scholarly journals A rapid cell-based assay for determining poloxamer quality in CHO suspension cell culture

BioTechniques ◽  
2019 ◽  
Vol 67 (3) ◽  
pp. 98-109
Author(s):  
Athmaram Thimmasandra Narayanappa ◽  
Sam Mwilu ◽  
Stacy Holdread ◽  
Kimesha Hammett ◽  
George Bu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
High Performance ◽  
Viable Cell ◽  
Viable Cell Density ◽  
Poor Quality ◽  
Water Soluble ◽  
Suspension Cell Culture ◽  
Adhesive Properties ◽  
Significant Drop

Poloxamers are water-soluble polymers that are widely used in cell culture bioprocessing to protect cells against shearing forces. Use of poor-quality poloxamers may lead to a drastic reduction in cell growth, viabilities and productivities in cell culture-based manufacturing. In order to evaluate poloxamer quality and promote more consistent performance, a rapid cell membrane adhesion to hydrocarbon assay was developed based on the adhesive properties of cell membranes to selective hydrocarbons. The assay can identify a poor-performing poloxamer characterized by significant drop in viable cell density and percent viability. The assay was verified across multiple good and bad poloxamer lots, and the results were in agreement with established cell growth and high-performance liquid chromatography assays.

scholarly journals Mapping the molecular basis for growth related phenotypes in industrial producer CHO cell lines using differential proteomic analysis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Laura Bryan ◽  
Michael Henry ◽  
Ronan M. Kelly ◽  
Christopher C. Frye ◽  
Matthew D. Osborne ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Density ◽  
Cell Lines ◽  
Viable Cell ◽  
Viable Cell Density ◽  
High Peak ◽  
Proteomic Profiling ◽  
Cho Cell ◽  
Extended Culture ◽  
Key Pathways

Abstract Background The ability to achieve high peak viable cell density earlier in CHO cell culture and maintain an extended cell viability throughout the production process is highly desirable to increase recombinant protein yields, reduce host cell impurities for downstream processing and reduce the cost of goods. In this study we implemented label-free LC-MS/MS proteomic profiling of IgG4 producing CHO cell lines throughout the duration of the cell culture to identify differentially expressed (DE) proteins and intracellular pathways associated with the high peak viable cell density (VCD) and extended culture VCD phenotypes. Results We identified key pathways in DNA replication, mitotic cell cycle and evasion of p53 mediated apoptosis in high peak VCD clonally derived cell lines (CDCLs). ER to Golgi vesicle mediated transport was found to be highly expressed in extended culture VCD CDCLs while networks involving endocytosis and oxidative stress response were significantly downregulated. Conclusion This investigation highlights key pathways for targeted engineering to generate desirable CHO cell phenotypes for biotherapeutic production.

scholarly journals Screening Naturally Occurring Phenolic Antioxidants for Their Suitability as Additives to CHO Cell Culture Media Used to Produce Monoclonal Antibodies

Antioxidants ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 159 ◽  
Author(s):  
Luis Toronjo-Urquiza ◽  
David James ◽  
Tibor Nagy ◽  
Robert Falconer
Keyword(s):  
Cell Culture ◽  
Cell Density ◽  
Culture Media ◽  
Viable Cell ◽  
Viable Cell Density ◽  
Specific Productivity ◽  
Cho Cell ◽  
Cell Culture Media ◽  
Igg Synthesis ◽  
Cho Cell Culture

This study identified several antioxidants that could be used in Chinese hamster ovary (CHO)cell culture media and benefit monoclonal antibody production. The flavan-3-ols, catechin, epicatechin, epigallocatechin gallate and gallocatechin gallate all had no detrimental effect on cell viability at the concentrations tested, and they reduced the final viable cell count with a resulting rise in the cell specific productivity. The flavone, luteolin behave similarly to the flavan-3-ols. Resveratrol at 50 μM concentration resulted in the most pronounced reduction in viable cell density with minimal decrease in IgG synthesis and the largest increase in cell specific productivity. Low concentrations of α-tocopherol (35 μM) reduced viable cell density and raised cell specific productivity, but at higher concentration it had little additional effect. As high concentrations of α-tocopherol are not toxic to CHO cells, its addition as an anti-oxidant has great potential. Kaempferol up to 50 μM, curcumin up to 20 μM and piceid up to 100 μM showed little effect on growth or IgG synthesis and could be useful as antioxidants. Caffeic acid phenethyl ester was toxic to CHO cell and of no interest. Seven of the phenolic compounds tested are potential cell cycle inhibitors as well as having intrinsic antioxidant properties.

scholarly journals High-Throughput Raman Spectroscopy Combined with Innovate Data Analysis Workflow to Enhance Biopharmaceutical Process Development

Processes ◽  
2020 ◽  
Vol 8 (9) ◽  
pp. 1179
Author(s):  
Stephen Goldrick ◽  
Alexandra Umprecht ◽  
Alison Tang ◽  
Roman Zakrzewski ◽  
Matthew Cheeks ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Raman Spectroscopy ◽  
Data Analysis ◽  
High Throughput ◽  
High Performance ◽  
Process Development ◽  
Viable Cell ◽  
Viable Cell Density ◽  
Exchange Chromatography ◽  
Analysis Workflow

Raman spectroscopy has the potential to revolutionise many aspects of biopharmaceutical process development. The widespread adoption of this promising technology has been hindered by the high cost associated with individual probes and the challenge of measuring low sample volumes. To address these issues, this paper investigates the potential of an emerging new high-throughput (HT) Raman spectroscopy microscope combined with a novel data analysis workflow to replace off-line analytics for upstream and downstream operations. On the upstream front, the case study involved the at-line monitoring of an HT micro-bioreactor system cultivating two mammalian cell cultures expressing two different therapeutic proteins. The spectra generated were analysed using a partial least squares (PLS) model. This enabled the successful prediction of the glucose, lactate, antibody, and viable cell density concentrations directly from the Raman spectra without reliance on multiple off-line analytical devices and using only a single low-volume sample (50–300 μL). However, upon the subsequent investigation of these models, only the glucose and lactate models appeared to be robust based upon their model coefficients containing the expected Raman vibrational signatures. On the downstream front, the HT Raman device was incorporated into the development of a cation exchange chromatography step for an Fc-fusion protein to compare different elution conditions. PLS models were derived from the spectra and were found to predict accurately monomer purity and concentration. The low molecular weight (LMW) and high molecular weight (HMW) species concentrations were found to be too low to be predicted accurately by the Raman device. However, the method enabled the classification of samples based on protein concentration and monomer purity, allowing a prioritisation and reduction in samples analysed using A280 UV absorbance and high-performance liquid chromatography (HPLC). The flexibility and highly configurable nature of this HT Raman spectroscopy microscope makes it an ideal tool for bioprocess research and development, and is a cost-effective solution based on its ability to support a large range of unit operations in both upstream and downstream process operations.

scholarly journals Kultur suspensi sel tanaman gajah beranak (Goniothalamus tapis Miq) terhadap kandungan zat goniotalamin

Jurnal Agro ◽  
2022 ◽  
Vol 8 (2) ◽  
pp. 247-261
Author(s):  
Imam Mahadi ◽  
Sri Wulandari ◽  
Wan Safii ◽  
Irda Sayuti
Keyword(s):  
Cell Culture ◽  
Callus Culture ◽  
High Performance ◽  
Callus Formation ◽  
Cell Suspension Cultures ◽  
Suspension Cell ◽  
Stem Explants ◽  
Suspension Cell Culture ◽  
Randomized Design ◽  
Quantitative Results

Zat goniotalamin pada tanaman gajah beranak (Goniothalamus tapis) merupakan obat alternatif penyembuhan kanker. Penelitian bertujuan untuk mendapatkan zat goniotalamin melalui kultur kalus dan kultur suspensi sel. Metode penelitian eksperimen Rancangan Acak Lengkap (RAL) dengan kombinasi 2,4-D (1-10 mgL-1) dan BAP (0,5-2 mgL-1) menggunakan eksplan batang muda, terdiri dari 17 perlakuan dan 3 kali ulangan. Analisis data menggunakan Analysis of Variances dan uji lanjut Duncan Multiple Range Test (DMRT) taraf 5%. Hasil menunjukkan bahwa kultur kalus G. tapis pada media 5,0 mg L-1 2,4-D + 1 mg L-1 BAP adalah yang terbaik dengan waktu muncul kalus 28,33 hari dan persentase pembentukan kalus 100%. Kalus untuk kultur suspensi sel bertekstur remah dan berwarna kuning kehijauan. Kultur suspensi sel menghasilkan pertumbuhan sel yang cepat, tidak lembek berair dan mudah dipisahkan. Hasil kualitatif Kromatografi Lapis Tipis kultur suspensi sel sangat jelas, bersih dan terdapat potensi kandungan zat goniotalamin pada perlakuan 2,4-D 5 mg L-1 + BAP 0,5 mg L-1, 2,4-D 5 mg L-1 + BAP 1 mg L-1, 2,4-D 5 mg L-1 + BAP 2 mg L-1, 2,4-D 10 mg L-1 + BAP 0,5 mg L-1 dan 2,4-D 10 mg L-1 + BAP 1 mg L-1. Hasil kuantitatif zat goniotalamin dengan Kromatografi Cair Prestasi Tinggi terdapat pada perlakuan 2,4-D 5,0 mgL-1 + BAP 1 mg L-1 yaitu 9,57 mg g-1.The goniothalamine compound on Goniothalamus tapis is an alternative cancer medicine. This study aimed to obtain gonotalamin through callus culture and suspension cell culture. The experiment research method was Completely Randomized Design (CRD) with a combination of 2.4-D (1-10 mg L-1) and BAP (0.5-2 mg L-1) using young stem explants consisting of 17 treatments with 3 replications. Data analysis used ANOVA and DMRT at 5%. The results showed that G. tapis callus culture on 5.0 mg L-1 2.4-D + 1 mg L-1 BAP was the best treatment medium with callus emergence time of 28.33 days and percentage of callus formation 100%. The callus used for suspension cell culture was friable and greenish-yellow in color. Suspension cell culture resulted in rapid cell growth, was not fleshy, and easily separated. The  quality test by Thin Layer Chromatography (TLC) from suspension cell culture resulted very clear, clean, and potential content of goniothalamin found in treatments 2.4-D 5.0 mg L-1 + BAP 0.5 mg L-1, 2.4-D 5.0 mg L-1 + BAP 1 mg L-1, 2.4-D 5.0 mg L-1 + BAP 2 mg L-1, 2.4-D 10 mg L-1 + BAP 0.5 mg-1 and 2.4-D 10 mg-1 + BAP 1 mg-1. The quantitative results of the best goniotalamine compounds in cell suspension cultures using High-Performance Liquid Chromatography (HPLC) on medium 2,4-D 5.0 mgL-1 + BAP 1 mg L-1 ie 9.57 g-1.

scholarly journals Adhesive Droplets of Glowworm Snares (Keroplatidae: Arachnocampa spp.) Are a Complex Mix of Organic Compounds

2021 ◽  
Vol 7 ◽  
Author(s):  
Jonas O. Wolff ◽  
Janek von Byern ◽  
Dakota Piorkowski ◽  
Jian Fang ◽  
Xungai Wang ◽  
...  
Keyword(s):  
New Zealand ◽  
Chemical Composition ◽  
Organic Compounds ◽  
High Performance ◽  
Prey Capture ◽  
Polar Compounds ◽  
Water Soluble ◽  
High Humidity ◽  
Adhesive Properties ◽  
Spider Webs

Adhesive snares built from silks are fascinating adaptations that have rarely evolved outside spiders. Glowworms (Arachnocampa spp.) are an iconic part of the fauna of Australia and New Zealand that combine the construction of a sticky snare with a bioluminescent lure. Recently, the structure and biomechanical properties of glowworm silk have been studied in detail, but the chemical composition of its adhesive coating, and how it varies between species of Arachnocampa remained unclear, limiting an understanding of the glue function. Here, we studied the chemical composition of the water-soluble fraction of the adhesive droplets from the snares in cave and epigaeic populations of three species of Arachnocampa from mainland Australia, Tasmania, and New Zealand, using a combination of nuclear magnetic resonance and mass spectrometry. We found that glowworm glues comprise a large variety of small organic compounds, with organic acids, amino acids, amino acid derivates, alcohols, urea, and urea derivates being the major fraction, supplemented by small amounts of sugars, fatty acids, and other organic compounds. While there was a general overlap in the compounds detected in the adhesives of all tested Arachnocampa species and populations, the relative amounts differed considerably. We expect that these differences are a product of diet rather than an adaptive response to different environments, but experiments are needed for clarification. The high amount of polar substances and compounds that are hygroscopic at high humidity explains the adhesive properties of the viscous solution and its stability in damp environments. These results contribute to our understanding of the unique prey capture strategy of glowworms. Further, the comparison with convergent spider webs highlights the use of small polar compounds as plasticizers of macro-molecular bioadhesives as a general principle. This may inspire the biomimetic design of novel pressure sensitive adhesives with high performance under high humidity conditions.

Platelet-Rich and Platelet-Poor Plasma Might Play Supportive Roles in Cancer Cell Culture: A Replacement for Fetal Bovine Serum?

2021 ◽  
Vol 21 ◽  
Author(s):  
Mehdi Talebi ◽  
Mousa Vatanmakanian ◽  
Ali Mirzaei ◽  
Yaghoub Barfar ◽  
Maryam Hemmatzadeh ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Cancer Cell ◽  
Human Cancer ◽  
High Price ◽  
Dependent Manner ◽  
Platelet Poor Plasma ◽  
Protein Levels ◽  
Healthy Donors ◽  
Regulatory Functions

Background: Platelet-rich (PRP) and Platelet-poor plasma (PPP) are widely used in research and clinical platforms mainly due to their capacities to enhance cell growth. Although short half-life (5 days) and the high price of platelet products pose challenges regarding their usage, they maintain the growth regulatory functions for weeks. Thus, we aimed to assess the supplementary values of these products in human CCRF-CEM cancer cells. Mechanistically, we also checked if the PRP/PPP treatment enhances YKL-40 expression as a known protein regulating cell growth. Methods: The PRP/PPP was prepared from healthy donors using manual stepwise centrifugation and phase separation. The viability of the cells treated with gradient PRP/PPP concentrations (2, 5, 10, and 15%) was measured by the MTT assay. The YKL-40 mRNA and protein levels were assessed using qRT-PCR and western blotting. The data were compared to FBS-treated cells. Result: Our findings revealed that the cells treated by PRP/PPP not only were morphologically comparable to those treated by FBS but also, they showed greater viability at the concentrations of 10 and 15%. Moreover, it was shown that PRP/PPP induce cell culture support, at least in part, via inducing YKL-40 expression at both mRNA and protein levels in a time- and dose-dependent manner. Conclusion: Collectively, by showing cell culture support comparable to FBS, the PRP/PPP might be used as good candidates to supplement the cancer cell culture and overcome concerns regarding the use of FBS as a non-human source in human cancer research.

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