Single Chinese Hamster Ovary Cells Research Articles

A complete workflow for single cell mtDNAseq in CHO cells, from cell culture to bioinformatic analysis.

Chinese hamster ovary (CHO) cells have a long history in the biopharmaceutical industry and currently produce the vast majority of recombinant therapeutic proteins. A key step in controlling the process and product consistency is the development of a producer cell line derived from a single cell clone. However, it is recognized that genetic and phenotypic heterogeneity between individual cells in a clonal CHO population tends to arise over time. Previous bulk analysis of CHO cell populations revealed considerable variation within the mtDNA sequence (heteroplasmy), which could have implications for the performance of the cell line. By analyzing the heteroplasmy of single cells within the same population, this heterogeneity can be characterized with greater resolution. Such analysis may identify heterogeneity in the mitochondrial genome, which impacts the overall phenotypic performance of a producer cell population, and potentially reveal routes for genetic engineering. A critical first step is the development of robust experimental and computational methods to enable single cell mtDNA sequencing (termed scmtDNAseq). Here, we present a protocol from cell culture to bioinformatic analysis and provide preliminary evidence of significant mtDNA heteroplasmy across a small panel of single CHO cells.

Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.

OPP Labeling Enables Total Protein Synthesis Quantification in CHO Production Cell Lines at the Single-Cell Level.

Accurate measurement of global and specific protein synthesis rates is becoming increasingly important, especially in the context of biotechnological applications such as process modeling or selection of production cell clones. While quantification of total protein translation across whole cell populations is easily achieved, methods that are capable of tracking population dynamics at the single-cell level are still lacking. To address this need, we apply O-propargyl-puromycin (OPP) labeling to assess total protein synthesis in single recombinant Chinese hamster ovary (CHO) cells by flow cytometry. Thereby we demonstrate that global protein translation rates slightly increase with progression through the cell cycle during exponential growth. Stable CHO cell lines producing recombinant protein display similar levels of total protein synthesis as their parental CHO host cell line. Global protein translation does not correlate with intracellular product content of three model proteins, but the host cell line with high transient productivity has a higher OPP signal. This indicates that production cell lines with increased overall protein synthesis capacity can be identified by our method at the single-cell level. In conclusion, OPP-labeling allows rapid and reproducible assessment of global protein synthesis in single CHO cells, and can be multiplexed with DNA staining or any type of immunolabeling of specific proteins or markers for organelles.

Dielectrophoresis study of temporal change in internal conductivity of single CHO cells after electroporation by pulsed electric fields.

Applying sufficiently strong pulsed electric fields to a cell can permeabilize the membrane and subsequently affect its dielectric properties. In this study, we employ a microfluidic dielectrophoresis cytometry technique to simultaneously electroporate and measure the time-dependent dielectric response of single Chinese hamster ovary cells. Using experimental measurements along with numerical simulations, we present quantitative results for the changes in the cytoplasm conductivity of single cells within seconds after exposure to 100 μs duration pulsed electric fields with various intensities. It is shown that, for electroporation in a medium with conductivity lower than that of the cell's cytoplasm, the internal conductivity of the cell decreases after the electroporation on a time scale of seconds and stronger pulses cause a larger and more rapid decrease. We also observe that, after the electroporation, the cell's internal conductivity is constrained to a threshold. This implies that the cell prevents some of the ions in its cytoplasm from diffusing through the created pores to the external medium. The temporal change in the dielectric response of each individual cell is continuously monitored over minutes after exposure to pulsed electric fields. A time constant associated with the cell's internal conductivity change is observed, which ranges from seconds to tens of seconds depending on the applied pulse intensity. This experimental observation supports the results of numerical models reported in the literature.

Dielectric model for Chinese hamster ovary cells obtained by dielectrophoresis cytometry.

We present a dielectric model and its parameters for Chinese hamster ovary (CHO) cells based on a double-shell structure which includes the cell membrane, cytoplasm, nuclear envelope, and nucleoplasm. Employing a dielectrophoresis (DEP) based technique and a microfluidic system, the DEP response of many single CHO cells is measured and the spectrum of the Clausius-Mossotti factor is obtained. The dielectric parameters of the model are then extracted by curve-fitting to the measured spectral data. Using this approach over the 0.6-10 MHz frequency range, we report the values for CHO cells' membrane permittivity, membrane thickness, cytoplasm conductivity, nuclear envelope permittivity, and nucleoplasm conductivity. The size of the cell and its nuclei are obtained using optical techniques.

Influence of the phase effect on gradient‐based and statistics‐based focus measures in bright field microscopy

Autofocusing is essential to high throughput microscopy and live cell imaging and requires reliable focus measures. Phase objects such as separated single Chinese hamster ovary cells are almost invisible at the optical focus position in bright field microscopy images. Because of the phase effect, defocused images of phase objects have more contrast. In this paper, we show that widely used focus measures exhibit an untypical behaviour for such images. In the case of homogeneous cells, that is, when most cells tend to lie in the same focal plane, both gradient-based and statistics-based focus measures tend to have a local minimum instead of a global maximum at the optical focus position. On the other hand, if images show inhomogeneous cells, gradient-based focus measures tend to yield typical focus curves, whereas statistics-based focus measures deliver curves similar to the case of homogeneous cells. These results were interpreted using the equation describing the phase effect and patch-wise analysis of the focus curves. Bioprocess engineering experts are also influenced by the phase effect. Forty-four focus positions selected by them led to the conclusion that they prefer to look at defocused images instead of those at the optical focus.

Investigating differential cell‐matrix adhesion by directly comparative single‐cell force spectroscopy

Tissue-embedded cells are often exposed to a complex mixture of extracellular matrix (ECM) molecules, to which they bind with different cell adhesion receptors and affinities. Differential cell adhesion to ECM components is believed to regulate many aspects of tissue function, such as the sorting of specific cell types into different tissue compartments or ECM niches. In turn, aberrant switches in cell adhesion preferences may contribute to cell misplacement, tissue invasion, and metastasis. Methods to determine differential adhesion profiles of single cells are therefore desirable, but established bulk assays usually only test cell population adhesion to a single type of ECM molecule. We have recently demonstrated that atomic force microscopy-based single-cell force spectroscopy (SCFS), performed on bifunctional, microstructured adhesion substrates, provides a useful tool for accurately quantitating differential matrix adhesion of single Chinese hamster ovary cells to laminin and collagen I. Here, we have extended this approach to include additional ECM substrates, such as bifunctional collagen I/collagen IV surfaces, as well as adhesion-passivated control surfaces. We investigate differential single cell adhesion to these substrates and analyze in detail suitable experimental conditions for comparative SCFS, including optimal cell-substrate contact times and the impact of force cycle repetitions on single cell adhesion force statistics. Insight gained through these experiments may help in adapting this technique to other ECM molecules and cell systems, making directly comparative SCFS a versatile tool for comparing receptor-mediated cell adhesion to different matrix molecules in a wide range of biological contexts.

In what time scale proton transfer takes place in a live CHO cell?

Excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulfonate, HPTS) in a live Chinese hamster ovary (CHO) cell is studied by time resolved confocal microscopy. The cytoplasm region of the cell is stained by a photoacid, HPTS (HA). The time constant of initial proton transfer (τ(PT)) in the cell is found to be ~10 times longer than that in bulk water, while the time constants of recombination (τ(rec)) and dissociation (τ(diss)) in the cell are ~3 times and ~2 times longer, respectively. The slower rate of proton transfer (~10 times) inside the CHO cell compared to that in bulk water is ascribed to slower solvation dynamics, lower availability of free water molecules, and disruption of hydrogen-bond network inside the cell. Translational and rotational diffusion of HPTS inside a single CHO cell have been investigated by fluorescence correlation spectroscopy (FCS) and picosecond anisotropy measurement, respectively. Both the translational and rotational diffusion slow down inside the live cell. FCS studies indicate that HPTS remains tightly bound to a macromolecule inside the cell.

Highly expressed recombinant human follicle-stimulating hormone from Chinese hamster ovary cells grown in serum-free medium and its effect on induction of folliculogenesis and ovulation

To develop efficient Chinese hamster ovary (CHO) cells that express recombinant human FSH (rhFSH) in serum-free conditions and to investigate the effect of this newly synthesized rhFSH on folliculogenesis and ovulation. Experimental study. Seoul National University, South Korea. Forty immature hypophysectomized rats and 40 androgen-sterilized mice. A stable single CHO cell that expresses rhFSH at a high level was obtained by introducing the human chorionic gonadotropin (hCG) alpha-subunit and FSH beta-subunit genes. After purification processing, we investigated the effect of this newly synthesized rhFSH on folliculogenesis in hypophysectomized rats and ovulation in androgen-sterilized mice. The ovary weight, uterine weight, number of follicles, and ovarian morphology were evaluated in immature hypophysectomized rats. The number of ovulated oocytes and ovarian morphology were examined in androgen-sterilized mice. After purification processing, we analyzed the new rhFSH using matrix-associated laser desorption ionization-time of flight and found that this new rhFSH increased both ovarian weight and uterine weight in hypophysectomized rats and induced ovulation in androgen-sterilized mice. This newly synthesized rhFSH might be safely used in anovulatory infertile woman as well as in ovulation induction protocols for subfertile women.

Nonlinear current–voltage relationship of the plasma membrane of single CHO cells

The application of electric field pulses to Chinese Hamster Ovary (CHO) cells causes membrane electroporation (MEP). If a voltage or current ramp is applied to the cellular membrane of a single CHO cell, the membrane conductance increases nonlinearly with field strength reaching saturation. In particular, the kinetics of the induced conductance changes represents the data basis for the interpretation in terms of underlying structural changes. The current/voltage characteristic is found to be continuous, but displays occasionally a sharp increase in the conductance. The step-like increases are interpreted to reflect the formation of one (or more) larger pore(s). The analysis of current clamp data yields pores of radius ( r p) in the range of 2.5 ≤ r p/nm ≤ 20; the pores of the voltage clamp data are in the range of 2.5 ≤ r p/nm ≤ 55. The larger pores occur predominantly during hyperpolarising and less frequently during depolarising conditions, respectively. The different kinetics of pore formation in the hyperpolarising condition, where the inward field increases, and the depolarising condition, where the inward field first decreases and then increases in the opposite direction, suggests structural asymmetry with respect to the direction of the electric membrane field. At the required higher voltage, the effect of the resting potential is negligibly small.

Proteomic studies support the use of multi‐product immunoassays to monitor host cell protein impurities

In the biopharmaceutical industry, recombinant protein drugs are commonly produced in Chinese hamster ovary (CHO) cells. During the development process, removal of CHO cell-derived proteins from the biopharmaceutical product is monitored using multi-product immunoassays. Such immunoassays are developed by raising antibodies to a single CHO cell protein preparation. However, these assays are utilized to monitor CHO cell protein impurities during the recovery of products from different CHO cell lines. To address whether underlying differences between CHO cell lines result in sufficient protein expression changes to exclude the suitability of multi-product immunoassays, a comparative proteomics study of three independently generated CHO cell lines was performed. Statistical analysis of over 1000 proteins resolved by 2-D PAGE demonstrated that the protein expression profiles of three different CHO cell lines exhibit very few differences in protein expression. Only 11 qualitative changes in protein expression and 26 quantitative changes greater than two-fold were observed. Identification of protein spots by mass spectrometry revealed that many of the observed changes were due to post-translational modifications rather than expression of novel proteins in each cell line. These results suggest that multi-product immunoassays are suitable for monitoring host cell proteins in biopharmaceuticals produced in different CHO cell lines.

IP3 receptor blockade fails to prevent intracellular Ca2+ release by ET-1 and alpha-thrombin.

The effect of inositol 1,4,5-trisphosphate (IP3) receptor blockade on platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), endothelin-1 (ET-1), or alpha-thrombin receptor-mediated intracellular Ca2+ (Ca2+i) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells and myoblasts. Blockade of the IP3 receptor was achieved by microinjection of heparin or monoclonal antibody (MAb) 18A10 into the IP3 type 1 receptor. Heparin completely inhibited Ca2+i release after flash photolysis with caged IP3 and after exposure to PDGF and FGF. In contrast, heparin failed to block Ca2+i release after alpha-thrombin and ET-1. After application of ligand, IP3 levels were five- to sevenfold higher for alpha-thrombin than for ET-1 or PDGF. IP3 levels after PDGF and ET-1 were comparable. Similar to heparin, MAb 18A10 blocked Ca2+i release after PDGF but failed to block Ca2+i release after ET-1 or alpha-thrombin. These data suggest that the mechanisms of Ca2+i release by tyrosine kinase and certain 7-transmembrane receptors may differ. Although both receptor types use the IP3-signaling system, the ET-1 and alpha-thrombin receptors may have a second, alternative mechanism for activating CA2+i release.

PHi and serum regulate AE2-mediated Cl-/HCO3- exchange in CHOP cells of defined transient transfection status

Anion exchanger (AE) protein-mediated anion exchange contributes to regulation of intracellular pH (pHi), Cl- concentration, and volume in vertebrate cells. We have extended the functional characterization of recombinant AE2-mediated Cl-/HCO3- exchange in single Chinese hamster ovary cells stably transfected with the polyoma large T antigen (CHOP cells) of defined transient transfection status using a novel surface marker coexpression vector. Marker expression and detection had minimal effect on the low endogenous Cl-/HCO3- exchange activity of CHOP cells, whereas coexpression of marker with AE2 elevated CHOP cell Cl-/HCO3- exchange activity 16-fold. Between pHi of 7.3 and 7.8, AE2-mediated flux of proton equivalents was activated > 11-fold by increasingly alkaline pHi without reaching saturation. This activation may be secondary to allosteric effects of pHi on AE2, in parallel with the obligatory increase in substrate intracellular HCO3- concentration. Nominal removal of CO2/HCO3- reduced AE2 activity by 90%. Addition of 10% calf serum slowly activated AE2 activity severalfold. This activation was slowly reversed after serum removal. Surface marker coexpression vectors improve both the efficiency and reliability of studies of recombinant protein function for a wide range of single cell assays in many cell types.

Mutagen treatment of single Chinese hamster ovary cells produces colonies mosaic for glucose-6-phosphate dehydrogenase activity.

When populations of single Chinesee hamster ovary (CHO) cells were exposed to the mutagen ethyl methane sulfonate (EMS), allowed to grow into colonies, and stained for glucose-6-phosphate dehydrogenase (G6PD) activity, two types of unstained colonies were observed at a frequency of about on per thousand stained colonies. These negative-staining colonies consisted of (1) colonies uniformly deficient in staining activity (pure); and (2) colonies containing both stained and unstained sectors (mosaic) in various relative sizes and patterns. Unstained cells isolated from mosaic colonies were genetically stable and had significantly reduced or absent G6PD activity. Random cell aggregation or chromosome segregation from tetraploid cells is not a significant cause of the sectoring phenomenon. Also, mosaic colonies are not principally caused by mutation at one of two replicated G6PD genes and their subsequent segregation during division. The simplest explanation for this phenomenon is that EMS induces a mutational change in one of the two DNA strands and DNA replication then produces normal and mutant double-stranded DNAs which segregate into wild-type and G6PD-deficient cell types, producing a mosaic colony.