Validation of an automated cell counting method for cGMP manufacturing of human induced pluripotent stem cells

Manzini, P.; Peli, Valeria; Rivera-Ordaz, Araceli; Budelli, Silvia; Barilani, Mario; Lazzari, Lorenza

doi:10.1016/j.btre.2022.e00708

Cited by 4 publications

(4 citation statements)

References 23 publications

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“…As no major deviations of the DF measured from the DF targeted could be measured (S1 the dilution factors, as is standard practice in other works as well [5,11,16,19]. The intercept, slope and R 2 of the straight-line fit of both methods are reported in Table 2.…”

Section: Results Related To Total Cell Densitymentioning

confidence: 99%

“…The measured TCDs of set A were plotted against the DF targeted for both methods in Fig 2 and a straight-line fit was applied. At this point, we would like to emphasize that the plotted measured TCDs do not represent the raw TCDs measured in reality, as they have been extrapolated using the dilution factors, as is standard practice in other works as well [5, 11, 16, 19]. The intercept, slope and R 2 of the straight-line fit of both methods are reported in Table 2.…”

Section: Resultsmentioning

confidence: 99%

“…In sets A May 28, 2024 3/15 and C, three dilution fractions were examined each (Table 1). The concentration range of A was oriented at the range of other works [4,11,[14][15][16]. The concentration range for B was chosen to cover the range of expected concentrations that occur during cell cultivation in the bioreactor.…”

Section: Methodsmentioning

confidence: 99%

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Semi-automated vs. manual: Comparative study of cell culture counting methods using validation parameters

Ramm,

Odefey,

Frahm

et al. 2024

Preprint

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Determining cell density and cell viability is fundamental for any cell cultivation process. In addition to the manual counting method using hemocytometers, (semi-)automated methods offer advantages such as lower variability and shortened analysis times. However, these methods should provide at least comparable results to the manual method, which is why a comparison of methods is essential. We conducted a dilution series experimental design according to ISO 20391-2:2019 and compared two cell counting methods based on validation parameters aligned with the ICH Q2(R1) guideline. Regarding specificity and linearity, the manual (hemocytometer) and semi-automated (Countstar BioTech®) method exhibited similar results in the two evaluated characteristics total cell density and cell viability of CHO-K1 cells. Regarding repeatability of determining total cell density, the semi-automated method achieved significant (α= 0.05) better results with average relative standard deviations of < 6 %, than the manual method with average relative standard deviations of > 9 %. Concerning repeatability of the cell viability measurement, no significant difference between the two methods were shown. These results show the suitabililty of the dilution series experimental design. For the applied example, they indicate that the investigated semi-automated method is an appropriate alternative to the manual method.

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Section: Results Related To Total Cell Densitymentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Semi-automated vs. manual: Comparative study of cell culture counting methods using validation parameters

Ramm,

Odefey,

Frahm

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…In this context, cell viability and cell numbers per target dose can be used as product purity parameters and associated potency based on the assumed mechanism of action 26 to monitor ATMP-manufacturing processes and is also often selected as one of the IPCs for critical steps. Various analytical methods are used for cell counting and viability, including manual trypan blue exclusion, 27 automatic cell counters, 28 , 29 flow cytometry-based methods using live/dead cell markers or Trucount beads, 30 and built-in fluidic-based system based on volumetric method that enable precise automated cell counting in a single-platform flow cytometry. 31 However, real-time analysis of cellular products remains difficult, especially for adherent cells.…”

Section: The Abc Of Analytical Qc Methods For Atmp Manufacturingmentioning

confidence: 99%

Basics of advanced therapy medicinal product development in academic pharma and the role of a GMP simulation unit

Johanna,

Daudeij,

Devina

et al. 2023

Immuno-Oncology and Technology

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Biopolymer-based emulsions for the stabilization of Trichoderma atrobrunneum conidia for biological control

Martínez

Heeb

Kalač

et al. 2023

Appl Microbiol Biotechnol

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Trichoderma spp. are ubiquitous soil-borne fungi that are widely used in biological control to promote and regulate healthy plant growth, as well as protect against plant pathogens. However, as with many biological materials, the relative instability of Trichoderma propagules limits its practical use in industrial applications. Therefore, there has been significant research interest in developing novel formulations with various carrier substances that are compatible with these fungal propagules and can enhance the shelf-life and overall efficacy of the Trichoderma. To this end, herein, we investigate the use of a variety of biopolymers and nanoparticles for the stabilization of Trichoderma atrobrunneum T720 conidia for biological control. The best-performing agents—agar and cellulose nanocrystals (CNC)—were then used in the preparation of oil-in-water emulsions to encapsulate conidia of T720. Emulsion properties including oil type, oil:water ratio, and biopolymer/particle concentration were investigated with respect to emulsion stability, droplet size, and viability of T720 conidia over time. Overall, agar-based formulations yielded highly stable emulsions with small droplet sizes, showing no evidence of drastic creaming, or phase separation after 1 month of storage. Moreover, agar-based formulations were able to maintain ~ 100% conidial viability of T720 after 3 months of storage, and over 70% viability after 6 months. We anticipate that the results demonstrated herein will lead to a new generation of significantly improved formulations for practical biological control applications. Graphical abstract Key points • Various biopolymers were evaluated for improving the stability of Trichoderma conidia • Oil in water emulsions was prepared using cellulose nanocrystals and agar as interface stabilizers • Agar-based emulsions showed ~ 100% viability for encapsulated conidia after 3 months of storage

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Validation of an automated cell counting method for cGMP manufacturing of human induced pluripotent stem cells

Cited by 4 publications

References 23 publications

Semi-automated vs. manual: Comparative study of cell culture counting methods using validation parameters

Semi-automated vs. manual: Comparative study of cell culture counting methods using validation parameters

Basics of advanced therapy medicinal product development in academic pharma and the role of a GMP simulation unit

Biopolymer-based emulsions for the stabilization of Trichoderma atrobrunneum conidia for biological control

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