Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression

Maarof, Manira; Anuar, Khairoji Khairul; Seet, Wan Tai; Irfan, Abd Wahab Ahmad; Ng, Min Hwei; Chua, Kien Hui; Yunus, Mohd Heikal Mohd; Aminuddin, B S; Ruszymah, B H I

doi:10.1007/s10561-013-9368-y

Cited by 25 publications

(17 citation statements)

References 16 publications

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“…Cell dissociation enzymes such as Trypsin‐EDTA have been used to detach adherent cells while maintaining high viability, by cleaving cell surface proteins . TrypLE is a recombinant, animal component free replacement for porcine trypsin and was investigated as a treatment for cells in suspension culture immediately prior to cloning.…”

Section: Resultsmentioning

confidence: 99%

Measuring the aggregation of CHO cells prior to single cell cloning allows a more accurate determination of the probability of clonality

Klottrup¹,

Miró-Quesada²,

Flack³

et al. 2017

Biotechnology Progress

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The manufacturing process for biotherapeutics is closely regulated by the Food and Drug Administration (FDA), European Medicines Agency (EMA) and other regulatory agencies worldwide. To ensure consistency of the product of a manufacturing cell line, International Committee on Harmonization guidelines (Q5D, 1997) state that the cell substrate should be derived from a single cell progenitor, i.e., clonal.Cell lines in suspension culture may naturally revert to cell adhesion in the form of doublets, triplets and higher order structures of clustered cells. We can show evidence of a single colony from limiting dilution cloning or in semi-solid media, but we cannot determine the number of cells from which the colony originated. To address this, we have used the ViCELL® XR (Beckman Coulter, High Wycombe, UK) cell viability analyzer to determine the proportion of clusters of two or more cells in a sample of the cell suspension immediately prior to cloning. Here, we show data to define the accuracy of the ViCELL for characterizing a cell suspension and summarize the statistical model combining two or more rounds of cloning to derive the probability of clonality. The resulting statistical model is applied to cloning in semi-solid medium, but could equally be applied to a limiting dilution cloning process. We also describe approaches to reduce cell clusters to generate a cell line with a high probability of clonality from a CHO host lineage. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:593-601, 2018.

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Section: Resultsmentioning

confidence: 99%

Measuring the aggregation of CHO cells prior to single cell cloning allows a more accurate determination of the probability of clonality

Klottrup¹,

Miró-Quesada²,

Flack³

et al. 2017

Biotechnology Progress

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“…The final reagent was TrypLE, a trypsin‐like substitute comprised of a proprietary recombinant protease and EDTA. TrypLE is a comparable, but gentler alternative to trypsin that is becoming widely used as it maintains surface protein expression .…”

Section: Resultsmentioning

confidence: 99%

Novel flow cytometric analysis of the blood–brain barrier

2015

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The blood–brain barrier (BBB) is primarily comprised of brain microvascular endothelial cells (BMVEC) and astrocytes and serves as a physical and chemical barrier that separates the periphery from the brain. We describe a flow cytometric method using our in vitro model of the human BBB to characterize BMVEC surface junctional proteins critical for maintenance of barrier function, cell viability, and leukocyte adhesion. For this methodology, BMVEC are cocultured with astrocytes in a transwell tissue culture insert to establish the barrier, after which time the BBB are treated with specific agents, and the BMVEC collected for flow cytometric analyses. We use a standard and optimized method to recover the BMVEC from the coculture model that maintains junctional protein expression and cell viability. A novel leukocyte adhesion assay enables a quantitative analysis of peripheral blood mononuclear cell (PBMC) interactions with the BMVEC and can be used to assess the adhesion of many cell types to the BBB. Furthermore, this method enables the concomitant analysis of a large number of adhesion molecules and tight junction proteins on both the BMVEC and adherent PBMC under homeostatic and pathologic conditions. Flow cytometry is an extremely powerful tool, and this technique can also be applied to assess variables not performed in this study, including cell cycle progression, and calcium flux.

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“…Skin tissue was processed according to Manira et al (2014) with a slight modification [22]. In brief, the skin was cleaned before cut into small pieces.…”

Section: Isolation Of Primary Human Keratinocytesmentioning

confidence: 99%

Effect of Kelulut Honey on the Cellular Dynamics of TGFβ-Induced Epithelial to Mesenchymal Transition in Primary Human Keratinocytes

Nordin

Chowdhury

Saim

et al. 2020

IJERPH

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Over-induction of epithelial to mesenchymal transition (EMT) by tumor growth factor beta (TGFβ) in keratinocytes is a key feature in keloid scar. The present work seeks to investigate the effect of Kelulut honey (KH) on TGFβ-induced EMT in human primary keratinocytes. Image analysis of the real time observation of TGFβ-induced keratinocytes revealed a faster wound closure and individual migration velocity compared to the untreated control. TGFβ-induced keratinocytes also have reduced circularity and display a classic EMT protein expression. Treatment of 0.0015% (v/v) KH reverses these effects. In untreated keratinocytes, KH resulted in slower initial wound closure and individual migration velocity, which sped up later on, resulting in greater wound closure at the final time point. KH treatment also led to greater directional migration compared to the control. KH treatment caused reduced circularity in keratinocytes but displayed a partial EMT protein expression. Taken together, the findings suggest the therapeutic potential of KH in preventing keloid scar by attenuating TGFβ-induced EMT.

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Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression

Cited by 25 publications

References 16 publications

Measuring the aggregation of CHO cells prior to single cell cloning allows a more accurate determination of the probability of clonality

Measuring the aggregation of CHO cells prior to single cell cloning allows a more accurate determination of the probability of clonality

Novel flow cytometric analysis of the blood–brain barrier

Effect of Kelulut Honey on the Cellular Dynamics of TGFβ-Induced Epithelial to Mesenchymal Transition in Primary Human Keratinocytes

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