In-cell NMR spectroscopy offers a unique approach to study protein structure and function directly in living cells and to monitor biologically relevant processes such as protein-ligand interactions. This NMR bioreactor can keep a high number of human cells viable and metabolically active for several days in the spectrometer, enable real-time in-cell NMR applications. This method can be applied to any soluble, freely tumbling intracellular protein undergoing conformational or chemical changes.
Isotope labeling further extends the applicability of the method. To begin, assemble the flow unit using a second flow unit NMR tube, which will later be replaced with the one containing cells. Refer to the flow unit operating instructions for correct assembly.
Set the water bath connected to the flow unit temperature control to 37 degrees Celsius and place the reservoir bottle in the water bath. Connect the reservoir bottle's FEP tubing to the pump. Turn the bioreactor valve to bypass and pre-fill the pump with medium.
Then, turn the bioreactor valve to flow and pre-fill the bioreactor with the medium at 0.1 milliliters per minute. Take a T75 flask of transfected HEK 293 T cells from the carbon dioxide incubator and remove the spent medium. Add two milliliters of trypsin EDTA to the cells and incubate for five minutes at room temperature to detach the cells.
Inactivate the trypsin with 20 milliliters of complete DMEM and thoroughly re-suspend the cells by pipetting up and down. Then, transfer them in a 50-milliliter centrifuge tube. Centrifuge the cells at 800X G for five minutes at room temperature and discard the supernatant.
Transfer the cell pellet to a 1.5-milliliter capped microcentrifuge tube. To embed the cells in agarose threads, melt one aliquot of solidified agarose at 85 degrees Celsius in a block heater, then keep it in solution at 37 degrees Celsius in the block heater. With a Pasteur pipette, fill the bottom of the flow unit NMR tube with 60 to 70 microliters of 1.5%agarose gel and place it on ice to create a five-millimeter-high bottom plug.
Heat the cell pellet for 15 to 20 seconds in the block heater and carefully re-suspend the cells in 450 microliters of agarose solution, avoiding the formation of bubbles. Aspirate the cell/agarose suspension into a 30-centimeter-long chromatography PEEK tubing of 0.75-millimeter inner diameter connected to a one-milliliter syringe. Let the tubing cool at room temperature for two minutes.
Pre-fill the flow unit NMR tube with 100 microliters of PBS at room temperature. Cast threads of cells embedded in agarose into the flow unit NMR tube by gently pushing the syringe. Remove the empty NMR tube from the flow unit and increase the flow rate to two milliliters per minute for a few minutes to remove residual gas bubbles in the inlet tubing.
Set the flow rate to 0.2 milliliters per minute and insert the NMR tube containing the cells by pushing it upwards slowly but steadily. Supply the bioreactor medium at a flow rate of 0.1 milliliters per minute. Set the temperature in the NMR spectrometer to 310 Kelvin and insert the flow unit in the spectrometer.
Once the bioreactor is inserted in the NMR spectrometer, wait a few minutes to allow the medium exchange. Adjust the matching and tuning of the proton channel, shim the magnet, and calculate the proton 90-degree hard pulse length. Adjust the proton power levels in each pulse sequence according to the proton hard pulse.
Record a first zgesgp proton NMR spectrum to record the sample content in field homogeneity. Copy the zgesgp and the p3919gp or the SOFAST-HMQC experiments to the desired number and queue them in the acquisition spooler. Inject a concentrated solution of the external molecule to the medium reservoir bottle by piercing the silicone tubing with a sterile, long-needle syringe.
At the end of the NMR experiment, replace the tube containing the cells with an empty tube and rinse the flow unit with water. For cells expressing unlabeled carbonic anhydrase, process the p3919gp spectra by applying zero-filling and exponential line-broadening window function. For cells expressing nitrogen-15 labeled, superoxide dismutase, process the SOFAST-HMQC spectra by applying zero-filling and squared-sign bell window function in both dimensions.
In MATLAB, import the spectral regions using the custom script Load_ascii_spectra. Run the Load_acqus script to extract the timestamps from the 1D spectra. Open MCR-ALS 2.0 GUI by running the mcr_main script, and in the Data selection tab, load the spectra matrix.
Check the data by plotting it. Evaluate the number of components, either by singular value decomposition or manually, and select a method for the initial estimation of the pure spectra. Either purest variable detection or evolving factor analysis can be used.
In the Selection of the dataset window, select Continue. Set the constraints for the concentrations in the Constraints:row mode window. Apply a non-negativity constraint, selecting fnnls as Implementation, and two as the number of species.
Then, apply one closure constraint, set the constraint to one, the closure constraint condition as equal to, and apply to all species. Set the constraints for the spectra in the Constraints:column mode window. Apply a non-negativity constraint, selecting fnnls as implementation, and two as the number of species.
In the final window, set 50 iterations and a 0.01 Convergence criterion. Specify the Output names for concentrations, spectra, and standard deviation. Click on Continue to run the MCR-ALS fitting.
The cells in the bioreactor can be maintained alive for up to 72 hours, as confirmed by a trypan blue test. The bioreactor was used for real-time monitoring of the binding of two inhibitors, acetazolamide and methazolamide to carbonic anhydrase overexpressed in the cytosol of HEK 293 T cells. The presence of signals from the overexpressed protein and the field homogeneity was assessed with the first excitation-sculpting proton spectrum.
In the case of carbonic anhydrase, the intracellular binding of the two inhibitors was monitored by observing proton signals in the region between 11 and 16 parts per million of the WATERGATE spectrum, arising from the zinc-coordinating histidines and other aromatic residues. Successful binding was confirmed by the appearance of additional signals. MCR-ALS was used to separate the NMR signals arising from free and bound carbonic anhydrase, and to provide the relative concentration profiles of the two species.
The bioreactor was applied to monitor the formation of zinc-bound superoxide dismutase intramolecular disulfide bond promoted by ebselen, a glutathione peroxidase mimic. MCR-ALS analysis on selected regions of the 2D spectrum separated the signals arising from the two species and provided their relative concentration profiles. It is important to insert and remove the flow unit NMR tube slowly and steadily to avoid pressure jumps that may cause the formation of bubbles.
The development of NMR bioreactors has paved the way to study atomic resolution time-dependent processes, such as plotting redux regulation and drug/target interactions in living cells.
