Comparison of potency assays using different read-out systems and their suitability for quality control

Zumpe, C.; Bachmann, Christiane; Metzger, Armin U.; Wiedemann, Nils

doi:10.1016/j.jim.2010.06.019

Cited by 8 publications

(3 citation statements)

References 33 publications

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“…The ATP bioluminescence signal detection system is the most sensitive, non-radioactive readout available for cell viability and function [ 42 ]. Previous studies had demonstrated the correlation between CFU colony counts and the measurement of iATP as a biochemical marker for hematopoietic cell proliferation [ 43 ].…”

Section: Discussionmentioning

confidence: 99%

Detecting primitive hematopoietic stem cells in total nucleated and mononuclear cell fractions from umbilical cord blood segments and units

et al. 2015

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BackgroundRare hematopoietic stem cell populations are responsible for the transplantation engraftment process. Umbilical cord blood (UCB) is usually processed to the total nucleated cell (TNC), but not to the mononuclear cell (MNC) fraction. TNC counts are used to determine UCB unit storage, release for transplantation and correlation with time to engraftment. However, the TNC fraction contains varying concentrations of red blood cells, granulocytes, platelets and other cells that dilute and mask the stem cells from being detected. This does not allow the quality and potency of the stem cells to be reliably measured.Methods63 UCB segments and 10 UCB units plus segments were analyzed for the response of both primitive lympho-hematopoietic and primitive hematopoietic stem cells in both the TNC and MNC fractions. The samples were analyzed using a highly sensitive, standardized and validated adenosine triphosphate (ATP) bioluminescence stem cell proliferation assay verified against the colony-forming unit (CFU) assay. Dye exclusion and metabolic viability were also determined.ResultsRegardless of whether the cells were derived from a segment or unit, the TNC fraction always produced a significantly lower and more variable stem cell response than that derived from the MNC fraction. Routine dye exclusion cell viability did not correspond with metabolic viability and stem cell response. Paired UCB segments produced highly variable results, and the UCB segment did not produce similar results to the unit.DiscussionThe TNC fraction underestimates the ability and capacity of the stem cells in both the UCB segment and unit and therefore provides an erroneous interpretation of the of the results. Dye exclusion viability can result in false positive values, when in fact the stem cells may be dead or incapable of proliferation. The difference in response between the segment and unit calls into question the ability to use the segment as a representative sample of the UCB unit. It is apparent that present UCB processing and testing methods are inadequate to properly determine the quality and potency of the unit for release and use in a patient.

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Section: Discussionmentioning

confidence: 99%

Detecting primitive hematopoietic stem cells in total nucleated and mononuclear cell fractions from umbilical cord blood segments and units

et al. 2015

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“…This assay was chosen because it is considered more sensitive, robust, and precise than colorimetric and fluorescence assays. It also has a high correlation between ATP detection and cell viability values, since ATP production is immediately interrupted during cell death (Maehara et al, 1987;Zumpe et al, 2010).…”

Section: Cytotoxicity and Phototoxicity Assaysmentioning

confidence: 99%

Hybrid Nanoparticles as an Efficient Porphyrin Delivery System for Cancer Cells to Enhance Photodynamic Therapy

Silva

Castro

Botteon

et al. 2021

Front. Bioeng. Biotechnol.

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Photodynamic therapy (PDT) is a potential non-invasive approach for application in oncological diseases, based on the activation of a photosensitizer (PS) by light at a specific wavelength in the presence of molecular oxygen to produce reactive oxygen species (ROS) that trigger the death tumor cells. In this context, porphyrins are interesting PS because they are robust, have high chemical, photo, thermal, and oxidative stability, and can generate singlet oxygen (1O2). However, porphyrins exhibit low solubility and a strong tendency to aggregate in a biological environment which limits their clinical application. To overcome these challenges, we developed hybrid nanostructures to immobilize 5,10,15,20-tetrakis[(4-carboxyphenyl) thio-2,3,5,6-tetrafluorophenyl] (P), a new third-generation PS. The biological effect of this system was evaluated against bladder cancer (BC) cells with or without light exposition. The nanostructure composed of lipid carriers coated by porphyrin-chitosan (P-HNP), presented a size of ca. 130 nm and low polydispersity (ca. 0.25). The presence of the porphyrin-chitosan (P-chitosan) on lipid nanoparticle surfaces increased the nanoparticle size, changed the zeta potential to positive, decreased the recrystallization index, and increased the thermal stability of nanoparticles. Furthermore, P-chitosan incorporation on nanoparticles increased the stability and enhanced the self-organization of the system and the formation of spherical structures, as observed by small-angle X-ray scattering (SAXS) analysis. Furthermore, the immobilization process maintained the P photoactivity and improved the photophysical properties of PS, minimizing its aggregation in the cell culture medium. In the photoinduction assays, the P-HNP displayed high phototoxicity with IC50 3.2-folds lower than free porphyrin. This higher cytotoxic effect can be correlated to the high cellular uptake of porphyrin immobilized, as observed by confocal images. Moreover, the coated nanoparticles showed mucoadhesive properties interesting to its application in vivo. Therefore, the physical and chemical properties of nanoparticles may be relevant to improve the porphyrin photodynamic activity in BC cells.

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“…2.24.2), with fluorescence output being proportional to the number of metabolically active and viable cells (O'Brien et al, 2000;Abdoli et al, 2015). The principal advantages of this assay are simplicity, versatility, reproducibility, sensitivity, and low cost (Voytik-Harbin et al, 1998), as well as the fact that it does not involve cell lysis and can be performed with other tests or kinetic measurements on the same set of cells (Zumpe et al, 2010). The main disadvantages of this method are possible fluorescent interference of 2.24.7 tested compounds, and intrinsic toxicity of resazurin for particular cell types (Riss et al, 2004), although this can be easily assessed by performing proper controls with alternative viability assays.…”

Section: Commentary Background Informationmentioning

confidence: 99%

Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay

et al. 2016

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The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding and excessive manipulations in assays that test different cell samples across plates. The procedure also reduces the amount of cells, test compound, and reagents required, as well as the time expended in conventional tests, thus resulting in a more confident prediction of toxic thresholds for the tested compounds. © 2016 by John Wiley & Sons, Inc.

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Comparison of potency assays using different read-out systems and their suitability for quality control

Cited by 8 publications

References 33 publications

Detecting primitive hematopoietic stem cells in total nucleated and mononuclear cell fractions from umbilical cord blood segments and units

Detecting primitive hematopoietic stem cells in total nucleated and mononuclear cell fractions from umbilical cord blood segments and units

Hybrid Nanoparticles as an Efficient Porphyrin Delivery System for Cancer Cells to Enhance Photodynamic Therapy

Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay

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