Shaul Reuveny | Agency of Science and Technology, Singapore (original) (raw)

Papers by Shaul Reuveny

PubMed, 1980

Pilot plant scale production of human lymphoblastoid (Namalva) interferon (IF) is described. Nama... more Pilot plant scale production of human lymphoblastoid (Namalva) interferon (IF) is described. Namalva cells were grown in a semicontinuous cultivation method in pilot plant scale fermentors having up to 40-liter culture volume. The harvested Namalva cells were suspended in a serum-free medium at a concentration of 10(7) cells per ml and were induced to produce IF by infection with Sendai virus. The content of IF and protein in the culture supernatant ranged between 2.5 to 5.0 x 10(4) IF units and 150 to 250 microgram protein per ml, respectively. Concentration and partial purification of the IF was done by trichloroacetic acid precipitation, followed by gel filtration through an Ultrogel AcA 54 column. The specific activities of the IF preparations ranged between 2 to 4 x 10(6) IF units per mg protein. Scaling-up the IF production system to 300 liter fermentor scale is under research and development.

PubMed, 1985

Factors that affected the production of monoclonal antibodies by a mouse-mouse hybridoma cell lin... more Factors that affected the production of monoclonal antibodies by a mouse-mouse hybridoma cell line, propagated in vitro in stirred vessels, were investigated. The purpose of the research was to estimate the efficiency of this system for large scale production of monoclonal antibodies. The antibody produced by these hybridoma cells was an IgG2a, specific for a surface antigen on Rhizobium japonicum NR-7 cells. Antibody content in the culture supernatant was determined by a radial-immunodiffusion assay using rabbit anti-mouse IgG antibodies in the immobile phase and mouse IgG (the monoclonal antibody) as the antigen in the mobile phase. This method was found to be more reproducible and reliable compared with an ELISA method. Cells were adapted to grow in an inexpensive, low protein content medium based on Dulbecco's Modified Eagle Medium (DMEM) supplemented with 0.25% Primatone RL, 0.01% Pluronic polyol F-68 and fetal bovine serum as low as 1%. Doubling time for the cells averaged 24 hrs, and final yields reached 2 X 10(6) cells per ml. The hybridoma cells were grown in the newly developed medium in 3 liter fermentors. Monoclonal antibody was produced during the early growth phase (3 days), however, most of the antibody was produced during a later growth phase (3-10 days) when 30 to 90% of the cells were dead. Final antibody yields were estimated to be 100-200 micrograms/ml. A low level of dissolved oxygen (25% air saturation) in the culture was found to increase the amount of antibody produced as compared with cells propagated at 60% air saturation (the optimal level for cell propagation) since the cells were kept alive for longer periods at the lower dissolved oxygen concentration. Using a fed-batch propagation method we were able to keep cells alive for long periods (up to 1 month) at a concentration of about 1 X 10(6) cells per ml, and thus to increase further monoclonal antibody production. Yields of 300-400 micrograms/ml were obtained.

PubMed, 1987

Factors that affect production of monoclonal antibodies (McAb) by a murine cell line were investi... more Factors that affect production of monoclonal antibodies (McAb) by a murine cell line were investigated. The goal was to estimate the efficiency of large scale production in stirred reactors. It was found that in batch cultures most McAb was produced after the log growth phase; final yield was 100-200 micrograms/ml. Yields of McAb were increased to 290 micrograms/ml by feeding cells glucose and glutamine. Lactic acid, which was produced in culture as a result of glucose metabolism, had no toxic effect on cells, while another waste product, ammonium ion, was probably accumulated at toxic levels during late stages of cell growth. The hybridoma cell line was propagated in four different systems: fed-batch, semi-continuous, two stage and perfusion. These systems were compared to batch cultures for their effect on cell viability and antibody production. Daily addition of fresh medium (fed-batch propagation) increased antibody productivity from 15 (batch culture) to 27 mg/l of culture/day. In the semi-continuous culture productivity was raised to 34 mg/l/day. Further increase in productivity to a level of 62 mg/l/day was achieved by applying a second batch stage to the semi-continuous culture. A perfusion culturing method was the most effective for production of McAb. Average concentrations of 2.2 X 10(7) live cells/ml and 390 micrograms of antibody/ml corresponding to productivity of 660 mg/l/day were achieved. Serum concentration in the medium was reduced to 0.125% resulting in a specific activity of 0.4 mg of McAb per mg of protein in the cell-free culture broth.

Elsevier eBooks, 1994

Summary Spodopdera frugiperda (Sf-9) cells were propagated in batch, fed-batch, perfusion and med... more Summary Spodopdera frugiperda (Sf-9) cells were propagated in batch, fed-batch, perfusion and medium replacement methods, achieving cell concentrations of 4.5, 6.7, 9.5 and 11.6×106 cells/ml respectively. Fed-batch cultures were carried out by continuous feeding of the culture with glucose, glutamine and yeastolate ultrafiltrate. Perfusion cultures were carried out by continuous medium perfusion at a rate of 0.4–0.5 vol/vol day using an external 0.2μm pore size hollow fiber microfilter for continuous cell-medium separation. In the medium replacement method the same hollow fiber microfilter was used for replacement of 80% of the spent medium with fresh medium when growth limiting conditions occured. Although the highest cell density was achieved by applying perfusion or periodical replacement of the medium, better medium utilization (6.7×106 cells per ml medium) was obtained in the fed batch methods. We have combined the medium replacement and fed-batch methods, in order to achieve both, high medium utilization (6.7×106 cells per ml medium) and high cell concentration (16×106 cells/ml). Maximal production of recombinant proteins (0.9 mg of E. coli b-galactosidase or 1.7 ug of glucocerebrosidase per 106 cells) in batch cultures was achieved when the Sf-9 cells were infected with the recombinant baculoviruses in the early or middle exponential phase of growth. Less than 10% of this productivity was obtained when cells were infected at the late exponential phase (cell concentration of about 4×106 cells/ml). Therefore, in order to obtain the maximal protein productivity, cells from the late exponential phase of growth were resuspended with fresh growth medium prior to the viral infection and fed with glucose glutamine and yeastolate ultrafiltrate during the infection. These conditions do not yield the maximal protein productivity when viral infection is done at cells concentration above 4×106. A continuous two stage reactor system for production of recombinant proteins is suggested. In the first stage the Sf-9 cells are propagated to a concentration of 16×106 cells/ml using the medium replacement-fed batch method. Every 4 days, 95% of the cell culture is transferred to the second stage reactor where it is diluted with fresh growth medium to cell concentration of 4×106 cells/ml. During the infection, the culture is continuously fed with nutrients.

Biotechnology Letters, Jun 1, 1980

... I. PRODUCTION OF CRUDE INTERFERON Avshalom Mizrahi*, Shaul Reuveny, Abraham Traub and Moshe M... more ... I. PRODUCTION OF CRUDE INTERFERON Avshalom Mizrahi*, Shaul Reuveny, Abraham Traub and Moshe Minai Israel Institute for Biological Research Ness-Ziona 70400, Israel. ... skilful technical assistance of Mrs. Miriam Gez, Lea Silberstein and Mr. Shlomo Habousha, ...

Biochemical Journal, Nov 1, 1995

The possible role of post-translational modifications such as subunit oligomerization, protein gl... more The possible role of post-translational modifications such as subunit oligomerization, protein glycosylation and oligosaccharide processing on the circulatory lifetime of proteins was studied using recombinant human acetylcholinesterase (rHuAChE). Different preparations of rHuAChE containing various amounts of tetramers, dimers and monomers are cleared at similar rates from the circulation, suggesting that oligomerization does not play an important role in determining the rate of clearance. An engineered rHuAChE mutant containing only one N-glycosylation site was cleared from the circulation more rapidly than the wild-type triglycosylated enzyme. On the other hand, hyperglycosylated mutants containing either four or five occupied N-glycosylation sites, analagous to those present on the slowly cleared fetal bovine serum acetylcholinesterase (FBS-AChE), were also cleared more rapidly from the bloodstream than the wild-type species. Furthermore, the two different tetraglycosylated mutants were cleared at different rates while the pentaglycosylated mutant exhibited the most rapid clearance

Biotechnology Letters, Jul 1, 1983

... 1981) using K562 cells as a target. DNA determination: Nick translation of Namalva IFN was pe... more ... 1981) using K562 cells as a target. DNA determination: Nick translation of Namalva IFN was performed according to Rigby et al. (1977). ... Safety tests. The first and the most important test to be performed on each IFN lot is for the presence of nucleic acids. The nick-translation-...

Enzymes of the Cholinesterase Family, 1995

Acetylcholinesterase is a serine hydrolase whose function at the cholinergic synapse, is the rapi... more Acetylcholinesterase is a serine hydrolase whose function at the cholinergic synapse, is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh). The recently resolved 3D structure of Torpedo californica AChE (TcAChE) revealed a deep and narrow ‘gorge’, which penetrates halfway into the enzyme and contains the catalytic site at about 4A from its base (Sussman et al., 1991). The active center interacts with ACh through several subsites including the catalytic triad (Ser203(200), His447(440), Glu334(327): Sussman et al., 1991; Gibney et al., 1990; Shafferman et al., 1992a, b), the oxyanion hole (Gly121(119), Gly 122(120), Ala204(201); Sussman et al., 1991), the acyl pocket (Phe295 (288) and Phe297(290); Vellom et al., 1993; Ordentlich et al., 1993a).

Developments in biological standardization, 1985

Differentiation of embryonic rat and chick myoblasts was investigated using a tridimensional supp... more Differentiation of embryonic rat and chick myoblasts was investigated using a tridimensional support made of positively charged, uncoated DEAE-cellulose microcarriers (MC). Following rapid cell attachment, the MC interconnected to form large cell-MC conglomerates which remained floating in the nutrient medium. Cells within the conglomerates fused to form myotubes which synthesized muscle-specific proteins such as: creatine kinase, acetylcholinesterase, acetylcholine receptors, and contracted in response to electrical stimulation. Myotubes, at different stages of differentiation, showed characteristic morphology (as observed by transmission and scanning electron-microscopies). Upon addition of dissociated spinal cord cells to these muscle-MC cultures, intensive sprouting of nerve fibres took place. After a few days an extensive network of nerve fibres was formed on the top of muscle myotubes and nerve-muscle contacts were established.

Novel Approaches for Bioremediation of Organic Pollution, 1999

ABSTRACT A closed loop perfusion system for cultivation of animal cells in polyurethane (PU) foam... more ABSTRACT A closed loop perfusion system for cultivation of animal cells in polyurethane (PU) foam matrix is described. The container for cell growth is packed with PU sponges in the form of 0.5-1 cm3 cubes. The porous matrix leaves a path for nutrients to flow through the packed bed. Medium is continuously circulated by an airlift pump mounted in the cell-free chamber. The system can be used for both anchorage dependent and independent cells, where the former grow attached and the latter immobilized in the foams. The foam matrix reactor has been tested with a variety of cell lines producing different natural products. These include HeLa-derived growth factor (HDGF) from a human cervical carcinoma, carcinoembryonic antigen (CEA) from a human colon carcinoma, B-cell stimulating factor (BSF) from a human hybridoma and monoclonal antibodies from mouse hybridoma cell lines.

Cytotherapy, May 1, 2020

Background & Aim In vivo mitochondrial transfer of mesenchymal stromal cells (MSCs) to immune cel... more Background & Aim In vivo mitochondrial transfer of mesenchymal stromal cells (MSCs) to immune cells, alveolar and bronchial epithelial cells resulted in metabolic and functional improvements. Thus, we hypothesized that naked mitochondria isolated from MSCs can be internalized by pulmonary structural cells and could be used as systemic therapy in a sepsis model. Methods, Results & Conclusion MSCs-derived mitochondria were incubated with septic murine lung endothelial and epithelial cells. Mitochondrial internalization was assessed by fluorescence microscopy, and its impact on oxidative stress and cellular respiration were evaluated. C57Bl6 mice were divided into control group, sepsis group (CLP, cecal ligation and puncture) and sepsis group with mito-treatment (CLPmito, intravenous administration of mitochondria derived from 3 £ 10 6 MSCs, 24 hours after CLP). The effects on mortality, pulmonary function and morphometry, liver and kidney histopathological and molecular changes were evaluated after 24 hours of mito-treatment. Mitochondria internalization by septic endothelial and epithelial cells led to reduce oxidative stress which was increased by 250% in endothelial cells and 150% in epithelial cells in CLP mice compared to control. Likewise, by oxygraphy, we analyzed the rate of oxygen consumption (OCR) of these cells. We have seen that, in epithelial cells, only maximal (MR) repolarization was reduced by about 30% after sepsis. In endothelial, both basal (BR) and maximal respiration (MR) were reduced by 50 and 48% respectivelly after sepsis, as well as a significant increase in the proton-leak (PL) rate in relation to the control group (33%). In both cell types, the altered parameters were re-established at control levels after mitocondrial treatment. Mito-treatment induced higher survival rates (60%) and reduced lung elastance (24%). Lung morphometry analysis demonstrated that mito-treatment reversed increases found in septic animals in alveolar collapse (230%), lung neutrophils (90%), lung collagen collagen (300%), elastogenesis (38%). Histological analysis of the liver demonstrates the increase of vacuolization (210%) and inflammatory infiltrate (105%) in septic animals, which were completely normalized after mito-treatment. Treatment also improved sepsis-induced kidney brush border damage (350%) and interstitial edema (205%). This work demonstrates the ability to internalize mitochondria isolated from MSCs by structural cells and the therapeutic potential of mito-treatment in a sepsis model.

Stem Cells and Development, Nov 1, 2010

Metabolic studies of human embryonic stem cells (hESCs) can provide important information for ste... more Metabolic studies of human embryonic stem cells (hESCs) can provide important information for stem cell bioprocessing. To this end, we have examined growth and metabolism of hESCs in both traditional 2-dimensional (2D) colony cultures and 3-dimensional microcarrier cultures using a conditioned medium and 3 serum-free media. The 2D colony cultures plateaued at cell densities of 1.1-1.5Â10 6 cells=mL at day 6 due to surface limitation. Microcarrier cultures achieved 1.5-2Â10 6 cells=mL on days 8-10 before reaching a plateau; this growth arrest was not due to surface limitation, but probably due to metabolic limitations. Metabolic analysis of the cultures showed that amino acids (including glutamine) and glucose are in excess and are not limiting cell growth; on the other hand, the high levels of waste products (25 mM lactate and 0.8 mM ammonium) and low pH (6.6) obtained at the last stages of cell propagation could be the causes for growth arrest. hESCs cultured in media supplemented with lactate (up to 28 mM) showed reduced cell growth, whereas ammonium (up to 5 mM) had no effect. Lactate and, to a lesser extent, ammonia affected pluripotency as reflected by the decreasing population of cells expressing pluripotent marker TRA-1-60. Feeding hESC cultures with low concentrations of glucose resulted in lower lactate levels (*10%) and a higher pH level of 6.7, which leads to a 40% increase in cell density. We conclude that the high lactate levels and the low pH during the last stages of high-density hESC culture may limit cell growth and affect pluripotency. To overcome this limitation, a controlled feed of low levels of glucose and online control of pH can be used.

Cell Proliferation, May 19, 2022

ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by lo... more ObjectivesInduced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges.Materials and MethodsFive sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures.ResultsImprovement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated.ConclusionsOur method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

PubMed, 1981

Microgranular DEAE-cellulose anion exchanger with exchange capacities ranging between 0-2.7 meq/g... more Microgranular DEAE-cellulose anion exchanger with exchange capacities ranging between 0-2.7 meq/gr dry materials were tested for their suitability to be used as microcarriers for culturing several anchorage-dependent-cells. It was found that cells from established cell lines (BHK, MDCK), grew as confluent monolayer on DEAE-cellulose microcarriers with exchange capacity ranging between 1.00-2.0 meq/gr dry materials. Primary cells (chick embryo fibroblast) grow as a cell-microcarriers aggregate on DEAE-cellulose microcarriers with an exchange capacity ranging between 1.59-2.0 meq/gr dry materials. Cells from human diploid cell strains grew poorly on these microcarriers. Cell yields obtained using the DEAE-cellulose microcarriers were similar to those obtained on commercially available DEAE-dextran microcarrier culturing systems. Several viruses were propagated on cells grown in DEAE-cellulose microcarrier culturing systems. Virus yields per cell were similar to those obtained in conventional monolayer cultures.

PubMed, 1983

Dissociated spinal cord cells from rat fetuses were successfully cultured up to the state of syna... more Dissociated spinal cord cells from rat fetuses were successfully cultured up to the state of synaptogenesis and myelin formation on DEAE cellulose cylindrical microcarriers. Nerve cells and microcarriers interconnected to form conglomerates offering optimal nutritional conditions which enhanced cell maturation. These conglomerates yielded large amounts of cells which could easily be harvested for biochemical analysis.

Antimicrobial Agents and Chemotherapy, Aug 1, 1981

Stem Cell Research, May 1, 2021

Mesenchymal stem cells (MSCs) are of great clinical interest as a form of allogenic therapy due t... more Mesenchymal stem cells (MSCs) are of great clinical interest as a form of allogenic therapy due to their excellent regenerative and immunomodulatory effects for various therapeutic indications. Stirred suspension bioreactors using microcarriers (MC) have been used for large-scale production of MSCs compared to planar cultivation systems. Previously, we have demonstrated that expansion of MSCs in MC-spinner cultures improved chondrogenic, osteogenic, and cell migration potentials as compared to monolayer-static cultures. In this study, we sought to address this by analyzing global gene expression patterns, miRNA profiles and secretome under both monolayer-static and MC-spinner cultures in serum-free medium at different growth phases. The datasets revealed differential expression patterns that correlated with potentially improved MSC properties in cells from MC-spinner cultures compared to those of monolayer-static cultures. Transcriptome analysis identified a unique expression signature for cells from MC-spinner cultures, which correlated well with miRNA expression, and cytokine secretion involved in key MSC functions. Importantly, MC-spinner cultures and conditioned medium showed increased expression of factors that possibly enhance pathways of extracellular matrix dynamics, cellular metabolism, differentiation potential, immunoregulatory function, and wound healing. This systematic analysis provides insights for the efficient optimization of stem cell bioprocessing and infers that MC-based bioprocess manufacturing could improve post-expansion cellular properties for stem cell therapies.

Springer eBooks, 2021

Human-induced pluripotent stem cells are known for their high proliferation capacity as well as t... more Human-induced pluripotent stem cells are known for their high proliferation capacity as well as their ability to differentiate to different lineages (Ban et al., Theranostics 7(7):2067-2077, 2017; Chen et al., Stem Cell Res 15(2):365-375, 2015; Serra et al., Trends Biotechnol 30(6):350-359, 2012). For stem-cell-derived cardiomyocytes to evolve into a scalable therapeutic source, a large quantity of highly pure cardiomyocytes is needed. Thus, lies the challenge of defining an efficient cardiomyocyte differentiation process. This chapter describes a method to evaluate multiple human-induced pluripotent stem cell lines for their cardiac differentiation potentials before evaluating their integrated proliferation and differentiation abilities in microcarrier cultures in a spinner culture format.