A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Summary Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm³ per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed. Offprint requests to: S. Mitsuda     

An oxygen supply strategy for the large-scale production of tissue plasminogen activator by microcarrier cell culture

An oxygen supply strategy involving agitation speed and aeration method for the large-scale production of tissue plasminogen activator (TPA) by a microcarrier cell culture was investigated by small-scale model experiments. A preliminary calculation indicated that diffusion limitation of dissolved oxygen (DO) could be caused in a microcarrier sedimentation layer more than 0.5 mm in thickness. Within an agitation speed range above 70 rpm, which was the critical speed for all of the microcarrier beads to remain suspended and thus for avoiding a deficiency of DO, the TPA productivity was higher at a lower agitation speed, while the cell concentration was not affected by the agitation speed. The addition of soluble starch to the culture medium prevented sedimentation of the microcarrier beads, even at the low agitation speed of 20 rpm, resulting in a TPA productivity higher than that at 70 rpm, which was the optimum speed without soluble starch. Use of an air spray system with an optimized air flow rate resulted in a k_La 2.35 times higher than that with simple surface aeration. Increasing the internal pressure of the culture from 0.2 kg/cm² (1209 hPa) to 1.5 kg/cm² (2483 hPa) had no effect on the cell growth but slightly increased the TPA production rates. However, based on the glucose consumption, both the cell and TPA yields were much improved by pressurization. As an optimum mixing and oxygen supply strategy for the production of TPA on a large scale, it is recommended that soluble starch be added to the culture medium to allow the microcarrier suspension to be maintained at a low agitation speed, while keeping a high oxygen transfer rate by means of an air spray system and pressurization.     

Isolation and characteristics of urokinase-type plasminogen activator from a culture of human embryo lung fibroblasts

Plasminogen activator from conditioned medium of human embryonal lung fibroblasts was purified by phosphocellulose P11 chromatography, followed by p-aminobenzamidine-agarose chromatography. Two forms of plasminogen activators were separated by chromatography on the heparin-sepharose. The high molecular weight form (53 kDa) with specific activity 130 000 IU/mg consists of two polypeptide chains (31 kDa and 20 kDa) and exhibits strong affinity for fibrin-celite, lysine-sepharose and heparin-sepharose. The low molecular weight form (32 kDa, 190 000 IU/mg) also binds to these sorbents, but more weakly, and its properties are very similar to those of low molecular weight urokinase. Activity of both forms of plasminogen activators are inhibited by monoclonal antibodies against urokinase. A number of enzymological chromatographic and immunological properties indicates, that the plasminogen activator from lung fibroblasts is of urokinase type.     

Anchorage independent growth and plasminogen activator production by bovine endothelial cells

Endothelial cells obtained from the aortae of 1- to 2-d-old calves were cloned at high efficiency using fibrin-coated dishes. Primary cultures as well as clones derived from them produced high fibrinolytic activity when grown on 125I-fibrin-coated dishes which was 90% dependent upon the presence of plasminogen. High plasminogen-dependent proteolytic activity was also demonstrated in endothelial cell lysates and in the culture medium of the cells. The production and secretion of the plasminogen activator(s) were found to increase during the log phase of cell growth and to reach a maximum level at confluence. These endothelial cells exhibited morphological phenotypes comparable to those of transformed cells when grown in the presence of acid-treated fetal calf, dog, or human serum. Furthermore, they demonstrated anchorage independent growth, and large colonies were formed in semisolid media. Spontaneous neoplastic transformation of these cells was excluded by karyotypic analysis, lack of tumorigenicity in athymic nude mice, and limited lifespan in culture. Cell clones isolated from colonies grown in agarose demonstrated the same growth characteristics and proteolytic activity as before plating in agarose. High fibrinolytic activity, morphological changes in the appropriate serum, and growth in semisolid media may therefore be indicative of the migratory and/or invasive capacity of both nontransformed endothelial cells as well as tumor cells.     

Stable production of an analog of human tissue plasminogen activator from culturedDrosophila cells

We have studied the expression of an analog of human tissue plasminogen activator, FK2P, inDrosophila Schneider 2 cells. A number of promoters were tested, including theDrosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE),Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1 promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 g FK2P/10⁶ cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 g FK2P/10⁶ cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominately as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.     

On-line determination of optimum time for switching from growth phase to production phase in tissue plasminogen activator production in a microcarrier cell culture

To maximize the productivity of tissue plasminogen activator (TPA) by a mammalian cell culture, on-line determination of the optimum time to switch from the cell growth phase to the TPA production phase was investigated. By measuring the TPA production activity of the cells during the cell growth culture, it was shown that this optimum time was not necessarily the same as the time at which the cell concentration was maximized, and that the optimum time varied with growth culture batch. The TPA production activity of the cells during the growth culture could be estimated by on-line regression analysis using physiological data of the current state, including the oxygen consumption rate (Io₂) and cell concentration, as well as data from past batches. Applying this on-line estimation, the optimum switching time was determined to be the time at which the TPA production activity of the cells in the growth culture became highest, or higher than a certain value according to determined criteria.     

Stimulation of tissue plasminogen activator production from epithelial cell lines

The aim of this study was to investigate the possibility of enhancing the yield of tissue plasminogen activator (tPA) from two epithelial cell lines of normal (non-malignant) derivation grown in tissue culture. The three agents used in this investigation were chosen because of their proven enhancing effect on analogous cells or products. The anabolic hormone stanozolol was found to have no significant stimulatory effect on these cell lines. A phorbol acetate (12-O-tetradecanoylphorbol 13-acetate) caused a twofold enhancement in tPA yield but the most significant results were obtained with 5-azacytidine. This agent increased the yield by up to fourfold in small stationary cultures and threefold in large-scale microcarrier cultures. A combination of azacytidine and phorbol acetate did not have an additive effect on total yield but did alter the kinetics of tPA expression with time. Indications were that the maximum yield with these types of potentiating agents was achieved as it could not be increased by using a combination of two different agents.     

Microcarrier culture of recombinant Chinese hamster ovary cells for production of human immune interferon and human tissue-type plasminogen activator

Summary Recombinant Chinese hamster ovary cells were successfully cultured semi-continuously on microcarriers of gelatin or modified dextran under non-selective conditions for up to three weeks. High and constant production rates for human immune interferon and tissue-type plasminogen activator were obtained. For cells that produced interferon, the highest cell concentration and interferon production was obtained with gelatin microcarriers though the specific production when grown in the presence of 0.2% fetal calf serum was slightly higher for cells cultured on dextran microcarriers (0.12 U/cell day versus 0.11 U/cell day). For cells that produced plasminogen activator, a slightly higher cell concentration was obtained for cells grown on dextran microcarriers (9x10⁵ cells/ml versus 7x10⁵ cells/ml). However, the specific and total production rates were significantly higher for cells cultured on gelatin microcarriers (6.7 pg/cell day versus 2.1 pg/cell day). The maximum cell concentration and specific production rate could be increased to 2.3x10⁶ cells/ml and 3.4 pg/cell day for dextran microcarriers by adding 6-aminohexanoic acid to the medium. For gelatin microcarriers, the addition of 6-aminohexanoic acid increased the specific production rate to 14.4 pg/cell day. Cell growth, however, was inhibited.     

Stimulation of human synovial fibroblast plasminogen activator production by mononuclear cell supernatants

Conditioned medium from concanavalin A-stimulated human peripheral blood mononuclear cells (c-MCCM) stimulates the plasminogen activator (PA) production of nonrheumatoid human synovial fibroblasts obtained from explant cultures. The effect of this synovial fibroblast-stimulating activity is observed within 2 to 4 hr and requires RNA and protein synthesis. Reversible morphological changes in the synovial cells can be observed as a result of c-MCCM action. These enzymatic and morphologic changes are similar to some of the effects of transforming viruses and tumor promoters on target cells. The possible significance of these data for an understanding of the cellular interactions involved in the formation and function of the rheumatoid "pannus" is discussed.     

Development of high density mammalian cell culture system for the production of tissue-type plasminogen activator

A high cell density culture system for the anchorage dependent CHO cells was developed based on the combination ofin situ removal of ammonium ion and microcarrier culture system, and semi-fed-batch feeding of glucose and glutamine was employed to the developed culture system. The glass bead was selected as an optimum microcarrier in terms of cell growth. An ammonium ion selective zeolite, Phillipsite-Gismondine, was packed in a dialysis membrane and equipped on the agitator of spinner reactor forin situ removal of ammonium ion. The semi-fed-batch operation was employed to the novel culture system for the high density cell culture, and the results showed the cell growth was improved by 32% and tPA productivity by 250%.     

Enhanced production and extracellular deposition of the endothelial- type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-beta

Cultured human embryonic lung fibroblasts were used as a model to study the effects of transforming growth factor-beta (TGF beta) on the plasminogen activator (PA) activity released by nontumorigenic cells into the culture medium. The cells were exposed to TGF beta under serum- free conditions, and the changes in PA activity and protein metabolism were analyzed by caseinolysis-in-agar assays, zymography, and polypeptide analysis. Treatment of the cells with TGF beta caused a significant decrease in the PA activity of the culture medium as analyzed by the caseinolysis-in-agar assays. The quantitatively most prominent effect of TGF beta on confluent cultures of cells was the induction of an Mr 47,000 protein, as detected by metabolic labeling. The Mr 47,000 protein was a PA inhibitor as judged by reverse zymography. It was antigenically related to a PA inhibitor secreted by HT-1080 tumor cells as demonstrated with monoclonal antibodies. The induced Mr 47,000 inhibitor was deposited into the growth substratum of the cells, as detected by metabolic labeling, immunoblotting analysis, and reverse zymography assays of extracellular matrix preparations. TGF beta also decreased the amounts of urokinase-type and tissue-type PAs accumulated in the conditioned medium, as detected by zymography. Epidermal growth factor antagonized the inhibitory effects of TGF beta by enhancing the amounts of the PAs. These results indicate that growth factors modulate the proteolytic balance of cultured cells by altering the amounts of PAs and their inhibitors.     

Phosphatidylinositol liposomes increase calcium uptake and tissue plasminogen activator secretion by fetal human lung fibroblasts

PtdIns liposomes, at a concentration of 40 microM, induced in FLF the synthesis of t-PA-Ag, and enhanced 45Ca2+ uptake. The induction of t-PA-Ag biosynthesis by PtdIns liposomes in FLF was inhibited by 5-15 microM verapamil, an inhibitor of Ca2+ uptake via the so-called "slow channels" by 0.5-10 microM TFP, an inhibitor of Ca2+ transport ATPase, and by 10-90 microM TMB-8, an inhibitor of intracellular Ca2+ mobilization. t-PA-Ag secretion was inhibited by decreasing the Ca2+ concentration less than 1.2 mM. On the other hand, addition of 0.08 microM of calcium ionophore A23187 increased t-PA-Ag biosynthesis after 72 hr of incubation by 247% (P less than 0.01). These data support previous results and indicate that the synthesis of t-PA in FLF is Ca2+ dependent. Thus, it is suggested that PtdIns liposomes increase t-PA biosynthesis by affecting calcium metabolism.     

Quantification of attached cells in tissue culture plates and on microcarriers

A method for quantification of anchorage-dependent cells in culture on plane surfaces or on microcarriers is proposed. It is based on Coomassie brilliant blue R-250 adsorption, followed by elution of the dye and measurement by spectrophotometry at 595 nm. A linear correlation (r = 0.988 to 0.996) was observed between absorbance and cell number along a large range of cell densities. This technique may be used for monitoring cell growth, from seeding of initial inoculi to scaling up of cultures in bioreactors.     

Effectors of the activation of human [Glu1]plasminogen by human tissue plasminogen activator

The activation of human [Glu1]plasminogen [( Glu1]Pg) by human recombinant (rec) two-chain tissue plasminogen activator (t-PA) is inhibited by Cl-, at physiological concentrations, and stimulated by epsilon-aminocaproic acid (EACA), as well as fibrin(ogen). Chloride functions as a result of its binding to [Glu1]Pg, with a Ki of approximately 9.0 mM, thereby rendering [Glu1]Pg a less effective substrate for two-chain rec-t-PA. EACA stimulates the activation in Cl-(-)containing solutions, with a Ka of approximately 4.0 mM, primarily by reversal of the Cl-(-)inhibitory effect. Fibrinogen appears to exert its stimulatory properties mainly through effects on the enzyme, two-chain rec-t-PA, with a Ka of approximately 3.7 microM in activation systems containing physiological levels of Cl-. Analysis of the results of this paper reveals that normal plasma components, Cl- and fibrinogen, exert major regulatory roles on the ability of [Glu1]Pg to be activated by two-chain rec-t-PA, in in vitro systems. The presence of Cl- inhibits the stimulation of [Glu1]Pg activation that would normally occur in the presence of fibrinogen, a result of possible importance to the observation that some degree of systemic fibrinogenolysis accompanies therapeutic use of tissue plasminogen activator.     

Binding of tissue plasminogen activator to human aortic endothelial cells.

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.     

Stimulation and desensitization of tissue plasminogen activator release from human endothelial cells

Tumor-promoting phorbol esters and histamine induce tissue plasminogen activator (tPA) release from human endothelial cells in a dose- and time-dependent manner. Phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu) increased tPA concentration in the culture medium by eight to 12 times after 24 h with half-maximal stimulation at 13 and 55 nM, respectively. Maximum release by histamine was only half that of the phorbol esters and required 18 microM for half-maximal response. Kinetics of enhanced release was similar with both types of agonists: a 4-h lag period followed by a period of rapid release (4 h in PMA-treated and 10 h in histamine-treated cultures) followed by a decline toward pretreatment rates. The PMA and histamine effects were additive while histamine and thrombin, which also stimulates tPA release in human endothelial cells, were no more effective together than they were alone. Exposure of the cells to PMA, PDBu, or phorbol 12,13-didecanoate caused a loss of responsiveness to second treatment of the homologous agent that was time- and dose-dependent, sustained, and specific to active tumor promoters (half-maximal desensitization = 52 nM PDBu). A partial desensitized state was also established by histamine which resulted in a 60% lower response to a second challenge dose. Histamine-induced desensitization did not interfere with the PMA response. However, PMA-induced desensitization caused a 75% loss of the histamine and a 67% loss of the thrombin effects. These studies indicate that tumor promoters are potent agonists of tPA release from human endothelial cells and establish a desensitized state to further stimulation. Treatment of these cells with histamine has similar effects which may be mediated at least in part by pathways common to phorbol ester stimulation.