Adherent Chinese Hamster Ovary Cell Research Articles

Contributions of Chinese hamster ovary cell derived extracellular vesicles and other cellular materials to hollow fiber filter fouling during perfusion manufacturing of monoclonal antibodies.

Hollow fiber filter fouling is a common issue plaguing perfusion production process for biologics therapeutics, but the nature of filter foulant has been elusive. Here we studied cell culture materials especially Chinese hamster ovary (CHO) cell-derived extracellular vesicles in perfusion process to determine their role in filter fouling. We found that the decrease of CHO-derived small extracellular vesicles (sEVs) with 50-200 nm in diameter in perfusion permeates always preceded the increase in transmembrane pressure (TMP) and subsequent decrease in product sieving, suggesting that sEVs might have been retained inside filters and contributed to filter fouling. Using scanning electron microscopy and helium ion microscopy, we found sEV-like structures in pores and on foulant patches of hollow fiber tangential flow filtration filter (HF-TFF) membranes. We also observed that the Day 28 TMP of perfusion culture correlated positively with the percentage of foulant patch areas. In addition, energy dispersive X-ray spectroscopy-based elemental mapping microscopy and spectroscopy analysis suggests that foulant patches had enriched cellular materials but not antifoam. Fluorescent staining results further indicate that these cellular materials could be DNA, proteins, and even adherent CHO cells. Lastly, in a small-scale HF-TFF model, addition of CHO-specific sEVs in CHO culture simulated filter fouling behaviors in a concentration-dependent manner. Based on these results, we proposed a mechanism of HF-TFF fouling, in which filter pore constriction by CHO sEVs is followed by cake formation of cellular materials on filter membrane.

Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics.

Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC-MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO-CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work and database described in our study will enable the CHO community to rapidly identify high-abundance HCPs in their cultures and therefore help assess process and purification methods used in the production of biologic drugs.

Release of Intracellular Proteins by Electroporation with Preserved Cell Viability

Extraction of intracellular proteins from cells is often an important first step for conducting molecular biology and proteomics studies. Although ultrasensitive detection and analytical technology at the single molecule level is becoming routine, protein extraction techniques have not followed suit and still call for complete lysis that leads to cell death. In principle, with refined extraction techniques, intracellular proteins can potentially be extracted without killing the cell. In this Letter, we demonstrate that electroporation is capable of releasing intracellular proteins from adherent Chinese hamster ovary cells while preserving the cell viability. By tuning the duration and intensity of an electric pulse, we were able to control the average amount of protein release and the percentage of viable cells after the operation. Our results indicate that a substantial fraction of the cell population was able to release proteins under electroporation and survive the procedure. Interestingly, at the single cell level, the probability for cell death does not increase with more protein release. This work paves the way to extracting and analyzing intracellular proteins while keeping cells live.

A Microfluidic Pipette for Single-Cell Pharmacology

We report on a free-standing microfluidic pipette made in poly(dimethylsiloxane) having a circulating liquid tip that generates a self-confining volume in front of the outlet channels. The method is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. The pipette is capable of carrying out a variety of complex fluid processing operations, such as mixing, multiplexing, or gradient generation at selected cells in cell and tissue cultures. Using an uptake assay, we show that it is possible to generate dose-response curves in situ from adherent Chinese hamster ovary cells expressing proton-activated human transient receptor potential vanilloid (hTRPV1) receptors. Using confined superfusion and cell stimulation, we could activate hTRPV1 receptors in single cells, measure the response by a patch-clamp pipette, and induce membrane bleb formation by exposing selected groups of cells to formaldehyde/dithiothreitol-containing solutions, respectively. In short, the microfluidic pipette allows for complex, contamination-free multiple-compound delivery for pharmacological screening of intact adherent cells.

Correlation between cell cycle phase of adherent Chinese hamster ovary cells and laser phase shift determined by phase-shifting laser microscopy

The simultaneous determination of the cell cycle phase of individual adherent Chinese hamster ovary cells using a fluorescence microscope after staining with 4',6-diamidine-2'-phenylindole dihydrochloride and bromodeoxyuridine and the laser phase shift by a phase-shifting laser microscopy revealed that the laser phase shift of cells in the G2/M phase was markedly higher than that of cells in the G1 and S phases.

Microfluidic Cell Electroporation Using a Mechanical Valve

A microfluidic electroporation technique is demonstrated based on the operation of an elastomeric valve in a poly(dimethylsiloxane) (PDMS) fabricated microchip and a common dc power supply. The pulse needed for permeabilization of the cell membrane is generated by temporarily interrupting the circuit using the valve. The electropermeabilization of suspended and adherent Chinese hamster ovary cells with green DNA dye SYTOX is demonstrated. The technique eliminates the cost and complexity associated with a pulse generator and microfabricated electrodes that are often involved in microscale electroporation devices. It also offers the potential of integrating electroporation as a unit operation in large-scale microfluidic systems with the increasing application of elastomeric valves in these systems.

Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro.

Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12--18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4--4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X-ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48--54 A. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA-surfactant complex.

The Molecular Adapter SLP-76 Relays Signals from Platelet Integrin αIIbβ3 to the Actin Cytoskeleton

Platelet adhesion to fibrinogen through integrin alpha(IIb)beta(3) triggers actin rearrangements and cell spreading. Mice deficient in the SLP-76 adapter molecule bleed excessively, and their platelets spread poorly on fibrinogen. Here we used human platelets and a Chinese hamster ovary (CHO) cell expression system to better define the role of SLP-76 in alpha(IIb)beta(3) signaling. CHO cell adhesion to fibrinogen required alpha(IIb)beta(3) and stimulated tyrosine phosphorylation of SLP-76. SLP-76 phosphorylation required coexpression of Syk tyrosine kinase and stimulated association of SLP-76 with the adapter, Nck, and with the Rac exchange factor, Vav1. SLP-76 expression increased lamellipodia formation induced by Syk and Vav1 in adherent CHO cells (p < 0.001). Although lamellipodia formation requires Rac, SLP-76 functioned downstream of Rac by potentiating adhesion-dependent activation of PAK kinase (p < 0.001), a Rac effector that associates with Nck. In platelets, adhesion to fibrinogen stimulated the association of SLP-76 with the SLAP-130 adapter and with VASP, a SLAP-130 binding partner implicated in actin reorganization. Furthermore, SLAP-130 colocalized with VASP at the periphery of spread platelets. Thus, SLP-76 functions to relay signals from alpha(IIb)beta(3) to effectors of cytoskeletal reorganization. Therefore, deficient recruitment of specific adapters and effectors to sites of adhesion may explain the integrin phenotype of SLP-76(-/-) platelets.

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells.

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) receptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (-)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells.

CHO transfectants produce large amounts of recombinant protein in suspension culture

Chinese hamster ovary (CHO) cells transfected with various genes are widely used as adherent cell monolayers to produce recombinant proteins. In this report we present a new culture technique for CHO cells transfected with the vector pPOL-DHFR-CD14 using a minifermenter (miniPERM, Heraeus) for the production of recombinant human endotoxin receptor CD14 (rCD14). The transfectants were cultured for 12–17 days under serum-free conditions and formed spheroids. From this system we harvested supernatants containing up to 3.1 mg/ml recombinant CD14 (rCD14). This represents a 200-fold increase of rCD14 yield compared to conventional adherent CHO cell culture.

Preparation and characterization of fluorescein-labeled plasma membrane vesicles from transfected Chinese hamster ovary cells

Plasma membrane vesicles have been used to study a wide variety of biological events including the nature of receptor-ligand interactions and the physiology of molecular transport across membranes. Plasma membrane vesicles were chemically induced from an adherent Chinese hamster ovary cell line expressing recombinant glycophorin A, a well-studied intrinsic membrane protein of the red blood cell. They were also prepared from Chinese hamster ovary cells in suspension culture. Biochemical and immunological assays demonstrated the equivalence of glycophorin A on cell membranes and vesicles. The transfected Chinese hamster ovary cell membrane was also labeled with a highly aliphatic fluorescent cell linker. Vesicles produced by the fluorescein-labeled cells demonstrated bright surface staining of the plasma membrane. They too expressed glycophorin A biochemically and immunologically indistinguishable from cellular-based glycophorin A. Plasma membrane vesicles are non-adherent in culture and stably retain the fluorescent probe in the cell membrane. Fluorescein-labeled vesicles will be particularly useful for studying cellular interactions in which both constituents of a receptor-ligand pair are expressed on adherent cell lines. Unlabeled vesicles may also prove to be useful as soluble immunoadsorbants in both the clinical laboratory and basic science research settings.

Development and monitoring of purification process for nerve growth factor fusion antibody

A purification process and an on-line process monitoring procedure for a human nerve growth factor (NGF) fusion antibody (NAK) was developed. The NAK protein was produced with a recombinant technique by an adherent Chinese hamster ovary cell line in a tissue culture system supplemented with 5–10% fetal bovine serum, then purified from tissue culture supernatant. The overall purity goal for the process was 90%. A capture step was developed and optimized on POROS 50A which served as a purification and concentration step for the NAK product. A second purification step to remove contaminating bovine IgGs was developed and optimized first on a POROS HS/M cation-exchange column using 20-μm particles. The process was then scaled to a POROS 50 HS (50-μm particle) column. Once an optimized process was developed the capture step on POROS 50A was scaled from an 8-ml column to a 0.3-1 column on PerSeptive Biosystems AutoPilot system.

Monoclonal antibodies directed against the galactose-binding lectin of Entamoeba histolytica enhance adherence.

The Entamoeba histolytica galactose-binding lectin is a surface glycoprotein composed of 170- and 35-kDa subunits. Inhibition of this lectin with galactose or anti-170 kDa subunit polyclonal antibody blocks amebic adherence to target cells and colonic mucin glycoproteins. We describe the properties of 10 mAb with specificity for the 170-kDa subunit. Based on competitive binding studies, six nonoverlapping antigenic determinants on the lectin were identified. The effect of the mAb on adherence of amebic trophozoites to both Chinese hamster ovary (CHO) cells and human colonic mucins was measured. Antilectin antibodies directed against epitopes 1 and 2 enhanced adherence, with the number of amebae having at least three adherent CHO cells increasing with the addition of epitope 1 mAb from 26 +/- 9 to 88 +/- 2% and the binding of colonic mucins increasing from 34 +/- 1 to 164 +/- 3 pg/10(5) amebae. Antibody-enhanced adherence remained 90 to 100% galactose inhibitable, occurred at 4 degrees C and was not Fc mediated. Univalent Fab fragments of epitope 1 mAb augmented mucin binding by 238% and CHO cell adherence by 338%. The binding of purified lectin to CHO cells was increased from 1.1 +/- 0.1 to 2.4 +/- 0.3 ng/10(3) CHO cells by mAb directed to epitope 1, demonstrating that enhanced adherence was due to direct activation of the lectin. mAb to epitope 3 bound to the lectin only upon its solubilization from the membrane and had no effect on adherence. Adherence to CHO cells and mucins was inhibited from 50 to 75% by mAb to epitopes 4 and 5; epitope 6 mAb inhibited amebic adherence to CHO cells but not mucins. The pooled sera from 10 patients with amebic liver abscess blocked the binding to the 170-kDa subunit of mAb directed to all six epitopes. Striking individual variations in the effects of immune sera on adherence were observed. Although the sera of all 44 South African patients with amebic liver abscess had high titer anti-lectin antibodies, 16 patients' sera significantly (more than 3 SEM) enhanced adherence whereas 25 patients' sera significantly inhibited adherence. Antilectin antibodies exert profound functional effects on the interaction of E. histolytica with target cells and human colonic mucins. Exploration of the clinical consequences of adherence-enhancing and inhibitory antibody responses may give insight into the role of antilectin antibodies in immunity to invasive amebiasis.

Flow cytometric studies of the survival of cytochalasin-induced polyploidy in Chinese hamster ovary cells

A method has been developed to estimate the post-irradiation survival of cytochalasin B-induced polyploidization of adherent Chinese hamster ovary cells using the flow cytometer. After exposure to radiation, surviving cells are allowed to become polyploid in the presence of cytochalasin, and are detached using trypsin, fixed by the addition of glutaraldehyde and stained using mithramycin. DNA content distributions are polymodal, and the absolute number of cells per culture in any given ploidy class is estimated by reference to a non-fluorescent bead internal standard, detected using forward scatter. Post-irradiation survival is defined as the ability to reach a given DNA content, and is reduced exponentially with dose. A bioassay to determine optimum cytochalasin concentrations can be derived from the relative size of the 2C ( G 0 G 1 ) peak in the DNA content distribution. At culture densities greater than about 8 × 10 4 cell/cm 2 the relative number of cells reaching at least 16C is reduced, but this inhibition is partially reversible by an increase in the medium glucose concentration, but not by the use of cytochalasin D or dihydro-B.

Effects of Prospidine on Survival and Growth of Mammalian Cells in Culture&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

The effects of prospidine, chemically known as 3, 12-diaza-6,9 diazoniadispiro[5.2.5.2]hexadecane,3,12-bis(3-chloro-2-hydroxypropyl)-dichloride (NSC-166100), on cell viability, growth, and colony formation were investigated in several mammalian cell lines. Cell cycle progression and the terminal point of action of the drug were monitored by flow cytometry. Prospidine was cytostatic for two suspension cultures (Friend leukemia and L1210 cells) at a concentration of 10 mg/ml during the first cell cycle after exposure to the drug. Cells were blocked in G2 at lower concentrations of prospidine (e.g., 1.0 mg/ml) but only after 12--24 hours of continuous exposure, i.e., during the second cell cycle in the presence of drug. Cells could be observed to accumulate in G2 by 24 hours even if prospidine (0.1 mg/ml) was removed after 12 hours. A short incubation with a liver cytosol fraction (30 min) or with cultured cells (12 hr) failed to enhance the potency of the drug. Formation of colonies of the adherent Chinese hamster ovary cell line was inhibited by 50% following 24-hour exposure to 1.1 mg prospidine/ml. Under culture conditions in which cells were blocked in G2, their RNA content increased only slightly, but the incorporation of [5-(3)H]uridine into RNA was suppressed by 15--20%. Incubation of cells with prospidine increased the stability of DNA in situ to acid-induced denaturation, which thus suggested that the drug may interact with cellular DNA.

Fibrin adherent CHO cell behavior in response to chelators and enterotoxin

Chinese hamster ovary (CHO) cells attach to fibrin with a round morphology and differentiate into stretched form when cell density increases. This morphological differentiation is accelerated and accentuated by striking elongation following the addition of dibutyryl cyclic AMP (db-cAMP) or cholera toxin (CT). In contrast to the prompt effect within 2–4 h after db-cAMP, there is a characteristic lag in the onset of CT effect. CHO cell attachment to fibrin was substantially more resistant to release by EDTA or EGTA than were cells attached to plastic. While cell attachment to fibrin could be gradually released by EDTA, adherence was relatively resistant to release by EGTA, suggesting that Mg 2+ may be important in cell adherence to fibrin.