Patent Application
20130316442
The invention relates to a system for monitoring a culture environment for the growth of mammalian and non-mammalian cells.
There is a need for academic researchers, applied clinicians or industrial scientists to have an incubating system that produces high yields while decreasing labour demands over traditional cell culture devices and without impact on culture process protocols.
For instance, stem cells generate great hopes for disease modelling, drug discovery or as a therapeutic tool for regenerative medicine. Stem cells can be obtained from different sources during the life of an individual (embryonic, cord blood, various adult tissues), and each type of stem cells has advantages and limits. Major advances in stem cell cultivation has provided the capability to robustly manipulate stem cell fate ex vivo, as demonstrated by the genetic reprogramming of adult stem cell to ground pluripotency (induced pluripotent stem cells—iPS) or by the reprogramming of thymic epithelial cells to multipotent stem cells of the hair follicle in response to an inductive skin microenvironment. However, manipulation of stem cell fate necessitates a strict control of culture conditions that can affect stem cell behavior. Current technology only provides approximate read-out and not in a real-time fashion.
Controlling the culture conditions of embryos is also a key to the success of assisted reproduction procedures aiming to single blastocyst implantation, in particular to decrease the probability for multiple pregnancies.
In contrast to conventional two-dimensional culture vessels, there is an advantage for certain applications in growing cells in suspension or adherent cultures, including organotypic culture, which involves growing cells in a three-dimensional environment that is biochemically and physiologically more similar to in vivo tissue.
Culture dishes are typically made of a transparent plastic, sometimes glass, and come in standard sizes. There would be an advantage in providing a culture system that is compatible or may be used with commercial off-the-shelf disposable single-use culture dishes.
In WO1998020108A1 an apparatus for holding cells comprises a mechanism for incubating cells, having a dynamically controlled environment in which the cells are grown, which are maintained in a desired condition and in which cells can be examined while the environment is dynamically controlled and maintained in the desired condition. The system is dedicated to individual cell analysis in a dynamically controlled environment but not to cell growth medium monitoring even if this functionality is mentioned (dynamically controlled environment).
In WO2007120619A2 an incubation condition monitoring device has a reader unit to measure selected characteristics within an incubator. The reader unit transmits the information to a receiver/transmitter within the incubator to receive the measurements and to transmit the measurements of the selected characteristics to a data logger outside the incubator. A monitor and display system monitors and display the measurements of the selected characteristics. The selected characteristics within the incubator can be temperature and pH. A cuvette contains a sample of the fluid which is the same as that in the culture dish. The measurement is thus indirect and may not correspond to the actual cell culture environment.
In US2006003441A1a cell culture system includes a monitoring system with predefined sensor modules. By means of this monitoring system, parameters in the relevant cell culture chamber can be measured using accordingly assigned sensors for the duration of a test. For this purpose, the monitoring system is connected to the individual cell culture chambers. The parameters measured by the sensors are transmitted by the monitoring system via a line to the computer-controlled monitoring and control system for further processing. The system however uses dedicated dishes, not commercially available single-use Petri dishes.
In HEER R ET AL “Wireless powered electronic sensors for biological applications” 2010 Annual International Conference of the IEEE Engineering in Medicine and Biology Society 31.08-04.09.2010 (ISBN 978-1-4244-4123-5) there is described an RFID sensor device consisting of an RFID tag, an impedance measurement sensor mounted on the RFID tag, and a culture well mounted on the tag and the sensor such that the sensor forms a bottom wall of a culture well. The sensor is formed of interdigitated electrode fingers that measure cell growth by way of the impedance of the cell culture, which is dependent on the proliferation and physiologic condition of the cell cultures adhering to the sensor. This sensor device does not however measure nor monitor any parameters in the cell culture medium that are relevant to cell culture growth (such as CO2, pH, O2, temperature, glucose and others). The tag has a diameter of 34 mm and is designed to fit into wells of a 6-well microtiter plate for standard handling, however the sensor device has its own culture well and is not designed for disposable single-use. The volume of the available culture medium is thus reduced compared to the volume of culture medium that may be placed directly in the well in a standard procedure. This known device is bulky, costly to manufacture and use, and poses sterility or safety problems in view of the difficulty in cleaning and sterilizing the device for re-use.
An object of this invention is to provide a system for monitoring a culture environment for the growth of mammalian and non-mammalian cells that enables economical yet high yield growth of cells and that has no or negligible impact on culture process protocols.
It is an advantage to provide a system for monitoring a culture environment for the growth of cells that requires minimal handling by personnel.
It is an advantage to provide a system for monitoring a culture environment for the growth of cells that is reliable and safe.
It is an advantage to provide a system for monitoring a culture environment for the growth of cells that reduces processing of culture growth parameters and data.
It is an advantage to provide a system for monitoring a culture environment for the growth of cells that allows easy adjustment of culture growth parameters to increase yield.
Objects of this invention have been achieved by providing a cell culture environment monitoring system according to claim 1.
Objects of this invention have also been achieved by providing a cell culture growth system according to claim 14.
Disclosed herein is a cell culture growth system and a culture environment monitoring system for monitoring parameters relevant to cell growth in at least one culture dish containing a cell growth medium, including at least one sensing device configured to measure environmental parameters relevant to cell growth comprising an RFID transponder, and a tray supporting said at least one culture dish comprising an RFID base station configured to interrogate the RFID transponder of the sensing device.
The sensing device is advantageously configured as a single use disposable element for mounting inside a variety of standard or non-standard culture recipients and may be immersed partially or totally within said cell growth medium for measuring at least one parameter within the cell growth medium. The sensing device, or an additional sensing device may also be positioned outside or partially outside the cell growth medium to measure parameters in the gaseous environment in the immediate vicinity of the culture medium within the culture dish.
The tray may advantageously comprise a support base configured to support and position thereon a plurality of culture recipients. More than one, or all of the culture recipients may have one or two sensing devices mounted therein.
The RFID base station includes an antenna that may advantageously be mounted in or on the support base configured to enable communication between the RFID base station and the plurality of sensing devices positioned in the plurality of culture recipients. The antenna may comprise a conductor loop surrounding the plurality of culture recipients configured for near-field communication with the sensing devices, the antenna embedded in or mounted on the support base.
The support base may advantageously comprise optical inspection ports positioned below the culture recipients to allow passage of light through the culture dish and growth medium for optical inspection or testing without having to remove the culture dish from the tray.
The RFID base station may include a signal processing circuit that includes an RFID interrogator, a microprocessor, a portable power source such as a battery, a communications interface configured for wireless and/or hardwire link to an external computing system and optionally a memory for storing or logging data received from the sensing devices.
The sensing device advantageously comprises a plurality of sensors responsive to different parameters relevant to cell growth, which may include temperature, pH, Ca++, CO2, glucose and other cell nutrients, O2 and light.
The sensing device may further include a microprocessor and energy harvesting and/or storage means for short term power supply of the RFID transponder, microprocessor and sensors.
The sensing device comprises a support or base on which, or within which, the sensors and signal processing circuitry are mounted. In an advantageous embodiment, the support comprises an adhesive base configured to allow the sensing device to be stuck on an inside surface of the culture dish and immersed partially or totally in the cell culture growth medium. The sensing device may be generally in the form of a thin sticker or label having a height less than 5 mm, possibly less than 3 mm, that allows it to be stuck on the bottom wall of a conventional Petri dish and completely covered by the growth medium contained in the Petri dish, or stuck on the underside of the cover of the Petri dish without contacting the cell growth medium when the cover is positioned on the container part of the Petri dish. The sensing device may also be configured as an element that may simply be dropped into a culture dish or a flask and totally immersed in the culture medium, the sensing device laying unattached on the bottom of the culture dish or flask.
In a variant, the sensing device may comprises sensor probes that extend at different lengths, certain said sensor probes configured for insertion in the cell growth medium and other said sensor probes configured to remain outside of the growth medium such that parameters within the growth medium and the gaseous environment surrounding the growth medium can be measured by the same sensing device. The sensing device may have mechanical fixing means such as a clip or hook or clasp for fixing to the culture dish side wall.
Advantageously, the system according to the invention enables non invasive continuous monitoring of multiple parameters relevant to cell growth (e.g. temperature, light, CO2, O2, pH, glucose concentration and other nutrients), to achieve high cell growth rates and yields in a safe, reliable and economical manner. In addition, the cells can further be examined by standard techniques, e.g. by standard imaging techniques, in this wirelessly monitored growth medium.
Further objects and advantageous aspects of the invention will be apparent from the claims, following detailed description and accompanying figures.
a to 3c are respectively perspective, top and side views of a cell culture environment monitoring system according to an embodiment of the invention;
a and 4b are respectively perspective and side views of a cell culture dish and sensing devices according to an embodiment of the invention;
a to 5c are respectively perspective, top and side views of the sensing device of
a, 6b are similar to
Referring to the figures, in particular first to
Culture recipients 4, 4′ which include well-known so-called “Petri dishes” 4 (cf
A cell culture environment monitoring system 6 according to an embodiment of this invention includes one or more sensing devices 22 mounted in each culture dish or flask and a communications and support tray 24 that communicates with one or more sensing devices 22. Referring to
The dish positioning means 46 may simply be in the form of recesses, for example circular recesses within the support base 40 each configured to snugly receive the periphery of the base of a container part 18. Instead of recesses, other positioning means such as protuberances projecting from the support base for positioning the culture dish container part thereon may be provided. The support base 40 may comprise a plurality of positioning means for positioning culture dishes of different sizes, for example by providing a large recess and co-axially therein a further smaller recess (not shown) within the base of the large recess for a smaller container. This enables the communications and support tray 24 to be used with culture dishes of different sizes. The communications and support tray advantageously comprise a plurality of culture dish positioning elements, for example 4, 6, 8 or more and 2, 3, 4 or more rows and/or columns of a size suitable for positioning on a shelf inside a conventional incubator.
The dish positioning means may be provided with other shapes and sizes, in particular configured to support culture recipients of other shapes and sizes, for instance a plurality of culture flasks 4′. Where applicable herein, the term “dish” shall also be meant to include other forms of culture recipients such as flasks.
The antenna 42 may be in the form of a conductor loop embedded in the support base 40 surrounding culture dishes 4, configured to allow near field RFID communication with the sensing devices 22 positioned in the culture dishes 4. The antenna conductor 42 may thus form a loop close to, or on the outer contour of the support base 4 surrounding the culture dishes. The antenna 42 may be configured as a single loop or a plurality of windings and may be formed as a coil embedded within the support base 40 or formed on a surface of the support base, either a top or bottom surface 40a, 40b or a lateral surface 40c. The antenna may also be in the form of a separate component that is mounted on the support base 40 by various known fixing means such as bonding with an adhesive, mechanical clasp or clipping means, or welded for example by ultrasonic welding.
The support base 40 may advantageously comprise optical inspection ports 47 positioned within the recess or positioning means of the culture dishes below the container part 18 base wall to allow passage of light through the culture dish and growth medium for optical inspection or testing, for example by means of microscope, spectrometer or other optical testing systems, either automatically or by manual manipulation, without removing the culture dishes from the communications and support tray. The optical inspection port may be formed by a passage or hole through the support base, corresponding to only a small portion of the surface area below the culture dish, or covering almost all the surface of the culture dish which is then supported on the base essentially only at the periphery for example. Alternatively, instead of a hole through the support base, the support base may comprise an optically transparent material, such as a transparent plastic material, or have a transparent portion for the inspection port underneath the culture dish or other type of culture recipient.
Referring to
Referring to
In a first embodiment illustrated in
The RFID transponder 34 may, as is per se known in RFID transponders, comprise a conductive coil 39 that acts as a resonance coil LR of the RFID transponder to capture and transmit wireless signals to the RFID base station 44 via the antenna 42. Referring to
One culture dish may be monitored by one or more multi-parameter transponders, each being identified by a unique serial number.
It is also possible, in the case of multi-dish configuration per tray (as illustrated in
The sensing devices may for instance comprise a near field RFID transponder operating at a frequency configured to avoid any interference with cell metabolism, preferably less that 30 MHz, for instance 125 KHz, 134.2 KHz, 13.56 MHz or 27 MHz, more preferably 125 KHz or 134.2 KHz, with an RFID antenna average diameter or width in the range of 5 to 20 mm, for instance approximately 10 mm.
The RFID base station interrogator 50 may comprise a selective address mechanism, for instance one reader may address up to 255 transponders. The RFID base station 44 may further comprise a far field communication transceiver for example operating at a frequency such as 2.4 GHz, for wireless communication with the computer system 26.
In a preferred embodiment, the sensing device 22, 22′ comprises a plurality of sensors for monitoring parameters relevant to cell growth, including a temperature sensor, a pH sensor and analyte sensors which may include a glucose sensor and a calcium ion (Ca++) sensor. Further sensors may be included that are relevant to growth of the specific cells that are grown including for example sodium and/or potassium ion sensors. Further sensors may be included to measure light radiation in particular for measuring light energy and possibly also light spectrum that is applied on the cell culture either for promoting, reducing or stabilizing cell growth. There may also be sensors for measuring parameters of the gaseous environment that are relevant to cell growth or indicative of cell culture activity, in particular carbon dioxide and/or oxygen. Sensors may also be provided to measure humidity and other gaseous components in the culture dish close to the growth medium. The sensors may advantageously be configured to measure these parameters intermittently at regular or predetermined intervals, or on user initiated request, by corresponding interrogation by the RFID interrogator to form a quasi continuous or on demand multi parameter monitoring system. The RFID base station thus energizes the multi-parameter sensing devices and interrogates them periodically or at any predetermined or user activated time to upload measured environmental parameters.
Depending on the application, the data may be recorded in the signal processing circuit of the sensing device 22, 22′, which may comprise a data logger function, in waiting for a further interrogation by the RFID base station. Otherwise, the data may be sent on the fly in real time towards the RFID base station, i.e. corresponding to an Interrogator function.
One RFID base station 44 may thus control several multi-parameter sensing devices, i.e. a single- as well as a multi-well configuration is possible. The RFID base station 44 may also comprise one or more sensing devices to measure overall environment parameters within the incubator or other environment surrounding the tray 24 and culture dishes 4, for instance to measure any one or more of the following parameters: temperature, O2, CO2, light and humidity.
The RFID base station may transmit by hard wiring (e.g. USB) or wirelessly (e.g. Bluetooth) collected data to a computing device 26 for display and processing purposes from the incubator where the culture batch is stored between manipulations. A logging of the selected parameters to provide an audit history throughout the cell culture cycle may be then enabled, and provide warnings if some of these parameters go outside of a specified range. The monitor and display system may include an alarm mechanism that provides an alarm (audible, visual, SMS or e-mail) when the measured characteristics are outside a selected range
The monitoring process may operate either when the culture batch is inside or outside the incubator, e.g. during the feeding process or the observation stage, when the dish is placed under an imaging system (e.g. microscope).
A normal RFID system is completely passive: the RFID base station sends a command to a RFID transponder, and the transponder answers, normally with its serial number. The sensing device transponder 22 is not connected to an internal power source: it is completely powered out of the RF field supplied by the RFID base station 44.
In addition to serial number, the RFID transponder 34 is able to control a multi-analyte sensing system: the transponder of the multi-parameter sensing device captures the radio-frequency power (125 KHz, 134.2 KHz, 13.56 MHz or 27 MHz) through the site antenna, energizes the sensor, performs the measurement, converts it to a digital value, and sends this information back to the RFID Base Station.
The on site microprocessor 36 of the sensing device 22, 22′ may perform some compensation computations beforehand, stored in a calibration EEPROM 29 programmed in factory.
Referring to
Examples of the functional and operational characteristics of the sensors comprised in the sensing devices 22, 22′ are described below and in relation to
Temperature may be measured by a temperature sensor generally internal to the microprocessor 36. For certain other parameters, two advantageous measurement methods allowing targeting minimal footprint and production costs compatible with disposability may be implemented as follows.
Measurement Based on Viscosity Change Detection (e.g. Glucose):
A chemico-mechanical method which aims at detecting viscosity changes of a solution with a selective affinity for the analyte of interest has been proposed in [Boss09] and [Boss11]. Referring to
Referring to
The sensing diaphragm output voltage is conditioned by a voltage amplifier providing voltage levels suitable for an Analog Digital Converter (ADC) that may be for instance implemented in the microprocessor 36.
Finally, an algorithm implemented in the microprocessor computes the phase shift between the actuation voltage and the sensing voltage from which the viscosity and further the analyte concentration is determined.
The purpose of chemical sensors consists in converting chemical information into signals suitable for electronic measuring processes. A typical chemical sensor consists of a material-recognizing element and a transducer. An advantageous implementation of such chemical sensors is to use hydrogel thin films as sensing elements. Hydrogels are cross-linked polymers which swell in solvents to appreciable extent. The amount of solvent uptake depends on the polymer structure, and can be made responsive to environmental factors, such as solvent composition, pH value, temperature, electrical voltage etc. Hydrogels are capable to convert reversibly chemical energy into mechanical energy and therefore they can be used as sensitive material for appropriate sensors. Such an approach per se has been described for instance by [Guenther07] and [Guenther08] where the transducer of the chemical sensors comprises a piezoresistive silicon pressure sensor forming a Wheatstone bridge 70 (
Also, a measurement concept has been realized by [Herber05] for the detection of carbon dioxide, where the CO2 induced pressure generation by an enclosed pH-sensitive hydrogel is measured with a micro pressure sensor.
Referring to
| Number | Date | Country | Kind |
|---|---|---|---|
| 11153590.2 | Feb 2011 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2012/050534 | 2/6/2012 | WO | 00 | 8/2/2013 |
