Weak cell cycle dependency but strong distortive effects of transfection with Lipofectamine 2000 in near‐physiologically synchronized cell culture

Fuge, Grischa; Zeng, An‐Ping; Jandt, Uwe

doi:10.1002/elsc.201600113

Cited by 6 publications

(9 citation statements)

References 13 publications

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“…Materials and methods are partially based on previously published studies for nonproducing mammalian cell lines (Castillo et al, ; Fuge et al, ; Jandt et al, ; Platas Barradas et al, ). These were adapted for the cultivation of antibody (IgG) producing CHO DP‐12 cells and new laboratory equipment, and optimized for efficiency, as described in the following.…”

Section: Methodsmentioning

confidence: 99%

“…Only physical separation techniques were considered for near‐physiological synchronization due to their minor influence on the metabolism of mammalian cells (Banfalvi, ; Jandt et al, ; Platas Barradas et al, ). Therefore, countercurrent centrifugal elutriation was used as shown in Fuge et al (), with slight modifications for the synchronization of higher cell numbers in a single run. The elutriation chamber (centrifuge: Avanti J‐265 XP, JE‐5.0 Beckmann coulter, Krefeld, Germany) was changed from 5 ml (standard chamber) to 40 ml (great chamber).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we implemented this combined approach to identify cell‐cycle‐dependent metabolic rates for the nonproducing cell lines

{AGE1}_{AAT}

and CHO‐K1 (Castillo, Fuge, Jandt, & Zeng, ; Jandt, Platas Barradas, Pörtner, & Zeng, ; Platas Barradas et al, ). On the other hand, strong cell‐cycle dependencies for transient transfection processes in HEK293s could be ruled out (Fuge, Zeng, & Jandt, ).…”

Section: Introductionmentioning

confidence: 99%

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Model‐based identification of cell‐cycle‐dependent metabolism and putative autocrine effects in antibody producing CHO cell culture

Möller

Korte

Pörtner

et al. 2018

Biotech & Bioengineering

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The understanding of cell-cycle-dependent population heterogeneities in mammalian cell culture and their influence on production rates is still limited. Furthermore, metabolic regulations arising from self-expressed signaling factors (autocrine/autoinhibitory factors) have been postulated in the past, but no determination of such effects have been made so far for fast-growing production Chinese hamster ovary (CHO) cells in chemically defined media. In this study, a novel approach combining near-physiological treatment of cells (including synchronization), population resolved mechanistic modeling and statistical analysis was developed to identify population inhomogeneities. Cell-cycle-dependent population dynamics and metabolic regulations due to a putative autocrine factor were examined and their impact on the metabolic rates and antibody production of near-physiologically synchronized CHO DP-12 cell cultures was determined. To achieve this, a population resolved model was extended to describe putative autocrine-dependentt and cell-cycle-related metabolic rates for glucose, glutamine, lactate, ammonia, and antibody production. The model parameters were estimated based on data of two repeated batch cultivations (three batches each), with main substrates in excess and potentially inhibiting waste products (lactate and ammonium) controlled within narrow ranges. Significant changes, due to a putative autocrine factor, were identified for lactate and ammonia formation and antibody production. The cell growth and the uptake of glucose and glutamine were only partially affected by the putative autocrine under the given conditions. The results indicate the presence of a self-expressed autocrine factor and its strong impact on the metabolism of CHO DP-12 cells. Furthermore, glucose and glutamine uptake, as well as the formation of ammonium and antibody were found to be significantly cell-cycle-dependent. The combined approach has a strong potential to improve the understanding of the interplay of population heterogeneities and signal factors in mammalian cell culture.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Recently, we implemented this combined approach to identify cell‐cycle‐dependent metabolic rates for the nonproducing cell lines

{AGE1}_{AAT}

Section: Introductionmentioning

confidence: 99%

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Model‐based identification of cell‐cycle‐dependent metabolism and putative autocrine effects in antibody producing CHO cell culture

Möller

Korte

Pörtner

et al. 2018

Biotech & Bioengineering

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“…This fact can explain the lower transfection rates obtained when lipofection was used. Moreover, the use of lipofection has been generally associated with transient transfection (Ooi et al, 2016;Fuge et al, 2017), a fact that limits its application in the production of transgenic animals (Bressan et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Efficiency of transgene expression in bovine cells varies according to cell type and gene transfer method

Gómez

2019

Rev Colom Cienc Pecua

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Background: Production of transgenic animals is still a low-efficiency biotechnology, and simple alternatives should be used to improve the rate of transgenic bovine production by nuclear transfer. One such alternative is selecting the appropriate donor cell type and transfection method. Objective: To investigate the effect of cell type (fetal or adult fibroblasts, and cumulus cells), and gene transfer method (lipofection and lentiviral transduction) on the incorporation, expression, and fluorescence intensity of transgene on bovine cells analyzed by flow cytometry. Methods: Fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) were transfected using lipofection, or transduced using lentiviral particles produced with Green Fluorescent Protein (GFP) expressing plasmids, and analyzed by flow cytometry. Results: Lentiviral transduction resulted in higher transgene expression rates for all cell types (FF: 88.8 ± 0.98; AF: 91.6 ± 2.96; CC: 60.7% ± 14.7) compared to lipofection (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65; CC: 3.9% ± 1.97). Cumulus cells showed lower transgene expression rates than the other cell types. Regarding fluorescence intensity, there was no significant difference between lipofection and lentiviral transduction; in both treatments, higher fluorescence intensity was obtained when adult cells were used instead of fetal cells. Conclusion: Gene transfer efficiency varies according to cell type, and gene transfer method, with lentiviral transduction achieving higher transgene expression rate, and adult fibroblasts showing better transgene expression.Keywords: cloning, epigenetics, lipofection, lentiviral transduction, nuclear reprogramming. Resumen Antecedentes: La producción de animalestransgénicossigue siendo una biotecnología de baja eficiencia, y se deberían utilizar alternativas sencillas para mejorar la tasa de producción de bovinos transgénicos mediante transferencia nuclear. Una de estas alternativas es la selección del tipo mas apropiado de célula donante y método de transferencia génica. Objetivo: Investigar el efecto del tipo celular (fibroblastos fetales o adultos, y celulas del cumulus), y el método de transferencia génica (lipofección y transducción lentiviral) en la incorporación, expresión génica, y la intensidad de fluorescencia del transgén en células bovinas analizadas por citometría de flujo. Métodos: Fibroblastos fetales (FF), fibroblastos adultos (AF), y células del cúmulo (CC) fueron transfectados a través de lipofección o transducidos utilizando partículas lentivirales producidas con plásmidos que expresan la proteína verde fluorescente (GFP). Resultados: La transducción lentiviral dio lugar a mayores tasas de expresión del transgen en todos los tipos de células (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96, CC: 60,7% ± 14,7) en comparación con la lipofección (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). Las células del cúmulus mostraron menores tasas de expresión del transgen que los otros tipos celulares. En cuanto a la intensidad de fluorescencia, no hubo diferencias significativas entre lipofección y transducción lentiviral; en ambos tratamientos, se obtuvo una mayor intensidad de fluorescencia cuando se usaron células adultas en lugar de células fetales. Conclusión: La eficiencia de la transferencia de genes varía según el tipo de célula y el método de transferencia génica, con la transducción lentiviral se logra una mayor tasa de transfección, y los fibroblastos adultos muestran una mejor expresión transgénica.Palabras clave: clonación, epigenética, lipofección, reprogramación nuclear, transducción lentiviral. Resumo Antecedentes: A produção de animais transgênicos é uma biotecnologia que ainda apresenta baixa eficiência e alternativas simples devem ser usadas para o aumento da produção de bovinos transgênicos por transferência nuclear. Uma destas alternativas compreende a seleção do tipo apropriado de célula doadora de núcleo e do método de transferência gênica. Objetivo: Investigar a influência do tipo celular (fibroblastos fetais ou adultos, e células do cumulus), e do método de transferência gênica (transfecção por lipofecção ou transdução lentiviral) na incorporação, expressão, e na intensidade de fluorescência do transgene em células bovinas analisadas por citometria de fluxo. Métodos: Fibroblastos fetais (FF), fibroblastos adultos (AF), e células do cumulus (CC) foram submetidas à lipofecção ou à transfecção lentiviral utilizando plasmídeos expressando a Proteína Fluorescente Verde – GFP). Resultados: A transdução lentiviral resultou em maiores taxas de expressão do transgene em todos os tipos celulares (FF: 88,8 ± 0,98; AF: 91,6 ± 2,96; CC: 60,7% ± 14.7) quando comparada com a lipofeccção (FF: 17,8 ± 2,82; AF: 10,66 ± 0,65; CC: 3,9% ± 1,97). As células do cumulus apresentaram menores taxas de expressão quando comparadas aos outros tipos celulares. Com relação à intensidade de fluorescência, não houve diferença significativa entre a lipofecção e a transdução lentiviral e em ambos os tratamentos as células adultas apresentaram maior intensidade de fluorescência do que as células fetais. Conclusão: A eficiência de transferência gênica varia de acordo com o tipo celular, e com o método de transferência gênica, sendo que a transdução lentiviral resultou em maiores taxas, e que os fibroblastos adultos mostraram melhor expressão do transgene.Palavras-chave: clonagem, epigenética, lipofecção, reprogramação nuclear, transdução lentiviral.

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“…[ 113 ] However, studies of cell cycle effects have their limitations, including the susceptibility of the chemical and physical methods of arresting cells in a specific growth phase to artifacts [ 114 ] and the potential distortion of the synchronous progression of the cell cycle. [ 115 ] Jandt and co‐workers reported a method of synchronizing mammalian suspension cell cultures under near‐physiological conditions, which were used to study transfection. [ 115,116 ] Using physiological synchronization, no obvious cell cycle dependency of transfection efficiencies of HEK293s cells using Lipofectamine 2000 was reported.…”

Section: Single Cell Levelmentioning

confidence: 99%

A Focus on “Bio” in Bio–Nanoscience: The Impact of Biological Factors on Nanomaterial Interactions

Cortez‐Jugo

Czuba-Wojnilowicz

Tan

et al. 2021

Adv Healthcare Materials

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Bio–nanoscience research encompasses studies on the interactions of nanomaterials with biological structures or what is commonly referred to as the biointerface. Fundamental studies on the influence of nanomaterial properties, including size, shape, composition, and charge, on the interaction with the biointerface have been central in bio–nanoscience to assess nanomaterial efficacy and safety for a range of biomedical applications. However, the state of the cells, tissues, or biological models can also influence the behavior of nanomaterials at the biointerface and their intracellular processing. Focusing on the “bio” in bio–nano, this review discusses the impact of biological properties at the cellular, tissue, and whole organism level that influences nanomaterial behavior, including cell type, cell cycle, tumor physiology, and disease states. Understanding how the biological factors can be addressed or exploited to enhance nanomaterial accumulation and uptake can guide the design of better and suitable models to improve the outcomes of materials in nanomedicine.

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Weak cell cycle dependency but strong distortive effects of transfection with Lipofectamine 2000 in near‐physiologically synchronized cell culture

Cited by 6 publications

References 13 publications

Model‐based identification of cell‐cycle‐dependent metabolism and putative autocrine effects in antibody producing CHO cell culture

Model‐based identification of cell‐cycle‐dependent metabolism and putative autocrine effects in antibody producing CHO cell culture

Efficiency of transgene expression in bovine cells varies according to cell type and gene transfer method

A Focus on “Bio” in Bio–Nanoscience: The Impact of Biological Factors on Nanomaterial Interactions

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