Patent Application
20190383787
N/A
N/A
Use of radio frequency (RF) to characterize of cell cultures presents a problem of measuring biologic materials nondestructively because the exposure of cultures at sufficient energy to perform spectroscopy or tomography causes degradation of the cells or other fragile biologic material such as proteins.
Biopharmaceutical process characterization focuses on parameters such as cell growth, yield, product quality, temperature, pH, pO2, pCO2, and metabolite levels. Biological systems are very sensitive to process changes, resulting in unviable bioproducts. Current manual measurement techniques are inadequate to provide accurate real-time automated viability testing, by either being limited by image analysis or involving destructive testing of the culture, by complex microfluidic chambers to isolate small samples of the specimen, or by the inability to non-destructively probe three-dimensional cell cultures. Adequate monitoring of cell cultures is needed in testing specificity and response time to provide the needed relative cellular health feedback that will be used to increase quality and decrease in production time and costs. Expensive carefully prepared cell lines may be lost due to the delays incurred in testing and performance mitigating responsive actions.
The better known transmission tomographic images are generally formed by illuminating an object with x-rays or microwaves and measuring the energy that passes in a straight ray propagation, the measurement can be the amplitude or the time of arrival of the received signal; with reflection tomography, the reflected, and refracted signal can be measured. The use of penetrating RF signals, and the reconstruction of the signals after passing through targets of interest to identify subsurface features has been proven a successful technique for biomedical imaging applications, such as Computed tomography (CT) of the heart, abdomen, and pelvis, as well as MRI. These tomographic images are formed by illuminating an object with energy such as x-rays, microwaves, or ultrasound; and measuring the energy that passes straight ray propagation. The antennas are typically arranged to surround the target with transmitting antenna moving through a circle.
Currently, the use of dielectric or reflective RF tomography has been limited to determining the presence or amount of biomass. At power levels currently used these applications are destructive, damaging cells, tissues, or other biologic materials such as proteins.
To address the need for real time monitoring of 2 dimensional (2D) and 3 dimensional (3D) cellular cultures, a combined radio frequency capacitive and reflective tomography device is used to monitor for tissue viability and purity. Cellular properties strongly influence their dielectric properties, across the vast range of DC to RF and millimeter wave frequencies. By monitoring both the dielectric response versus frequency in the near field, as well as measuring the far field propagation and reflection across an ultra-wide bandwidth, the overall cellular health can be rapidly and non-invasively measured. The use of a variety of capacitive sensors, a far field antenna array, and an embedded analysis system for rapid analysis feedback of the condition of the sample. These systems will require minimal training and can be used by ordinary users.
The proposed technology will also provide the identification of contamination through identification of other cells and debris from their different dielectric responses, as well as the cell viability. Physical properties of samples change as their its relative health degrades.
The device uses highly sensitive RF detectors characterizing biomaterial, chemical species, and RF spectroscopy. The increase in sensitivity of RF sensors allows for non-destructive cellular culture and biologic material analysis for real time measurements of cell health and viability.
In order to perform real time monitoring of 2D and 3D cellular samples a device combining radio frequency capacitive and tomography device to monitor for tissue viability and purity is described. Cellular properties strongly influence their dielectric properties, across the range of DC to RF and millimeter wave frequencies, and by monitoring both the dielectric response versus frequency in the near field, as well as measuring the far field propagation and reflection across an ultra-wide bandwidth, overall culture health can be rapidly and non-invasively measured. One embodiment of a standalone device and system that receives biomass samples. Another embodiment is configured to measure larger 3D cell cultures in special conditions. The device has a variety of capacitive sensors, a far field antenna array, and an embedded analysis system for rapid analysis feedback. These systems will require minimal training and can be used by low tech users.
The described technology will also provide the identification of contamination through identification of other cells and debris from differences in its dielectric responses compared to the targeted biological sample, as well as the cell viability as the physical properties of the cells change as their relative health degrades. A sensitive Radio Frequency (RF) sensor system, such as the Nokomis Hiawatha™ will be used to collect electromagnetic emissions and responses, convert the signals into the frequency domain, and perform spectral analysis. This technology offers extensive capability and sensitivity in RF sensing of chemical species, biomaterial characterization, and RF spectroscopy. The described technology provides a reliable, non-destructive cellular culture analytical device for real time measurements of cell health and viability.
Dielectric spectroscopy is a near field measurement technique that is based on the passive electrical properties of a material, the capacitance C, and the conductance G. The sample is placed between two electrodes with an oscillating electric field; the material properties of the sample between the two electrodes respond with the changing field to amplify or attenuate the signal depending of the frequency of oscillation. The relative permittivity et and conductivity k describe the relation independent from the electrodes given by the following equations:
These apply for parallel plate electrodes with a surface area A with a separation d. Specifically, for a suspension of ideal spherical cells with radius r and the capacitance per membrane unit are Cm, the capacitance is:
Where P is the volume fraction of the cells in the suspension volume between the parallel plate electrodes, and the cell volume is given as 4/3πr3. With a cell density per unit volume N the capacitance can be made a function of the cell radius and correlates linearly with the cell density per membrane unit area:
Using these fundamentals, tissue health can be identified using these principles.
The solid lines correspond to the current flow due to low frequency signals, while the dashed lines correspond to current paths at higher frequency signals. At higher frequencies the current is able to permeate cell membranes, which effectively probe their physical properties. Low frequencies do not permeate the cell membranes due to the polarized nature of cells. At low frequencies the cells are able to rotate and align with the fields. At higher frequencies the RF field is able to permeate the cell membrane and allows for measurements inside the cell. For bulk cellular cultures physical properties of the sample will change as the cells become unviable or deviate from the intended bioproduct. In
Near field dielectric spectroscopy measurements can be performed on both 2D and 3D cell cultures. First, broadband RF spectral data is acquired. Broadband data is collected with the intact cell culture. Wide band spectral measurements will be collected with frequencies ranging from 1 MHz to 1000 MHz to cover the range of measurements relevant to cellular environments. These parameters including: biomass, cell size, intracellular matrix, cellular membrane health, subcellular matrix properties.
Potential harm can come to cell cultures, particularly 3D cultures, if the cells try to align with the electric fields. The dielectric spectroscopy measurements described will utilize very low power, typically less than 10 mW. The ability to use low power for these measurements is enabled by use a very sensitive Radio Frequency (RF) sensor system, such as the Nokomis Hiawatha™, capable of sensitivities of −170 dB.
An advantage to using reflective tomography is that it can operate at a lower emission power in that it is not necessary to completely penetrate the target sample.
In reflective tomography, unlike transmission tomography, the antenna or antennas are arranged to receive the reflections. Where more than one receiving antenna is used one embodiment is a generally linear alignment of the antennae. Another embodiment is to arrange the antennae in a generally semicircular fashion.
The signals reflect, refract, and deflect at specific angles, dependent on the material properties at each interface. See
The RF tomography approach, utilizing 1-40 GHz, will enable property measurements at all levels of the culture, layer by layer.
A perspective view of a resonant cell is shown in
A sensitive detector preferably has a sensitivity of −170 dB at room temperature, 4 dB higher than the theoretical limit. A standard range is between 30 MHz to 1000 MHz, but the detector is not limited to this range and can be modified by replacing the tuning system for different frequency range. A FPGA/CPU 556 receives the signal from the ADC 514 and preferably, a clock 570. The FPGA has a display controller 510 and a keyboard controller 512. The FPGA communicates with RAM memory 518 and preferably an EPROM 538. The FPGA outputs to USB physical layer 550 at output port 552. The unit 500 has an internal and/or external power supply 560.
Another embodiment is shown in
The biologic property measurement device 630 receives the signals from the sensitive detector and applies wavelet denoising 622. It then integrates the time or frequency bins. The frequency domain pattern is enhanced with noise cancelation 618 and a signature is created 616. The frequency domain signal processing pattern recognition 624 is performed against a known signature base line pattern. A threshold is applied against the processed signal to determine whether the sample status is abnormal.
The apparatus has at least one logic circuit to combine a plurality of signals received from an array of a plurality of antennas. The combining of signals and windowing the signals creates of more coherent signal suitable for analysis.
The presently preferred embodiment of the device is benchtop system as shown. It can be configured to assess 3D cell cultures, tissue samples and proteins.
Another embodiment would be to have the apparatus continuously monitoring cell cultures in an industrial setting.
Another embodiment would be to have the apparatus monitoring food for the presence of contamination by bacteria.
Another embodiment of the device is configured to monitor existing cell culture incubators to provide real-time assessment of cellular health.
