scholarly journals Growth and eGFP Production of CHO-K1 Suspension Cells Cultivated From Single Cell to Laboratory Scale

2021 ◽  
Vol 9 ◽  
Author(s):  
Julian Schmitz ◽  
Oliver Hertel ◽  
Boris Yermakov ◽  
Thomas Noll ◽  
Alexander Grünberger
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Mammalian Cell Culture ◽  
Laboratory Scale ◽  
Cellular Heterogeneity ◽  
Suspension Cells ◽  
Stirred Tank Bioreactor ◽  
Mammalian Cell Lines

Scaling down bioproduction processes has become a major driving force for more accelerated and efficient process development over the last decades. Especially expensive and time-consuming processes like the production of biopharmaceuticals with mammalian cell lines benefit clearly from miniaturization, due to higher parallelization and increased insights while at the same time decreasing experimental time and costs. Lately, novel microfluidic methods have been developed, especially microfluidic single-cell cultivation (MSCC) devices have been proved to be valuable to miniaturize the cultivation of mammalian cells. So far, growth characteristics of microfluidic cultivated cell lines were not systematically compared to larger cultivation scales; however, validation of a miniaturization tool against initial cultivation scales is mandatory to prove its applicability for bioprocess development. Here, we systematically investigate growth, morphology, and eGFP production of CHO-K1 cells in different cultivation scales ranging from a microfluidic chip (230 nl) to a shake flask (125 ml) and laboratory-scale stirred tank bioreactor (2.0 L). Our study shows a high comparability regarding specific growth rates, cellular diameters, and eGFP production, which proves the feasibility of MSCC as a miniaturized cultivation tool for mammalian cell culture. In addition, we demonstrate that MSCC provides insights into cellular heterogeneity and single-cell dynamics concerning growth and production behavior which, when occurring in bioproduction processes, might severely affect process robustness.

scholarly journals Growth and eGFP-production of CHO-K1 suspension cells cultivated from single-cell to lab-scale

2021 ◽  
Author(s):  
Julian Schmitz ◽  
Oliver Hertel ◽  
Boris Yermakov ◽  
Thomas Noll ◽  
Alexander Gruenberger
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Process Development ◽  
Mammalian Cell Culture ◽  
Cellular Heterogeneity ◽  
Suspension Cells ◽  
Cell Dynamics ◽  
Mammalian Cell Lines

Scaling down bioproduction processes became a major driving force for more accelerated and efficient process development over the last decades. Especially expensive and time-consuming processes like the production of biopharmaceuticals with mammalian cell lines benefit clearly from miniaturisation, due to higher parallelisation and increased insights while at the same time decreasing experimental time and costs. Lately, novel microfluidic methods have been developed, especially microfluidic single-cell cultivation (MSCC) devices proofed to be valuable to miniaturise the cultivation of mammalian cells. So far growth characteristics of microfluidic cultivated cell lines were not systematically compared to larger cultivation scales, however validation of a miniaturisation tool against initial cultivation scales is mandatory to proof its applicability for bioprocess development. Here, we systematically investigate growth, morphology, and eGFP-production of CHO-K1 cells in different cultivation scales including microfluidic chip (230 nL), shake flask (60 mL), and lab-scale bioreactor (1.5 L). Our study shows a high comparability regarding growth rates, cellular diameters, and eGFP production which proofs the feasibility of MSCC as miniaturised cultivation tool for mammalian cell culture. In addition, we demonstrate that MSCC allows insights into cellular heterogeneity and single-cell dynamics concerning growth and production behaviour which, when occurring in bioproduction processes, might severely affect process robustness. Eventually, by providing insights into cellular heterogeneity, MSCC has the potential to be applied as a novel and powerful tool in the context of cell line development and bioprocesses implementation.

NanoLiterBioReactor: Monitoring of Long-Term Mammalian Cell Physiology at Nanofabricated Scale

2004 ◽  
Vol 820 ◽  
Author(s):  
Ales Prokop ◽  
Zdenka Prokop ◽  
David Schaffer ◽  
Eugene Kozlov ◽  
John Wikswo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Cell Counting ◽  
Cell Physiology ◽  
Mammalian Cell Lines ◽  
Cell Counts ◽  
Multiple Cell

AbstractThere is a need for microminiaturized cell-culture environments, i.e., NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. Reduced NBR volumes would not only shorten the time required for diffusive mixing, for achieving thermal equilibrium, and for cells to grow to confluence, but also simplify accurate cell counting, minimize required volumes of expensive analytical pharmaceuticals or toxins, and allow for thousands of culture chambers on a single instrumented chip. These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. The challenge, beyond that of optimizing the NBR physically, is to detect cellular response, provide appropriate control signals, and, eventually, facilitate closed-loop adjustments of the environment--e.g., to control temperature, pH, ionic concentration, etc., to maintain homeostasis, or to apply drugs or toxins followed by the adaptive administration of a selective toxin antidote. To characterize in a nonspecific manner the metabolic activity of cells, the biosensor elements of the NBR might include planar pH, dissolved oxygen, and redox potential sensors, or even an isothermal picocalorimeter (pC) to monitor thermodynamic response. Equipped with such sensors, the NBR could be used to perform short- and long-term cultivation of several mammalian cell lines in a perfused system, and to monitor their response to analytes in a massively parallel format. This approach will enable automated, parallel, and multiphasic monitoring of multiple cell lines for drug and toxicology screening. An added bonus is the possibility of studying cell populations with low cell counts whose constituents are completely detached from typical tissue environment, or populations in controlled physical and chemical gradients.

scholarly journals Mammalian Cell Culture Clarification: A Case Study Using Chimeric Anti-CEA Monoclonal Antibodies

2011 ◽  
Vol 12 (4) ◽  
Author(s):  
Mohamed Ali Abol Hassan ◽  
Abdul Wahab Mohammad ◽  
And Badarulhisam Abdul Rahman
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibodies ◽  
Host Cell ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Host Cell Proteins ◽  
Depth Filtration

The extracellular expression of monoclonal antibodies (mAbs) in mammalian cell culture provides both opportunities and restrictions for the design of robust harvest and clarification operations. With advances in cell culture media and cell lines, it is now possible to achieve high titers of over 5 g/l for mAbs. However, Mammalian cells are sensitive to breakage due to shear stress that can result in release of proteases and other host cell proteins (HCPs) which eventually affects product stability and purity. There is larger number of mAbs undergoing clinical development and it has placed significant importance on platform technologies of process development. Generally, Centrifugation and microfiltration are the primary harvest techniques used in the industry and depth filtration is also used as a step operation on clarification. This study compares the unit operations; centrifugation, microfiltration and depth filtration for maximum recovery of monoclonal antibodies. The results have shown that the depth filtration as more suitable operation for mammalian cell culture clarification since it gives 96% recovery of mAbs in comparison to centrifugation and microfiltration. ABSTRAK: Pengungkapan luar sel dari antibodi monoklon (monoclonal antibodies ((mAbs) dalam kultur sel mamalia memberi ruang dan batasan terhadap reka bentuk penuaian yang cekap dan penerangan operasi. Dengan kemajuan dalam media sel kultur dan cell lines (produk yang berupa sel kekal yang digunakan untuk tujuan kajian biologi), kini adalah berkemungkinan untuk memperolehi titer tinggi melebihi 5g/l untuk mAbs [2]. Walaupun begitu, sel mamalia sensitif terhadap retakan disebabkan tegasan ricih yang menyebabkan pengeluaran protease dan hos sel protein yang lain, (host cell proteins (HCPs)) akhirnya mempengaruhi kestabilan dan keaslian produk. Terdapat mAbs dalam jumlah besar yang masih menjalani pembangunan klinikal dan sesungguhnya ini penting sebagai satu landasan teknologi dalam proses pembangunan. Umumnya pengemparan dan mikropenurasan merupakan teknik asas tuaian dalam industri dan penurasan dalam juga digunakan sebagai satu pengendalian langkah dalam penjelasannya. Kajian ini membandingkan operasi unit: pengemparan, mikropenurasan dan penurasan dalam untuk perolehan antibodi monoklon yang maksima. Keputusan menunjukkan penurasan dalam adalah operasi yang lebih sesuai untuk penjelasan kultur sel mamalia kerana ia memberikan perolehan 96 % mAbs berbandingkan dengan cara pengemparan dan mikropenurasan.

scholarly journals Genetic screens in isogenic mammalian cell lines without single cell cloning

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Peter C. DeWeirdt ◽  
Annabel K. Sangree ◽  
Ruth E. Hanna ◽  
Kendall R. Sanson ◽  
Mudra Hegde ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Genetic Screens ◽  
Cell Cloning ◽  
Mammalian Cell Lines ◽  
Single Cell Cloning

NanoLiterBioReactor: Monitoring of Long-Term Mammalian Cell Physiology at Nanofabricated Scale

2004 ◽  
Vol 823 ◽  
Author(s):  
Ales Prokop ◽  
Zdenka Prokop ◽  
David Schaffer ◽  
Eugene Kozlov ◽  
John Wikswo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Cell Counting ◽  
Cell Physiology ◽  
Mammalian Cell Lines ◽  
Cell Counts ◽  
Multiple Cell

AbstractThere is a need for microminiaturized cell-culture environments, i.e., NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. Reduced NBR volumes would not only shorten the time required for diffusive mixing, for achieving thermal equilibrium, and for cells to grow to confluence, but also simplify accurate cell counting, minimize required volumes of expensive analytical pharmaceuticals or toxins, and allow for thousands of culture chambers on a single instrumented chip. These devices would enable the development of a new class of miniature, automated cell- based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. The challenge, beyond that of optimizing the NBR physically, is to detect cellular response, provide appropriate control signals, and, eventually, facilitate closed-loop adjustments of the environment–e.g., to control temperature, pH, ionic concentration, etc., to maintain homeostasis, or to apply drugs or toxins followed by the adaptive administration of a selective toxin antidote. To characterize in a nonspecific manner the metabolic activity of cells, the biosensor elements of the NBR might include planar pH, dissolved oxygen, and redox potential sensors, or even an isothermal picocalorimeter (pC) to monitor thermodynamic response. Equipped with such sensors, the NBR could be used to perform short- and long-term cultivation of several mammalian cell lines in a perfused system, and to monitor their response to analytes in a massively parallel format. This approach will enable automated, parallel, and multiphasic monitoring of multiple cell lines for drug and toxicology screening. An added bonus is the possibility of studying cell populations with low cell counts whose constituents are completely detached from typical tissue environment, or populations in controlled physical and chemical gradients.

scholarly journals Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9

2019 ◽  
Author(s):  
Christopher J Giuliano ◽  
Ann Lin ◽  
Jason Sheltzer
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Protein Function ◽  
Mammalian Cells ◽  
Gene Loss ◽  
Loss Of Function ◽  
Guide Rna ◽  
Mammalian Cell Lines ◽  
Clonal Cell Lines ◽  
Rna Design

CRISPR/Cas9 technology enables the rapid and efficient generation of total loss-of- function mutations in a targeted gene in mammalian cells. A single cell that harbors those mutations can be used to establish a new cell line, thereby creating a CRISPR-induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of various biological reagents. However, the successful derivation of knockout clones may be technically challenging and can be complicated by multiple factors, including incomplete target ablation and inter-clonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation and identification of true knockout clones and that are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells.

scholarly journals Physiological phenotyping of mammalian cell lines by enzymatic activity fingerprinting of key carbohydrate metabolic enzymes: a pilot and feasibility study

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Julian Geiger ◽  
Rebecca Doelker ◽  
Sofia Salö ◽  
Thomas Roitsch ◽  
Louise T. Dalgaard
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
High Throughput ◽  
Mammalian Cells ◽  
Analysis Tool ◽  
Mammalian Cell Lines ◽  
Plant Samples ◽  
Experimental Parameters ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Selection Of

Abstract Objective Enzymatic fingerprinting of key enzymes of glucose metabolism is a valuable analysis tool in cell physiological phenotyping of plant samples. Yet, a similar approach for mammalian cell line samples is missing. In this study, we applied semi-high throughput enzyme activity assays that were originally designed for plant samples and tested their feasibility in extracts of six frequently used mammalian cell lines (Caco2, HaCaT, C2C12, HEK293, HepG2 and INS-1E). Results Enzyme activities for aldolase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, phosphofructokinase could be detected in samples of one or more mammalian cell lines. We characterized effects of sample dilution, assay temperature and repeated freeze–thaw cycles causing potential biases. After careful selection of experimental parameters, the presented semi-high throughput methods could be established as useful tool for physiological phenotyping of cultured mammalian cells.

scholarly journals Mutations in the Neuraminidase-Like Protein of Bat Influenza H18N11 Virus Enhance Virus Replication in Mammalian Cells, Mice, and Ferrets

2019 ◽  
Vol 94 (5) ◽  
Author(s):  
Gongxun Zhong ◽  
Shufang Fan ◽  
Masato Hatta ◽  
Sumiho Nakatsu ◽  
Kevin B. Walters ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Mammalian Cell Lines ◽  
Organ Tropism ◽  
Darby Canine Kidney ◽  
Canine Kidney

ABSTRACT To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets. IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals.

scholarly journals In Vitro and In Vivo Toxicity Evaluation of Natural Products with Potential Applications as Biopesticides

Toxins ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 805
Author(s):  
Felicia Sangermano ◽  
Marco Masi ◽  
Amrish Kumar ◽  
Ravindra Peravali ◽  
Angela Tuzi ◽  
...  
Keyword(s):  
Natural Products ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Toxic Effects ◽  
Mammalian Cell Lines ◽  
Dittrichia Viscosa ◽  
Potential Applications

The use of natural products in agriculture as pesticides has been strongly advocated. However, it is necessary to assess their toxicity to ensure their safe use. In the present study, mammalian cell lines and fish models of the zebrafish (Danio rerio) and medaka (Oryzias latipes) have been used to investigate the toxic effects of ten natural products which have potential applications as biopesticides. The fungal metabolites cavoxin, epi-epoformin, papyracillic acid, seiridin and sphaeropsidone, together with the plant compounds inuloxins A and C and ungeremine, showed no toxic effects in mammalian cells and zebrafish embryos. Conversely, cyclopaldic and α-costic acids, produced by Seiridium cupressi and Dittrichia viscosa, respectively, caused significant mortality in zebrafish and medaka embryos as a result of yolk coagulation. However, both compounds showed little effect in zebrafish or mammalian cell lines in culture, thus highlighting the importance of the fish embryotoxicity test in the assessment of environmental impact. Given the embryotoxicity of α-costic acid and cyclopaldic acid, their use as biopesticides is not recommended. Further ecotoxicological studies are needed to evaluate the potential applications of the other compounds.

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