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Unraveling the metabolism of HEK-293 cells using lactate isotopomer analysis

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Abstract

HEK-293 is the most extensively used human cell line for the production of viral vectors and is gaining increasing attention for the production of recombinant proteins by transient transfection. To further improve the metabolic characterization of this cell line, we have performed cultures using 13C-labeled substrates and measured the resulting mass isotopomer distributions in lactate by LC/MS. Simultaneous metabolite and isotopomer balancing allowed improvement and validation of the metabolic model and quantification of key intracellular pathways. We have determined the amounts of glucose carbon channeled through the PPP, incorporated into the TCA cycle for energy production and lipids biosynthesis, as well as the cytosolic and mitochondrial malic enzyme fluxes. Our analysis also revealed that glutamine did not significantly contribute to lactate formation. An improved and quantitative understanding of the central carbon metabolism is greatly needed to pursue the rational development of engineering approaches at both the cellular and process levels.

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Abbreviations

ACCOA:

Acetyl-CoA

ACCOAm:

Acetyl-CoA in the mitochondria

AKG:

Alpha-ketoglutarate

ASN:

Asparagine

ASP:

Aspartate

ATP:

Adenosine triphosphate

BHK:

Baby hamster kidney

CHO:

Chinese hamster ovary

CIT:

Citrate

CITm:

Citrate in the mitochondria

CoA:

Coenzyme A

CYS:

Cysteine

E4P:

Erythrose-4-phosphate

F6P:

Fructose 6-phosphate

G3P:

Glyceraldehyde 3-phosphate

G6P:

Glucose 6-phosphate

GLY:

Glycine

HEK-293:

Human embryonic kidney

i-13C:

Singly 13C-labeled substrate in the “i” position

ILE:

Isoleucine

LAC:

Lactate

LC/MS:

Liquid chromatography mass spectrometry

LC-SFM:

Low-calcium serum-free medium

LEU:

Leucine

LYS:

Lysine

MAL:

Malate

MET:

Methionine

NADPH:

Reduced nicotinamide adenine dinucleotide phosphate oxidase

NMR:

Nuclear magnetic resonance

OAA:

Oxaloacetate

OAAm:

Oxaloacetate in the mitochondria

PHE:

Phenylalanine

PPP:

Pentose phosphate pathway

PYR:

Pyruvate

PYRm:

Pyruvate in the mitochondria

R5P:

Ribulose-5-phosphate

S7P:

Sedoheptulose-7-phosphate

SER:

Serine

TCA:

Tricarboxylic acid cycle

THR:

Threonine

TYR:

Tyrosine

U-13C:

Uniformly 13C labeled substrate

VAL:

Valine

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Acknowledgments

The authors wish to acknowledge the following contributors: Hugues-Anderson Kamga-Wambo and Steve Hisiger from École Polytechnique de Montreal for 13C LC/MS analysis.

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Authors and Affiliations

  1. Chemical Engineering Department, École Polytechnique de Montréal, C.P. 6079, Succ. Centre-ville, Montréal, QC, H3C 3A7, Canada

    Olivier Henry & Mario Jolicoeur

  2. National Research Council Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montréal, QC, H4P 2R2, Canada

    Amine Kamen

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  1. Olivier Henry
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  2. Mario Jolicoeur
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  3. Amine Kamen
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Corresponding author

Correspondence to Olivier Henry.

Appendix

Appendix

Metabolic network for HEK293 cells and the corresponding carbon atom transitions (Table 3).

Table 3 Metabolic model and atomic transitions for HEK-293 cells
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Henry, O., Jolicoeur, M. & Kamen, A. Unraveling the metabolism of HEK-293 cells using lactate isotopomer analysis. Bioprocess Biosyst Eng 34, 263–273 (2011). https://doi.org/10.1007/s00449-010-0468-9

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