Large-scale animal cell cultures: design and operational considerations.

The manufacture of biologicals, especially proteins, using large-scale culture of animal cells is becoming popular. There is a need for a rational approach to the design and scale-up of bioreactors for these applications. The ultimate requirement of any scale-up strategy should be to preserve the biological activity of these high-value molecules. With this as the central theme, the design and operation of animal cell processes has been discussed. Equal importance has been given to both the biological and the engineering aspects which need to be considered for a successful scale-up. An integrated systems approach has been stressed.

Regulation of lac operon expression in mixed sugar chemostat cultures.

Expression of the lactose (lac) operon in the Escherichia coli chromosome has been studied in mixed-sugar chemostat cultures under steady-state and transient conditions. A unified model has been formulated which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression. The model of the lac operon control system focuses on the molecular interactions among the regulatory species and the genetic control elements for the initiation of transcription. The role of catabolite modulator factor (CMF) in the regulation of transcription is described. The modeling of glucose-mediated regulation of intracellular cyclic adenosine monophosphate (cAMP) and inducer exclusion is based on the recently elucidated mechanisms of the involvement of the PTS (phosphoen-olpyruvate dependent sugar transport system) enzymes, in the presence of glucose, in regulation of adenylate cyclase and non-PTS sugar transport proteins (i.e. per-meases). The adequacy of the unified model was verified with experimental data.

Binding of thymopoietin to the acetylcholine receptor.

Thymopoietin is a polypeptide hormone of the thymus with physiological effects on the immune system and on acetylcholine-mediated transmission at the neuromuscular synapse. Elucidation of the structure and function of the nicotinic acetylcholine receptor has been facilitated by the use of the electric organs of Torpedo ray or Electrophorus eel as rich sources of the receptor and by the use of snake polypeptide toxins such as alpha-bungarotoxin as highly selective labels of the acetylcholine binding site. We now show that thymopoietin binds with high affinity (Ka approximately equal to 2.5 X 10(9) M-1) to the acetylcholine binding region of the acetylcholine receptor of Torpedo californica, as evidenced by similar and complete inhibition of the binding of radiolabeled thymopoietin or alpha-bungarotoxin by either of these polypeptides. These findings raise intriguing questions concerning the mechanisms whereby alpha-bungarotoxin and the thymopoietin affect acetylcholine receptor function, since these two polypeptides with such similar binding properties have very different functional effects.

Steady-state and transient behavior in microbial methanification: I. Experimental results.

The process of methanification of volatile fatty acids (VFA) was studied to elucidate its kinetics. An upflow anaerobic sludge bed (UASB) system was used to perform the experiments. At residence times of less than 2.5 h the UASB system was found to exhibit hysteresis with respect to acetic and propionic acid consumption but not with respect to butyric acid consumption. These hysteretic effects could be attributed to the manner in which the various VFA-consuming cultures were structured inside the flocculated biomass in light of the cross-inhibitory effects of the acetic- and propionic-acid-consuming fractions of the total culture. (Butyric acid proved to be non-interactive.) Production of methane was found to respond almost instantaneously to changes in the inlet conditions of the UASB system. This indicated that methane is not primarily growth associated, as has often been assumed, but is related to changes in the culture's maintenance energy requirements. Reactor operation was found to be stable even when the concentration of each VFA in the feed was simultaneously changed by 50%. Even at very high organic throughput rates (35 kg COD/day m(3)-reactor) conversions of 82% were observed.

Steady-state and transient behavior in microbial methanification: II. Mathematical modeling and verification.

It has been shown that the Upflow Anaerobic Sludge Bed (UASB) system data reported earlier(1) cannot be explained by simple Monod-type substrate consumption patterns. An autoinhibition model was also ruled out because the substrate concentration range over which hysteresis was observed was much larger than such a model would predict. However, propionic and acetic acids were found to inhibit each other's conversion machineries. Since in the UASB system the biocatalyst is flocculated, it was found that a model additionally incorporating this facet of the reactor set-up could explain the steady-state data very well. Using the parameters generated from steady-state data and data from butyric acid step change,(1) i.e., the entire set of parameters (Table I), a very good agreement between predicted and observed data was found. International Mathematical and Statistical Libraries (IMSL) and Upjohn's NONLIN library combined with various root-finding and integrating subroutines were used for parameter estimation. The model thus described was used to predict the response of the UASB system when acetic acid and propionic acid influent concentrations were stepped-up/down. The agreement between the predicted and observed data was found to be excellent in each case during the step-up schedule. During the step-down the data seemed to indicate that the UASB system, like any other chemostat, responded faster than predicted. This could be due to the fact that when the culture has to "gear up" part of the lag time is the time required for the cell to produce the requisite amount of enzymes. In the case of "gearing down" this time is not required and the system responds faster.

Immobilized live cell reactor dynamics following dilution rate shift to growth conditions: Cell synchrony effects.

Nutrient deprivation was used to synchronize an immobilized live cell culture of Acetobacter suboxydans. The substrate supply was increased by a step change in the dilution rate to the reactor. Oscillations in cell, substrate, and product concentrations were observed. A population balance model was developed to explain the observed reactor dynamics. Simulation results based on the model were used to substantiate the premise that cell synchrony is the likely phenomenon responsible for the observed oscillations. The implications of cell synchrony in immobilized cell systems are discussed briefly.

Enzyme adsorption in porous supports: local thermodynamic equilibrium model.

Enzyme adsorption from a finite bath (batch adsorption) onto porous spherical supports is investigated both experimentally and theoretically using beta-galactosidase and Duolite ion-exchange resin as a model system. Efficient numerical techniques are presented that have been used in conjunction with a parameter estimation routine to evaluate adsorption isotherm constants. Results show that even for adsorption processes lasting almost 10 h, the majority of the enzyme is confined to the outer half of the support and, for high initial enzyme concentrations in the bath, this loading takes place as a slowly moving front. Information on the enzyme distribution has practical importance in the design of immobilized enzyme reactors that in previous works have almost always been analyzed assuming a uniform catalyst distribution.

Role of neurotransmitters and teratogens on palate development.

Neurotransmitters regulate palate shelf reorientation. Acetylcholine and serotonin stimulate, whereas GABA inhibits reorientation. Serotonin stimulates cell movement in an in vitro chemotactic system. Diazepam may cause cleft palate by mimicking GABA. Diazepam sensitivity may be caused by genotypic differences in a GABA-ergic system in the embryo.

Adhesive and migratory behavior of normal and sulfate-deficient sea urchin cells in vitro.

Dissociated cells from different stage embryos of the sea urchin Lytechinus pictus were compared in their adhesion to various substrates. Micromeres from 16-cell stage embryos bind to tissue culture and Petri dishes but not to Petri dishes coated with human plasma fibronectin. Other cell types did not adhere to any of the substrates tested. By hatched blastula stage, about 28% of the cells adhered to fibronectin as well as to tissue culture dishes. By the mesenchyme blastula stage, there was a further increase in the proportion of cells adhering to these substrates. At no stage did cells adhere to native rat tail collagen. Primary mesenchymal cells were isolated by their selective adhesion to tissue culture dishes in the presence of horse serum. These cells were then examined for their migratory capacity. Cell spreading and migration followed adhesion and occurred on fibronectin but not on the other substrates tested. Based on analysis of video tapes, greater than 60% of these cells moved faster than 1 micron/min. On the other hand, cells from sulfate-deprived embryos, in which primary mesenchyme migration is blocked in situ, failed to spread and migrated little on the same substratum. This defect was reversed by a 6 h pretreatment of the cells in normal sea water. Thus, the in vitro migratory behavior parallels that observed in vivo. These results support the hypothesis that the primary mesenchymal cells produce a sulfate-dependent component that is required for cell spreading and migration.

Chemotactic behavior of myoblasts.

Earlier studies have suggested that myogenic cells of somite origin migrate into the developing limb, but little is known about the factors affecting the pattern of migration. In order to understand the migratory behavior of myogenic cells, embryonic skeletal muscle cells were tested for their ability to migrate chemotactically using a modified Boyden chamber assay system. It is shown here, for the first time, that embryonic skeletal muscle cells have the capacity to migrate toward a gradient of platelet-derived growth factor (PDGF) and PDGF-like factors present in serum and chick embryo extract (CEE). On the other hand, nonmyogenic limb mesenchyme cells do not exhibit such a response. A hypothesis is proposed here that chemotactic factors from the already patterned vasculature might influence the distribution of skeletal muscle cells during early limb development.

Palate cell motility and substrate interaction.

To elucidate the mechanism by which palate shelves reorient during embryogenesis, migration of palate mesenchymal cells has been studied employing various substrates. When palate explants were cultured in a hydrated collagen lattice, it was observed that bipolar spindle-shaped cells migrated out of each explant toward the other. These cells were aligned parallel to each other and to the fibrous tracks that formed. The cells appeared to move along and through the fibrous tracks. Cell migration was dependent on the presence of serum and fibronectin. The fibrous tracks viewed by phase microscopy were sensitive to collagenase. Scanning electron microscopy revealed that the collagen fibers of the hydrated lattice had coalesced into larger bundles. Pretreatment of explants with serotonin stimulated cell migration out of the explant into the hydrated collagen lattice. This effect was specific, since the antagonist methysergide blocked the stimulation produced by serotonin. Employing other substrates, it was noted that palate cells migrating out of double explants toward each other produced large wrinkles in a polysiloxane substratum. Similarly, cultured monolayer cells also produced wrinkles that disappeared as cells rounded up after trypsin treatment. Finally, monolayer cells pulled on and distorted collagen films when cultured on the substrate. It is concluded that migrating palate cells can interact with their substrate producing tractional forces. Serotonin-induced modulation of cell motility and its relationship to palate reorientation are discussed.

Serotonin regulation of palatal cell motility and metabolism.

Serotonin has been previously shown to stimulate palate reorientation. To elucidate the mechanism by which the neurotransmitter may be regulating palate morphogenesis, the effects of serotonin on cell motility and various metabolic reactions have been measured in vitro. To monitor cell motility, a chemotactic system was employed in which cultured palate mesenchymal cells in a modified Boyden chamber migrate toward the chemoattractant(s) in N-18 neuroblastoma conditioned medium. Serotonin stimulated cell motility and 10(-5) M was optimal with nearly 100% stimulation achieved. With N-18 conditioned medium diluted 1:100, serotonin stimulated cell motility 4.9-fold. Serotonin itself was not chemotactic but modulated cell movement in the presence of the chemoattractant. Protein carboxyl methylation was stimulated by serotonin about 100% at concentrations ranging from 3 X 10(-7) M to 3 X 10(-6) M in different experiments. The net stimulation may have been elicited by an indirect effect since serotonin also inhibited demethylation of protein methyl esters. Serotonin was shown to inhibit cyclic AMP in cultured palate cells: 10(-5) M agonist depressed levels to 19% of control in 3 h. Further, prostaglandin E1, which stimulated cyclic AMP levels, markedly inhibited cell motility in the chemotactic assay. Thus there is an inverse relationship between cyclic AMP levels and cell motility in fetal palate cells. Finally it was observed that serotonin stimulated cyclic GMP levels; 10(-5) M serotonin optimally stimulated cyclic GMP with a spike of stimulation (6.1-fold) within 30 sec. In summary, serotonin in palate cells stimulates both protein carboxyl methylation and cyclic GMP. Modulation of these reactions could be regulating cell motility and/or protein secretion, which in turn could function in palate reorientation.

Enhancement of starch conversion efficiency with free and immobilized pullulanase and alpha-1,4-glucosidase.

Glucoamylase and pullulanase were immobilized on reconstituted bovine-hide collagen membranes using the covalent azide linkage method. A pretanning step was incorporated into the immobilization procedure to enable the support matrix to resist proteolytic activity while accommodating an operating temperature of 50 degrees C. The immobilized glucoamylase and pullulanase activities were 0.91 and 0.022 mg dextrose equivalent (DE) min(-1) cm(-2) of membrane, respectively. Immobilized glucoamylase had a half-life of 50 days while the immobilized pullulanase had a half-life of 7 days. This is a considerably improved stability over that reported by other researchers. The enzymes were studied in their free and immobilized forms on a variety of starch substrates including waxy maize, a material which contains 80% alpha-1-6-glucosidic linkages. Substrate concentrations ranged from 1% to a typical commercial concentration of 30%. Conversion efficiencies of 90-92% DE were obtained with free and immobilized glucoamylase preparations. Conversion enhancements of 4-5 mg of DE above this level were obtained by the use of pullulanase in its free or immobilized forms. Close examination of free pullulanase stability as a function of pH indicated improved thermal stability at higher pH values. At 50 degrees C and pH 5.0, the free enzyme was inactivated after 24 h. At pH 7.0, the enzyme still possessed one-half its activity after 72 h. Studies were conducted in both batch and continuous total recycle reactors. All experiments were conducted at 50 degrees C. Experiments conducted with coimmobilized enzymes proved quite promising. Levels of conversion equivalent to those obtained with the individually immobilized enzymes were realized.

Prenatal and postnatal development of protein carboxymethylation activity in the mouse.

The carboxymethylation of endogenous methyl-accepting proteins in intact cells of whole embryos decreased between days 9.5 and 11.5 of gestation and thereafter increased. This fluctuation was not due to a change in the level of protein carboxymethylase (PCM) which remained constant during this period. Embryonic PCM was located predominantly in the cytosol of the embryonic cells, as is the case in adult tissues. Brain PCM specific activity was the same as the rest of the body from gestational days 13.5 to 19.5, but thereafter underwent a two- to threefold increase so that by the end of weaning (about 30 days), it had reached the high adult level. This time course suggests that increasing PCM may be part of the brain maturation process occurring during the neonatal period.

A comparative study of platelet reactivity in arthritis.

We utilized a standardized in vitro method which employs transmission electron microscopy to monitor the degree of surface activation (cytoplasmic spreading) and amount of aggregation displayed by platelet populations from 314 patients with one of five distinct rheumatic diseases and from 72 normal subjects. The percentage of patients in each group whose platelet populations were hyperactive was as follows: polymyalgia rheumatica, 75 percent; scleroderma, 65 percent; primary gout, 61 percent; rheumatoid arthritis, 57 percent; and degenerative joint disease, 40 percent. Pair-wise contrasts performed after an analysis of variance suggest the following differences and similarities: (1) the mean differential platelet count of the normal subjects differed from that in each disease state; (2) the platelet responsivity in patients with degenerative joint disease most closely resembled that in normal subjects; (3) the platelet response in polymyalgia rheumatica plus temporal arteritis was the most abnormal; and (4) platelet response in scleroderma, rheumatoid arthritis, and gout closely resembled each other. The increased platelet response in vitro may reflect the in vivo presence of disease-related "risk factors" (hyperuricemia, immune complexes, and atherosclerosis). Those patients with "triggered" platelet populations may be appropriate candidates for antiplatelet therapy.