Apparatus and methodology for microcarrier cell culture.
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Animals↗
Biomedical subjects
S Reuveny
Publications and source records attributed to S Reuveny.
Newly developed microcarrier culturing systems--an overview.
Microcarrier (MC) culturing systems are widely employed for the production of biological products required for human and veterinary uses and serve as a tool for basic research in cell biology. Three types of MCs were developed by our group: cellulose, polyacrylamide and polystyrene based MCs. These newly developed MCs were used for the propagation of primary cells, cells from a human diploid cell strain and cells from established cell lines, for the production of biological products (various viruses, mouse interferon and carcinoembryonic antigen) as well as for studying cell differentiation. Basic questions regarding the effect of the chemical nature of MCs on cell attachment, spreading and growth were investigated throughout the development of these MCs.
Acrylic Resins↗
Ion exchange capacity of DEAE microcarriers determined the growth pattern of cells in culture.
Transformed embryonic avian and mammalian cells, as well as cells from established cell lines grow as a monolayer on DEAE-cellulose based microcarriers (MC): the DE-52 and DE-53 MC. Normal, non-transformed, embryonic avian and mammalian cells do not grow on DE-52 MC (having an exchange capacity of 1 meq/g dry material) but grow well on DE-53 MC (having an exchange capacity of 2 meq/g dry material). On DE-53 MC embryonic non-transformed cells grow in multilayers, while embryonic viral-transformed cells and cells from established cell lines grow in a monolayer form. Possible explanations for the differences in cell growth on various MC and variation in the mode of growth in monolayer vs multilayer are discussed. These differences may serve as a valuable tool for separation and distinction between normal and transformed cells. In addition, the described novel MC culturing system provides a support for tridimensional growth on which cell growth mimics the in vivo growth conditions. Therefore, this system is suitable for the study of cell recognition, cell to cell connection and cell orientation.
Animals↗
Differentiation of myoblasts with nerve cells on microcarriers in culture.
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.
Animals↗
Production of monoclonal antibodies in culture.
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.
Animals↗
Role of substrata in determining the growth topology of transformed and nontransformed cells in culture.
Cylindrical DEAE cellulose anion exchangers (DE-53), generally used for chromatography, were found suitable as a substratum for cultivating cells. Embryonic avian and mammalian cells cultured on DE-53 microcarriers (MC) grow in multilayers, while the same embryonic cells when transformed by avian sarcoma virus (ASV) grow in monolayers. These patterns of cell growth differ from those of normal and transformed cells grown on conventional glass or plastic Petri dishes, or on beaded MC. Cells derived from established cell lines such as BHK, HeLa, L-929, MDCK, and VERO grow in monolayer on these MC. A human adenocarcinoma cell line is the only exception growing in a multilayer form. These results indicate that the ability of cultured cells to grow in multilayers, is determined not only by their state of transformation but also by the properties of the support on which they are cultured.
Adenocarcinoma↗
Synaptogenesis and myelination in dissociated cerebral microcarrier cell cultures.
A new approach for cultivating dissociated cerebral neurons is described. It is based on a rapid attachment of neurons to the DEAE-cellulose cylindrical MC and their subsequent interconnection to form cell-MC conglomerates. Intensive fiber growth followed by synaptogenesis and progressive myelin formation are indicative of optimal conditions of nutrition and oxygenation.
Acetylcholinesterase↗
Myogenesis on microcarrier cultures.
The capacity of embryonic chick myoblasts to grow in vitro on DEAE-cellulose microcarriers (MC) has been investigated biochemically and morphologically. The cells attached to the MC, replicated and fused to form elongated myotubes. These myotubes synthesized muscle-specific proteins, such as creatine kinase (CK) and acetylcholine receptors (AChR), and they contracted spontaneously. Some of the advantages of this technique are: a) Tridimensional development of myotubes on MC with orientation of fibers parallel to each other; b) Muscle cells can be cultured on MC for long periods (months); c) Easy harvesting of samples at any time during cultivation; d) DEAE-cellulose MC are commercially available, inexpensive and easy to handle.
Animals↗
New microcarriers for culturing mammalian cells.
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Acrylic Resins↗
Mammalian cell propagation on derivatized polyacrylamide microcarriers.
A working system for studying the effects of factors involved in the chemical nature of microcarrier on cell attachment, spreading and growth, was established. The system is based on polyacrylamide beads, prepared by the emulsion polymerization technique. Sieved beads of desirable mean diameter were derivatized to generate controlled amounts of primary and tertiary amino groups. These microcarriers were used for propagation of vive different cell strains: BHK, MDCK, CEF, MRC-5 and FS. It was found that BHK cells attach and spread significantly faster on primary amino derivatized beads than those with tertiary amino groups, and at a lower degree of charging. As a result of introducing hydrophobicity to the side chain carrying the primary amino group, higher kinetic rates of BHK cell attachment were obtained. Optimal cell growth of all the tested cells was recorded for the butyl and the hexyl side chains. On the other hand the introduction of hydrophobicity into the polymeric backbone of the microcarrier led to lower cell yields. Primary amino derivatized MCs with an optimal degree of hydrophobicity city exhibit a higher cell yield of MDCK cells (with pronounced epithelial morphology), as compared to the tertiary amino derivatized MCs. CEF and FS cells (with pronounced fibroblast morphology) attained cell yields comparable to those obtained with the tertiary amino derivatized MCs. The diaminohexane derivatized polyacrylamide microcarriers seem to be a potential alternative for the commonly used tertiary amino derivatized micro-carriers.
Acrylic Resins↗
Human lymphoblastoid interferon for clinical trials: large scale purification and safety tests.
Human lymphoblastoid cell lines are one of the best sources for the production of large amounts of human interferon for clinical trials. The potential risk of using transformed cell lines for production of a substance for human use can be overcome by an extensive purification of the interferon to a high degree, and by carefully testing the final product for DNA content and toxic materials. In previous reports we described a method for large scale production of interferon by a human lymphoblastoid cell line (Namalva). In this report a system for large scale purification of Namalva interferon and safety tests is presented. The procedure consists of three main steps: 1. Concentration of crude interferon by ultrafiltration. II. Gel filtration on Sephadex G-75 or Ultrogel AcA 54 columns. III. Affinity chromatography on anti-interferon antibody column. All steps were adapted for the processing of batches of hundreds of litres and can be easily further scaled-up. The final highly purified product undergoes a series of quality control tests such as purity, sterility, toxicity, pyrogenicity and DNA content. After passing all tests the IFN is released for clinical trials.
Cell Line↗
Neuronal cultures on microcarriers: dissociated spinal cord cells.
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.
Animals↗
DE-52 and DE-53 cellulose microcarriers. I. Growth of primary and established anchorage-dependent cells.
DEAE cellulose anion exchangers having small ion exchange capacity (0.5 to 2.0 meq/g dry material) were tested for cell attachment kinetics and capacity to support growth anchorage-dependent cells. It was found that cells from established cell lines (BHK and MDCK) can grow to confluency on DEAE cellulose particles having exchange capacity of 1 and 2 meq/g dry materials, DE-52 and DE-53, respectively. On the other hand, chick embryo fibroblasts (primary cells) can grow only on DE-53 particles.
Animals↗
Isolation of two species of human lymphoblastoid (Namalva) cell-derived interferon that differ in rates of clearance, size, and cell specificity.
Chromatography of human lymphoblastoid (Namalva) interferon on blue dextran Sepharose has separated the interferon activity into two species which differ in affinity to the dye Cibacron Blue F3GA. The separated species differ also in rates of clearance from the circulation of mice, size, and specificity toward human and bovine cells. The species retained by the dye manifests a slower clearance rate, is about 20,000 mol. wt., and is equally active on both human fibroblasts FS-11A and bovine cell line MDBK. The unretained species disappears from the circulation at a faster rate, is 16,000 mol. wt., is active on bovine cells, but almost inactive on human cells. It is suggested that a comparative study of the two species may aid in the elucidation of the causes responsible for the rapid clearance or bioinactivation of interferon.
Animals↗
Interferon-albumin conjugate with conserved biological activity.
The heterobifunctional reagent N-succinilimidyl 3-(2-pyridylthio)propionate (SPDP) was used for the preparation of a disulphide-linked conjugate between Namalwa lymphoblastoid interferon and serum albumin. The linkage of interferon to albumin did not reduce the ability to protect MDBK cells against infection by vesicular stomatitis virus (VSV). The conjugate did not dissociate into free interferon and albumin under conditions prevailing in the assay for interferon activity. The rate of clearance of the interferon-albumin conjugate from the mouse circulatory system was somewhat slower than that of free interferon.
Animals↗
Interferon production by a human lymphoblastoid cell line (DG-75) free of the Epstein-Barr genome.
A new lymphoblastoid cell line, DG-75, was investigated for its ability to produce interferon. DG-75 cells, previously shown to be free of Epstein-Barr virus genome and receptors, could be grown in submerged culture and could produce interferon in titers comparable to interferon produced by Namalva cells. The interferon produced was similar in size to the Namalva interferon as determined by gel filtration in Ultrogel AcA54. The DG-75 cells present a new source of large quantities of interferon which may be safer for human use than the Namalva interferon.
Cell Line↗
Factors affecting the large scale production of human lymphoblastoid interferon.
Large scale production of human lymphoblastoid (Namalva) interferon (IFN) is carried out in pilot-plant-scale fermentors having up to 250 liter culture volume. Cells are grown in a semi-continuous culture. For IFN production, which is an IFN-alpha (Ly), harvested cells were induced by Sendai virus. Variations in IFN yields of up to ten-fold were observed during long-term cell cultivation. Several factors such as cell concentration, virus concentration, "age" of induced cells and serum substitutions, were investigated for their effect on the fluctuation in the IFN yields. Optimization of the above factors were found to be of great importance in controlling the IFN production and maintaining a stable and high IFN yield.
Cell Division↗
Cell and virus propagation on cylindrical cellulose based microcarriers.
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.
Animals↗