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 exogenous reagents are necessary for metabolite analysis. Förster Resonance Energy Transfer (FRET) biosensors consist of two fluorescent proteins connected by a ligand binding domain. FRET is a nonradiative transfer of energy from a photo-excited donor to an acceptor fluorescent molecule located in close proximity (<100). Ligand cleavage or binding causes a conformational change in the sensor, which, in turn, induces a change in the proximity of the fluorophores, leading to a change in FRET efficiency measured by the change in the emission spectrum. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line and their spectral characteristics change based on the concentration of metabolite within the cell. FIBS have fast response times making them ideal for taking measurements for dynamic models⁶. Previous applications include monitoring single^7-9 and multiple¹⁰ metabolites and providing data on spatiotemporal distribution¹¹. FIBS can be created in two configurations: Apo-Max, where ligand binding disrupts the proximity of the fluorophores lowering energy transfer and Apo-Min, where ligand binding brings the two fluorophores into closer contact (Figure 1).

In this work, a protocol is presented for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for a metabolite, glucose or glutamine. A methodology is established for the calibration of the sensor in vivo to enable future quantitative measurements of intracellular metabolite concentration, as presented in Figure 2. Based on this, the intracellular concentration of glucose or glutamine can be determined in fed-batch CHO cell cultures, to which the two nutrients were added in high concentrations in-process. The results demonstrate that using the fluorescent signal from the FIBS and the previously constructed calibration curve, accurately prediction of the intracellular concentration is possible, as confirmed by an independent enzymatic assay. This method offers substantial advantages over current analytical technologies because it is noninvasive, low-cost and fast, giving a real-time signal of the FIBS that can be monitored throughout the culture.

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, on an orbital shaking platform rotating at 120 rpm.
7. Subculture every 3-4 days in the complete growth medium at a seeding density of 2 x 10⁵ cells/ml.

2. Transfection of the Cell Line with the Plasmid Containing the Biosensor Gene

1. Prepare plasmid DNA using a large-scale plasmid purification kit.
2. Maintain cells at the appropriate conditions (37 °C, 5% CO₂) 12-24 hr prior to transfection to ensure the cells are actively dividing at the time of transfection.
3. Count the cells using the trypan blue dye exclusion method, then prepare a working volume of 20 ml of cell culture in a 125 ml shake flask at a concentration of 1 x 10⁶ cells/ml.
4. Use a suitable transfection kit for the cell line in use. The plasmid DNA to transfection reagents ratio will be dependent on the cell line and vector. It is recommended that this is optimized by testing a range of ratios within the range specified by the manufacturer prior to conducting the final transfections. Also maintain at least one negative control culture containing the cells but no DNA.
5. Incubate for 4 days in static mode and then transfer to a shaking platform rotating at 125 rpm.
6. Add the appropriate antibiotic for plasmid selection to a suitable concentration as determined by a kill curve. In this study, zeocin was added to a final concentration of 400 μg/ml for selection of transfected cells.
7. Change media at an appropriate time interval for the cell line, adding antibiotic each time, until cells in control well have died.
8. Use the most confluent wells to progress to shaking cultures.
9. Establish a cell bank by freezing the cells in cryogenic vials containing 10⁷ viable cells in 1 ml of freeze mix. In this work, this consists of 92.5% growth medium and 7.5% dimethyl sulfoxide.

3. Batch and Fed-batch Cell Growth Curve

1. Follow instructions for cell maintenance above to establish triplicate cell cultures of the transfected CHO cells in 250 ml shake flasks with a working volume of 50 ml.
2. Maintain the cultures in a humidified cell incubator at 37 °C, with 5% CO₂, on an orbital shaking platform rotating at 125 rpm, and remove 4.1 ml samples from each growing culture at 24 hr intervals.
3. Use 100 μl of the sample to determine the viable cell concentration and cell viability with the trypan blue dye exclusion method.
4. Repeat until the viable cell concentration is reduced to zero.
5. Repeat for the fed-batch cell cultures supplementing with the appropriate amount of glucose or glutamine on day 6 to restore their concentrations to their initial values of 36 mM and 4 mM, respectively. It is recommended that additional control cultures are also maintained, which are to be fed with the same volume (12 ml in this case) of pure water.

4. FRET Ratio Measurements

1. Take 2 ml of the daily samples removed in step 3.2 above and centrifuge at 100 x g for 5 min at 4 °C.
2. Resuspended the cell pellet in 2 ml of ice cold phosphate buffered saline (PBS). Transfer this into a 6-well plate and add a blank sample containing no cells to one well.
3. Measure the levels of blue and yellow fluorescence immediately at an excitation wavelength of 430/435 nm and emission wavelengths of 465/435 nm (blue) and 520/510 nm (yellow).
4. Calculate the FRET ratios (ratio of yellow fluorescence detected over blue fluorescence detected).

5. Metabolite Assays

1. Take 2 ml of the daily samples removed in step 3.2 above and centrifuge at 100 x g for 5 min at 4 °C. Remove the supernatant.
2. Resuspend the cell pellet in 3 ml ice cold PBS and centrifuge again at 100 x g for 5 min.
3. Remove the supernatant and resuspend the cell pellet in 3 ml ice cold PBS as above. Sonicate the sample 5x for 3 min each at a pulse of 15 sec on and 15 sec off. At this point, the cell extracts may be frozen if desired.
4. Use an appropriate glucose assay kit as per the manufacturer’s instructions on samples of cell extracts to determine the intracellular glucose concentration (see table of materials).
   Note: Use appropriate standards to construct a standard curve. In this work, the concentration of standards ranged between 0-100 mM.
5. Using the readings for the standards, construct the standard curve and find the linear equation of best fit (in this case: y = 1310.51x - 723.43 where y is the absorbance reading and x is the glucose concentration).
6. Calculate the glucose concentrations using the standard curve and taking the dilution into account. The intracellular glucose concentration can then be calculated using this equation:
   
   For CHO cells, the single cell volume was calculated using a cell diameter of 12 μM ¹².
7. Use an appropriate glutamine assay kit as per the manufacturer’s instructions on samples of cell extracts to determine the intracellular glutamine concentration (see table of materials).
   Note: Use appropriate standards to construct a standard curve. In this work, the concentration of standards ranged between 0-2 mM.
8. As in 5.5 above, construct the calibration curve (in this case y = 203.1x + 17.1 where y is the absorbance reading and x is the glucose concentration)
9. Using the standard curve, calculate the glutamine concentration of the samples, taking the dilution into account. The intracellular glutamine concentration can be calculated using Equation 1 above.

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