In search of an effective cell disruption method to isolate intact mitochondria from <scp>C</scp>hinese hamster ovary cells

Bahnemann, Janina; Kayo, Sabrina; Wahrheit, Judith; Heinzle, Elmar; Pörtner, Ralf; Zeng, An‐Ping

doi:10.1002/elsc.201200182

Cited by 9 publications

(12 citation statements)

References 36 publications

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“…An optimum composition was found to consist of a concentration of 0.122% m/v digitonin, 12.5 mL of lysis buffer and 43.65 mL of quenching buffer. The identified digitonin concentrations were about ten times higher than in comparative publications [42,43]. Notably, these approaches exposed cells to digitonin for several minutes while in our approach digitonin contact took only a few seconds.…”

Section: Selectively Separating the Cytosol With The Help Of Digitonincontrasting

confidence: 51%

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

Matuszczyk

Teleki

Pfizenmaier

2015

Biotechnology Journal

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Mammalian cells show a compartmented metabolism. Getting access to subcellular metabolite pools is of high interest to understand the cells' metabolomic state. Therefore a protocol is developed and applied for monitoring compartment-specific metabolite and nucleotide pool sizes in Chinese hamster ovary (CHO) cells. The approach consists of a subtracting filtering method separating cytosolic components from physically intact mitochondrial compartments. The internal standards glucose-6-phosphate and cis-aconitate were chosen to quantify cytosolic secession and mitochondrial membrane integrity. Extracts of related fractions were studied by liquid chromatography-isotope dilution mass spectrometry for the absolute quantification of a subset of glycolytic and tricarboxylic acid cycle intermediates together with the adenylate nucleotides ATP, ADP and AMP. The application of the protocol revealed highly dynamic changes in the related pool sizes as a function of distinct cultivation periods of IgG1 producing CHO cells. Mitochondrial and cytosolic pool dynamics were in agreement with anticipated metabolite pools of independent studies. The analysis of adenosine phosphate levels unraveled significantly higher ATP levels in the cytosol leading to the hypothesis that mitochondria predominantly serve for fueling ATP into the cytosol where it is tightly controlled at physiological adenylate energy charges about 0.9.

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Section: Selectively Separating the Cytosol With The Help Of Digitonincontrasting

confidence: 51%

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

Matuszczyk

Teleki

Pfizenmaier

2015

Biotechnology Journal

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“…This wide presence of ER can be explained by physical proximity and in many cases the contacts with other organelles [ 3 , 58 – 60 ], with the exception of the cytosol, where its detection indicated some breakage of organelles by mechanical homogenization [ 24 ]. The location of mitochondria (Hsp60) in nuclear and mitochondrial precipitates was probably due to overlapped centrifugation velocities [ 19 , 36 , 59 , 61 – 63 ], different mitochondria populations [ 35 , 36 , 56 ], nuclei breakage and cytoskeleton aggregation [ 19 , 24 , 56 , 63 , 64 ].…”

Section: Discussionmentioning

confidence: 99%

Enrichment of microsomes from Chinese hamster ovary cells by subcellular fractionation for its use in proteomic analysis

et al. 2020

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Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.

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“…Selective plasma membrane permeabilization using digitonin generates functioning mitochondria enclosed in a cell ghost . Since CHO cells have not commonly been used as biological system in mitochondrial studies, we performed different stainings to characterize the permeabilization of CHO suspension cells as quality control for the preparation of intact, functional mitochondria.…”

Section: Resultsmentioning

confidence: 99%

“…Selective permeabilization is faster, easier, and requires less cell material than the isolation of mitochondria. Potential artifacts due to the isolation procedure itself, for example mitochondrial damage, biased loss of mitochondrial subpopulations, are avoided . Most importantly, many cellular structures and interactions, for example with the cytoskeleton, are preserved after permeabilization, representing a situation that is closer to the actual in vivo situation than isolated mitochondria .…”

Section: Introductionmentioning

confidence: 99%

“…In classical studies, respiration is measured in an oxygraph involving a closed, stirred reaction vessel and a Clark electrode. Mitochondrial function is usually assessed by testing the functionality of different respiratory chain complexes using a substrate‐titration approach . Such closed systems provide the highest precision and sensitivity, yet require substantial experimental effort.…”

Section: Introductionmentioning

confidence: 99%

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High‐throughput respiration screening of single mitochondrial substrates using permeabilized CHO cells highlights control of mitochondria metabolism

Wahrheit

Nonnenmacher

Sperber

et al. 2015

Engineering in Life Sciences

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Respiration analysis using isolated mitochondria and electrochemical oxygen sensing has contributed significantly to the knowledge about mitochondrial metabolism, which is involved in energy generation but also in ageing and numerous diseases. Here, we present a high‐throughput respiration screening for functional in situ mitochondrial studies in permeabilized Chinese hamster ovary cells. The determination of oxygen uptake rates allowed a quantitative comparison between different conditions and a distinction of substrates into three groups providing an insight into tricarboxylic acid (TCA) cycle regulation. The mitochondrial metabolization of citrate, isocitrate, glutamine, and glutamate was highly stimulated by ADP supply. In contrast, the metabolization of α‐ketoglutarate, succinate, fumarate, and malate was little controlled by the energy and redox state. Metabolization of pyruvate was very strictly regulated by several independent mechanisms: phosphorylation, feedback inhibition, but also by the availability of CoA. A moderate stimulation of pyruvate metabolization was accomplished by feeding both pyruvate and aspartate simultaneously. The presented high‐throughput respiration screening provides comprehensive information about the effect of single or mixed substrates on mitochondrial metabolic activities, including transport and TCA cycle regulation, and metabolic bottlenecks. This supports the design of efficient mammalian producer strains or feeding strategies, but also the investigation of pathological and toxicological effects related to mitochondrial metabolism.

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In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells

Cited by 9 publications

References 36 publications

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

Enrichment of microsomes from Chinese hamster ovary cells by subcellular fractionation for its use in proteomic analysis

High‐throughput respiration screening of single mitochondrial substrates using permeabilized CHO cells highlights control of mitochondria metabolism

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