Abstract:
An improved cell culture monitoring system and method that detects cell growth and concentration in a dynamic environment of incubator/shaker. In order to reduce power consumption and make a wireless cell culture monitoring system practical, several methods of temperature compensation are used to replace a method of controlling the temperature of sensing module. Furthermore its power consumption can be significantly reduced by using an adaptive and synchronized light pulse detection technique.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This patent application claims priority from U.S. Provisional Patent Application Ser. No. 62/317,644, filed on Apr. 4, 2016, which is incorporated by reference herein in its entirety. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    Apparatuses and methods as described in U.S. Pat. No. 7,339,671, entitled: “Apparatus and Method for Monitoring Biological Cell Culture”, presented a system which can perform real time and on-line monitoring of a biological cell culture in an incubator/shaker. Such system employs a light scattering technique to detect biology cell concentration (in form of turbidity or optical density) or other measurable properties of the biological culture medium in a transparent container such as an Erlenmeyer flask. With progress of wireless technologies, internet cloud and smart phone, the invention of this cell culture monitoring system initiated in a decade ago can have some new feasible improvements. When U.S. Pat. No. 7,339,671 was filed a decade ago, a practical way in term of technologies and costs for the cell culture monitoring system was to use wire to power culture detection sensor and temperature control circuit in a sensor head as well as to send measured signal or data to a control module or a computer via an intermediate control module. The sensor head is a sensing device which can be put in an incubator/shaker and can attach to a cell culture medium container in operation. However, wire connection from the sensor head to the control module can be difficult for many existing shakers because of their sealed enclosure for temperature control. Also the shaking environment can make wire connection unstable so that extra care for wire selection and wire arrangement in shakers are required. To overcome the problem of wire connection, wireless embodiment as described in the initial invention has to deal with some critical issues such as power consumption, measurement accuracy and reliable RF wireless connection. For a wireless and battery powered sensor head, the crucial challenge is to monitor biologic cell continuously and accurately for many hours or days in some case without changing or charging the battery. 
         [0003]    With respect to the sensor head or probe defined in U.S. Pat. No. 7,339,671, the major power consumption comes from temperature control module and light sensing module. The light sensing module consists of at least one light source such as a LED or laser diode and at least one photodetector such as a photodiode. The radiation intensity of the light source and the sensitivity of the photodetector are temperature dependent. Usually incubator/shaker can operate at a temperature from ambient+5 C to 80 C. To have an accurate measurement in such temperature range, the monitoring system needs temperature control or temperature compensation for its light source and photodetector. A temperature control with peltier element dissipates a lot of electrical power and is not feasible for a battery powered sensor head. Therefore a power saving temperature compensation becomes a necessary method for constructing a wireless sensor head. 
         [0004]    Temperature compensation methods for LED, laser diodes and photodetectors have been reported in many patent publications. Some publications presented analog compensation circuits with temperature sensing thermistor for automatically adjusting current applied to LED and laser diode or adjusting voltage applied to photodetector. This type of temperature compensation is analog and has low power consumption. But it is not easy to find good match in temperature characteristic among thermistors and a variety of LED, laser diode or photodetector for a wide temperature range. Some publication presented software compensation with pre-measured and pre-calculated temperature coefficients of combined light source and photodetector. Because both light source and photodetector have non-linear relationship with temperature, their superimposed temperature coefficients becomes so complicated that its temperature correction could require 4th degree polynomial regression. Also for different light intensity detected in photodetector, the coefficients of polynomial are different. 
         [0005]    In recent years, many wireless technologies (Wifi, Bluetooth, Zigbee, etc) have been used for various wireless applications. The power consumption and reliability for the wireless technologies has been improved. Bluetooth Low Energy (BLE) appears to be a technology with much low power consumption comparing with Wifi and classic Bluetooth. BLE is designed to run for months or years with a button cell battery such as CR2032. With such wireless technology, the major challenges for the wireless cell culture monitoring system are to make accurate measurement without temperature control and to prolong battery life in usage for days or weeks. 
         [0006]    The object of this invention is to improve the cell culture monitoring system presented in U.S. Pat. No. 7,339,671 with low power consumption methods and devices. The innovated methods and devices make the cell culture monitoring system with a wireless sensor head feasible and practical. The wireless monitoring system gets rid of the wire connection problem and makes its sensor head to be easily mounted in incubator/shakers. Furthermore, the wireless monitoring system enables it to be easily integrated with not only PCs but also modern wireless devices such as smart phones and tablets. 
       SUMMERY OF THE INVENTION 
       [0007]    This invention presents a plurality of embodiments to improve the cell culture monitoring system with a wireless sensor head. In one aspect, without temperature control for the light sensing module, a method of two step temperature compensations are used to improve over all measurement accuracy of the cell culture monitoring system. The two step compensations comprise 1) a solely analog circuit or an analog circuit controlled by a microprocessor to reduce light intensity variation of light source due to temperature change and 2) a microprocessor to make a correction on final detection signal such as turbidity or optical density (OD) with pre-measured, pre-calculated and saved temperature coefficients. In another aspect, the power consumption in the sensor head can be significantly reduced with a method that the light source is controlled by a light driving pulse signal which has a low duty cycle. Using this method, the time and duration of turning on the light source are synchronized with trigger pulses for data acquisition in A/D converter. Instead of a fixed duty cycle or fixed time duration of tuning off the light source, the time duration of tuning off the light source can be adaptive to cell culture growth level and growth rate. As an example, the turning off time duration can change with the change of cell culture turbidity or turbidity change rate. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0008]      FIG. 1 . A schematic diagram of a standalone cell culture monitoring system with a wireless sensor head for flask biological culture applications. 
           [0009]      FIG. 2  A schematic and block diagram of a wireless sensor head. 
           [0010]      FIG. 3  A schematic and block diagram of light source driving circuit. 
           [0011]      FIG. 4  A schematic diagram of a basic light driving pulse signal with a constant duty cycle. 
           [0012]      FIG. 5  A schematic diagram of time sequence of light driving signal and pulse triggers for A/D conversion. 
           [0013]      FIG. 6  Typical growth curve and growth rate in term of the scattering turbidity of biological culture. 
       
    
    
     DESCRIPTION OF THE INVENTION 
       [0014]    New development in wireless and IoT (Internet of Thing) technologies provides opportunities for improving the cell culture monitoring system described in U.S. Pat. No. 7,339,671.  FIG. 1  shows one embodiment of such improved cell culture monitoring system. Sensor head  105  of the cell culture monitoring system can be a wireless probe which includes at least one wireless transceiver, A/D and D/A converter, microprocessor, temperature sensor, motion sensor, memory and battery. In one embodiment, battery can be a separated part which is not mounted in the sensor head enclosure. The battery can be mounted outside or attached to sensor head  105  with power supply wires. Sensor head  105  has basic functions for monitoring cell culture medium such as scattering light intensity, turbidity or OD and sending data wirelessly to other devices such as a control module  201  outside of incubator/shaker  900 . In one embodiment, sensor head  105  and module  201  compose a standalone cell culture monitoring system. Module  201  can be a user interface device which has at least a wireless transceiver, microprocessor, memory, LCD display, alarm and keypad/button. Module  201  can perform data process, data storage, data display, calibration and control of the cell monitoring system. In one option, module  201  can also be a networked device via DSL or Wifi or other technologies so that module  201  can be controlled by other computer  601  or smart device  701  such as smart phone and the data from module  201  can be put in cloud  800  and shared by other devices. In some embodiments, module  201  is replaced by a computer  600  or a smart device  700 . Sensor head  105  can directly connect to a computer  600  or smart device  700  via Bluetooth or other wireless technology. With help of software, computer  600  or smart device  700  can not only process and display the data from probe  105  but also control the data acquisition of sensor head  105  like module  201  does. Because a motion sensor and temperature sensor are built in sensor head  105 , module  201 , computer  600  or smart device  700  can show and record not only the culture turbidity/OD but also the orbital rotation speed of incubator/shaker  900  as well as cell culture temperature. 
         [0015]    In one embodiment, wireless sensor head  105  as shown in  FIG. 2  comprises sensing module  150 , light source drive circuit  130 , sensing circuit  140 , temperature sensor  121 , motion sensor  122 , pulse circuit  161 , A/D converter  162 , microprocessor  160 , wireless transceiver  163  and memory  164 . The data collected and processed by microprocessor  160  can be transmitted to module  201 , computer  600  or smart device  700  through a wireless transceiver  163 . Module  201 , computer  600  or smart device  700  can also send command to sensor head  105  through transceiver  163 .  FIG. 2  doesn&#39;t show a plurality of light sources, photodetectors, MCUs, and A/D converters, etc for the sake of description simplification. However in some embodiments, a plurality of component devices such as light source  110 , photodetector  120  and A/D converter  162  can be used in sensor head  105 . 
         [0016]    Sensing module  150  is a key part of sensor head  105 . Module  150  comprises at least one light source  110 , at least one photodetector  120  and at least one temperature sensor  121 . To have fast (low time constant) and accurate temperature measurement of light source  110  and photodetector  120 , in one embodiment, module  150  comprises a good thermo-conductive housing for light source  110 , photodetector  120  and temperature sensor  121 . Temperature sensor  121  is positioned between light source  110  and photodetector  120  so as to have accurate temperature measurement of the both devices. Sensing module  150  is also designed to align the radiation beam of light source  110  and the sensing area and wavelength of photodetector  120  for scattering light detection of cell culture medium  550 . For such reason, module  150  can comprise collimators, lens and optical filter to avoid or reduce light reflection influence of culture medium container  500  such as a flask. 
         [0017]    Without temperature control, the light intensity of light source  110  such as a LED or a laser diode changes with the change of temperature. Light source drive circuit  130  becomes an important part of this invention for temperature compensation and power conservation.  FIG. 3  shows one embodiment of light source drive circuit  130  which comprises power supply  131 , transistor  132  and current control circuit  133  for temperature compensation. Input port  134  is connected to a pulse supply circuit  161 . A high voltage pulse input at port  134  turns on light source  110  and light source  110  is in “light-on” status. A low or zero voltage input turns off light source  110  and light source  110  is in “light-off” status. Power supply  131  is a constant DC voltage supply. Because the output voltage of battery decreases with its usage, power supply  131  can have a step up or down regulator to keep the output DC voltage to be constant. Transistor  132  can be any kind of transistors such as BJT, JFET and MOSFET. Current control circuit  133  for temperature compensation can use different techniques. The technique can be different for LED or laser diode. For a laser diode, its lasing threshold current and output power is temperature dependent. The lasing threshold increases and the output power decreases when temperature increases. There are some circuit methods for temperature compensation. The popular one is to use a photodiode to detect the output power of the laser diode and then make adjustment of driving current to the laser diode automatically. This method is widely used in laser pointer. However this APC control can&#39;t compensate the change of the lasing threshold current. Another method is to use a thermistor circuit to control the driving current of the laser diode. The principle is that a selected thermistor can have approximately same exponential temperature characteristic as that of the lasing threshold current of the laser diode. This analog method can make the temperature compensation less complicated and also having a wide temperature range. 
         [0018]    In one embodiment of this invention, current control circuit  133  can use a thermistor circuit method for the temperature compensation of light source  110 , especially for laser diode. Besides solely analog circuit current control, in another embodiment, current control circuit  133  is controlled by microprocessor  160  based on the measurement input of temperature sensor  121 . Generally, microprocessor  160  makes a variable voltage output via its DAC port to circuit  133 . In this case, circuit  133  is a voltage controlled current source. Microprocessor  160  has a pre-saved compensation function of the driving current of light source  110  versus temperature for a specific and constant output power of light source  110 . This method can be used for both LED and laser diode.  FIG. 3  discloses just one embodiment of light driving circuit  130  with both pulse switching on/off and temperature compensation capabilities. To those skilled in the art, there are many circuit designs which can have both pulse switching on/off and temperature compensation capabilities. 
         [0019]    Although light driving circuit  130  and sensing circuit  140  use some techniques to make temperature compensation, a further step of temperature compensation technique is needed to improve over all optical detection accuracy of the cell culture monitoring system. This second step of temperature compensation is accomplished by microprocessor  160  and software. In one embodiment, microprocessor  160  and memory  164  are used to store a plurality of pre-measured and pre-calculated temperature coefficients for an array of different turbidity of standard medium. Those temperature coefficients are superimposed coefficients of photodetector  120  and light source  110  with circuit compensation. Because the light source  110  has already had a circuit temperature compensation, the temperature coefficients can be obtained with 2 nd  degree polynomial regression instead of 4 th  degree polynomial regression from measured turbidity of a standard turbidity medium at different temperature. To cover a full range of turbidity of culture medium, an array of standard medium such as Formazin with various turbidity values need to be measured at different temperature using sensor head  105 . With those pre-stored coefficients, microprocessor  160  can calculate a correction for the output signal of photodetector  120  based on measured temperature from temperature sensor  121  and turbidity detected from culture medium. This two step temperature compensation technique is different from existing one step techniques. 
         [0020]    Pulse generation circuit  161  is controlled by microprocessor  160 . In one embodiment, circuit  161  can comprise part of digital I/O of microprocessor  160  and a crystal oscillator/clock for pulse and trigger synchronization. The basic function of circuit  161  is to generate a low duty cycle pulse as shown schematically in  FIG. 4  to drive light source  110 . Then drive light source  110  can generate pulsed light based on the driving signal. In one embodiment, the light driving signal has a constant and preset period. The period is equal to the sum of one cycle of light-on time duration t on  and light-off time duration t off  (period=t on +t off ). Because cell culture process in incubator/shaker can take many hours and the change of scattering turbidity of culture medium  550  is slow, the duty cycle can be set to low level. For instance, t on =1 s and t off =19 s, its duty cycle is 5%. This means 95% power can be saved in comparison with a continuous driving light source  110  in prior state of the art without a power saving issue. To conserve power, bandwidth and memory, in one embodiment, A/D converter  162  is externally triggered by pulses from generator  161 . The A/D converter  162  starts analog to digital conversion after receiving the first trigger and stops the conversion after receiving the second trigger. A/D converter  162  can include multiple A/D converters for light signal, temperature signal and motion signal. In one embodiment, pulse circuit  161  generates light driving signal and a plurality of trigger pulses in a sequence as shown in  FIG. 5 . This sequence occurs in each light-on and light-off cycle. Pulse  161   a  is the light driving signal. Pulse  161   b  is a trigger signal for photodetector signal A/D conversion. When light source  110  is on, time t 4  starts A/D conversion and time t 5  stops A/D conversion. There is always a delayed time (t 4 −t 3 ) to allow the output power of light source  110  to be stabilized after it is switched on at t 3 . When light source  110  is switched off at t 6 , shortly, time t 7  starts A/D conversion and time t 8  stops A/D conversion. When light is off, the photodetector signal during the time of t 7 −t 8 , can be used to subtract common noise like ambient light or electrical noise from the light-on signal. Pulse  161   c  is a trigger signal for A/D conversion of both temperature and motion signal. This conversion occurs shortly before the light source is tuned on. Time t 1  starts A/D conversion and time t 2  stops A/D conversion. The temperature measured in duration t 1 −t 2  can be used for the temperature compensation of following light signal measurement in duration t 4 −t 5 . This is critical for the low duty cycle pulse method. Generally, duty cycle can be preset and changed by changing the duration t off . Duration t on  is kept to be constant so that all trigger times (t 1 , t 2 , t 4 , t 5 , t 7  and t 8 ) relative to t 3  and t 6  are fixed. This sequence is synchronized with a clock. All data acquisition of light signal, temperature and motion are timely stamped and can be saved in memory  164  or memory in control module  201 . 
         [0021]    Sensing circuit  140  comprises low noise amplifiers and signal conditioning circuit for photodetector  120 , temperature sensor  121  and motion sensor  122 . Circuit  140  may have differential amplification design which uses a second photodetector as reference to reduce temperature caused drift in output signal. The second photodetector is placed near the first photodetector  120  so that they always have the same temperature. The second photodetector with the same characteristic specs of photodetector  120  is isolated from sensing incident light. 
         [0022]    Wireless transceiver  163  can be constructed with different wireless technologies which are Bluetooth, BLE, Zigbee, or proprietary wireless technology such as ANT. In one embodiment, wireless transceiver  163  is constructed with BLE (Bluetooth Low Energy). Because the power consumption of BLE is low for the cell culture monitoring application. Also piconet of BLE allows control module  201 , computer  600  or smart device  700  to control and monitor up to eight of sensor heads  105 . 
         [0023]    For batch microbial culture such as shaking flask culture, biological cells such as microorganisms experience typical four phases as shown in  FIG. 6 , lag phase, log phase, stationary phase and death phase. In the lag phase, microorganisms grow slowly and are acclimated to their new habitat. In the log phase, the number of microorganisms increases exponentially. In the stationary phase, the viable number of microorganisms becomes stabilized. In the death phase, the viable number of microorganisms decreases. The different biological cells, their growth rate or curves can be different at different shaking speed or temperature. Based on this fact, in some embodiments, the duration of light-off t off  can be adaptive to the growth curve or growth rate of biological cells instead of having a preset and fixed period of the pulse for turning on/off light source  110 . This means that the pulse period is a variable which depends on the biological cell growth level or growth rate. In one embodiment, t off  adjustment is based on the change of calibrated scattering turbidity (T). In another embodiment, t off  adjustment can be based on the change of culture medium OD. In another embodiment, t off  adjustment can be based on light intensity change detected by photodetector  120  such as the voltage output of photodetector amplifier. In this case, the monitoring system doesn&#39;t need to convert photodetector output to turbidity or culture OD. In following embodiment description, only turbidity is mentioned. However the photodetector signal output or culture OD can also be used similarly for the adjustment of light-off duration t off . 
         [0024]    With respect to the t off  adjustment, in one embodiment, the maximum light-off duration t max  and the minimum duration t min  need to be defined and preset before cell culture process. The cell culture will start with t max . In one simple option, the duration t off  can change from t max  to t min  when a growing turbidity value reaches a preset threshold turbidity T t  as shown in  FIG. 6 , for example, such as 120% of initial turbidity value T 0  when cell culture starts. After t off  reaches t min , light-off duration t off  will keep the value of t min  in the rest of cell culture process. In another option, the light-off duration t off  can be a linear function of the turbidity T before the turbidity reaches threshold turbidity T t  and t off  becomes t min  as shown in equation, 
         [0000]    
       
         
           
             
               t 
               off 
             
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                 t 
                 max 
               
               - 
               
                 
                   
                     T 
                     - 
                     
                       T 
                       0 
                     
                   
                   
                     
                       T 
                       t 
                     
                     - 
                     
                       T 
                       0 
                     
                   
                 
                  
                 
                   ( 
                   
                     
                       t 
                       max 
                     
                     - 
                     
                       t 
                       min 
                     
                   
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         [0000]    where T 0  is an initial turbidity of the culture medium. In this case, the light-off duration t off  will also keep the minimum duration t min  even the cell culture is in stationary and death phase. To address this drawback and prolong light-off duration t off  in the stationary and death phase, in one embodiment, the light-off duration t off  can change according to turbidity change rate dT/dt as shown in  FIG. 6 . In the same simple (t max  or t min ) option, the sleep time interval will change from t max  to t min  when the turbidity change rate dT/dt is equal to or larger than a preset threshold rate R t . When cell growth rate decreases and turbidity change rate dT/dt drops below the threshold rate R t  or a different rate, the light-off duration t off  will switch back from t min  to t max  again. In another embodiment, light-off duration t off  changes when the turbidity rate dT/dt changes, t off =t max −C*dT/dt, where C is a preset coefficient which makes C*dT/dt always less than t max . When cell culture starts, dT/dt=0, t off =t max . In one option, light-off duration t off  keeps to be the minimum limit t min . When (t max −C*dT/dt) is equal to or less than t min . 
         [0025]    Motion sensor  122  can be an accelerometer or a vibration sensor. In one embodiment, an accelerometer is used for measuring the shaking speed of incubator/shaker  900 . The shaking speed information can be used for power conservation of sensor head  105 . A shaking Incubator/shaker  900  can be stopped or suspended often for various reasons such as making manual OD measurement, adding drug, or adding another culture flask, etc. When the shaking speed becomes zero during a shaking cell culture process, the operation of turbidity detection in sensor head  105  can be suspended until the incubator/shaker starts to shake again. During the suspension, there is no light emission from the light emission source and there is no the A/D conversion for the photodetector. 
         [0026]    While the invention has been described in conjunction with the preferred embodiments, features and methods, it should be noted that many alternatives, novel features, novel combination, modifications and variations are apparent to those skilled in the art. Accordingly, the preferred embodiments and description in the invention set forth above are intended to be illustrative and not limiting. Various changes may be made without departing from the spirit and scope of the application.