The overall goal of this procedure is to monitor the level of intracellular glucose or glutamine non-invasively using genetically encoded protein-based biosensors. This is accomplished by first transecting the cells of interest with the biosensor gene containing plasmid and selecting for those cells expressing the biosensor. In the second step, cell culture samples are taken from batch or fed batch cultures and their fret ratios are measured.
The intracellular glucose or glutamine concentration of the samples can then be determined by an independent assay. Ultimately, the fret ratio of the samples can be used to calculate the intracellular concentration of glucose or glutamine non-invasively and in real time. The implications of this technique extend toward the improvement of bioprocesses because this is a reliable method for obtaining real time metabolic information that can be used for online optimization and control.
Though the method can be used to provide insight into bioprocessing, it can also be used in other systems, including the sensing of disease markers. Begin by reviving CHO cells In nine milliliters of complete growth medium. Then spin down the cells, resuspend the pellet in 10 milliliters of fresh growth, medium, and count the cells by tripe in blue die exclusion.
Next, initiate a culture at a seeding density of three times 10 to the fifth cells per milliliter in 125 milliliters. Shaker flasks at 37 degrees Celsius and 5%CO2 on an orbital shaking platform. With a 125 RPM rotation, subculture the cells every three to four days in the complete growth medium at a seating density of two times 10 to the Fifth cells per milliliter for cell transfection.
Maintain the Cells at 37 degrees Celsius and 5%CO2 for 12 to 24 hours prior to the transfection to ensure that the cells are actively dividing. Then after preparing the plasmid DNA, count the cells by tripe and blue die exclusion and dilute the cells to a working volume of 20 milliliters of cell culture media. In a 125 milliliter, shake flask at a concentration of one times 10 to the sixth cells per milliliter.
Use a suitable transfection kit for the cell line in use, including at least one negative control well containing cells, but no DNA. Incubate the transfected cells for four days in static mode. Now add the appropriate antibiotic for plasmid selection to a suitable concentration as determined by a kill curve.
For example, in this study, zin was added to a final concentration of 400 grams per milliliter for selection of the transfected cells then transfer the cultures to a shaking platform rotating at 125 RPM. Change the media at an appropriate time interval for the cell line, adding antibiotic each time until the cells in the control well have died. Use the most confluent wells to progress to the shaking cultures.
Then freeze the cells in cryogenic vials containing one times 10 to the seventh viable cells in one Milliliter of freeze mix to generate batch and fed Batch growth curves. First, establish triplicate cell cultures of the transfected CHO cells in 250 milliliter Shake flasks with a working volume of 50 milliliters. Then place the cultures in a humidified cell incubator with shaking and remove 4.1 milliliter samples from each growing culture at 24 hour intervals For the Fed batch cultures, supplement the cells with the appropriate amount of glucose or glutamine on day six to restore their concentrations to their initial values.
Feeding the control cultures with the same volume of pure water use 100 microliters of the 4.1 milliliter samples from 4.2 daily to count the cells by trip and blue die exclusion until the viable cell Concentration is reduced to zero. To determine the fret ratio Measurements, centrifuge two milliliters of the daily samples and resuspend the pellet in two milliliters of ice cold PBS transfer half of the cell suspension into a six well plate and add a blank sample containing no cells to one. Well then immediately measure the levels of blue and yellow fluorescence at an excitation wavelength of 430 with a bandwidth of 35 nanometers and a mission wavelengths of 465 with a bandwidth of 35 nanometers and 520 with a bandwidth of 10 nanometers for the blue and yellow fluorescence respectively.
Calculate the fret ratios by determining the ratio of the yellow fluorescence detected to that Of the blue fluorescence. Now spin down the final two milliliters of The daily samples and then wash the pellet twice in ice cold PBS after the second wash. Sonicate the sample five times for three minutes each time at a pulse of 15 seconds on and 15 seconds off.
Finally, perform the metabolite assays according to the manufacturer's instruction, and use the resulting standard curves to calculate the intracellular glucose or Glutamine levels as appropriate. In this representative Experiment, batch overgrown cultures of two cell lines were maintained as just demonstrated with daily samples used for the in vivo calibration of each fibs. A representative viable cell growth profile of the two cell lines in relation to their intracellular glucose or glutamine levels is demonstrated in this graph and the corresponding fret measurements and intracellular metabolite concentrations determined by the enzymatic assays are presented in this table.
It was determined that the low cell numbers present in the first four days of culture led to an unreliable fret signal. Similarly, the high level of cell lysate present after day eight of culture produced a high amount of light scattering. Therefore, only data for days four to eight is used to construct the calibration curves for the glucose and glutamine fibs.
The results indicate that the FIB signal produces a reliable correlation with the intracellular concentrations between one and five millimolar for glucose and 0.3 and two millimolar for glutamine. To validate the fibs findings, fed bash overgrown cultures of the two cell lines were performed. A feed containing a high substrate concentration was supplemented on day six of the cell culture to elevate the extracellular concentrations of the substrate.
As the grafts depict the fret ratios on each day exhibit a clear response to the feeding. By reversing the trend they followed up to day six. For example, in the glucose fed cultures, the extracellular glucose concentration increased back up to 36 millimolar.
While in the glutamine fed cultures, the glutamine concentration increased to four millimolar. In the glucose experiment, the fret ratio for the glucose fibs chose cell line decreased on day seven in response to the addition of glucose. Since the glucose fibs follows the APO on configuration.
Similarly, in the glutamine experiment, the fret ratio for the glutamine fibs chose cell line increased in response to the addition of glutamine in line with the principle of the APO off sensors. These fret measurements were finally used to calculate the corresponding intracellular glucose and glutamine concentrations based on the aforementioned calibration curves. They're compared to the actual intracellular concentrations of these substrates as determined with the glucose and glutamine enzymatic assays, And show adequate agreement After its development.
This technique paves the way for researchers in the field of to explore the effect of different medium formulations on protein productivity. After watching this video, you should have a good understanding of how to use genetically encoded biosensors to monitor intracellular metabolite concentrations.
