Encapsulated animal cell culture for the production of monoclonal antibody (MAb)

Biopolymer membrane was prepared using two oppositely charged natural biopolymer. The biopolymer membrane was used for the encapsulation of two hybridoma cell (ATCC CRL-1606, ATCC BH-8852) to produce monoclonal antibodies. In order to reduce the down stream steps, the pore size of the membrane was controlled to retain the monoclonal antibodies in the capsules based on the diffusion experiments with standard proteins. T-flask culture showed cell densities of 8×10⁷ cells/mL and 3×10⁷ cells/mL, and MAb concentrations of 506 μg/mL and 109 μg/mL for encapsulated ATCC CRL-1606 and HB-8852, respectively. Two liter perfusion culture with encapsulated ATCC HB-8852 was performed to enhance the MAb production. The MAb production of the encapsulated hybridoma increased considerably comparing to the culture using silicone tubing for oxygen transfer.     

Initial attachment of baby hamster kidney cells to an epoxy substratum. Ultrastructural analysis

In the presence of serum-containing medium, BHK cells attached and spread during a 1-h period onto a 3-5 nm thick serum layer absorbed on the substratum surface. The closest approach of the plasma membrane to the serum layer was observed to be about 9nm, which was determined by tilting the sectioned cells in a goniometer holder. Bundles of microfilaments or other cytoplasmic specializations were not observed in association with the regions of close contact. However, in the space between the plasma membrane and the adsorbed serum layer, a diffusely stained material could be visualized after fixation/staining by the tannic acid-glutaraldehyde technique. This technique also permitted increased clarity of visualization of trilaminar appearance of the plasma membrane. The distribution and mobility of anionic sites on the surfaces of attached and spreading cells was determined by labeling with polycationic ferritin. We observed movement of polycationic ferritin into large clusters on the cell surface, collapse of cell surface microextensions, and endocytosis, all of which were similar to our previous findings utilizing cells in suspension. However, the absolute amount of ferritin bound to the upper cell surface was less than that previously observed when suspended cells were put under similar labeling conditions. Also, polycationic ferritin did not appear to penetrate between the lower cell surface and the substratum.     

The different faces of mass action in virus assembly

The spontaneous encapsulation of genomic and non-genomic polyanions by coat proteins of simple icosahedral viruses is driven, in the first instance, by electrostatic interactions with polycationic RNA binding domains on these proteins. The efficiency with which the polyanions can be encapsulated in vitro, and presumably also in vivo, must in addition be governed by the loss of translational and mixing entropy associated with co-assembly, at least if this co-assembly constitutes a reversible process. These forms of entropy counteract the impact of attractive interactions between the constituents and hence they counteract complexation. By invoking mass action-type arguments and a simple model describing electrostatic interactions, we show how these forms of entropy might settle the competition between negatively charged polymers of different molecular weights for co-assembly with the coat proteins. In direct competition, mass action turns out to strongly work against the encapsulation of RNAs that are significantly shorter, which is typically the case for non-viral (host) RNAs. We also find that coat proteins favor forming virus particles over nonspecific binding to other proteins in the cytosol even if these are present in vast excess. Our results rationalize a number of recent in vitro co-assembly experiments showing that short polyanions are less effective at attracting virus coat proteins to form virus-like particles than long ones do, even if both are present at equal weight concentrations in the assembly mixture.     

Modulation by polyelectrolytes of canine cardiac microsomal calcium uptake and the possible relationship to phospholamban

Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in canine cardiac microsomes were found to be stimulated by heparin and various other polyanions. Prior treatment of the microsomes with the ionophores alamethicin or A23187 produced no change in the extent of stimulation of the ATPase activity by heparin yet eliminated net calcium uptake. This finding and a lack of change in the stoichiometric ratio of mol of calcium transported/mol of ATP hydrolyzed (calcium:ATP) suggest that the effect of heparin is on the calcium pump rather than on a parallel calcium efflux pathway. Certain polycationic compounds including poly-L-arginine and histone inhibited both cardiac and fast skeletal muscle microsomal calcium uptake and also produced no change in the stoichiometric ratio of calcium to ATP. Several lines of evidence indicate that the polyanionic compounds tested stimulate calcium uptake by interacting with phospholamban, the putative phosphorylatable regulator of the cardiac sarcoplasmic reticulum calcium pump, whereas polycationic compounds appear to interact with the pump. (i) Heparin stimulated calcium uptake to the same extent as protein kinase A or trypsin, whereas prior phosphorylation or tryptic cleavage of phospholamban from the membrane abolished the stimulatory effect of heparin. (ii) Calcium uptake and (Ca2+ + Mg2+)-ATPase activity in fast skeletal muscle microsomes, which lack phospholamban, were unaffected by heparin. (iii) Purified cardiac (Ca2+ + Mg2+)-ATPase activity was no longer stimulated by heparin yet was still inhibited by polycationic compounds. The heparin-induced stimulation of calcium uptake was dependent on the pH and ionic strength of the heparin-containing preincubation medium, hence electrostatic interactions appear to play a significant role in heparin's stimulatory action. The data are consistent with an inhibitory role of the positively charged cytoplasmic domain of phospholamban with respect to calcium pump activity and the relief of the inhibition upon reduction in phospholamban's positive charge by phosphorylation or binding of polyanions.     

Antigen-induced proliferation and immunoglobulin A secretion by a human-human hybridoma

The capacity of membrane immunoglobulin A (IgA)-bearing B cells to respond to specific antigen in the absence of T cell influences has not been defined. A human-human hybridoma, constructed from an Epstein-Barr virus transformed tonsil B cell that secreted IgA anti-phosphorycholine (PC) and a human plasmacytoma cell, was utilized to examine this issue. The cloned hybridoma expressed membrane IgA and secreted IgA specific for PC. Stimulation of the hybridoma cells with PC conjugated to Sepharose beads (PC-Sepharose) but not glycine-conjugated Sepharose resulted in an increase in DNA synthesis. Affinity purified goat anti-human IgA bound to Sepharose also augmented DNA synthesis. Soluble PC did not increase DNA synthesis and inhibited the increase in DNA synthesis resulting from PC-Sepharose. IgA secretion was augmented in response to PC-Sepharose, as demonstrated by an increase in the number of Ig-secreting cells detected by a reverse hemolytic plaque assay and by quantitation of the IgA secreted per cell by enzyme-linked immunosorbent assay. Mitogen-stimulated T cell supernatants increased IgA secretion of the hybridoma cells but did not cause synergistic stimulation of the cells in the presence of PC-Sepharose. These data indicate that Sepharose-bound antigen was sufficient to induce proliferation and augment IgA secretion by this membrane IgA anti-PC-bearing hybridoma. The results suggest that cross-linking of membrane IgA by specific antigen may be a sufficient stimulus for proliferation and differentiation of B cells at this stage of maturation.     

Heterogeneity of planarian stem cells in the S/G2/M phase

The planarian adult stem cell (pASC) population has a specific molecular signature and can be easily visualized and isolated by flow cytometry. However, the lack of antibodies against specific surface markers for planarian cells prevents a deeper analysis of specific cell populations. Here, if we describe the results of the immunoscreening of pASC plasma membrane proteins (PMPs). A novel papain-based method for planarian cell dissociation enabling both high yield and improved cell viability was used to generate single cell preparations for PMP purification. PMPs were used for intraperitoneal immunization of mice and thus about 1000 hybridoma clones were generated and screened. Supernatants collected from the hybridoma clones were first screened by ELISA and then by live immuno-staining. About half of these supernatants stained all the planarian cells, whereas the other half specifically labeled a subfraction thereof. A detailed analysis of two hybridoma supernatants revealed that large subfractions of the X1, X2 and Xin populations differentially express specific membrane markers. Quantitative PCR data disclosed a correlation between the immunostaining results and the expression of markers of the early and late progeny, also for those pASCs in the S/G2/M phase of the cell cycle (X1 population). Thus, about two thirds of the cycling pASCs showed a specific membrane signature coupled with the expression of markers hitherto considered to be restricted to differentiating, post-mitotic progeny. In summary, a library of 66 monoclonal antibodies against planarian PMPs was generated. The analysis of two of the clones generated revealed that a subset of cells of the X1 population expresses early and late progeny markers, which might indicate that these cells are committed while still proliferating. The findings demonstrate the usefulness of our PMP antibody library for planarian research.     

Mitochondrial membrane potential selects hybridomas yielding high viability in fed-batch cultures

Prior research (Follstad, B. D.; Wang, D. I. C.; Stephanopoulos, G. Mitochondrial membrane potential differentiates cells resistant to apoptosis in hybridoma cultures. Eur. J. Biochem. 2000, 267, 6534-6540.) identified mitochondrial membrane potential (MMP) as a marker of hybridoma subpopulations resistant to apoptosis caused by a variety of apoptosis inducers. In this study, we investigated the viability of hybridoma cell cultures inoculated with cells of varying MMP in regular fed-batch operation. A hybridoma cell population was separated using FACS into subpopulations based on their mean mitochondrial membrane potential (MMP) as measured using the common mitochondrial stain, Rhodamine 123 (Rh123). These subpopulations showed dramatic differences in their apoptotic death kinetics. Fed-batches inoculated with a high MMP subpopulation reached higher viable cell concentrations and viabilities that were maintained for prolonged periods of time relative to fed-batches inoculated with low MMP subpopulations. These results underline the heterogeneous nature of hybridoma cell cultures and suggest that mitochondrial physiology is a critical parameter determining culture performance.     

Identification and characterization of a novel cell-penetrating peptide

Cell-penetrating peptides (CPPs) are short amino acid sequences that promote their own translocation across cell plasma membrane. When linked with cargo such as polypeptides, nucleic acid, or liposomes, CPPs can facilitate the transport of these entities across the cell membrane. Therefore, CPPs are receiving increased interest in drug delivery and gene therapy. The majority of CPPs identified so far are polycationic peptides which interact with heparin sulfate chains of plasma membrane for internalization. Here, we report the identification and characterization of a conformationally constrained 13 amino acid peptide (CVQWSLLRGYQPC, designated as S41) which is clearly distinct from classical polycationic peptides. Immunofluorescence assay was employed to test the cellular uptake of S41 in mouse neuroblastoma cell line Neuro2A (N2A) and rat cerebellar granule neurons (CGNs). Internalization of S41 was further examined in N2A cells by means of mutational analysis, flow cytometry and confocal microscopy. Our results demonstrate that S41 can enter cells through lipid rafts dependent endocytosis.     

MOBILIZATION OF B AND T LYMPHOCYTES AND HAEMOPOIETIC STEM CELLS BY POLYMETHACRYLIC ACID AND DEXTRAN SULPHATE

The leucocytosis which can be evoked by the polyanions dextran sulphate (DS), polymethacrylic acid (PMAA) and the copolymer of PMAA and styrene (PMAA—STYR) was studied in mice. After intravenous administration of these polyanions peak numbers of leucocytes were found in the peripheral blood 3 hr after injection. All three types of polyanions increased the number of lymphocytes, granulocytes and monocytes. Dose—response studies revealed that the nature of the polyanion determined the degree of leucocyte mobilization. The most potent mobilizer was found to be DS. This polyanion could evoke a six-fold increase of the number of peripheral blood leucocytes. By means of the membrane fluorescence technique it could be demonstrated that optimal doses of DS, PMAA and PMAA—STYR mobilized both B and T lymphocytes. The ratio between the number of B and T cells mobilized was greater for DS than for the other two polyanions. Intravenous injection of DS, PMAA and PMAA—STYR also increased the number of circulating haemopoietic stem cells (CFU-S). The most potent stem cell mobilizer appeared to be PMAA—STYR. This polyanion evoked a twenty-five-fold increase in the number of CFU-S.     

Mobilization of B and T lymphocytes and haemopoietic stem cells by polymethacrylic acid and dextran sulphate.

The leucocytosis which can be evoked by the polyanions dextran sulphate (DS), polymethacrylic acid (PMAA) and the copolymer of PMAA and styrene (PMAA--STYR) was studied in mice. After intravenous administration of these polyanions peak numbers of leucocytes were found in the peripheral blood 3 hr after injection. All three types of polyanions increased the number of lymphocytes, granulocytes and monocytes. Dose--response studies revealed that the nature of the polyanion determined the degree of leucocyte mobilization. The most potent mobilizer was found to be DS. This polyanion could evoke a six-fold increase of the number of peripheral blood leucocytes. By means of the membrane fluorescence technique it could be demonstrated that optimal doses of DS, PMAA and PMAA--STYR mobilized both B and T lymphocytes. The ratio between the number of B and T cells mobilized was greater for DS than for the other two polyanions. Intravenous injection of DS, PMAA and PMAA--STYR also increased the number of circulating haemopoietic stem cells (CFU-S). The most potent stem cell mobilizer appeared to be PMAA--STYR. This polyanion evoked a twenty-five-fold increase in the number of CFU-S.     

Methods for determination of growth-rate-dependent changes in hybridoma volume,shape and surface structure during continuous recycle

Hybridoma volume and surface membrane structure were found to vary as a function of specific growth rate using a method of cell recycle with continuous medium perfusion to vary growth rate. Mean hybridoma volume determined at constant osmolality by both electronic particle counting and scanning electron microscopic (SEM) methods indicated that rapidly growing cells are significantly larger than very slowly growing cells. We have previously determined that during both rapid and slow growth over a range of L-glutamine provision rates (Gln PR) that specific monoclonal antibody (MoAb) secretion rate was not changed. In this study a constant MoAb secretion rate per unit of membrane area was found which may indicate that changing membrane area is not a rate-determining factor in MoAb secretion. SEM methods were of limited use for accurate determination of cell volume due to cell shrinkage and large coefficients of variations. In spite of this limitation, SEM stereology methods were useful in confirming that cells remained spherical over a wide range of specific growth rates and that hybridoma cells were not circular. Sequential SEM observations also revealed that surface membrane structure of the 9.2.27 murine hybridoma investigated was correlated with growth rate. Under conditions of very slow growth, hybridoma surface microvilli density appeared to be significantly reduced.     

Use of fluorescence anisotropy determinations for indicating the physiological status of hybridoma cell cultures

The evolution of lipid compartment fluidity during culture of hybridoma cells was studied by fluorescence polarization measurements. The probe partition between the plasma membrane and intracytoplasmic compartments was determined by a quenching fluorescence method. A progressive decrease of the plasma membrane fluidity was observed during the growth phase with an increase during stationary and degeneration phases of the culture. These data suggest that fluidity parameters could be used to follow the behaviour of hybridoma cell cultures.     

Poly(ethylenimine)-reinforced liquid-core capsules for the cultivation of hybridoma cells

The mechanical stability of liquid-core alginate-poly-L-lysine (PLL) capsules used for encapsulation of hybridoma cells can be greatly enhanced by the inclusion of poly(ethylenimine) (PEI) in the hardening solution containing calcium chloride. The PEI can also reinforce the PLL-coated carboxymethyl-celluose liquid-core capsules. The cultivation of murein hybridoma CT04 in these two capsules was carried out. Cell concentrations higher than 10(8) cells/mL per capsule were obtained with ca. 80% of the specific antibody productivity as the freely suspended cells. These capsules could withstand severe agitation and aeration in an air-lift reactor over a period of 3 weeks with minimal damage. (c) 1992 John Wiley & Sons, Inc.     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

Electrostatic interactions in the early events of VSV infection

The inportance of electrostatic interactions in the early phases of vesicular stomatitis virus (VSV) infection has been investigated in susceptible cells of different origin, human (HeLa) and avian (CER), by using some polyanions (heparin, polygalacturonic acid and mucin) and polycations (polymyxin B sulphate, poly-L-lysine, protamine, histone and polybrene). In HeLa cells, the attachment of VSV was enhanced by polymers having a positive charge and inhibited by those having a negative charge. In CER cells, all the polyanions tested reduced virus infection. Among the polycations, histone, polymyxin B sulphate and poly-L-lysine enhanced virus plaque forle protamine and polybrene reduced virus attachment. The effect of polyions on VSV particles and on cell membrane receptors has also been investigated. The analysis of the results obtained suggest that, although electrostatic interactions play an essential role in the binding of VSV to the cell membrane, more specific structural features appear to be required for viral attachment to occur.     

Membrane-cytoskeleton interactions: Inhibition of odontoblast differentiation by a monoclonal antibody directed against a membrane protein

It is known that high-molecular-weight (HMW) membrane proteins mediate interactions with constituents of the extracellular matrix and/or with cytoskeletal elements. To study participation of HMW membrane proteins in odontoblast or ameloblast differentiation, an immunological approach has been adopted. Antibodies directed against membrane proteins (Mr, 110-190) from mouse embryos have been produced by the hybridoma technique. Supernatants of hybridoma cultures were screened for their ability to stain dental tissues and also tested for their biological activities on dental cells in primary culture or on developing tooth germs in organ culture. An IgM monoclonal antibody, MC16A16, directed against a 165-kDa antigen present in plasma membrane preparations, reacted strongly with the dental epithelium and weakly with the mesenchyme. MC16A16 also reacted with the cell surface of nonpermeabilized cultured dental cells and could detach epithelial cells cultured on glass, but not mesenchymal cells which maintained vinculin-containing focal contacts. This antibody, which affected the organization of dental-cell microfilaments in primary culture, also inhibited the polarization of odontoblasts, but not that of ameloblasts.     

Kinetic studies and unstructured models of lymphocyte metabolism in fed-batch culture

The growth of two lymphocyte cell lines, a hybridoma cell line and a human cutaneous T cell lymphoma (HuT78), was studied in fed-batch culture, and unstructured models of growth developed. A criteria was established to insure that the growth rate varied by less than a specified tolerance throughout the culture period. Glutamine and serum were growth-limiting nutrients for both cell lines with half-maximal growth rates at 0. 53 mM glutamine and 0. 55%(v/v) serum for the hybridoma cells and 0. 21 mM glutamine and 1. 5% serum for the HuT-78 cells. Over the range of glucose concentrations from 5. 5 mM to 28 mM, the specific growth rate of hybridoma cells was independent of glucose concentration, whereas glucose concentrations above 5. 5 mM inhibited HuT-78 growth. For both cell lines, the growth rate was significantly inhibited by the addition of ammonium, although the hybridoma cell line was more affected by ammonia than was the HuT-78 cell line. Growth of HuT-78 cells increased in the presence of interleukin-2. Unstructured models for the hybridoma cells were similar to other models presented in the literature. Applications of these models to adoptive immunotherapy are discussed.     

In vivo and in vitro regulation of IgE production in murine hybridomas

Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.     

Binding of antigen-specific, H-2-restricted T cell hybridomas to antigen-pulsed adherent cell monolayers

Two methods were investigated for studying the binding of radiolabeled hybridoma T cells to antigen (Ag) and H-2 products for which they bore receptors. In both cases hybridoma T cells were labeled with tritiated thymidine. In one method labeled cells were added to adherent splenic cells prepulsed with antigen, and the mixture was incubated overnight at 37 degrees C before nonadherent cells were gently washed away. The percent of adherent hybridoma T cells was then estimated by harvesting the adherent monolayers and measuring tritium counts bound. In a second method radiolabeled hybridoma T cells were added to adherent antigen-pulsed B cell lymphomas or hybridomas for between 15 min and 1 hr at 37 degrees C before removal of nonadherent cells and harvesting of the adherent monolayers. In both cases binding was both antigen- and I-region specific. In the second case binding was also rapid; significant binding could be measured after 15 min incubation. These techniques were used to study subclones of one of our T cell hybridomas that were thought by a functional assay (interleukin 2 release) to have lost receptors for Ag/H-2. It was found that subclones of the hybridoma that no longer secreted interleukin 2 in response to Ag/H-2, even though they continued to secrete interleukin 2 in response to concanavalin A, also no longer bound specifically to Ag-pulsed monolayers of the appropriate H-2 type. This confirmed the idea that these subclones had lost the ability to synthesize receptors for Ag/H-2. It is hoped that assays of this type will be useful in the future for the study of Ag/H-2 receptors on T cells.     

Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability

Interactions of polycationic polymers with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and live cell membranes (KB and Rat2) have been investigated using atomic force microscopy (AFM), cytosolic enzyme assays, confocal laser scanning microscopy (CLSM), and a fluorescence-activated cell sorter (FACS). Polycationic polymers poly-L-lysine (PLL), polyethylenimine (PEI), and diethylaminoethyl-dextran (DEAE-DEX) and sphere-like poly(amidoamine) (PAMAM) dendrimers are employed because of their importance for gene and drug delivery. AFM studies indicate that all the polycationic polymers cause the formation and/or expansion of preexisting defects in supported DMPC bilayers in the concentration range of 1-3 microg/mL. By way of contrast, hydroxyl-containing neutral linear poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) do not induce hole formation or expand the size of preexisting defects in the same concentration range. All polymers tested are not toxic to KB or Rat2 cells up to a 12 microg/mL concentration (XTT assay). In the concentration range of 6-12 microg/mL, however, significant amounts of the cytosolic enzymes lactate dehydrogenase (LDH) and luciferase (LUC) are released. PEI, which possesses the greatest density of charged groups on its chain, shows the most dramatic increase in membrane permeability. In addition, treatment with polycationic polymers allows the small dye molecules propidium idodide (PI) and fluorescein (FITC) to diffuse in and out of the cells. CLSM images also show internalization of PLL labeled with FITC dye. In contrast, controls of membrane permeability using the neutral linear polymers PEG and PVA show dramatically less LDH and LUC leakage and no enhanced dye diffusion. Taken together, these data are consistent with the hypothesis that polycationic polymers induce the formation of transient, nanoscale holes in living cells and that these holes allow a greatly enhanced exchange of materials across the cell membrane.