Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID₅₀) titers of the two cell lines reached 4.51×10⁶ cells/mL, 2.94Log₁₀(HAU/50 μL) and 8.49Log₁₀(virions/mL), and 5.97×10⁶ cells/mL, 3.88Log₁₀(HAU/50 μL), and 10.34Log₁₀(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.     

l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies)

The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate.In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light.With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.     

低密度和条件培养对红豆杉细胞生长的影响

红豆杉种胚来源的细胞,在改良Ｂ５液体培养基中继代培养的临界接种密度为鲜重４０ｇ／Ｌ．低密度培养下,１０－１６ｄ的条件培养液（ＣＭ）与新鲜培养液按５７：４３的比例混合时,能显著缩短细胞生长的延迟期,提高生长率,１００Ｌ生物反应器中,按４５．５％体积分数添加条件培养液,在鲜重２７ｇ／Ｌ低接种密度下培养５周,生物量增长９倍,达干重１４．３ｇ／Ｌ．对内源植物激素、精胺、维生素和氨基酸等的比较分析表明,吲哚     

Increased production of human granulocyte-macrophage colony stimulating factor (hGM-CSF) by the addition of stabilizing polymer in plant suspension cultures

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that stimulates the production of granulocytes, macrophages, and white blood cells. Secretion of human GM-CSF from cell suspension cultures of genetically modified tobacco has been facilitated using natural mammalian leader sequences. At the mid-exponential growth phase (day 4 after the initiation of cell suspension culture), GM-CSF was detected in the medium at a maximum concentration of 180 microg l(-1). However, the secreted GM-CSF was unstable in the medium, and rapidly degraded after day 5. In order to stabilize the secreted GM-CSF, three stabilizing polymers were tested, polyethylene glycol, polyvinylpyrrolidone and gelatin. Gelatin was the most effective in stabilizing the secreted GM-CSF. Following the addition of 5% (w/v) gelatin, the maximum GM-CSF concentration reached 783 microg l(-1), a 4.6-fold increase over control.     

Enhanced human thrombopoietin production by sodium butyrate addition to serum-free suspension culture of bcl-2-overexpressing CHO cells

When sodium butyrate (NaBu) was added to serum-free suspension culture of recombinant CHO (rCHO) cells for enhanced expression of human thrombopoietin (hTPO), apoptotic cell death of rCHO cells was induced in a dose-dependent manner and hTPO quality was deteriorated in regard to sialic acid and acidic isoform contents. To overcome these problems, we overexpressed Bcl-2 protein, an antiapoptotic protein, in rCHO cells producing hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bc12-14 cells) and 3 mM NaBu addition. Both the enhanced specific productivity endowed by NaBu and the extended culture longevity provided by the antiapoptotic effect of Bcl-2 overexpression contributed to the enhancement of maximum hTPO concentration. The problem of quality reduction of hTPO induced by NaBu was not solved by Bcl-2 overexpression, but it was not that significant. Compared to the culture in the absence of NaBu, the percentage of hTPO isoforms in pI 3-5 with high in vivo biological activity produced by R-bc12-14 cells was decreased by approximately 18% in the presence of 3 mM. As a result, a more than 6-fold increase in the production of hTPO isoforms in pI 3-5 was achieved in R-bcl2-14 cell culture with 3 mM NaBu addition. Taken together, the data obtained suggest that Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO.     

The improvement of polyethylene glycol-induced cell fusion by incubation of cells in a medium with increased NaCl concentration

Incubation of cells in a medium with NaCl concentration increased by 0.2 mol/l for one hour prior to and one hour after the polyethylene glycol induced cell fusion improves the performance of fusion. This procedure increases the percentage of fused cells, the formation of massive fusion products (in case of fusion performed in suspension) and allows the improvement of cell viability. The possible mechanisms involved in such effects of increased NaCl concentration are discussed.     

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/gamma2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 +/- 0.12 day(-1) (+/-standard deviation), while that in medium containing 1% serum was approximately 0.73 +/- 0.12 day(-1). The specific MAb productivity was almost constant at 3.69 +/- 0.57 mug/10(6) cells/day irrespective of serum concentration reached a maximum at ca. 1.8 x 10(6) cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.     

Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cells

Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

碳源对迷迭香悬浮培养细胞的生长、迷迭香酸积累及抗氧化酶活性的影响

以迷迭香悬浮培养细胞为材料,详细研究了基本培养基中添加蔗糖、麦芽糖和葡萄糖对细胞生长及次生代谢产物积累的影响,同时对不同蔗糖浓度处理的悬浮培养细胞抗氧化酶活性进行了研究。研究结果表明：在不同的糖处理中,30 g·L^-1的蔗糖、70 g·L^-1的麦芽糖及40 g·L^-1的葡萄糖最有利于迷迭香悬浮培养细胞生长。30 g·L^-1蔗糖和70 g·L^-1麦芽糖处理中悬浮培养细胞的生长率分别为74.08%和72.33%,高出40 g·L^-1葡萄糖处理接近3倍之多。30 g·L^-1蔗糖处理的悬浮培养细胞迷迭香酸含量高出70 g·L^-1麦芽糖处理228倍,略低于40 g·L^-1葡萄糖处理。在不同蔗糖的处理中,随着蔗糖浓度的增加,迷迭香酸含量均呈现增加趋势,表明高浓度的蔗糖有利于悬浮培养细胞迷迭香酸的积累。在高浓度的蔗糖处理中,悬浮培养细胞H₂O₂和MDA含量明显增加,同时抗氧化酶SOD、POD及CAT的活性也明显增强,表明高浓度的蔗糖产生了渗透胁迫,这种渗透胁迫虽不利于迷迭香悬浮培养细胞的生长,但有利于次生代谢产物的积累。综合迷迭香悬浮细胞的生长率和迷迭香酸的含量,我们最终得出30 g·L^-1的蔗糖最有利于迷迭香悬浮细胞的培养。     

Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor

Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture.     

The role of lipid peroxidation in aluminium toxicity in soybean cell suspension cultures

The primary reactions leading to Al toxicity in plant cells have not yet been elucidated. We used soybean (Glycine max [L.] Merr.) cell suspension cultures to address the question whether lipid peroxidation plays an important role in Al toxicity. Upon transfer to an Al-containing culture medium with a calculated Al3+ activity of 15 microM soybean cells showed a distinct and longtime increase in lipid peroxidation within 4 h. At the same time a drastic loss of cell viability was observed. Butylated hydroxyanisole (BHA) and N,N'-diphenyl-p-phenylenediamine (DPPD), two lipophilic antioxidants, were able to almost completely suppress lipid peroxidation in Al-treated cells at a concentration of 20 microM. This effect was dose-dependent for DPPD and was observed at minimum concentrations of 1-2 microM. When lipid peroxidation was suppressed by DPPD or BHA cell viability remained high even in the presence of toxic Al concentrations. These results suggest that Al-induced enhancement of lipid peroxidation is a decisive factor for Al toxicity in suspension cultured soybean cells.     

Cell suspensions from porcine olfactory mucosa. Changes in membrane potential and membrane fluidity in response to various odorants

A suspension of olfactory epithelial cells was prepared from porcine olfactory mucosa and the physiological functions of the suspension were examined. The membrane potential of the cell suspension, which was monitored by measuring the fluorescence changes of rhodamine 6G, was depolarized by an increase in the K+ concentration in the external medium. Various odorants depolarized the cell suspension in a dose-dependent fashion. The magnitude of depolarization by odorants was either unchanged or slightly increased by a reduction of the concentration of Na+, Ca2+, and Cl- in the external medium, which suggests that changes in the permeabilities of specific ions are not involved in depolarization by odorants. The application of various odorants to the cell suspension induced changes in the membrane fluidity at different sites of the membrane that were monitored with various fluorescent dyes [8-anilino-1-naphthalene sulfonate, n-(9-anthroyloxy) stearic acids, 12-(9-anthroyloxy) oleic acid, and (1,6-diphenyl-1,3,5-hexatriene)], which suggests that the odorants having different odors are adsorbed on different sites in the membrane. On the basis of these results, a possible mechanism of odor discrimination is discussed.     

钙离子对293细胞结团和生长的影响

分别在有血清和无血清条件下、方瓶和转瓶中考察了Ca²⁺ 对2 93细胞结团和生长的影响。通过实验发现,Ca²⁺ 浓度在0 1～1 0mmol L范围内对2 93细胞的贴壁和结团性质有显著影响,而对生长影响不大。结果表明:有血清贴壁培养时,较高的Ca²⁺ 浓度有利于细胞贴壁;无血清悬浮培养中,Ca²⁺ 浓度越高,细胞结团越严重,细胞结团达到平衡后的平均粒径(D ,μm)与Ca²⁺ 浓度(c,mmol L)在0.1～0.5mmol L范围内可用一次函数D =58.65c +16.96描述,细胞结团尺寸是可调控的;而细胞在不同的Ca²⁺ 浓度下有相似的生长规律。     

Co-expression of proteinase inhibitor enhances recombinant human granulocyte-macrophage colony stimulating factor production in transgenic rice cell suspension culture

The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte–macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production.     

Effects of synthetic lysophosphatidylcholines on suspension cultures of the Chinese hamster ovary DG44 cells in protein-free media

This study described the effects of synthetic lysophosphatidylcholines on the growth of recombinant CHO-DG44 cells in suspension. Overall, cell growth characteristics were improved when cultivated in suspension in a protein-free medium supplemented with natural soybean lysophosphatidylcholines. To substitute synthetic lysophosphatidylcholines for the naturally occurring lysophosphatidylcholines, we implemented a systematic approach in which twelve synthetic lysophosphatidylcholines were grouped into three lipid mixtures according to the length of their acyl chains. We found that synthetic lysophosphatidylcholines with medium acyl chain lengths (C14-C18), including oleoyl lysophosphatidylcholine (C18:1) could increase cell growth in the protein-free media. The fortified protein-free medium with medium acyl chain length lysophosphatidylcholines (C14-C18) maintained growth of CHO-DG44 cells over five consecutive passages, whereas the cell growth in a CHO protein-free medium was decreased gradually after four passages. We also observed that the restorative effect of oleoyl lysophosphatidylcholine was comparable to that of natural lysophosphatidylcholine in batch and long-term cultivation. These results show that synthetic lysophosphatidylcholines can be used as lipid supplements in either protein-free media or chemically defined media for CHO cell suspension cultures.     

Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions

A cell suspension of grape, Vitis vinifera L. cv Gamay Fréaux, was grown under different conditions of water stress (high external osmotic potential) induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Best growth (cell density) was achieved in the low osmotic potential medium. Increasing the osmotic potential of the medium from –0.5 MPa to –0.9 MPa medium resulted in a significant increase in accumulation of anthocyanins in pigmented cells. Regulation of the osmotic potential of culture medium may be useful in controlling anthocyanin production.     

Cellular determinants affecting the rate of cytokine in cultures of human hematopoietic cells

The present study was undertaken to define parameters that may limit the cytokine-mediated expansion of primitive hematopoietic cells in stirred suspension cultures of normal human marrow cells. In a first series of experiments, parallel measurements of the rate and extent of progenitor expansion and cytokine depletion from the medium were made for such cultures in which the cells were exposed to different cytokine concentrations. Supplementation of the medium with 2 ng/mL of interleukin-3 (IL-3), IL-6 and IL-11 plus 10 ng/mL of Flt-3 ligand (FL) and Steel factor (SF) allowed a 45-fold expansion of directly clonogenic cell (CFC) numbers within 2 weeks along with a 2.5-fold expansion of their precursors, detectable as longterm culture-initiating cells (LTC-IC). The addition of 5-fold higher levels of these cytokines enhanced the 2 week output of both CFC and LTC-IC numbers (to 66-fold and 9-fold above input respectively). However, this was also associated with an increase in the individual average rates of depletion of immunoreactive IL-3, SF and FL. As a result, even biweekly addition of fresh medium supplemented with the highest concentrations of cytokines tested failed to prevent a continuing decline in their levels relative to the input medium levels. A similar dependence of the IL-3 depletion rate on its extracellular concentration was demonstrable in suspension cultures of Mo7e cells, an IL-3-dependent human leukemic cell line.Additional experiments with various highly purified marrow cell fractions showed that the rate of cytokine depletion varied according to the type of responding cell as well as the specific cytokine. CD34(+)CD38(-) cells exhibited the greatest average cell-specific cytokine depletion rates (35-fold higher than unseparated bone marrow cells). These findings establish new principles that will be important for the optimization of hematopoietic cell bioreactors. In addition, they suggest that cytokine depletion may provide a novel feedback control mechanism in vivo which would contribute to the control of primitive hematopoietic cell proliferation and differentiation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 58-66, 1997.     

Growth kinetics of Chinese hamster ovary cells in a compact loop bioreactor. 3. Selection and characterization of an anchorage-independent subline and medium improvement.

Four sublines of Chinese hamster ovary (CHO) cells were selected or cloned on a 10% fetal calf serum supplemented MEM-alpha medium. Three of them were monolayer cultures and could proliferate by 2000 times a week (mu = 1.1 d 1) in T-flasks. The other subline, S1, could grow in suspension even in static T-flask cultures. The stability in chromosome number of these cell lines was investigated. By evaluating the kinetic growth parameters, i.e. the specific rates of growth, glucose consumption and lactic acid production, and the yields of cells and lactic acid from glucose, the S1 cells were considered to be the most suitable subline for the bioreactor suspension culture. The S1 cells reached the greatest maximum of cell concentration among all cell lines tested because of their efficient glucose utilization. Observed nutrient limitations in the S1 cell culture was overcome by modification of the medium composition, that is addition of 10 mg l-1 hypoxanthine, 1 mg l-1 FeSO4.7H2O, and 0.1 mg l-1 sodium putrescine, elimination of glutamine, supplementation of 6 mM asparagine and double amount of isoleucine, leucine, methionine and vitamins other than ascorbic acid, cyanocobalamin and biotin, increase of NaHCO3 concentration from 26 to 40 mM, and finally decrease of NaCl concentration from 122 to 100 mM. With this modified medium, 7.2 X 10(6) ml-1 of the maximum cell concentration was observed in a glucose fed-batch culture, the cell concentration which was twice as much as in batch cultures with the original medium.     

Effects of sex steroids and antihormones on growth, adhesiveness and receptors of L-929 cells cultured in serum-containing and serum-free media.

L-929 cells contain distinct steroid hormone receptors for glucocorticosteroids, for androgens and for estrogens. We studied the effects of different hormones at physiological concentrations on androgen and estrogen receptor protein accumulation and on cell multiplication. The cells were cultured in steroid-free serum-containing medium, either in Petri dishes or in suspension cultures, and in serum-free medium in Petri dishes. The presence of androstanolone (30 nM) in suspension cultures decreased the concentration of estradiol receptor-binding sites in the cytoplasmic fraction. This decrease was progressive following 3, 5 or 10 days of suspension culture in the presence of the androgen; simultaneously a parallel increase in cell multiplication and DNA was observed. The estradiol receptor decrease was approx. 50% after 10 days of treatment and was unaltered after a further 5 days. It was verified that the low androstanolone concentration in the medium did not provoke the translocation of the estradiol receptor into the nucleus. Progesterone 50 nM also decreased the cytoplasmic estradiol binding sites but had no influence on cell growth and no cytoplasmic progesterone receptor could be found. Diethylstilbestrol (30 nM) did not decrease the concentration of androgen receptor.Cell multiplication was stimulated after several days of suspension culture in the presence of either diethylstilbestrol, estradiol or androstanolone at a concentration of 10–30 nM. The specific anti-hormones, tamoxifen and cyproterone acetate, inhibited selectively the growth effects of estrogens and androgen, respectively. L-929 cells could be cultured for a long period of time in serum-free medium in Petri dishes. Cell adhesiveness was increased in the presence of 40 nM androstanolone or 40 nM estradiol, as well as cell multiplication. Dexamethasone had a negative effect on cell adhesiveness and cell growth. The experimental data suggest that at low concentrations the different steroids operated each through its own receptor and were active on cell growth even in serum-free medium.     

The effect of polyoma virus,serum factors,and dibutyryl cyclic AMP on dihydrofolate reductase synthesis,and the entry of quiescent cells into S phase

Three procedures were used to induce dihydrofolate reductase synthesis in quiescent cultures of methotrexate resistant mouse fibroblasts: (1) lytic infection with polyoma virus, (2) growth stimulation by replating cells at lower density in fresh cell culture medium, and (3) the addition of fresh medium to confluent cells. Following polyoma infection, an increase in the percentage of S-phase cells began at approximately 20 hours; dihydrofolate reductase synthesis also increased following a lag of 20 hours or more, and continued to increase throughout the late phase of lytic infection, reaching values nearly fivefold greater than that originally present in the quiescent cells. When quiescent cells received fresh medium (with or without replating), the percentage of cells in S phase began to increase by 10 hours and was accompanied by an increase in dihydrofolate reductase synthesis which reached a maximum by approximately 25 hours. These observations show that the initial entry of cells into S phase following mitogenic stimulation is associated with an induction of dihydrofolate reductase synthesis. Dibutyryl cyclic AMP blocked the stimulation of dihydrofolate reductase synthesis and the increase in the percentage of S-phase cells that resulted from the addition of fresh medium to confluent cells. When dibutyryl cyclic AMP was added at various times following the addition of fresh medium, the block in the induction of dihydrofolate reductase synthesis was correlated with a corresponding block in the increase in S-phase cells. These results suggest that dibutyryl cyclic AMP blocks cells at a point in Gl prior to either the induction of dihydrofolate reductase synthesis or the beginning of S phase. The relationship between the control of dihydrofolate reductase synthesis and entry into S phase suggests some form of coordinate control over these two parameters.