FIBS-enabled Noninvasive Metabolic Profiling

Summary

A description of how to calibrate Förster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented. The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

Abstract

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on Förster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

Introduction

Metabolite monitoring has various applications in bioprocessing, including process development, media and feed design, and metabolic engineering. Various methods are available for concentration measurements through the use of enzymatic¹, chemical², or binding assays³. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of the intracellular concentration of key metabolites. In ultra-low volume assays, fluorescence is a convenient tool as miniaturization actually improves the signal-to-noise ratio^4,5 and protein-based sensors can be genetically encoded meaning that no....

Protocol

1. Cell Line Revival and Maintenance

1. Revive CHO cells in 9 ml CD-CHO medium supplemented with 8 mM L-Glutamine and 10 ml/L 100x hypoxanthine/thymidine supplement (complete growth medium).
2. Centrifuge at 100 x g for 5 min.
3. Resuspend the cells in 10 ml of fresh complete growth medium.
4. Remove a 1 ml sample and determine the viable cell concentration by light microscopy using the trypan blue dye exclusion method in a hemocytometer.
5. Initiate a culture at a seeding density of 3 x 10⁵ cells/ml in 125 ml shake flasks.
6. Maintain the culture in an incubator at 37 °C, a humidified atmosphere of 5% CO₂

Results

An overview of the methodology is presented in Figure 2. In the work presented herein, CHO cells were transfected with the FIBS vector and stable cell lines were selected at an antibiotic pressure of 400 μg/ml zeocin. Two separate stable cell lines constitutively expressing the glucose and glutamine sensors were therefore created. Figure 1 depicts the configurations of the two biosensors used in this study. The glucose sensor is based on the Apo-Max principle and the corresponding plasmi.......

Discussion

The FIBS enable in vivo and in situ quantitation of key molecules, in this case growth-limiting nutrients, removing uncertainties arising from quenching and extraction methods. The findings suggest that there is a good correlation between the FIBS signal and the intracellular concentrations in the range of 1-5 mM for glucose and 0.3-2 mM for glutamine. In batch CHO cell culture, these concentrations are encountered in the exponential, stationary, and early decline phases. The exponential and stationary .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CHO-S Cells	Life Tecnologies	11619-012	Cell line will vary depending on the goals of the study
pCDNA4/TO vector	Life Tecnologies
TransIT-PRO Transfection Reagent	Mirus Bio	MIR 5700	Transfections can be accomplished using any method suitable for the cell line under study
Zeocin	Invivogen
CD-CHO medium	Life Technologies	10743-011	Cell growth medium is dependent upon the cells under study
100x HT Supplement	Life Technologies	11067-030
L-Glutamine 200 mM (100x), Liquid	Life Technologies	25030032
InfinitePRO 200 plate reader	Tecan	FLx800TBI	Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted
Filters for plate reader	Tecan	30000463
Maxiprep Plasmid Purification Kit	Qiagen	12163	Any suitable kit can be substituted
Amplex Red glucose/glucose oxidase assay kit	Invitrogen	A22189	Any suitable kit can be substituted
EnzyChrom  Glutamine Assay Kit	BioAssay Systems	EOAC-100	Any suitable kit can be substituted
Improved Neubauer hemocytometer	Fisher Scientific	MNK-420-010N
Incubator	Nuaire	NU-5510E