Using cell engineering and omic tools for the improvement of cell culture processes

Significant strides have been made in mammalian cell based biopharmaceutical process and cell line development over the past years. With several established mammalian host cell lines and expression systems, optimization of selection systems to reduce development times and improvement of glycosylation patterns are only some of the advances being made to improve cell culture processes. In this article, the advances pertaining to cell line development and cell engineering strategies are discussed. An overview of the cell engineering strategies to enhance cellular characteristics by genetic manipulation are illustrated, focusing on the use of genomics and proteomics tools and their application in such endeavors. Included in this review are some of the early studies using the ‘omic’ technique to understand cellular mechanisms of product synthesis and secretion, apoptosis, cell proliferation and the influence of the physicochemical environment. The article highlights the significance of integrating genomics and proteomics data with the vast amounts of bioprocess data for improved analysis of the biological pathways involved. Further improvements of the techniques and methodologies used are needed but ultimately, the new cell engineering strategies should provide great insight into the regulatory networks within the cell in a bioprocess environment and how to manipulate them to increase overall productivity.     

Fine structure of cell wall surfaces in the giant-cellular xanthophycean alga <Emphasis Type="Italic">Vaucheria terrestris</Emphasis>

The mechanical strength of cell walls in the tip-growing cells of Vaucheria terrestris is weakened by treatment with proteolytic enzymes. To clarify the morphological characteristics of the components maintaining cell wall strength, the fine structures of the cell walls, with and without protease treatment, were observed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Observations indicated that cellulose microfibrils were arranged in random directions and overlapped each other. Most of the microfibrils observed in the inner surface of the cell wall were embedded in amorphous materials, whereas in the outer surface of the cell wall, microfibrils were partially covered by amorphous materials. The matrix components embedding and covering microfibrils were almost completely removed by protease treatment, revealing layers of naked microfibrils deposited deeply in the cell wall. Topographic data taken from AFM observations provided some additional information that could not be obtained by TEM, including more detailed images of the granular surface textures of the matrix components and the detection of microfibrils in the interior of the cell wall. In addition, quantitative AFM data of local surface heights enabled us to draw three-dimensional renderings and to quantitatively estimate the extent of the exposure of microfibrils by the enzymatic treatment.     

Effects of long-term serial cell passaging on cell spreading,migration, and cell-surface ultrastructures of cultured vascular endothelial cells

The effects of serial cell passaging on cell spreading, migration, and cell-surface ultrastructures have been less investigated directly. This study evaluated the effects of long-term serial cell passaging (totally 35 passages) on cultured human umbilical vein endothelial cells which were pre-stored at −80 °C as usual. Percentage- and spread area-based spreading assays, measurements of fluorescently labeled actin filaments, migration assay, and measurements of cell-surface roughness were performed and quantitatively analyzed by confocal microscopy or atomic force microscopy. We found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments. Recovery from cold storage and effects of cell passaging were potentially responsible for the increases and decreases of the values, respectively. In contrast, the average roughness of cell surfaces (particularly the nucleus-surrounding region) first dropped at early passages and then rose after passage 15, which might be caused by cold storage- and cell passaging-induced endothelial microparticles. Our data will provide important information for understanding serial cell passaging and implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies.     

Circumventing the Crabtree Effect: A method to induce lactate consumption and increase oxidative phosphorylation in cell culture

Most cells grown in glucose-containing medium generate almost all their ATP via glycolysis despite abundant oxygen supply and functional mitochondria, a phenomenon known as the Crabtree effect. By contrast, most cells within the body rely on mitochondrial oxidative phosphorylation (OXPHOS) to generate the bulk of their energy supply. Thus, when utilising the accessibility of cell culture to elucidate fundamental elements of mitochondria in health and disease, it is advantageous to adopt culture conditions under which the cells have greater reliance upon OXPHOS for the supply of their energy needs. Substituting galactose for glucose in the culture medium can provide these conditions, but additional benefit can be gained from alternate in vitro models. Herein we describe culture conditions in which complete autonomous depletion of medium glucose induces a lactate-consuming phase marked by increased MitoTracker Deep Red staining intensity, increased expression of Kreb’s cycle proteins, increased expression of electron transport chain subunits, and increased sensitivity to the OXPHOS inhibitor rotenone. We propose these culture conditions represent an alternate accessible model for the in vitro study of cellular processes and diseases involving the mitochondrion without limitations incurred via the Crabtree effect.     

NucleoCounter—An efficient technique for the determination of cell number and viability in animal cell culture processes

The NucleoCounter is a novel, portable cell counting device based on the principle of fluorescence microscopy. The present work establishes its use with animal cells and checks its reliability, consistency and accuracy in comparison with other cytometric techniques. The main advantages of this technique are its ability to handle a large number of samples with a high degree of precision and its simplicity and specificity in detecting viable cells quantitatively in a heterogeneous culture. The work addresses and overcomes the problems of subjectivity, and some of the inherent sampling errors associated with using the traditional haemocytometer and Trypan Blue exclusion method. NucleoCounter offers reduced intra- and inter-observer variation as well as consistency in repetitive analysis that establishes it as an efficient and highly potential device for at-line monitoring of animal cell processes. Furthermore, since the only manual steps required are sample aspiration and mixing with two reagents, it is feasible that the whole method could be automated and brought on-line for process monitoring and control.     

Proteome analysis of mouse model systems: A tool to model human disease and for the investigation of tissue-specific biology

The molecular dissections of the mechanistic pathways involved in human disease have always relied on the use of model organisms. Among the higher mammalian organisms, the laboratory mouse (Mus musculus) is the most widely used model. A large number of commercially-available, inbred strains are available to the community, including an ever growing collection of transgenic, knock-out, and disease models. Coupled to availability is the fact that animal colonies can be kept under standardized housing condition at most major universities and research institutes, with relative ease and cost efficiency (compared to larger vertebrates). As such, mouse models to study human biology and disease remains extremely attractive. In the current review we will provide an historic overview of the use of mouse models in proteome research with a focus on general tissue and organelle biology, comparative proteomics of human and mouse and the use of mouse models to study cardiac disease.     

小鼠原代呼吸道上皮细胞分离培养的新方法

现有的呼吸道上皮细胞系大多来源于肿瘤组织或成系时与肿瘤细胞融合,其生物学行为与正常呼吸道上皮细胞差异较大.为更准确反映呼吸道疾病条件下该类细胞的生物学效应,本文对小鼠呼吸道上皮细胞分离的新技术和培养方法进行了探索.利用链霉蛋白酶消化法分离获得小鼠呼吸道上皮细胞,利用特殊的完全培养基和Ⅰ型胶原包被的培养皿进行原代培养.镜...     

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution,new validation tools,and implications for the future

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) “Resolution Revolution” made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.     

Trypanosoma cruzi: location of a specific antigen on the surface of bloodstream trypomastigote and culture epimastigote forms.

The nature of surface antigens of culture epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi was investigated by light and electron microscopy using indirect immunofluorescence and peroxidase labeling techniques and antisera against unique, common, and contaminant antigens. A specific antigen, identified by monospecific rabbit antiserum (anti-component 5 antiserum), is the major constituent of the cell surface and flagellar membrane of both the culture epimastigote and bloodstream trypomastigote forms. Antigens of heterologous stercorarian trypanosomes (Trypanosoma rangeli) and of culture medium proteins could not be detected on the cell surface of culture epimastigote forms and bloodstream trypomastigote forms.     

Human blood-derived fibrin releasates: Composition and use for the culture of cell lines and human primary cells

We have evaluated the capacity of two human blood fractions to substitute for FBS as growth medium supplement for human and animal cell cultures. Non-anticoagulated blood from volunteer donors (N = 13) was centrifuged to isolate a supernatant serum (SS) and a platelet-rich fibrin (PRF) clot which was squeezed to extract the releasate (PRFR). Both materials were characterized for the content in PDGF-AB, TGF-β1, VEGF, bFGF, EGF, IGF, total protein, albumin, IgG, IgM IgA, fibrinogen, cholesterol, triglycerides, various chemistry analytes and hemoglobin. Cell growth promoting activity of pooled SS and PRFR at 1, 5, and 10% in growth medium was evaluated over 7 days using human (HEK293, MG-63) and animal (SIRC, 3T3) cell lines and two human primary cultures (gingival fibroblasts and periodontal ligaments). Viable cell count was compared to that in cultures in FBS free-medium and 10% FBS supplement. SS and PRFR at 1-10% stimulated cell growth significantly more than FBS-free medium and in a way similar to 10% FBS in all cultures apart from 3T3. These two human blood-derived fibrin releasates are equally efficient to substitute for FBS as supplement for cell cultures and could be useful for specialized applications in regenerative medicine, dentistry and oral implantology, or cell therapy.     

Novel approach for the prediction of cell densities and viability in standardized translucent cell culture biochips with near infrared spectroscopy

Near infrared spectroscopy is a rapid and nondestructive method for compositional analysis of biological material. The technology is widely used within bioreactors and possesses potential as a standardized method for quality control in miniaturized microfluidic cell culture systems. Here, we established a method for quantification of cell density and viability of adherent HepaRG cells cultured in a translucent, miniaturized cell culture biochip. The newly developed statistical models for interpretation of near infrared spectroscopy from biochips are the basis for a novel method of fast, continuous, and contact‐free analysis of cell viability and real‐time monitoring of cell growth. The technique thus paves the way for a robust and reliable high‐throughput analysis of biochip‐embedded cell cultures.     

N-terminal acylation of the SV40 nuclear localization signal peptide enhances its oligonucleotide binding and membrane translocation efficiencies

Octanoyl and palmitoyl groups were coupled to the N-terminus of an analog of the SV40 nuclear localization signal peptide, SV126-133(Ser128), to study the effect of the fatty acid chain length on the complex formation with a single-stranded antisense oligodeoxynucleotide (ODN) and on the cellular uptake of the complex. The strongest binding affinity was observed for the palmitoylated peptide, indicating the better accessibility of the positively charged lysyl and arginyl side-chains to the phosphate groups due to the turn structures stabilized by the palmitoyl group. On increase of the peptide to ODN molar ratio (rM), gradual unstacking of the bases was observed, the maximal rate being reached at rM=10. At rM>10 restacking of the nucleotide bases was detected and the ODN was completely encapsulated in a liposome-like structure made up of palmitoylated peptides. Cell translocation experiments revealed a highly efficient cell transport of the ODN by palmitoylated SV40 peptide at rM>10.     

A fluidised bed vessel for the culture of immobilised plant cells and its application for the continuous production of fine cell suspensions

With the expansion of immobilised plant cell technology the need has arisen for a suitable vessel in which systems can be efficiently manipulated.Described is a vessel which incorporates many of the features of a fluidised bed, together with some of those from airlift technology to enable immobilised plant cells to be cultured at high biomass concentrations while maintaining a high mass transfer and controlled aeration under continuous flow conditions. In the case described, the vessel has been used for the continuous production of fine plant cell suspensions, although it is easily adaptable for use in cell mediated biotransformation or de novo synthesis studies.Abbreviations 2,4D 2.4 dichlorophenoxyacetic acid     

An investigation of the fine structure, cell surface carbohydrates, and appeal of the diatom Extubocellulus sp. as prey for small flagellates

Summary. The fine structure and surface exopolymers of a coastal planktonic nanodiatom of the sparsely reported genus Extubocellulus were studied respectively by scanning electron microscopy and confocal microscopy in conjunction with fluorescent lectins. Monitoring the suitability of the species as prey food for other protists was also investigated by video microscopy coupled with digital film. Cells are rectangular in girdle view, with a pervalvar axis longer than the apical axis. Valves are almost circular with a diameter of 2.8 to 3.6 μm. The valve face bears randomly distributed areolae (ca. 50 in 10 μm), which may be either open or occluded. Two small raised ocelluli occur at the apices, with a rim devoid of perforations and about 6–7 porelli. Glucose and N-acetyl-glucosamine moieties present on the surface of the live diatom were labelled with fluorescent lectins, and a differential pattern of distribution for both carbohydrates was observed. The potential role of fluorescent lectins as cellular probes of taxonomic value in small diatoms is compared with that of nucleotide and antibody probes. We provide the first illustrative evidence of the presence of Extubocellulus sp. in the cytoplasm of the nanoflagellate Goniomonas amphinema and of the egestion of diatom frustules. Results obtained are discussed in the light of the present knowledge of the role of carbohydrate–protein interactions in phagocytosis of prey by free-living protozoa. Correspondence (present address): M. Martin-Cereceda, Department of Ecology and Evolutionary Biology, Haworth Hall, 1200 Sunnyside Avenue, University of Kansas, Lawrence, KS 66045, U.S.A.     

Cytochalasin D and cationized ferritin as probes for the morphological investigation of blebbing in two human cell lines

Summary The potent fungal metabolite cytochalasin D (CD) and cationized ferritin (CF) are used in combination to test for negative charge distribution on blebs (knobs). Two established human epithelial cell lines, WISH and HeLa, that display blebs in various phases of the cell cycle or under certain culture conditions (37,46) are investigated. CD alone, applied at a low concentration (1.0 μg/ml) and for a short time period (3 min), causes blebs to appear as the prevalent surface feature. These are filled mainly with free ribosomes. Additionally, feltlike mats, presumed to be disorganized, compacted microfilaments, are formed directly beneath the cell membrane. These are especially evident in the cortical cytoplasm below the blebs or bleb clusters. CF (0.345 mg/ml), applied for a 5-min period after CD administration (1.0 μg/ml) for 3 min, appears along the surface of microvilli, at the base of blebs, and in vesicles beneath the bleb clusters. In some cases, microfilaments (6 nm in diameter) are closely related to the vesicles. CF does not preferentially bind to the apical cell membrane of blebs. Above areas of the subplasmalemmal microfilaments, CF membrane binding is apparent, even under circumstances where the filaments are disorganized by cytochalasin treatment. These results seem to show the following: (a) bleb membranes are different from the remainder of the cell and do exhibit a loss of negative charge and (b) surface charge may be dependent on the presence or structural integrity of membrane-related 6-nm microfilaments. The support of this research by a grant from the Baylor College of Dentistry and The Oklahoma College of Osteopathic Medicine and Surgery is gratefully acknowledged. The assistance of Dr. J. H. Martin, Department of Pathology, Baylor University Medical Center, is also greatly appreciated.     

A new criterion for the global stability of simultaneous cell replication and maturation processes

 We analyze a population model of cells that are capable of simultaneous and independent proliferation and maturation. This model is described by a first order partial differential equation with a time delay and a retardation of the maturation variable, both due to cell replication. We provide a general criterion for global stability in such equations. Received: 26 August 1996 / Revised version: 22 March 1997     

Primary culture of germ cells that portray stem cell characteristics and recipient preparation for autologous transplantation in the rhesus monkey

Fertility preservation for prepubertal cancer patients prior to oncologic treatment is an emerging issue, and non‐human primates are considered to constitute suitable models due to the limited availability of human testicular tissues. However, the feasibility of spermatogonial stem cell (SSC) propagation in vitro and autologous testicular germ cell transplantation in vivo requires further exploration in monkeys. Herein, we characterized germ cells in macaque testes at 6 months (M), 18 M and 60 M of age, and effectively isolated the spermatogenic cells (including the spermatogonia) from macaque testes with high purity (over 80%) using combined approaches of STA‐PUT separation, Percoll gradients and differential plating. We also generated recipient monkey testes with ablated endogenous spermatogenesis using the alkylating agent busulfan in six macaques, and successfully mimicked autologous cell transplantation in the testes under ultrasonographic guidance. The use of trypan blue led to successful intratubular injection in 4 of 4 testes. Although SSCs in culture showed no significant propagation, we were able to maintain monkey testicular germ cells with stem cell characteristics for up to 3 weeks. Collectively, these data provided meaningful information for future fertility preservation and SSC studies on both non‐human primates and humans.     

Characterization of a new insect cell line that is derived from the neonate larvae of Papilio xuthus (Lepidoptera: Papilionidae) and its susceptibility to AcNPV

The cell line RIRI-PX1 was established from neonate larval tissues of Papilio xuthus by performing primary cultures in the modified Grace medium that was supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of spindle-shaped and spherical cells which attached themselves to the flask. The population-doubling times (PDTs) at the 50th and 60th passage were 42.5 h and 42.1 h respectively. The average chromosome numbers of RIRI-PX1 cell line from passage 5 to passage 50 ranged from 103 to 199. It was confirmed that RIRI-PX1 cell line was derived from P. xuthus by comparing the mitochondrial cytochrome c oxidase subunit I gene (COI) of RIRI-PX1 cells and P. xuthus eggs. This cell line was susceptible to the Autographa californica nucleopolyhedrovirus (AcNPV) and produced high yield of polyhedral occlusion bodies (43.9 OBs/cell) after 10 days of infection by AcNPV. The virus titer of AcNPV infected RIRI-PX1 cells was 3.25 × 10⁷ TCID₅₀/ml. We concluded that the RIRI-PX1 cell line is established from the neonate larvae tissues successfully and the cells of the cell line are sensitive to AcNPV.