Patent Application
20210071129
Disclosed is a cell culture incubator system having a sensor strip configured to be placed within a reaction vessel (e.g., incubator vessel) so as to position the sensor within media covering the cells. A reader is placed outside but adjacent to the vessel to read the sensor so as to detect changes in dissolved O2 and pH. The system is used to determine if the incubator environment has too much CO2 and is therefore trending towards hypoxia and/or acidity, or has too much dissolved O2 and is therefore trending toward oxygen toxicity via the cell nutrient media. In some embodiments, the system includes a rocker unit configured to rock the reaction vessel to enhance cell growth. In some embodiments, the system includes a gas circulation system to adjust the CO2 and dissolved O2 levels in the reaction vessel.
Measuring dissolved oxygen (“DO”) and pH can provide a reliable approach to monitoring cell cultures. For instance, the biomass of growing cells can cause an increase in lactic acid within the cell growth media (e.g., nutrient media), which can be due to an end-product of cell metabolism. An increase in lactic acid can cause a steady decrease of pH level in the cell growth media, and the presence of DO can be an indicator of the health of the cell culture environment. Generally, optimal levels of DO in the cell growth media provide a healthy environment for cell growth. Thus, pH levels and DO levels can be indicators of conditions within the cell culture. For example, DO saturation with a positive rate of change can be indicative of equilibrium, whereas decreasing level of pH below a certain amount or at a certain rate can be indicative of over-growth in the cell culture (i.e., may require a subculture). Other conditions that can be determined may include, but are not limited to, an onset of programmed cell death (e.g., apoptosis), growth toward equilibrium, deviations from equilibrium, etc.
Changes in DO levels and pH levels can be monitored by placing a substrate within the cell growth media, the substrate being configured to generate light via fluorescence techniques. For example, a fluorescence-based patch (e.g., a sensor patch) containing fluorescent dye immobilized in a matrix can be placed within a reaction vessel containing the culture sample. Elicitation of fluorescence from chemicals within the patch can be performed, where emitted light due to induced fluorescence can be monitored via a reader (e.g., electro-optics and/or other system components) located outside the reaction vessel. Thus, electro-optical measurements can be conducted on the cell culture in a minimally invasive and/or non-invasive manner. Furthermore, the patch can be structured such that emitted light due to the fluorescence can occur at different wavelengths, the wavelengths being a function of changes in DO levels and/or pH levels occurring within a growth media.
Conventional cell culture incubator systems can be appreciated from U.S. Pat. No. 4,839,292; US 2008/0024779; US 2011/0188053; RA.GUPATHY, V. et al., Non-Invasive Optical Sensor Based Approaches for Monitoring Virus Culture to Minimize BSL3 Laboratory Entry, Sensors, 2015, 14864-14870, Vol 15, MDPI, Basel, Switzerland; GE, X. et al., Low-Cost Noninvasive Optical CO2 Sensing System for Fermentation and Cell Culture, Biotechnology and BioEngineering, 2005, 329-334, Vol 89, No. 3, Wiley Periodicals, Inc.; GE, X. et al., Validation of An Optical Sensor-Based High-Throughput Bioreactor System for Mammalian Cell Culture, Journal of Biotechnology, 2006, 291-306, Vol. 122, Elsevier.com; RAO, G et al., Disposable Bioprocessing: The Future Has Arrived, Biotechnology and BioEngineering, 2009, 348-356, Vol 102, No. 2, Wiley Periodicals, Inc.; HANSON, M. A., Comparisons of Optical pH and Dissolved Oxygen Sensors with Traditional Electrochemical Probes During Mammalian Cell Culture, 2007, 833-841, Vol. 4, No. 4, Wiley Periodicals, Inc.; and Schiefelbein, et. al “Oxygen supply in disposable shake-flasks: prediction of oxygen transfer rate, oxygen saturation and maximum cell concentration during aerobic growth”, Springer Science+Business Media. Jan. 10, 2013. Conventional systems are limited in that they generally require a reaction vessel to include a sensor patch already secured or attached to the reaction vessel, which in turn may require the sensor patch to be sterilized before use. In addition, such a set up requires proper alignment between the sensor patch and the reader. Conventional systems are further limited in that they fail to provide any means to effectively perturb the media within which the cells are grown. Perturbing the media, however, can enhance growth of the cells. Conventional systems are further limited in that they fail to provide an adequate means to adjust the DO and CO2 levels if they are detected to be at undesired levels.
Embodiments can relate to a cell culture incubator system having a sensor strip configured to be placed within a reaction vessel. The sensor strip can be configured so that when inserted, the sensor is positioned within media covering the cells. A reader is placed outside but adjacent to the vessel to read the sensor so as to detect changes in dissolved O2 and pH. The system is used to determine if the incubator environment has too much CO2 and is therefore trending towards hypoxia and/or acidity, or has too much dissolved O2 and is therefore trending toward oxygen toxicity via the cell nutrient media. Other conditions that can be determined may include the onset of apoptosis, growth toward equilibrium, deviations from equilibrium, etc.
In some embodiments, the system includes a gas circulation system to adjust the CO2 and dissolved O2 levels in the reaction vessel.
Some embodiments can include an incubator system in which a plurality of reaction vessels is being monitored. The incubator system can be a controlled housing configured to adjust lighting, temperature, pressure, etc. for the plurality of reaction vessels. The incubator system can house the plurality of reaction vessels so that each reaction vessel is in fluid communication with the gas circulation system. The gas circulation system can adjust the CO2 and dissolved O2 levels of each individual reaction vessel separately or in unison.
In some embodiments, the system includes a rocker unit configured to rock the reaction vessel to enhance cell growth.
In some embodiments, the system includes a rotation unit configured to rotate the reaction vessel to further enhance cell growth.
In some embodiments, the system can be part of or connected to a computer device or a computer system. With such embodiments, a feedback loop can be created between the reader, the gas circulation system, the incubator housing, and/or the rocker to monitor the CO2 and dissolved O2 levels in real-time and to adjust the CO2 and dissolved O2 levels, the rocking, the rotation, the temperature, the lighting, the pressure, etc. in response thereto.
Further features, aspects, objects, advantages, and possible applications of the present invention will become apparent from a study of the exemplary embodiments and examples described below, in combination with the Figures, and the appended claims.
The above and other objects, aspects, features, advantages and possible applications of the present invention will be more apparent from the following more particular description thereof, presented in conjunction with the following drawings, in which:
The following description is of an embodiment presently contemplated for carrying out the present invention. This description is not to be taken in a limiting sense, but is made merely for the purpose of describing the general principles and features of the present invention. The scope of the present invention should be determined with reference to the claims.
Referring to
The sensor strip 1 can be a rigid or semi-rigid member (e.g., polymer, plastic, glass, metal, etc.) configured to be removably inserted into the reaction vessel 115. It is contemplated for the sensor strip to be made from clear (e.g., transparent or translucent) plastic material. The sensor strip can have at least one sensor 2 disposed or attached to a portion of the strip. The shape and configuration of the strip, as well as the placement of the sensor thereon, can be such that the sensor resides within the media when the sensor strip is inserted within a reaction vessel. For instance, the sensor strip can have a first end 3 and a second end 4, with an elongated shaft extending between the first end and the second end. The elongated shaft can be straight, bent, angled, etc. so that after the first end 3 (e.g., a distal end) is inserted into the reaction vessel, it rests on a bottom portion of the reaction vessel 5. It is contemplated for the sensor to be located at or near the first end, but it can be located at any portion of the strip. The details of the sensor and the reader will be explained later, but it is contemplated for light to be imparted on the sensor and for the sensor to emit light based on the levels of dissolved O2 and/or pH of the media within which it is submerged. Thus, the strip portion where the sensor is located should be transparent to the anticipated (discussed in detail later) light that will be imparted to the sensor and emitted therefrom.
Referring to
As a non-limiting example, the sensor strip can be an elongated shaft with a first end having a sensor disposed thereon and a second end having a stopper disposed thereon. The elongated shaft can extend straight from the second end and form an L-shape that leads to the first end. Such an L-shape sensor strip 12 can be used for a vertically arranged reaction vessel 9 (see
In operation, the sensor strip can be sterilized and packaged into a sealed container or bag. When it is desired to have the dissolved O2 and/or CO2 level(s) of an incubator environment of a reaction vessel measured, a user can un-package the already sterilized sensor and insert it within the reaction vessel (obviating the need to buy expensive vessels with sensors already attached and obviating the need to sterilize the sensor before use). A reader 200 can be placed adjacent, but outside the reaction vessel. The reader 200 can be used to accurately detect the dissolved O2 or CO2 (or pH levels) by receiving and analyzing light being emitted from the sensor. The inventive system can provide in a quick, accurate, quantitative measurement of dissolved O2 or CO2 in an efficient and cost-effective manner (the details of how the sensor and the reader and can be used to generate a quantitative measurement of dissolved O2 or CO2 from light being emitted from the sensor will be discussed later). It should be noted that the inventive system can also obviate the need to properly align a reader with a sensor that has already been attached to the reaction vessel, as is required for existing systems.
Referring to
For instance, the incubator system 14 can include a rotation unit 15 having a jig upon which the reaction vessel is placed and/or secured thereto. The incubator system 14 can be configured so that when the rotation unit 15 is activated, it causes the jig to rotate in a circular or precession-like manner 16. The jig can be a dual-clamp assembly configured to clamp to an outside portion of the reaction vessel at the bottom of the reaction vessel. The jig can also clamp a reader 200 so that it is secured to the reaction vessel at the bottom of the reaction vessel, thereby facilitating unison rotation of the jig, the reaction vessel, and the reader 200 when the rotation unit 15 is activated. When the sensor strip is inserted into the reaction vessel and the reaction vessel is caused to rotate, the reader 200 is at-all times aligned with the sensor (due to the jig(s)) so as to impart light thereon and receive light therefrom.
Some embodiments can include a rocker unit. The rocker unit can be a motor in connection with a rocker arm assembly, a cam assembly, etc. that causes the reaction vessel to rock or oscillate so as to perturb the media. The perturbation of the media can involve causing the media to slosh in a reciprocating motion in a linear direction of the rocking motion. It is contemplated to perturb the media gently so the rocking or oscillatory motion should be at a low level of angular frequency and angular displacement (e.g., rock the reaction vessel within a range 17 from −8 to +8 degrees from a horizontal position 18—the horizontal position being a 0-degree angle of displacement). Some embodiments can include a platform configured to hold a plurality of reaction vessels so as to rock a plurality of reaction vessels (all being rocked in unison, any one or combination being rocked at different angular frequencies and/or angular displacements, etc.). Again, a jig can be used to secure a reader 200 to an outside of a reaction vessel (or a plurality of jigs for a plurality of reaction vessels) so that the reader(s) 200 can effectively impart and receive light to and from the sensor(s) of the sensor strip(s) that has/have been placed within the rocking reaction vessel(s). When the sensor strip is inserted into the reaction vessel(s) and the reaction vessel(s) is/are caused to rock, the reader(s) is/are at-all times aligned with the sensor(s) (due to the jig(s)) so as to impart light thereon and receive light therefrom.
Perturbing the media gently can enhance the growth of the cells by causing the cells to be exposed to more dissolved O2 than they otherwise would be. This can be attributed to the sloshing of the media so as to temporarily uncover the cells from the media as the reaction vessel is rocked. CO2 is heavier or more dense than dissolved O2 so the CO2 generally resides as a bed of gas on the media, thereby reducing the amount of dissolved O2 being exposed to the cells. Gently perturbing the media, however, can disturb this CO2 bed. For instance, gently perturbing the media can cause the media (taking some or all of the CO2 bed with it) to flow towards an end or edge of the reaction vessel, allowing for more dissolved O2 to be exposed to the cells than otherwise would have been if the CO2 bed would not be disturbed. This can lead to a 1.8× increase (e.g., an increase within a range from 0.1× to 1.8×) in cell growth. It should be noted that such a rocker system would benefit from using adherence cells (cells that adhere to the bottom of the reaction vessel) so that the cells remain in place at the bottom of the reaction vessel when the reaction vessel is rocked.
Referring to
Some embodiments can include a platform configured to hold a plurality of reaction vessels. The gas circulation system can include a gas manifold and/or multiplexer configured to facilitate introducing and/or removing gas to and from any one or combination of the plurality of the reactions vessels. For instance, the gas supply lines from the gas inlet/outlets of the individual reaction vessels can be connected to the gas supply or supplies via the gas manifold/multiplexer. Other components such as filters, humidifiers, heaters, etc. can be used to purify and/or condition the gas before it is introduced into the reaction vessel(s). The gas in all of the reaction vessels can be adjusted in unison, the gas in any one or combination of the reaction vessels can be adjusted at different rates, concentrations, volumes, etc.).
Some embodiments can include a discharge tank equipped with pumps, compressors, line or hoses, values, couplings, regulators, etc. to provide a pressurized system (e.g., negative pressure) that is a gas reservoir in fluid communication with the reaction vessel and further configured for receiving gas from the reaction vessel. This can facilitate removal of gas from a reaction vessel. Gas supply lines can extend from the gas inlet/outlet to the discharge tank.
In one embodiment, the incubator system includes a housing 23 that may comprise a lid. The lid can be attached to the housing via a hinge. In some embodiments, the lid can include a seal or gasket to provide a fluid-tight seal between the lid and the housing. The housing can define a volume of space within which at least one reaction vessel is held. The housing can have the gas manifold and/or multiplexer to facilitate directional flow of the gas from the gas source(s) to the reaction vessel(s). In some embodiments, the housing can be configured to provide a positive pressure within the volume of space so as to inhibit or prevent unwanted gases from entering the housing and possibly entering the incubation environment of the reaction vessel(s). The housing can also be equipped with a heater, a humidifier, lighting, etc. to provide a conditioned environment for the cell cultures within the reaction vessel(s) placed therein.
It should be noted that embodiments of the system can be configured to have any one or combination of the platform to accommodate a plurality of reaction vessels, the rotation unit, the rocker unit, or the gas circulation system. In addition, any embodiment of the system can include a sensor strip and a reader for detecting and measuring levels of CO2 and dissolved O2. As will be explained herein, the incubator system can include a computer device. This can be done to generate a computer system. With the computer device and/or computer system, a feedback loop can be created between the reader, the gas circulation system, the incubator housing, the rotary unit, and/or the rocker unit to monitor the CO2 and dissolved O2 levels in real-time and to adjust the CO2 and dissolved O2 levels, rocking, rotation, temperature, humidity, lighting, pressure, etc. in response thereto. This can include monitoring the CO2 and dissolved O2 levels of each reaction vessel individually or in unison, as well as adjusting adjust the CO2 and dissolved O2 levels, rocking, rotation, temperature, humidity, lighting, pressure, etc. of each reaction vessel individually or in unison.
Some embodiments can be configured as a kit. For instance, a kit can include one or more sensor strip(s) with one or more of reaction vessel(s) and/or one or more reader(s).
A detailed discussion of embodiments of the sensor and the reader will be discussed net.
Referring to
In some embodiments, at least one hub box 118 can be used to acquire data from a plurality of reaction vessels 112. In further embodiments, the hub box 118 can be used to determine a life of the patch 102, which may be defined as the time frame by which the patch 102 can effectively fluorescence. This can be done by acquisitioning code data from the patch 102. For instance, the code can be manufacturing data, for example, that may be transmitted to the computer device 110 to calculate expiration times. Other data, such as an expected offset of a patch 102 can be encoded within the code that is associated with the patch 102. This code can include information such as a date of manufacture of the patch 102, a date at which the expected offset was determined, a date the patch 102 was packaged, etc. Chemicals within the patch 102 can photo-bleach after a statistically pre-determined use has elapsed (e.g., imparting excitation light beams on the patch 102 every fifteen seconds for ninety days can cause chemicals impregnated into the patch 102 to fail to effectively fluoresce and radiate emitted light), and thus the code can include an expected expiration date based on the date of manufacture. In another embodiment, the computer device 110 can calculate the expected expiration date based on such data.
The code can further include a “calibration” date, which can include a pre-set time period after which a date the expected offset was determined. In some embodiments, the “calibration” date can indicate when the expected offset should be determined again. For example, if the date the expected offset is determined by a computer device 110 to be greater than six months from a current date of use, then the computer device 110 may transmit a signal to the hub box 118 indicating that the patch 102 should be “calibrated.” The hub box 118 can further be structured to have at least one useful life and/or calibration light indicator, indicating gradations of approaching expiration and/or calibration dates for the patch 102. For example, a green indicator light can be used to indicate that the patch 102 has at least two months before replacement and/or calibration. An amber light can be used to indicate that the patch 102 has less than one month before replacement and/or calibration. A red light can be used to indicate that the patch 102 has less than one week before replacement and/or calibration. Other indicator light schemes and/or time frames can be used.
In some embodiments, the computer device 110 can be communicatively and operably associated with a computer system 106 via a computer network. (See
At least a portion of a reaction vessel 112 can be transparent to form a reaction vessel window 122. In some embodiments, light 115a, 115b generated by a beam combiner assembly 104 can be directed to be incident upon a patch 102 located within a reaction vessel 112. The reaction vessel window 122 can be further configured to allow emitted light 117a, 117b from induced fluorescence of the patch 102 to be passed through the reaction vessel 112 so that it can be incident upon a detector 124 of the reader 200 that may be located outside of the reaction vessel 112, which may include being located on an outside surface of the reaction vessel 112 or on an outside surface of the beam combiner assembly 104. The detector 124 can be a device that is configured to detect various emitted light 117a, 117b based on wavelength and/or intensity. In some embodiments, the detector 124 can be a photodiode. In further embodiments, the detector 124 can also include a processor configured to convert the detected light into emitted light data, which can be representative of the light being detected. The detector 124 may also be configured to transmit the emitted light data to another component of the system.
Referring to
The beam combiner assembly 104 may house illumination sources, filters, mirrors, detectors, and other electro-optics. Generation of excitation light beams 115a, 115b may be achieved through use of at least one light emitting diode (“LED”), laser, or other illumination source 114a, 114b capable of generating coherent light and/or light within a very narrow bandwidth of wavelengths. Direction of excitation light beams 115a, 115b and/or emitted light 117a, 117b within a beam combiner assembly 104 can be achieved through use of waveguides, reflectors, refractors, etc.
In at least one implementation, the beam combiner assembly 104 can be positioned outside a reaction vessel 112 with a reaction vessel 112 placed adjacent thereto (e.g., placed on top) so that excitation light beams 115a, 115b generated by a beam combiner assembly 104 can be incident on the patch 102 within a reaction vessel 112 when caused to transmit through the reaction vessel window 122. For example, the beam combiner assembly 104 can facilitate resting a beam combiner assembly 104 on a flat, stable surface, which may enable placing a reaction vessel 112 on top and adjacent the beam combiner assembly window 128. For example, the reaction vessel 112 can be placed on top of the beam combiner assembly 104, where at least one excitation light beam 115a, 115b can be directed through a bottom of the reaction vessel 112 to be incident upon the patch 102 that may be attached to an inside surface of a reaction vessel 112.
The system may include a plurality of beam combiner assemblies 104. Whether there is one beam combiner assembly 104 or more than one, each beam combiner assembly 104 can be placed into electrical connection with an electrical power source to enable operation of the illumination sources 114 and/or other electro-optics. Any beam combiner assembly 104 can be further placed into electrical connection with the hub box 118. The hub box 118 can be separately placed into electrical connection with an electrical power source. As excitation light beams 115a, 115b are generated and directed to the patch 102 to induce emitted light 117a, 117b due to fluorescence, the emitted light 117a, 117b can be detected by the detector(s) 124. Excitation light beams 117a, 117b can be generated in a controlled manner via the computer device 110 (e.g., via algorithms and/or command data from users of a computer device). Emitted light data can be generated by the detector 124 and transmitted via the hub box 118 to the computer device 110 for data processing, data manipulation, and/or data analysis. In some embodiments, the detector 124 can include a processor to facilitate digitization of the emitted light 117a, 117b and transmission of digitized signals to the hub box 118 and/or the computer device 110.
In the non-limiting exemplary embodiment shown in
The combine light beam 119 can be directed toward the first filter/mirror arrangement 116a, which may be a dichroic filter/mirror. The combined light beam can be further sharpened before being incident upon the first filter/mirror arrangement 116a by a beam sharpener 121. The first filter/mirror arrangement 116a can reflect the combined light beam 119 and cause it to travel through the beam combiner assembly window 128. The combined light beam 119 can then be directed through the reaction vessel window 122 to be incident upon the patch 102 that may be located within the reaction vessel 112. The combined light 119 being incident upon the patch 102 can induce fluorescence of chemicals within the patch 102. This may cause at least one emitted light 117a, 117b to be generated and radiate from the patch 102. The emitted light 117a, 117b be can at a certain wavelength, which may depend on the pH level and/or DO level of the environment within which the patch 102 is exposed. For example, with a combined light beam 119 comprising violet and blue light, chemicals impregnated into the patch 102 can be configured such that they emit green light 117a from the patch 102 so as to be indicative of changes in pH of a growth medium. In some embodiments, emitted light 117a of a certain wavelength (e.g., green light) can be generated when the pH level is below or above a threshold level. As another example, chemicals impregnated into the patch 102 can be configured such that emitted light 117b of a certain wavelength (e.g., red light) from the patch 102 can be generated so as to be indicative of changes in DO of a growth medium. In some embodiments, emitted red light 117b can be generated when the DO level is below or above a threshold level. Thus, a first emitted light 117a of a certain wavelength can be generated that is indicative of pH level, and a second emitted light 117b of a certain wavelength can be generated that is indicative of DO level.
An example of a DO sensor patch 102 can include a use of ruthenium-based oxygen sensing films such as Ru(II) tris (4,7-diphenyl-1,10-phenanthroline) complex, immobilized in a silicone rubber membrane (Bambot, S. B. et al., Biotechnol. Bioeng. 43: 1139-1145 (1994)). Another example of a DO sensor patch 102 can include impregnating a material with an indicator dye such as a porphyrin dye, for example, or a metalloporphyrin such as platinum(II)-octaethyl-porphyrin combined with, e.g., encapsulated within, a polymer matrix such as polystyrene. The matrix layer may then be applied to a polystyrene support using, for example a toluene-based solvent (Liebsch, G. I. et al., Appl. Spectroscopy 54: 548-559 (2000)).
An example of a pH sensor patch 102 can include impregnating a material with any known ratiometric pH sensitive dye, such as 1-hydroxypyrene-3,5,7-sulfonic acid (HPTS). A sterilized solution of the dye can be directly introduced into a bioreactor media and detected via fluorescence. Fluorescence detection can be determined using front face geometry. For example, HPTS has two excitation peaks-400 and 450 nm. When excited at either 400 or 450 nm, HPTS can emit light at approximately 520 nm. The longer excitation peak can be excited using a blue LED (460 nm), for example, and the shorter excitation peak can be excited using an UV LED (375 nm), for example. The intensity ratio of the 520 nm fluorescence emissions from excitation at each of the two excitation peaks can be affected by the pH of the media. Thus, the pH can be calibrated by measuring the intensity ratio of the 520 nm fluorescence emissions at each of the two excitation peaks as the pH changes. pH can be optionally verified on a benchtop pH meter. This ratiometric approach may avoid interference from turbidity changes and provides accurate measurements of pH.
Other examples of pH and DO sensor patches 102 can be based on techniques disclosed in U.S. Pat. No. 6,673,532, filed Aug. 14, 2001, titled “Bioreactor and Bioprocessing Technique,” which is incorporated herein by reference in its entirety.
Emitted light 117a, 117b from the patch 102 can travel back through the reaction vessel window 122 and further through the beam combiner assembly window 128. The emitted light 117a, 117b can be further directed to be incident upon the first filter/mirror arrangement 116a. It is contemplated for the emitted light 117a, 117b (e.g., light emitted due to fluorescence) to be generally at a wavelength that is higher than a wavelength of any of the excitation light beams 115a, 115b required to elicit the fluorescence effect. Thus, emitted light traveling 117a, 117b back toward the first filter/mirror arrangement 116a may have wavelengths that are greater than both of the first and second excitation light beams 115a, 115b. In at least one embodiment, a surface of first filter/mirror arrangement 116a can be configured to reflect the combined excitation light beams 119, but to pass emitted light beams 117a, 117b. For example, the surface of the first filter/mirror arrangement 116a can be configured to pass emitted red and/or green light coming in-through the beam combiner assembly window 128, but reflect combined violet-blue light beams so as to be directed out-through the beam combiner assembly window 128.
The emitted light 117a, 117b can be further directed to be incident upon a second filter/mirror arrangement 116b. As shown in
While various embodiments disclose use of two excitation light beams 115a, 115b and two emitted light beams 117a, 117b, these are one exemplary. There can be any number of excitation 115 and emitted light beams 117 used. Further, the excitation 115 and emitted light beams 117 are not limited to the specific wavelengths disclosed, but the specific wavelengths are only exemplary of what can be used.
The sensor patch 102 can be a fluorescent-based optical patch. In one embodiment, the patch 102 can include a substrate with a polymeric backing. The substrate can be filter paper, which may be a monomer (e.g., methyl methacrylate) or cellulose-based filter paper. In some embodiments, the backing can include a silicon-based backing. The backings and/or the substrate can be configured to facilitate quick absorption of the excitation light beams 115a, 115b and quick radiation of emitted light beams 117a, 117b. One way to achieve this is by combining at least two fluorescent dyes in a monomer while depositing a measured amount of the same onto an inner surface of the reaction vessel 112. The measured amount can also be polymerized. It should be noted that the water solubility of polymers may vary, and thus polymer selection for the backing may be difficult. It should be further noted that the activity of chemical constituents of polymers can vary widely. Because of this potential variability, pre-calibration of the fluorescent response of a polymetric spot can be difficult to perform. However, using filter paper as a matrix for the reactive chemicals and dyes can facilitate easier calibration. Using filter paper as a matrix for the reactive chemicals and dyes can further increase the rate at which a response may be generated.
In one exemplary embodiment, a sterile optical patch 102 can be placed aseptically in the reaction vessel 112. The back of the patch 102 can be coated with an adhesive for adhering it to an inside surface of the reaction vessel 112. The adhesive can be a biocompatible adhesive. For example, silicone based adhesives with no support binders have been shown to be biocompatible. The patch 102 can be placed within the reaction vessel 112 so as to enable at least one excitation light beam 115a, 115b generated from the beam combiner assembly 104 to be incident upon it through a transparent portion of a reaction vessel 112. For example, the patch 102 can be placed adjacent the reaction vessel optical window 122 and/or at a position covering the reaction vessel optical window 122. In one embodiment, the patch 102 can be placed at a position subtending the reaction vessel optical window 122.
The transparent portion of the reaction vessel 112 can be the optical window 122 of the reaction vessel 112 configured to allow at least certain wavelengths of light (e.g., wavelengths associated with excitation light beams 115a, 115b and/or emitted light beams 117a, 117b) to transmit there-through. This may include blocking all other light from passing there-through, or blocking certain bands of light (wavelength bands). The patch 102 can include chemicals configured to generate at least one emitted light beam 117 when caused to fluoresce due to at least one excitation light beam 115b eing incident upon it. This can be achieved by, for example, impregnating the patch 102 with a blend of chemicals to generate emitted light 117 when subjected to at least one excitation light beam 115 and/or a combined light beam 119 of at least two excitation beams 115. Chemicals within the patch 102 can be further configured to radiate at least one the emitted light 117 in response to changes in oxygen partial pressure and/or pH levels. In some embodiments, the patch 102 can be at least one of a pH patch and a DO patch. A pH patch can be configured to radiate emitted light 117 at a certain wavelength in response to changes in pH levels. A DO patch can be configured to radiate emitted light 117 at a certain wavelength in response to changes in DO levels.
The pH patch can be structured to generate a ratio-metric response. For example, the chemicals impregnated into the pH patch may be excited by two different, but close, excitation light beams 117 (e.g., different with respect to wavelengths). This can cause generation of two different emission light beams 117 (e.g., each having a different wavelength). Each wavelength of the different emitted light beams 117 can differ depending on changes in pH levels the pH patch is exposed to. The ratio between wavelengths of the different emission light beams 117 can be used as an indicator of the pH level of a growth media in the reaction vessel 112.
The DO patch can be a fluorescent oxygen-sensing patch, which can be structured to use oxygen as a quenching agent to quench a chemical response of chemicals impregnated into the DO patch while exposed to a presence of oxygen. The DO patch can be further structured to radiate emitted light when excited by a single excitation light beam 117. The DO patch can be further structured to generate an emitted light beam 117 as a function of the chemical response. In some embodiments, the DO patch can be structured such that the more oxygen that is present in the environment within which the DO patch is located, the less the chemical response occurs. This may lead to a weaker emission light beam signal. Thus, the less oxygen that is present, the greater the chemical response occurs. This may lead to a stronger emission light beam signal generated by the system. Thus, the lower the levels of DO within a culture sample, the stronger the signal that can be detected from the emitted light. Generally, oxygen content of the growth media in a culture growth bioprocess is less than that of ambient air. Therefore, enabling generation of strong signals in environments where oxygen content is within a range from greater than 0% to 21% can be beneficial for cell culture monitoring.
The system can be configured so that a wavelength of an excitation light 115a beam to elicit a fluorescence response from a DO patch can be lower than a wavelength of an excitation light beam 115b to elicit a fluorescence response from a pH patch. For example, a wavelength of an excitation light beam 115a to elicit a response from a pH patch can be blue light beam (e.g., at or near 470 nm) and a wavelength of an excitation light beam 115b to elicit a response from a DO patch can be violet light beam (e.g., at or near 405 nm).
A mini-fluorometer can be used to generate excitation light beams 115a, 115b and to detect wavelengths of emitted light 117a, 117b. For example, the mini-fluorometer can be built into an integrated circuit board 134, which can be placed into communication with the computer device 110 via a hub box 118. Optoelectronics, such as a photodiode (e.g., the detector 124) for example, can be used to interrogate the patch 102 via modulation of excitation light beams 115a, 115b to detect the emitted light 117a, 117b. In some embodiments, a mini-fluorometer can be built into an integrated circuit board 134, both of which can be attached to and/or placed within the casing 126 forming the beam combiner assembly 104.
In at least one embodiment, an illumination source 114 for generating at least one excitation light beam 115 can be an LED. A band filter can be used to produce a narrow bandwidth of excitation light 115 coming from the illumination source 114. The band filter can be further used so that the gain of an LED may be adjusted to generate a desired intensity.
The sensor patch 102 can be mediated by changes in pH levels and/or changes in DO levels in growth media that support growth of cells within the reaction vessel 112. Detected emitted light 117 radiating from the patch 102 can be captured as signals and digitized by the detector 124. The digitized signals can be transmitted as reaction vessel data to the computer device 110. The computer device 110 can be located within and/or outside of a bio-safety laboratory. With reaction vessel data, the computer device 110 can be programmed to calculate fluorescence lifetimes and decay rates associated with oxygen concentration. This can be done to calculate DO concentration within a growth medium of cell culture sample. A first user interface 132′ can be displayed on the computer device 110, which may be programmed to cause the computer device 110 to display instantaneous values of DO concentration (See
The computer device 110 can be programmed to influence electrical, mechanical, and optical components of a system. This may include, but is not limited to, influencing controlling valves, pumps, mixers, detection devices, etc. For example, software can be stored on the memory 146a, 146b of the computer device 110, which may be programmed to cause the computer device 110 to accept reaction vessel data and/or to accept command data from users via the user interface 132′, 132″. Command data can include threshold levels and operating parameters. The software can be further programmed cause the computer device 110 to drive components of the system automatically within user-defined thresholds and user-defined parameters. In addition, a user can set pre-established rules to influence threshold levels and operating parameters to generate variations within each reaction vessel 112. In some embodiments, the system can be configured so that each reaction vessel 112 only responds to one set of parameters and thresholds, each of which can be pre-determined by a user. Thus, a group of parameters and thresholds can be set for a plurality of reaction vessels 112. For example, a group of parameters and thresholds can be set for as many as twelve reaction vessels 112. Any of the reaction vessels 112 can be arranged in parallel with another reaction vessel 112.
Some embodiments can include use of firmware as an alternative or in addition to software. In at least one embodiment, a digital sensing board (“DSB”) in connection with the system can include firmware programmed such that at least one computation is performed on the DSB. This can be done to have the DSB perform certain calculations and/or functions (e.g., addition of a gas to the reaction vessel 112) as opposed to the computer device 110 running software performing that calculation and/or function. In at least one embodiment, the DSB can be incorporated into a bioprocessor so that a signal can be transmitted from the DSB to a pump, a valve, or other system component. The signal can be transmitted directly from the DSB to the system component. Thus, actions performed by the system component can be initiated by the DSB and/or the computer device 110. In some embodiments, certain actions by the system component can be initiated by the DSB without being initiated by the software. This may be done to eliminate use of a computer device 110 for some or all aspects of the system. In some embodiment, monitoring functions and/or initiated action by certain monitoring functions can be customized by use of the software.
In at least one embodiment, the combined excitation light beam 119 can be collimated and coaxial. Further, the patch 102 can be placed adjacent the beam combiner assembly 104 so that it is subtending the detector 124. The beam combiner assembly 104 may be structured to combine at least two different excitation light beams 115. The beam combiner assembly 104 can be further structured to compare two emitted light 117 waves for pH analysis, and use intensity of a third emitted light 117 wave for DO analysis. The system can be further configured to detect emitted light 117 with reference to an expected offset. This may allow use of the system without a user knowing and/or without a user having to pre-setting the system to accommodate: 1) which excitation light beams 115 are being generated and/or which emitted light beams 117 are being induced; 2) whether the patch 102 is a pH patch or a DO patch; and/or, 3) whether the reaction vessel 112 with both a pH patch and a DO patch is being used. Hence, a reaction vessel 112 containing a pH patch and/or a reaction vessel 112 containing a DO patch can be used at any time without having to calibrate and/or re-calibrate the system.
Referring to
Setting a filter/mirror within a base 135 of a circuit board 134 and shifting a mirror of a filter/mirror arrangement 116 can increase available space within the circuit board block 134, which can enable improvements within the system. Referring to
In at least one embodiment, the system can include a filter/mirror arrangement 116 with a shifted mirror, where shifting the mirror can facilitate use of the cylindrical filter 137. In some embodiments, the shifted mirror 160 configuration can further allow the cylindrical filter 137 to protrude into the base 135 of the circuit board block 134 to which it may be attached. In some embodiments, the cylindrical filter 137 may extend below the base 135. This may enable reducing a height of the circuit board 134 even further, which may create additional room at the top of the circuit board 134. Reducing space can be beneficial, because the system and/or the reaction vessel 112 may be placed within an incubator. The space within an incubator may be compromising, and thus a reduction in volume occupied by the system can be beneficial.
Referring to
Determining an expected offset can be achieved as follows. An empty reaction vessel 112 can be examined via the system. The red reflector can be placed against the beam combiner assembly window 128 so that red light can be detected by the system. Alternatively, a red LED can be included within the circuit board block 134 of the beam combiner assembly 104. The beam combiner assembly 104 can generate excitation light beams 115 so as to detect red light by the detector 124, the red light being generated by the offsetting generator 180. This detected red light can be used as an expected offset. As noted above, the offset generator need not be red, but the detected red light can be used as an expected offset with embodiments where the presence of DO is expected to radiate red light by a DO patch. It should be noted that the chemical response for a DO patch can be linear. Thus, the red light of the red LED or red reflector can simulate the expected red emitted light of a DO patch when in use within the reaction vessel 112 and when DO is present within the reaction vessel 112.
Using an expected offset can enable use of any reaction vessel 112 without knowing beforehand whether the reaction vessel 112 has a pH patch or a DO patch. This can further enable use of the system without performing calibration of filter/mirror positions and/or angles. For example, the system can be encoded with the expected offset before using any patch 102 to conduct monitoring (e.g., the expected offset can be encoded to be used by software operated by the computer device 110). During use with the patch 102, the system can detect emitted light 117, and if the emitted light 117 is at or near the expected red light (i.e., the expected offset) then the detection system 100 can automatically determine that a DO patch is being used. If during use with the patch 102, the system detects emitted light 117 that is not at or near the expected offset, then the system can automatically determine that a pH patch is being used. The expected offset can be determined for each patch 102, or a set of patches 102, and encoded to be associated with each patch 102, or set of patches 102. Each patch 102, or set of patches 102, can be associated with the expected offset before being used in a system for monitoring cell cultures.
In at least one embodiment, the system can be used in the following manner. An empty reaction vessel 112 without the patch 102 can be placed in line with an excitation path of the system such that there is no reflected (fluoresced) light reaching the detector 124. The red reflector 180 can be placed against a beam combiner assembly window 128. Alternatively, a red LED 180 can be included within the circuit board block 134 of the beam combiner assembly 104. The reaction vessel 112 can then be placed adjacent the beam combiner assembly 104 so as to be between the beam combiner assembly 104 and the red reflector 180 and/or red LED 180. The beam combiner assembly 104 can be caused to generate excitation light beams 115 so as to detect red light by the detector 124, the red light being generated by the reflector offsetting generator 180. Alternatively, the LED offsetting generator 180 can be used to generate the red light. The detected red light can be used to determine an expected offset. The expected offset can then be record. The recorded expected offset can be associated with a plurality of patches 102, the plurality including at least one of a pH patch and a DO patch, so that an expected offset can be known for users of a set of patches 102. The expected offset associated with each patch 102 and/or set of patches 102 can be recorded and coded. A coded expected offset associated with each patch 102 and/or set of patches 102 can be placed within a barcode associated (e.g., attached thereto) with the reaction vessel 112 and/or patch 112, stored as a scatter code, stored within software of the computer device 102, transmitted to the computer device 102 from another computer device 102 via the computer system 106, etc.
A pH patch and/or a DO patch can be affixed to an inside of the reaction vessel 112 with the reaction vessel 112 being empty otherwise. A cell culture sample can be placed within the reaction vessel 112 for which an expected offset for the patch 102 associated therewith has been calculated. The red reflector offsetting generator 180 can be removed from a beam combiner assembly 104 and/or the red LED offsetting generator 180 can be turned off. At least one of a violet excitation light beam 115a (e.g., at or near 405 nm) and a blue excitation light beam 115b (e.g., at or near 470 nm) can be generated to be combined into a combined violet-blue excitation light beam 119 by the beam combiner 130. The combined violet-blue excitation light beam 119 can include the combined violet excitation light beam 115a and a blue excitation light beam 115b, which may be combined to be collimated and coaxial. The combined violet-blue excitation light beam 119 can be directed through the beam combiner assembly window 128 and further through the reaction vessel window 122 to be incident upon at least one of a pH patch and a DO patch. The combined violet-blue excitation light beam 119 can induce fluorescence from chemicals within at least one of the pH patch and the DO patch so that emitted light 117a, 117b is radiated therefrom and directed back through the reaction vessel window 122 and further through the beam combiner assembly window 128 to be incident upon the pass filter. The pass filter can pass light with wavelengths at or greater than 525 nm so as to be incident upon the detector 124, but block light with wavelengths less than 525 nm.
The detector 124 can digitize the emitted light 117a, 117b being incident upon it. The detector 124 can also record at least one of wavelength and intensity of the emitted light. The detector 124 can also be configured for generating a representative data detection signal therefrom. The detection signal can be transmitted to the hub box 118. The hub box 118 can be in communication with at least one beam combiner assembly 104, which can be in further communication with at least one computer device 110 (see
If during detection of emitted light 117a, 117b, an expected offset is detected, the computer device 110 can be programmed to determine that a DO patch is being used, a threshold level of DO is present, and/or a DO level is increasing or decreasing. If during detection of emitted light 117a, 117b, no expected offset is detected, the computer device 110 can be programmed to determine that a pH patch is being used, a threshold level of pH is present, and/or a pH level is increasing or decreasing. The reaction vessel 112 can be removed from the beam combiner assembly 104 and a different reaction vessel 112 can be placed on the beam combiner assembly 104 for DO level and pH level monitoring without calibration of filter/mirror positions and/or angles.
The sensor strip 1 from a same set can be switched multiple of times without calibration of filter/mirror position and/or angle and without entering a coded expected offset. If a reaction vessel 112 from a different set is used, monitoring can be done without calibration of filter/mirror positioning and/or angle, but a coded expected offset associated with those set of patches 102 and/or reaction vessels 112 may have to be entered.
Referring to
In further embodiments, a feedback loop can be generated via the system and at least one computer device 110 so that reaction vessel data can be transmitted to the computer device 110 and command data can be to be transmitted to at least one beam combiner assembly 104 from a user of the computer device 110 via the user interfaces 132′, 132″ of the system (see
Other configurations and uses for the reader 200 can be any of the embodiments disclosed in U.S. Pat. No. 10,379,047, the entire contents of which is incorporated herein by reference.
As shown in
The computer system 106 may include a plurality of computer devices 110, computer servers 136, databases 138, communication networks 148, and/or communication path/connections. A user of the system may use at least one processor device 144, memory storage 146a, 146b, and communications interface 142 to communicate and execute commands. Each computer server 136 may be connected to at least one database 138, where software executed by each computer device 110 may carry out functions of storing, coalescing, configuring, and/or transmitting data. Software may be stored on any type of suitable computer-readable medium or media. This may be a non-transitory computer-readable medium or media, such as a magnetic storage medium, optical storage medium, or the like.
The computer system 106 architecture shown in
The processor device 144 may be a single processor, a plurality of processors, or combinations thereof. The processor device 144 may have one or more processor cores. The processor device 144 may be a special purpose or a general purpose processor device. The processor device 144 may be connected to a communication infrastructure. The communication infrastructure may include, but is not limited to, a bus, message queue, network, multi-core message-passing scheme, etc.
The computer device 110 may include a main memory 146a. The main memory 146a may include, but is not limited to, a random access memory, a read-only memory, etc. The computer device 110 may include a secondary memory 146b. The secondary memory 147b may include, but is not limited to, a hard disk drive, a removable storage drive, a floppy disk drive, a magnetic tape drive, an optical disk drive, a flash memory, etc. Any of the main and the secondary memories 146a, 146b can be a non-volatile memory.
Computer program media, non-transitory computer readable media, and computer usable media may refer to tangible media, such as, for example, a removable storage unit and a hard disk installed in a hard disk drive. The removable storage drive may read from and/or write to a removable storage unit. The removable storage unit can include a removable storage media that can be read by, and written to, a removable storage drive. For example, if a removable storage drive is a floppy disk drive, the removable storage unit may be a floppy disk. The removable storage unit can also be non-transitory computer readable recording media.
In some embodiments, the secondary memory 146b may include alternative means for allowing computer programs or other instructions to be loaded into the computer device 110 and/or computer system 106. This may be, for example, a removable storage unit and/or an interface. Examples of such means may include, but are not limited to, a program cartridge and cartridge interface (e.g., as found in video game systems), a removable memory chip (e.g., Electronic Erasable Readable Programmable Read-Only Memory (“EEPROM”), Programmable Read-Only Memory (“PROM”)), etc. and associated socket, and/or other removable storage units and interfaces.
The computer system 106 may include a communications interface 142. The communications interface 142 may be configured to allow software and data to be transferred between computer devices 110 within the computer system 106 and/or the computer system 106 and external devices. Communications interfaces 142 may include, but are not limited to, a modem, a network interface (e.g., an Ethernet card), a communications port, a Personal Computer Memory Card International Association (“PCMCIA”) slot and card, etc. Software and data transferred via a communications interface may be in a form of signals, which may be electronic, electromagnetic, optical, or other signals. Signals may travel via a communications path, which may be configured to carry signals and may be implemented using wire, cable, fiber optics, a phone line, a cellular phone link, a radio frequency link, etc.
Computer program medium and computer usable medium may refer to memories, such as a main memory and a secondary memory, which may be memory semiconductors (e.g., Dynamic Random-Access Memory (“DRAM”)). These computer program products may be means for providing software to the network. Computer programs (e.g., computer control logic) may be stored in a main memory 146a and/or a secondary memory 146b. Computer programs may also be received via the communications interface 142. Such computer programs, when executed by a processor device 144, may enable a computer device 110 to execute commands and act upon the various components of the system 106. Accordingly, such computer programs may represent controllers of the computer system 106, where software may be stored in a computer program product and loaded into the computer device 110 using a removable storage drive, an interface, a hard disk drive, and/or a communications interface 142.
In some embodiments, the computer device 110 include a processor, a microprocessor, minicomputer, server, mainframe, laptop, personal data assistant, a cellular phone, smartphone, pager, or any other programmable device configured to enable transmission and/or reception of data, which may be over a network. The computer device 110 may include a peripheral device, such as an input/output device. The peripheral device may include, but is not limited to, a keyboard, a mouse, a screen display, a touch screen, a stylus pen, a monitor, a printer, a hard disk drive, a floppy disk drive, a joystick, an image scanner, etc.
One or more electronic communication networks 148 may be utilized by the computer system 106 to promote communication among different components, transfer data, and/or share resource information. Such communication networks 148 may be embodied as, but not limited to, at least one of Ethernet, wireless Local Area Network (“LAN”), Mobile Area Network (“MAN”), Wide Area Network (“WAN”), Virtual Private Network (“VPN”), Storage Area Network (“SAN”), Global Accelerator Network (“GAN”), Home Phoneline Network Alliance (“HomePNA”), etc.
In some embodiments, the computer system 106 may include a computer device 110 configured as a processor 144 operatively associated with at least one module 140, which may be programmed to display panels 150 and/or screen displays 152 on a monitor of a computer device 110. The processor 144 may be programmed to execute computer-readable instructions included within the module 140. Computer-readable instructions may be in a form of software stored on a non-transitory computer readable medium operatively associated with the processor 144. Each module 140 may be configured to generate the user interface (“UI”) 132′, 132″, which may enabling at least one user to issue commands, access data stored on a data storage media operatively associated with the processor, and/or transmit data to and from the data storage media. The module 140 may include software, firmware, hardware, or any reasonable combination thereof.
Any of the panels 150 may be programmed to display information and grant access to data related to certain aspects and functionalities of the computer system 106 and/or a system. Through the various modules 140 and panels 150, the computer system 106 can provide a communication network to orchestrate interaction between a user, the computer system 106, and the various components of the system. For instance, different panels 150 of each module 140 may be programmed to facilitate differentiated display of information and differentiated interaction between users, components of a computer system, and components of the system. This may be achieved by each module 140 generating different UIs 132′, 132″ for control of different aspects of the system.
Various embodiments of the present disclosure can be described in terms of the example computer system 106 described herein; however, other embodiments of the computer system 106, along with other embodiments of computer architectures, can be used. Although operations may be described as a sequential process, some of the operations may be performed in parallel, concurrently, and/or in a distributed environment, and with program code stored locally or remotely for access by single or multi-processor machines.
It will be apparent to those skilled in the art that numerous modifications and variations of the described examples and embodiments are possible in light of the above teachings of the disclosure. The disclosed examples and embodiments are presented for purposes of illustration only. Other alternate embodiments may include some or all of the features disclosed herein. Therefore, it is the intent to cover all such modifications and alternate embodiments as may come within the true scope of this invention, which is to be given the full breadth thereof. Additionally, the disclosure of a range of values is a disclosure of every numerical value within that range, including the end points.
This application is a non-provisional utility application which is related to and claims the benefit of U.S. Provisional Application No. 62/896,211 filed on Sep. 5, 2019, the entire contents of each being incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62896211 | Sep 2019 | US |
