Validation of three viable-cell counting methods: Manual, semi-automated, and automated

Cadena-Herrera, Daniela; Lara, Joshua E. Esparza-De; Ramírez-Ibañez, Nancy D.; López‐Morales, Carlos A.; Pérez, Nestor O.; Flores-Ortiz, Luis F.; Medina‐Rivero, Emilio

doi:10.1016/j.btre.2015.04.004

Cited by 159 publications

(124 citation statements)

References 11 publications

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“…Table contains the expected and observed growth rates for each of the light exposure times tested. Given the variability in cell counting methodology, and that the calculated growth rates were extrapolated, the growth rates for the light exposed medium were consistent with the theoretical values, confirming the predictability of the equation and the hypothesis of zero‐order kinetics for the chemical of interest. Additionally, the control samples from this experiment had growth consistent with medium stored under standard conditions (data not shown) and therefore any changes in cell culture performance can be attributed to light exposure.…”

Section: Resultssupporting

confidence: 67%

Tryptophan oxidation catabolite,N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance

McElearney

Ali

Gilbert

et al. 2015

Biotechnol Progress

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Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance.

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Section: Resultssupporting

confidence: 67%

Tryptophan oxidation catabolite,N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance

McElearney

Ali

Gilbert

et al. 2015

Biotechnol Progress

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“…The number of cells was routinely checked with the trypan blue exclusion assay (Cadena-Herrera et al 2015). Cells were incubated in poly-L-lysine precoated 12-well culture plates at a density of 1 3 10 6 /mL at 37C with 95% humidity and 5% CO2.…”

Section: Primary Retinal Cell Culturementioning

confidence: 99%

Protective Effect of Astaxanthin on Primary Retinal Cells of the GerbilPsammomys ObesusCultured in DiabeticMilieu

Baccouche

Mbarek

Dellaa

et al. 2016

Journal of Food Biochemistry

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Astaxanthin is a major marine carotenoid with powerful antioxidant and neuroprotective properties. The in vitro protective effect of astaxanthin in adult retinal cells of the type-2 diabetic model Psammomys obesus in hyperglycemic conditions was investigated. Primary retinal cells were cultured in normal (5 mM) or high concentrations of glucose (25 and 40 mM) for 5 days and treated with 1-20 μM astaxanthin for the last 48 h of culturing. Mitochondrial dehydrogenase activity and cell viability were assessed using MTT test and trypan blue exclusion dye. Retinal cells were characterized by immunohistochemistry. The results showed that mitochondrial function increased significantly (P<0.05) with 5-20 μM astaxanthin in 25-40 mM glucose. At 10 μM astaxanthin, cell viability recovered to 78.51±5.81% and 54.37±9.64% of control in 25 and 40 mM, respectively. Neurons and glial cells expression were enhanced in elevated glucose conditions. These finding suggest that astaxanthin exerted neuroprotection against high glucose induced- retinal damage. Practical Applications: The xanthophyll astaxanthin is found in many marine food sources such as shrimp. The cytoprotective and antioxidant capacity of astaxanthin in retinal disease has been pointed out and presented as a promising therapeutic strategy. The present study shows that astaxanthin can interfere with hyperglycemia-induced retinal cell death and that primary retinal culture of Psammomys obesus may represent an effective system to explore astaxanthin antioxidant mechanisms in diabetic retinopathy

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“…All eight mAb C cryopreserved cell lines showed no statistically significant difference in growth rates ( P > 0.44; Supporting Information Table S1) between the fourth passage after thaw and the passage used to source the first production culture assay (Experiment 1). Although the Student's t ‐test showed a statistically significant difference in viability ( P < 0.05; Supporting Information Table S1) between these two non‐consecutive seed train passages for the mAb C cell lines (spaced ∼1 month apart), the actual difference was small (∼1%) and within the variability associated with culture viability measurements by the Vi‐Cell XR instrument …”

Section: Resultsmentioning

confidence: 96%

“…Although the Student's t-test showed a statistically significant difference in viability (P < 0.05; Supporting Information Table S1) between these two non-consecutive seed train passages for the mAb C cell lines (spaced 1 month apart), the actual difference was small (1%) and within the variability associated with culture viability measurements by the Vi-Cell XR instrument. 19 By the start of the first production culture assay (Experiment 1) 1 month after recovery from thaw, the seed train viabilities were similarly high (>96%) for all 24 cryopreserved cell lines and their 24 non-cryopreserved counterparts ( Figure 3A). The growth rates for the 48 seed train cultures used to source Experiment 1 were also similar between the cryopreserved cell lines and their non-cryopreserved counterparts ( Figure 3B).…”

Section: Seed Train Performancementioning

confidence: 93%

Short‐ and long‐term effects on mAb‐producing CHO cell lines after cryopreservation

Subramanian¹,

Aulakh²,

Grewal³

et al. 2018

Biotechnology Progress

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Cryopreservation provides the foundation for research, development, and manufacturing operations in the CHO-based biopharmaceutical industry. Despite its criticality, studies are lacking that explicitly demonstrate that the routine cell banking process and the potential stress and damage during cryopreservation and recovery from thaw have no lasting detrimental effects on CHO cells. Statistics are also scarce on the decline of cell-specific productivity (Q ) over time for recombinant CHO cells developed using the glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection system. To address these gaps, we evaluated the impact of freeze-thaw on 24 recombinant CHO cell lines (generated by the GS/MSX selection system) using a series of production culture assays. Across the panel of cell lines expressing one of three monoclonal antibodies (mAbs), freeze-thaw did not result in any significant impact beyond the initial post-thaw passages. Production cultures sourced from cryopreserved cells and their non-cryopreserved counterparts yielded similar performance (growth, viability, and productivity), product quality (size, charge, and glycosylation distributions), and flow cytometric profiles (intracellular mAb expression). However, many production cultures yielded lower Q at increased cell age: 17 of the 24 cell lines displayed ≥20% Q decline after ∼2-3 months of passaging, irrespective of whether the cells were previously cryopreserved. The frequency of Q decline underscores the continued need for understanding the underlying mechanisms and for careful clone selection. Because our experiments were designed to decouple the effects of cryopreservation from those of cell age, we could conclusively rule out freeze-thaw as a cause for Q decline. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:463-477, 2018.

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Validation of three viable-cell counting methods: Manual, semi-automated, and automated

Abstract: Graphical abstract

Cited by 159 publications

References 11 publications

Tryptophan oxidation catabolite,N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance

Tryptophan oxidation catabolite,N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance

Protective Effect of Astaxanthin on Primary Retinal Cells of the GerbilPsammomys ObesusCultured in DiabeticMilieu

Short‐ and long‐term effects on mAb‐producing CHO cell lines after cryopreservation

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