Bowes human melanoma cell line. An immunocytochemical study

Bowes human melanoma cell line was investigated immunocytochemically to localize S-100 protein, HMB-45 and intermediate filament proteins. The majority of the cells showed strong positive staining with antibodies directed against S-100 protein, HMB-45 and vimentin filaments. Antibodies directed against the other cytoskeletal proteins failed to show any reactivity. These findings suggest that this transformed cell line does not dedifferentiate in culture and continues to express the specific antigens of human melanoma cells. Thus, Bowes cell line provides a useful model for studying the cellular biology and pathology of malignant melanoma.     

Effect of ammonia on the glycosylation of human recombinant erythropoietin in culture

Recombinant human erythropoietin (EPO) was produced by a stable transfected CHO-K1 cell clone (EPO-81) grown in serum-free medium. Our previous work showed that there was a significant increase in the heterogeneity of the glycoforms of EPO and a reduction of the sialylation at 20 mM NH(4)Cl. In the work presented here, the effects of ammonia on EPO N-linked oligosaccharides were analyzed. EPO was purified from culture supernatants by immunoaffinity chromatography. The N-linked oligosaccharides were released enzymatically and analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and HPLC. The FACE N-linked oligosaccharide profile showed that the sialylated glycans contain one prominent band at a position corresponding to eight glucose units. The density of the major band was greatly diminished and the width was significantly increased in cultures containing added ammonia. The proportion of tetraantennary structures was reduced by 60%, while the tri- and biantennary structures were increased proportionally in the presence of ammonia. Glycan analysis by HPLC using a weak anion exchange column showed that the most significant characteristic effect of ammonia was a reduction of the proportion of glycans with four sialic acids from 46% in control cultures to 29% in ammonia-treated cultures. Analysis of the desialylated glycans by normal phase chromatography indicated a distribution of tetra-, tri-, and biantennary structures similar to that shown by FACE. The N-linked glycan sequence was determined by sequential exoglycosidase digestion followed by FACE. The results indicated a typical N-linked complex oligosaccharide structure. Glycans from ammonia-containing cultures showed the same sequence pattern. In conclusion, we showed that ammonia in the culture medium affected EPO glycosylation, which was observed as a reduction of the tetraantennary and tetrasialylated oligosaccharide structures. However, the presence of ammonia in the cultures did not change the oligosaccharide sequence.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

Production of recombinant human granulocyte macrophage-colony stimulating factor in rice cell suspension culture with a human-like N-glycan structure

The rice α-amylase 3D promoter system, which is activated under sucrose-starved conditions, has emerged as a useful system for producing recombinant proteins. However, using rice as the production system for therapeutic proteins requires modifications of the N-glycosylation pattern because of the potential immunogenicity of plant-specific sugar residues. In this study, glyco-engineered rice were generated as a production host for therapeutic glycoproteins, using RNA interference (RNAi) technology to down-regulate the endogenous α-1,3-fucosyltransferase (α-1,3-FucT) and β-1,2-xylosyltransferase (β-1,2-XylT) genes. N-linked glycans from the RNAi lines were identified, and their structures were compared with those isolated from a wild-type cell suspension. The inverted-repeat chimeric RNA silencing construct of α-1,3-fucosyltransferase and β-1,2-xylosyltransferase (Δ3FT/XT)-9 glyco-engineered line with significantly reduced core α-1,3-fucosylated and/or β-1,2-xylosylated glycan structures was established. Moreover, levels of plant-specific α-1,3-fucose and/or β-1,2-xylose residues incorporated into recombinant human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced from the N44 + Δ3FT/XT-4 glyco-engineered line co-expressing ihpRNA of Δ3FT/XT and hGM-CSF were significantly decreased compared with those in the previously reported N44-08 transgenic line expressing hGM-CSF. None of the glyco-engineered lines differed from the wild type with respect to cell division, proliferation or ability to secrete proteins into the culture medium.     

Production of tissue plasminogen activator (tPA) in two and three dimensionally growing cultures of Bowes melanoma cells

Bowes melanoma cells synthesize more tissue plasminogen activator (tPA) in monolayer cultures than in multicell spheroids. Cellular production of tPA in these cells was measured during a cultivation period of 800 h. Without changing the cell culture assay, we were able to obtain monolayers, multilayers, and multicell spheroids (cell aggregates) by stirring microcarrier beads in 500-mL spinner flasks operated at 50 rpm. Thus, the medium conditions in the liquid were similar for cells in monolayers and in multicell spheroids. Probes for measurements of intracellular and extracellular parameters were taken from the same culture at distinct times; therefore, their variations during cultivation can directly be compared. Because cells were cultured in an unregulated (with regard to pH, glucose, etc.) spinner flask, their concentration was kept below 10(6) cells/mL, thus avoiding too fast and too severe depletion of oxygen and other medium factors. Nevertheless, the tPA productivity decreased from 8 ng/h/10(6) cells (monolayer) to 4 ng/h/10(6) cells (multicell spheroids with microcarrier nucleus, 800 mum diameter), matching the decrease of total cellular protein. Due to medium depletion, the cell cycle distribution changed from 45% to 68% G(1) cells in a characteristic way during growth of multicell spheroids. This is accompanied by changes in amino acids, glucose, lactate, and pH, which may account for the reduction of tPA productivity. (c) 1996 John Wiley & Sons, Inc.     

Stable expression of recombinant human coagulation factor XIII in protein-free suspension culture of Chinese hamster ovary cells

The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture system with a spin filter is employed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells

Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimentation equilibrium experiments showed the recombinant erythropoietin preparation to be essentially a single macromolecular component with a molecular weight of 30,400 and a carbohydrate content of 39%. The Stokes radius of recombinant erythropoietin was estimated to be 32 A from gel filtration, much larger than the 20-A radius calculated for a sphere of the observed molecular weight. This difference may be ascribed to the extensive glycosylation. The fluorescence and phosphorescence spectra showed that the luminescent tryptophan(s) is (are) solvent-exposed and can be quenched by I- and acrylamide but not by Cs+. On acid titration, the recombinant erythropoietin showed a conformational transition with a midpoint of pH 4.1. This suggests that the net charges on the protein moiety rather than on the whole molecule play a role in protein structure stability.     

Production of human recombinant beta-interferon in the avian cell culture

The avian recombinant adenovirus of serotype 1 (CELO) was obtained. The recombinant adenovirus of serotype 1 (CELO) induces expression of human beta-interferon (IB). The expression cassette containing IB gene was placed at the right end of the CELO genome under control of hybrid promoter hEF-1alpha/HTLV. The resulting recombinant adenovirus CELO-IB transduced the avian cell culture LMH. The level of production of the recombinant IB was 0.15 micro/ml. The IB protein yield after affine chromatography purification using Ni-NTA agarose was 50%. The biological activity of the purified IB was high (7.8 x 10(8) MU/microg protein). The purified IB inhibited replication of murine encephalomyocarditis virus (VMEC) in cell culture of human diploid fibroblasts (HDF). Thus, expression system based on avian cell culture is an effective system for producing biologically active protein of human interferon beta.     

Production of recombinant human erythropoietin from Pichia pastoris and its structural analysis

AIMS: To design and investigate a recombinant expression system producing a therapeutically important glycoprotein, human erythropoietin (rHuEPO), by Pichia pastoris. METHODS AND RESULTS: EPO cDNA was cloned into pPICZalphaA for expression under control of AOX1 promoter and fused, on the amino-terminal end, with a polyhistidine tag for rapid purification. A target site for factor Xa protease was also introduced, such that cleavage in vitro produced a mature form of rHuEPO having the native N- and C-termini. RHuEPO was characterized as to the extent and nature of N-linked glycosylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and western blotting. The rHuEPO produced was approximately 30 kDa. All three N-linked glycosylation sites were occupied dominantly by Man(17)(GlcNAc)(2). N-glycanase-treated rHuEPO purified but not digested with factor-Xa-protease, showed a spectral peak centered about m/z 20400 Da. CONCLUSIONS: The native polypeptide form of human EPO (c. 18 kDa) was obtained for the first time in P. pastoris expression system, after affinity purification, deglycosylation and factor-Xa-protease digestion. The amount of sodium dodecyl sulfate used prior to deglycosylation was found to be crucial in determining the dominant form of glycan in glycoproteins. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel approaches to protein expression and purification system and structural analysis presented, would be important especially for therapeutic proteins expressed in P. pastoris.     

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.     

Biological properties of human melanoma cells in culture.

Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and that metastatic melanoma seems to display a higher degree of malignant transformation than the primary.     

Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago

In the last two decades, the use of plants to produce recombinant proteins and particularly biopharmaceuticals has become an attractive alternative to established systems. This is due to advantages in scalability, economy and safety. In addition the expression of recombinant proteins in plants can also be achieved utilizing in vitro cell suspensions with all the advantages such systems confer, such as product consistency, production ??on demand?? and the ability to perform the entire process according to good manufacturing practices. In this study we have produced the glycosylated human hormone Erythropoietin (EPO), in Medicago truncatula and Arabidopsis thaliana plants and also in cultured cell lines of tobacco, Medicago and Arabidopsis. We have also tested two different versions of the protein, one with a KDEL tag for targeted expression in the Endoplasmic Reticulum, and an untagged version expected to be secreted to the apoplast. The recombinant protein was detected in the plant leaf extracts and in the cultured cell lines. In the latter, the rEPO was detected in the cell extracts and in the spent culture medium. It was possible to recover the KDEL version of rEPO from crude cell extracts by nickel affinity chromatography, however the secreted form did not bind to the Ni- agarose beads which may indicate a possible internalization of the his-tag in the folded protein. Although the yield of rEPO obtained in cell suspensions was not as high as expected, the protein was successfully produced and secreted into the culture medium, reinforcing that plant cell suspension cultures are a promising system for production of human biopharmaceuticals.     

Scalable expansion of human pluripotent stem cells in suspension culture

Routine commercial and clinical applications of human pluripotent stem cells (hPSCs) and their progenies will require increasing cell quantities that cannot be provided by conventional adherent culture technologies. Here we describe a straightforward culture protocol for the expansion of undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) in suspension culture. This culture technique was successfully tested on two hiPSC clones, three hESC lines and on a nonhuman primate ESC line. It is based on a defined medium and single-cell inoculation, but it does not require culture preadaptation, use of microcarriers or any other matrices. Over a time course of 4-7 d, hPSCs can be expanded up to sixfold. Preparation of a high-density culture and its subsequent translation to scalable stirred suspension in Erlenmeyer flasks and stirred spinner flasks are also feasible. Importantly, hPSCs maintain pluripotency and karyotype stability for more than ten passages.     

Transfected insect cells in suspension culture rapidly yield moderate quantities of recombinant proteins in protein-free culture medium

Methodology to rapidly express milligram quantities of recombinant proteins through the Lipofectin-mediated transfection of insect cells in small-scale, protein-free suspension culture is presented. The transfection phase in suspension culture was first optimized using the green fluorescence protein coupled with FACs analysis to examine the effect of variables such as the transfection media, duration, and cell density on transfection efficiency and expression level. The recombinant protein production phase was optimized using secreted alkaline phosphatase (SEAP) as a reporter protein to evaluate the cell seeding density and harvest time. Using this method, 5 secreted, 2 intracellular, and 1 chimeric protein were expressed at levels ranging from 6 to 50 mg/L. Furthermore, the ability to purify over 2 mg of His(6)-tagged SEAP by immobilized metal affinity chromatography from 50 mL insect cell culture medium to greater than 95% purity was also demonstrated. This method is suitable for scale-up and high-throughput applications.     

Production of recombinant rotavirus VP6 from a suspension culture of transgenic tomato (Lycopersicon esculentum Mill.) cells

Recombinant rotavirus VP6 expressed in transgenic tomato cells was found primarily in the intracellular fraction and had a molecular weight of 44 kDa. In a shake flask, transgenic tomato cells produced 0.33 mg recombinant VP6 l^–1 after 18 days of incubation. In a high aspect rotating-wall vessel designed by NASA to simulate microgravity, the transgenic cells produced up to 0.15 mg recombinant VP6 l^–1.     

Production of fertile transgenic maize by electroporation of suspension culture cells

Fertile, transgenic maize plants were generated by electroporation of suspension culture cells that were treated with a pectin-degrading enzyme. Electroporation of cells from two different suspension cultures, one derived from A188 X B73 and one derived from a B73-related inbred, with a plasmid containing the bar gene, resulted in high-frequency recovery of stably transformed callus lines. Plants were regenerated from thirteen transformed callus lines and transmission of bar to progeny was demonstrated.     

Continuous production of human erythropoietin by immobilized recombinant L-929 cells

Recombinant L-929 cells transfected with the human erythropoietin (EPO) gene were immobilized in a macroporous cellulosic support and its derivatives in which charged groups or cell attachment factors were introduced. The immobilized cells were cultured in serum-containing and serum-free media. Comparable production of EPO was observed even in the serum-free medium when a support modified by polyethyleneimine was used for immobilization. The cells immobilized on the supports were cultured in fluidized-bed and inner-loop type air-lift bioreactors for continuous production of EPO. A high cell density of more than 2 × 10⁷ cells/cm³-support and high EPO productivity were achieved and maintained for 50 d through the use of the inner-loop type air-lift bioreactor. The productivity was 13.4-fold higher than that of conventional static cultures in petri-dishes.