same extent. The number of hematopoietic progenitors produced in our culture system should be sufficient for successful engraftment following myelosuppressive therapy if produced on a scale of about one liter.

Antigens, CD34↗
PubMed1997

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.

Antigens, CD34↗
PubMed1996

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.

Animals↗
PubMed1996

Hematopoietic cell culture therapies (Part II): Clinical aspects and applications.

High-dose chemotherapy, followed by hematopoietic stem cell transplantation, holds significant promise for increasing the probability of long-term remission and possibly cure in a variety of cancers. Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, may play a significant role in reducing the danger and expense associated with the transplantation procedure. Phase I clinical trials have shown that ex vivo expanded cells have no significant toxicities, and some benefits. Ex vivo expansion of hematopoietic cells is likely to find other applications in gene therapy, tumor purging, production of dendritic cells for immunotherapy and the production of mature blood cells for transfusion therapies.

Antigens, CD34↗
PubMed1996

Ex vivo culture systems for hematopoietic cells.

During the past few years, hematopoietic cell culture technologies for transplantation therapies have progressed significantly on several fronts. Advances include the discoveries of the growth factors thrombopoietin and Flt-3 ligand, the development of a variety of bioreactor systems, and results from preliminary clinical trials that demonstrate the efficacy of ex vivo expanded hematopoietic cells for transplantation therapy.

Blood↗
PubMed1996

Comparison of whole serum-deprived media for ex vivo expansion of hematopoietic progenitor cells from cord blood and mobilized peripheral blood mononuclear cells.

A whole serum-deprived (WSD) medium was developed and optimized for expansion of colony-forming cells (CFC) in cord blood (CB) mononuclear cell (MNC) cultures. This medium was compared with four commercially available WSD media (commercial media), three WSD media whose compositions have been publicly disclosed (public media), two serum-containing media, and two basal media, for cell and CFC expansion in 10-day CB and mobilized peripheral blood (PB) MNC cultures supplemented with interleukin-3 (IL-3), IL-6, and stem cell factor (SCF). Selected WSD media and both serum-containing media gave significant CFC expansion in CBMNC and PBMNC cultures. The serum-containing human long-term medium gave the greatest cell (3.0-fold) and CFC (25-fold) expansions in CBMNC cultures, whereas our medium maintained the most cells (93% of input) and gave the greatest CFC expansion (6.1-fold) for PBMNC cultures. Of the commercial media, Progenitor-34 gave the greatest cell expansion (1.2-fold) and X VIVO-10 gave the greatest CFC expansion (11-fold) for CBMNC cultures, and Progenitor-34 maintained the most cells (83% of input) and gave the greatest CFC expansion (3.1-fold) for PBMNC cultures. Of the public media (including ours), our medium gave the greatest cell (1.4-fold) and CFC (6.1-fold) expansion for CBMNC cultures. Although there were slight correlations between cell and CFC expansion in 10-day CBMNC and PBMNC cultures (r2 of 0.848 and 0.594, respectively), the correlations did not give reliable predictions for medium selection. In addition, the different media favored expansion of different CFC types and performed differently for cultures using different cell sources (CB versus PB). Taken together, these results suggest that media must be carefully screened for the cell source to be cultured and the cell type(s) to be produced (e.g. total cells, CFC).

Cell Division↗
PubMed1995

Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells.

Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony-forming units granulocyte-monocyte (CFU-GM) and long-term culture-initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony-forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.

Antigens, CD↗
PubMed1995

First-order toxicity assays for eye irritation using cell lines: parameters that affect in vitro evaluation.

First-order toxicity assays can be used to rapidly screen test agents. Investigators in many laboratories have used cultured cell lines to obtain correlations between first-order assay end-points and in vivo eye irritation (Draize test) for a wide variety of compounds. Since validation is a key step in assay acceptance, it is important to understand which factors alter the responses of cell-line-based assays. In this study we examine: (1) the presence and configuration of a type I collagen gel; (2) the responses of epithelial (Sf-1-Ep) and fibroblast (Sirc and 3T3) cell lines; (3) the total glutathione content, ATP content, methionine incorporation, and neutral red absorption endpoint assays; (4) alcohol (C2-C8), surfactant (Tween 20), and heavy metal (NiCl) test agents; and (5) test agent exposure time (1 to 24 hr). The presence of a collagen gel and the cell type did not significantly affect endpoint assay R50 (test agent concentration that decreases assay response by 50%) values for a 1-hr exposure to hexanol. The ATP and glutathione endpoints (after 1-hr exposure) are able to distinguish between the relative in vivo toxicities of C2-C8 normal alcohols. All four endpoint assays detected sublethal damage, with the ATP and methionine endpoints being the most sensitive. The type of test agent affects the endpoint response, as shown by the lack of a glutathione R50 value for a 1-hr exposure to Tween 20 or NiCl. Even for a single test agent, endpoint assay R50 values may decrease continuously (ATP), decrease and then stabilize (glutathione), or remain unchanged (methionine incorporation) during a 24-hr exposure.

3T3 Cells↗
PubMed1994

Diversity in the ability of cultured cells to elongate and desaturate essential (n-6 and n-3) fatty acids.

Essential fatty acids (EFAs) cannot be synthesized by mammalian cells. Once taken in with the diet, they can undergo desaturations/saturations and chain elongations/shortenings to yield a variety of polyunsaturated fatty acids of the same family. Cells in vitro from a variety of tissues are capable of processing EFAs to varying extents. Conversion of the parent EFAs, linoleic (LA, n-6) and alpha-linolenic (LNA, n-3) acids, to the 20-carbon polyunsaturated fatty acids, arachidonic (AA, n-6) and eicosapentanoic (EPA, n-3), requires chain elongation and delta 6 and delta 5 desaturations. AA and EPA are required by many tissues for optimal biological function and are precursors of biologically active eicosanoid hormones. All cultured cells are able to elongate exogenous LA and LNA, and most can perform delta 5 desaturation, so delta 6 desaturation is the limiting step in AA and EPA production. Longer fatty acids that have more double bonds than AA or EPA are less frequently produced due to a deficiency in delta 4 desaturating ability. The process of retroconversion (chain shortening) is less extensively studied, but evidence from a variety of cells suggests that this type of metabolic conversion is normally active. The example of MCF-7 (human breast cancer cell line) and MCF-10A cells (human noncancerous breast cell line) is discussed in order to emphasize the diversity in EFA processing ability of cultured cells. Under identical culture conditions, MCF-10A cells perform extensive desaturations, elongations, and retroconversions, whereas MCF-7 cells can only elongate and retroconvert exogenous EFAs. Given the great diversity in the ability of cultured cells to process EFAs, no conclusions can be drawn regarding the mechanisms responsible for the effects of exogenous EFAs on a particular cell until that cell's EFA processing patterns have been evaluated.

Animals↗
PubMed1994

Discrimination of fluorinated uridine metabolites in N-417 small cell lung cancer cell extracts via 19F- and 31P-NMR.

31P-NMR extract spectra of N-417 Small Cell Lung Cancer (SCLC) cells cultured with fluorouridine (FUrd) reveal new peaks with chemical shifts in the diphosphodiester and nucleoside triphosphate regions. These peaks were identified as FUTP, FUDP, FUDP-glucose, FUDP-glucuronate, FUDP-GlcNAc, and FUDP-GalNAc via enzymatic conversion and 19F- and 31P-NMR analysis. Distinct 19F chemical shifts were assigned for FUTP, FUDP, and the FUDP-sugars.

Carcinoma, Small Cell↗
PubMed1994

Diverse effects of essential (n-6 and n-3) fatty acids on cultured cells.

Fatty acids (FAs) have long been recognized for their nutritional value in the absence of glucose, and as necessary components of cell membranes. However, FAs have other effects on cells that may be less familiar. Polyunsaturated FAs of dietary origin (n-6 and n-3) cannot be synthesized by mammals, and are termed 'essential' because they are required for the optimal biologic function of specialized cells and tissues. However, they do not appear to be necessary for normal growth and metabolism of a variety of cells in culture. The essential fatty acids (EFAs) have received increased attention in recent years due to their presumed involvement in cardiovascular disorders and in cancers of the breast, pancreas, colon and prostate. Many in vitro systems have emerged which either examine the role of EFAs in human disease directly, or utilize EFAs to mimic the in vivo cellular environment. The effects of EFAs on cells are both direct and indirect. As components of membrane phospholipids, and due to their varying structural and physical properties, EFAs can alter membrane fluidity, at least in the local environment, and affect any process that is mediated via the membrane. EFAs containing 20 carbons and at least three double bonds can be enzymatically converted to eicosanoid hormones, which play important roles in a variety of physiological and pathological processes. Alternatively, EFAs released into cells from phospholipids can act as second messengers that activate protein kinase C. Furthermore, susceptibility to oxidative damage increases with the degree of unsaturation, a complication that merits consideration because lipid peroxidation can lead to a variety of substances with toxic and mutagenic properties. The effects of EFAs on cultured cells are illustrated using the responses of normal and tumor human mammary epithelial cells. A thorough evaluation of EFA effects on commercially important cells could be used to advantage in the biotechnology industry by identifying EFA supplements that lead to improved cell growth and/or productivity.

Animals↗
PubMed1994

n-3 and n-6 fatty acid processing and growth effects in neoplastic and non-cancerous human mammary epithelial cell lines.

The type rather than the amount of dietary fat may be more important in breast carcinogenesis. While animal studies support this view, little is known about the effects of essential fatty acids (EFAs) at the cellular level. The MCF-7 breast cancer and the MCF-10A non-cancerous human mammary epithelial cell lines are compared in terms of growth response to EFAs and ability to incorporate and process the EFAs. Eicosapentaenoic (EPA, n-3) and docosahexaenoic (DHA, n-3) acids, presented bound to albumin, inhibited the growth of MCF-7 cells by as much as 50% in a dose-dependent manner (6-30 microM) in medium containing 0.5% serum. alpha-Linolenic (LNA, n-3) and arachidonic (AA, n-6) acids inhibited growth less extensively, while linoleic acid (LA, n-6) had no effect. In contrast, MCF-10A cells were not inhibited by any of the EFAs at levels below 24 microM. The differential effects of AA, EPA and DHA on MCF-7 and MCF-10A cells support a protective role of highly unsaturated essential fatty acids against breast cancer. The EFAs were primarily incorporated into phosphoglycerides. MCF-7 cells showed chain elongations and possibly delta 8 desaturation, but no AA was formed from LA, nor EPA or DHA from LNA. In contrast, MCF-10A cells desaturated and elongated the exogenous EFAs via all the known pathways. These findings suggest defects in the desaturating enzymes of MCF-7 cells. LNA, DHA and AA presented to MCF-7 cells in phospholipid liposomes inhibited growth as extensively as albumin-bound free acids, but were less extensively incorporated, suggesting different mechanisms of inhibition for the two methods.

Breast↗