Human TE671 cells express functional motilin receptors

In our search for a cell line expressing endogenous human motilin receptor, we have discovered that theTE671 cell line, a neuron-derived medulloblastoma human line, expresses functional motilin receptors. The cDNA of the receptor was isolated from the cells and its sequence was confirmed to be identical to the previously reported cDNA sequence isolated from human thyroid. The function of the receptor protein was evaluated both for its ability to inhibit the binding of ¹²⁵I-motilin to a crude membrane preparation of TE671 cells and for activation of the phospholipase C signal transduction pathway by calcium mobilization assay. The precise numbers of motilin receptor RNA molecule in TE671 cell and 24 human tissues were quantitatively determined by real-time PCR. TE671 cell line should be a useful tool for the study of motilin receptor-involved signal transduction in humans.     

Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line

Functional properties of acetylcholine receptors from intact TE671 human medulloblastoma cells were examined using tracer ion flux, ligand competition against 125I-labeled alpha-bungarotoxin binding, and single channel recording measurements. 125I-Labeled alpha-bungarotoxin binds to surface receptors with the forward rate constant 1.8 X 10(5) M-1 s-1 and dissociates with the rate constant 4.6 X 10(-5) s-1, at 21 degrees C; the apparent dissociation constant is 2.6 X 10(-10) M. alpha-Bungarotoxin binds to at least two sites/receptor, but blocks agonist-induced 22Na+ uptake when bound to only one site. The reversible antagonists, dimethyl-d-tubocurarine and gallamine, occupy two sites which exhibit nearly equivalent affinities, but block agonist-induced uptake by occupying only one site. Strong agonists activate rapid sodium uptake with relatively low affinity, but desensitize with a much higher affinity; among agonists, the ratio of low to high affinity dissociation constants ranges from 1600 to 4000. By using the estimated dissociation constants, the allosteric model of Monod, Wyman, and Changeux (MWC) can be fitted to the concentration dependencies of both steady-state agonist occupancy and desensitization. The fitting analysis discloses an allosteric constant of 3 X 10(-5), which is the ratio of activatable to desensitized receptors in the absence of agonist. The rate of recovery from desensitization can exceed the rate of onset of desensitization elicited by low concentrations of agonist, further supporting the general MWC framework. Single channel recordings show that the channel opening probability is greater than 0.7 at high agonist concentrations. Favorable channel opening is shown to only slightly oppose strong desensitization.     

Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine

Acetylcholine sensitive TE 671 cells were cultured on nanoporous membranes and chemically stimulated by localized application of i), calcein-AM and ii), acetylcholine, respectively, onto the bottom face of the membrane employing an ink jet print head. Stimulus correlated response of cells was recorded by fluorescence microscopy with temporal and spatial resolution. Calcein fluorescence develops as a result of intracellular enzymatic conversion of calcein-AM, whereas Ca(2+) imaging using fluo-4 dye was employed to visualize cellular response to acetylcholine stimulation. Using 25 pl droplets and substance concentration ranging from 10 microM to 1 mM on Nucleopore membranes with pore diameters between 50 nm and 1 microm, a resolution on the order of 50 microm was achieved.     

Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.     

Human interferon production with diploid fibroblast cells grown on microcarriers.

A variety of diploid human fibroblast lines have been successfully grown to high densities (greater than 10(6) cell/ml) on recently developed microcarriers. Interferon induction using poly I.poly C and a superinduction procedure resulted in yields greater than 10,000 units/ml with one cell line. A direct comparison of microcarrier cultures to roller bottle cultures showed equivalent interferon yields on a per cell basis and some apparent differences relating to optimum inducer concentrations and kinetics of interferon accumulation.     

Death rate in a small air-lift loop reactor of vero cells grown on solid microcarriers and in macroporous microcarriers

The death rate of Vero cells grown on Cytodex-3 microcarrierswas studied as a function of the gas flow rate in a smallair-lift loop reactor. The death rate may be described byfirst-order death-rate kinetics. The first-order death-rateconstant as calculated from the decrease in viable cells, theincrease in dead cells and the increase in LDH activity islinear proportional to the gas flow rate, with a specifichypothetical killing volume in which all cells are killed ofabout 2.10(-3)m(3) liquid per m(3) of air bubbles.In addition, an experiment was conducted in the sameair-lift reactor with Vero cells grown inside porous Asahimicrocarriers. The specific hypothetical killing volumecalculated from this experiment has a value of 3.10(-4)m(3) liquid per m(3) of air bubbles, which shows thatthe porous microcarriers were at least in part able to protectthe cells against the detrimental hydrodynamic forcesgenerated by the bubbles.     

Death rate in a small air-lift loop reactor of vero cells grown on solid microcarriers and in macroporous microcarriers

The death rate of Vero cells grown on Cytodex-3 microcarriers was studied as a function of the gas flow rate in a small air-lift loop reactor. The death rate may be described by first-order death-rate kinetics. The first-order death-rate constant as calculated from the decrease in viable cells, the increase in dead cells and the increase in LDH activity is linear proportional to the gas flow rate, with a specific hypothetical killing volume in which all cells are killed of about 2·10^–3 m³ liquid per m³ of air bubbles. In addition, an experiment was conducted in the same air-lift reactor with Vero cells grown inside porous Asahi microcarriers. The specific hypothetical killing volume calculated from this experiment has a value of 3·10^–4 m³ liquid per m³ of air bubbles, which shows that the porous microcarriers were at least in part able to protect the cells against the detrimental hydrodynamic forces generated by the bubbles.     

Differentiation of myoblasts and CNS cells grown either separately or as co-cultures on microcarriers

Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively charged cylindrical microcarriers (MCS), to a stage which simulatein vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously. Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation, for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated, injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers.     

Spatial distribution of mammalian cells grown on macroporous microcarriers with improved attachment kinetics.

Vero and HepG2 cells were cultivated on macroporous gelatin microcarriers prepared by the calcium carbonate inclusion method. Cell attachment to these microcarriers was slow. For HepG2 cells the subsequent growth was poor. Modification of the microcarriers by incorporation of (diethylamino)ethyl-HCl improved HepG2 attachment and subsequent growth. Optical sectioning with confocal microscopy allowed visualization of the distribution of cells within microcarriers. In most microcarriers, cells were found to preferentially populate regions close to the external surface and some cavities in the interior. Despite the incomplete occupancy of the interior of the microcarriers, high cell concentrations were achieved.     

Production of human immune interferon by recombinant mammalian cells cultivated on microcarriers

Recombinant Chinese hamster ovary (rCHO) cells were cultivated on microcarriers for the production of human immune (Gamma) interferon. The effect of basal medium, serum, and microcarrier concentration on interferon production was investigated. The specific interferon productivity in the post-confluent stage was similar to that in the growth stage. Control of the pH results in a significant improvement in the volumetric interferon production. The volumetric production rate of interferon by these rCHO cells did not decrease after one month of cultivation on microcarriers.     

Continuous dialysis of cultures of human diploid cells (MRC 5) grown on microcarriers

Summary We have shown that if the medium supplemented with foetal calf serum (FCS) is continuously dialysed against a large volume of medium without FCS, the growth of human diploid fibroblasts on microcarriers is more rapid and yields are more consistent. This approach allows a considerable saving of serum.     

ESR studies on membrane fluidity of Chinese hamster ovary cells grown on microcarriers and in suspension

Electron spin resonance (ESR) spin label methods were used to study membrane fluidity of Chinese hamster ovary (CHO) cells grown on microcarriers and in suspension using 5-doxylstearic acid spin label as a probe. CHO cells grown on microcarriers had a more rigid cell membrane compared to CHO cells grown in suspension culture. CHO cells removed from the surface of the microcarriers by either trypsinization, EDTA treatment or osmotic shock had a membrane fluidity similar to that of CHO cells grown in suspension culture. Conversely, when the cells grown in suspension culture were attached and flattened on the surface of the microcarriers the fluidity decreased. Moreover, membrane fluidity of CHO cells grown on microcarriers changed as a function of the population density, whereas that of the cells in suspension did not. This implies that cell adhesion and/or cell-cell interactions influence the fluidity of the cell surface membrane.     

Selection of mitotic Chinese hamster ovary cells from microcarriers

We describe a method of collecting large quantities of mitotic cells from a population of Chinese hamster ovary cells which were exponentially growing on positively charged dextran microcarriers in suspension culture. These cells were treated for 2.5 h with colcemid, and mitotic cells were harvested from the oicrocarriers by increasing spinner velocity. A yield of 2–3% of the total population was obtained using this method; of the cells collected, 85–95% were in metaphase as determined by microscopic inspection. Both synchrony and cell viability were excellent in the selected population.     

Lack of function of histamine H1 and muscarinic acetylcholine receptors of mouse neuroblastoma cells grown in serum-free medium

Summary Mouse neuroblastoma cells (Clone N1E-115) were grown in serum-free (defined) medium, defined medium supplemented with serum, and control medium to determine whether serum-free medium could substitute for serum-containing medium in our studies of the histamine H₁ and muscarinic acetylcholine receptors of these cells. The function of these receptors as determined by measurement of receptor-mediated cyclic [³H]GMP formation was absent in cells grown in serum-free medium and increased as the percentage of serum was increased in the defined medium, but never attained the levels found with control cells. Muscarinic receptor number for cells grown in defined medium was 60% above that found for control cells with no change in the affinity of the receptor for the radioligand (−)[³H]quinuclidinyl benzilate. Guanylate cyclase and acetylcholinesterase activities for cells grown in defined medium were 23 and 66% of those found in control cells, respectively. This marked reduction of guanylate cyclase activity in large part explains the lack of function of these receptors. Supported by Mayo Foundation and U.S. Public Health Service Grants MH27692, DA1490, and AA4443.     

Expansion of mouse sertoli cells on microcarriers

Background: Sertoli cells (SCs) have been described as the ‘nurse cells’ of the testis whose primary function is to provide essential growth factors and create an appropriate environment for development of other cells [for example, germinal and nerve stem cells (NSCs), used here]. However, the greatest challenge at present is that it is difficult to obtain sufficient SCs of normal physiological function for cell transplantation and biological medicine, largely due to traditional static culture parameter difficult to be monitored and scaled up. Objective: Operational stirred culture conditions for in vitro expansion and differentiation of SCs need to be optimized for large‐scale culture. Materials and methods: In this study, the culturing process for primary SC expansion and maintaining lack of differentiation was optimized for the first time, by using microcarrier bead technology in spinner flask culture. Effects of various feeding/refreshing regimes, stirring speeds, seed inoculum levels of SCs, and concentrations of microcarrier used for expansion of mouse SCs were also explored. In addition, pH, osmotic pressure and metabolic variables including consumption rates of glucose, glutamine, amino acids, and formation rates of lactic acid and ammonia, were investigated in culture. Results: After 6 days, maximal cell densities achieved were 4.6 × 10⁶ cells/ml for Cytodex‐1 in DMEM/FBS compared to 4.8 × 10⁵ cells/ml in static culture. Improved expansion was achieved using an inoculum of 1 × 10⁵ cells/ml and microcarrier concentration of 3 mg/ml at stirring speed of 30 rpm. Results indicated that medium replacement (50% changed everyday) resulted in supply of nutrients and removal of waste products inhibiting cell growth, that lead to maintenance of cultures in steady state for several days. These conditions favoured preservation of SCs in the undifferentiated state and significantly increased their physiological activity and trophic function, which were assessed by co‐culturing with NSCs and immunostaining. Conclusion: Data obtained in this study demonstrate the vast potential of this stirred culture system for efficient, reproducible and cost‐effective expansion of SCs in vitro. The system has advantages over static culture, which has major obstacles such as lower cell density, is time‐consuming and susceptible to contamination.     

A factor from neurons induces partial immobilization of nonclustered acetylcholine receptors on cultured muscle cells

A factor or factors released by cultured NG108-15 neuroblastoma X glioma hybrid cells and added to the medium of rat myotube primary cultures was found to immobilize some of the previously mobile acetylcholine receptors in the myotube membrane. Partial receptor immobilization occurred within 3 h after the beginning of treatment with the NG108-15-conditioned medium factor and persisted for at least 24 h of continuous treatment. A similarly derived conditioned medium concentrate from the non-neuronal parent glioma cell line did not immobilize receptors, relative to untreated controls. Acetylcholine receptors were visualized by fluorescent alpha-bungarotoxin and their lateral motion was observed by the technique of fluorescence photobleaching recovery.     

Production of erythropoietin by BHK cells growing on the microcarriers trapped in alginate gel beads

Baby hamster kidney (BHK) cells engineered to produce recombinant human erythropoietin (EPO) were cultured at high density on microcarriers entrapped by calcium alginate gel particles. In this system, the BHK cells proliferated not only on the microcarriers but also in vacant spaces in the alginate gel particles. These spaces contributed greatly to high-density cultivation of the cells and a high productivity of EPO.Abbreviations BHK Baby Hamster Kidney - EPO Erythropoietin     

Serial propagation of mammalian cells on microcarriers

For the large-scale operation of microcarrier culture to be successful, a technically feasible method for sequential inoculation is essential. Using human foreskin fibroblasts, FS-4, we have achieved this by detaching cells viably from microcarriers employing a selection pH trypsinization technique. Cells thus detached are able to reattach to microcarriers and grow normally after subsequent reinoculation into new cultures. However, after reinoculation cells attach to new microcarriers at a higher rate than to used microcarriers on which cells have previously grown. The effect of this differential cell attachment was analyzed and overcome by employing a low inoculum concentration. FS-4 cells could thus be serially propagated on microcarriers and subsequently used for beta-interferon production. This technique has also been applied to the cultivation of a monkey kidney cell line, Vero. We have also shown that Vero cells directly inoculated from a seed microcarrier culture could be used for virus production.