Impact of lipopolysaccharides on cultivation and recombinant protein expression in human embryonal kidney (HEK‐293) cells

Faust, Christine; Beil, Christian; Dittrich, Winand H.; Rao, Ercole; Langer, Thomas

doi:10.1002/elsc.202100065

Cited by 5 publications

(5 citation statements)

References 33 publications

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“…It is worth noting that bactericidal activity was observed for P. aeruginosa ATCC 27853 at 32.50 μg ml −1 and P. aeruginosa P2307 at 65.00 μg ml −1 , which were the highly virulent strains against the G. mellonella infection model (). In the case of Gram-negative bacteria, the bacteriostatic effect can be desirable since the release of LPS in the body can be avoided [65, 66]. These results highlight the potential of this SAR-endolysin against a broad range of MDR and virulent Gram-negative bacteria.…”

Section: Discussionmentioning

confidence: 99%

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Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5

Gontijo,

Pereira Teles,

Martins Correia

et al. 2024

Microbiology

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Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 μg ml−1 against Pseudomonas aeruginosa ATCC 27853, 16.25 μg ml−1 against S. enterica Typhimurium ATCC 13311, and 32.50 μg ml−1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 μg ml−1) and P. aeruginosa P2307 (65.00 μg ml−1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

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Section: Discussionmentioning

confidence: 99%

“…3 ). In the case of Gram-negative bacteria, the bacteriostatic effect can be desirable since the release of LPS in the body can be avoided [ 65 66 ]. These results highlight the potential of this SAR-endolysin against a broad range of MDR and virulent Gram-negative bacteria.…”

Section: Discussionmentioning

confidence: 99%

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5

Gontijo,

Pereira Teles,

Martins Correia

et al. 2024

Microbiology

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“…In addition, DNA should be of high quality, purity, integrity and concentration [ 7 , 8 ]. For example, DNA preparation can be contaminated with endotoxins, which can alter the growth of some cell lines [ 8 , 9 ]. Lastly, the transfection method plays a critical role not only on the efficiency parameter but also on the cell survival.…”

Section: Discussionmentioning

confidence: 99%

“…The quality of DNA depends, among other factors, on the presence of endotoxins or lipopolysaccharides (LPS), which are byproducts of plasmid DNA preparation [ 7 ]. The presence of LPS is unlikely to significantly affect the transfection efficiency or cell proliferation of commonly used cell lines, but it may pose challenges for specific cell lines [ 7 , 8 , 9 ]. Nonetheless, the two major drawbacks of DNA-based transient transfection are the inability to carefully control the expression level of the foreign construct even by using weak versus strong promoters [ 10 ] and, for most cell lines, the inability to reach up to 95% of transfected cells [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

The Prodigious Potential of mRNA Electrotransfer as a Substitute to Conventional DNA-Based Transient Transfection

Juncker,

Chatton,

Donzeau

2023

Cells

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Transient transfection of foreign DNA is the most widely used laboratory technique to study gene function and product. However, the transfection efficiency depends on many parameters, including DNA quantity and quality, transfection methods and target cell lines. Here, we describe the considerable advantage of mRNA electroporation compared to conventional DNA-based systems. Indeed, our methodology offers extremely high transfection efficiency up to 98% regardless of the cell line tested. Protein expression takes place a few hours post-transfection and lasts over 72 h, but overall, the electrotransfer of mRNAs enables the monitoring of the level of protein expressed by simply modulating the amount of mRNAs used. As a result, we successfully conducted cell imaging by matching the levels of expressed VHHs and the antigen present in the cell, preventing the necessity to remove the excess unbound VHHs. Altogether, our results demonstrate that mRNA electrotransfer could easily supplant the conventional DNA-based transient expression system.

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“…A detailed analysis of different HEK293 cell lines can be found in the studies by Malm et al [ 19 ] and Tan et al [ 3 ]. HEK293 cells growing in suspension typically have mean cell diameters ranging from 14

to 16

[ 20 , 21 , 22 ] and a typical maximum specific growth rate

ranging from 0.020

–0.029

[ 23 , 24 , 25 , 26 ]. Jang et al [ 24 ] were able to demonstrate comparable growth rates between adherent growing and in suspension growing HEK293 cells.…”

Section: Introductionmentioning

confidence: 99%

Improvement of HEK293 Cell Growth by Adapting Hydrodynamic Stress and Predicting Cell Aggregate Size Distribution

et al. 2023

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HEK293 is a widely used cell line in the fields of research and industry. It is assumed that these cells are sensitive to hydrodynamic stress. The aim of this research was to use particle image velocimetry validated computational fluid dynamics (CFD) to determine the hydrodynamic stress in both shake flasks, with and without baffles, and in stirred Minifors 2 bioreactors to evaluate its effect on the growth and aggregate size distribution of HEK293 suspension cells. The HEK FreeStyleTM 293-F cell line was cultivated in batch mode at different specific power inputs (from 63W/m3–451W/m3), whereby ≈60W/m3 corresponds to the upper limit, which is what has been typically described in published experiments. In addition to the specific growth rate and maximum viable cell density VCDmax, the cell size distribution over time and cluster size distribution were investigated. The VCDmax of 5.77±0.02e6cells/mL was reached at a specific power input of 233W/m3 and was 23.8% higher than the value obtained at 63W/m3 and 7.2% higher than the value obtained at 451W/m3. No significant change in the cell size distribution could be measured in the investigated range. It was shown that the cell cluster size distribution follows a strict geometric distribution whose free parameter p is linearly dependent on the mean Kolmogorov length scale. Based on the performed experiments, it has been shown that by using CFD-characterised bioreactors, the VCDmax can be increased and the cell aggregate rate can be precisely controlled.

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Impact of lipopolysaccharides on cultivation and recombinant protein expression in human embryonal kidney (HEK‐293) cells

Cited by 5 publications

References 33 publications

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5

The Prodigious Potential of mRNA Electrotransfer as a Substitute to Conventional DNA-Based Transient Transfection

Improvement of HEK293 Cell Growth by Adapting Hydrodynamic Stress and Predicting Cell Aggregate Size Distribution

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