Patent Publication Number: US-2011065084-A1

Title: Integrated oxygen measurement and control for static culture vessels

Description:
This application claims priority to U.S. Provisional Application Ser. No. 61/127,343, filed May 12, 2008, whose entire disclosure is incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to the sensing and control of bioprocess parameters and, more particularly, to non-invasive, integrated sensing and control of oxygen in static culture vessels. 
     2. Background of the Related Art 
     Bioprocesses are important in a wide variety of industries such as pharmaceutical, food, ecology and water treatment, as well as to ventures such as the human genome project (Arroyo, M. et al.,  Biotechnol. Prog.  16: 368-371 (2000); Bakoyianis, V. and Koutinas, A. A.,  Biotechnol. Bioeng.  49: 197-203 (1996); Bylund, F. et al,  Biotechnol. Bioeng.  69: 119-128 (2000); Handa-Corrigan, A. et al.,  J. Chem. Technol. Biotechnol.  71: 51-56 (1998); López-López, A. et al.,  Biotechnol. Bioeng.  63: 79-86 (1999); McIntyre, J. J. et al.,  Biotechnol. Bioeng.  62: 576-582 (1999); Pressman, J. G. et al.,  Biotechnol. Bioeng.  62: 681-692 (1999); Yang, J.-D. et al.,  Biotechnol. Bioeng.  69: 74-82 (2000)). 
     In particular, stem cell research provides an increasingly important path to treating many human diseases. To fully realize the benefits of the research, large quantities of regenerative material will be needed. These quantities can only be obtained by effective and efficient in vitro cultivation, using means which most closely simulate in vivo growth. In vivo, stem cells live in specialized niches and pO 2  is critical in determining their growth and differentiation. 
     To simulate such growth in vitro, techniques are required which accurately monitor and control pO 2  in the vessels used in the cultivation of stem cells. To date, the most common technique is to manipulate pO 2  externally by varying gas phase levels of oxygen in an incubator gas supply under the assumption that pO 2  in the culture vessels will track the external supply. However, this assumption may not be accurate, in that there is a need for more precise control and measurement of the oxygen level in the medium, thereby allowing growth conditions in vitro to more closely match the normal physiological stem cell environment. 
     SUMMARY OF THE INVENTION 
     An object of the invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter. 
     Therefore, an object of the present invention is to provide a system and method for non-invasively monitoring and independently controlling, in real time, pO 2  levels around the cells in individual culture vessels, such as T-flasks. 
     To achieve at least the above objects, in whole or in part, there is provided a system that includes a culture vessel for holding a culture medium, an oxygen sensor positioned inside the culture vessel for detecting dissolved oxygen in the culture medium, wherein the oxygen sensor is adapted to be monitored non-invasively, an agitator attached to the culture vessel for providing agitation to the culture medium, and a controller for determining a dissolved oxygen content of the culture medium based on data from the oxygen sensor and for controlling the agitator based on the dissolved oxygen content of the culture medium. 
     To achieve at least the above objects, in whole or in part, there is also provided a system that includes an incubator, at least two culture vessels positioned inside the incubator for holding respective culture media, an oxygen sensor positioned in each of the at least two culture vessels for detecting dissolved oxygen in each culture vessel, wherein the oxygen sensors are adapted to be monitored non-invasively, an agitator attached to each of the at least two culture vessels for providing agitation to each culture medium, and a controller for determining a dissolved oxygen content of the culture medium in each culture vessel based on data from the oxygen sensors and for independently controlling each agitator based on the dissolved oxygen content of the respective culture medium. 
     To achieve at least the above objects, in whole or in part, there is also provided a method that includes non-invasively monitoring dissolved oxygen levels in at least two culture media, and selectively and independently agitating the at least two culture media based on the dissolved oxygen levels in each culture medium. 
     To achieve at least the above objects, in whole or in part, there is also provided a method that includes non-invasively monitoring dissolved oxygen levels in a culture medium, and selectively agitating the culture medium based on the dissolved oxygen levels the culture medium. 
     Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and advantages of the invention may be realized and attained as particularly pointed out in the appended claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention will be described in detail with reference to the following drawings in which like reference numerals refer to like elements wherein: 
         FIG. 1  are plots illustrating several hypothetical profiles that can be achieved by varying the gas environment in an incubator; 
         FIG. 2  is a schematic diagram showing the principles of operation of an oxygen sensor patch used in the present invention; 
         FIG. 3  is a plot showing gas phase and liquid phase medium oxygen measurements in hybridomas; 
         FIG. 4  is a plot showing gas phase and liquid phase medium oxygen measurements in hybridomas while the T-flask was being agitated, in accordance with the present invention; 
         FIG. 5  is a schematic diagram of a system for non-invasive measurement and control of pO 2  in a culture vessel, in accordance with one embodiment of the present invention; 
         FIG. 6  is a plot showing oxygen control in three wells of a 24 well plate using agitation of each well, in accordance with the present invention; 
         FIG. 7  is shows an oxygen sensor module and an ADC used in one preferred embodiment of the present invention; 
         FIG. 8  shows a vibration motor used in one preferred embodiment of the present invention; 
         FIGS. 9A-9D  illustrate examples of other types of agitators that may be used to agitate the culture medium, in accordance other embodiments of the present invention; and 
         FIG. 10  is a schematic diagram illustrating how the system for non-invasive measurement and control of pO 2  in a culture vessel can be placed inside an incubator, in accordance with the present invention. 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     By way of example, the present invention will be described in connection with the monitoring and control of pO 2  levels in a stem cell cultivation environment. However, it should be appreciated that the present invention can be used to monitor and control pO 2  levels in any type of static culture vessel. 
     The description below cites numerous technical references, which are listed in the Appendix below. The numbers shown in parenthesis at the end of some of the sentences refer to specific references listed in the Appendix. For example, a “(1)” shown at the end of a sentence refers to reference “1” in the Appendix below. All of the references listed in the Appendix below are incorporated by reference herein in their entirety. 
     Adult stem cells have attracted enormous attention due to their promise as an unlimited source of regenerative material to treat a large number of human disease conditions (1-8). In particular, the multilineage potential of adult cells is attractive as it sidesteps controversies related to embryonic stem cells ( 43 ). 
     While many advances have been made in our ability to isolate adult stem cells, their culture, differentiation and expansion remains more art than science. It is believed that in order to fully understand and control stem cell behavior, the in vivo niches that they originate from need to be replicated in vitro ( 9 - 11 ). Recently, it has become evident that the culture conditions and, in particular, the oxygen level under which the cells are grown result in varying phenotypes ( 12 - 15 ). Thus, it is critical to control the oxygen level in in vivo stem cell cultures. 
     It has been assumed that the concentration of gaseous O 2  supplied into the incubator is the same that is achieved in the liquid medium in which cells grow. However, this critical premise is inaccurate in the case of both static and shaken culture vessels, and the oxygen levels that the cells experience in the liquid medium are quite different than the gas concentration. 
     In the description that follows, 100% oxygen is referred to as equivalent to air saturation. This stems from the bioprocessing practice of calibrating oxygen sensors to read 0% in equilibrium with nitrogen and 100% in equilibrium with air. All literature data referred to henceforth has been converted to these units. Therefore, a reviewer going back to a cited paper may find that what we refer to as 100% is referred to as 21% (or in some cases as 20% when 5% CO 2  is present) in the original citation. It is also important to understand that the statement % oxygen refers to equilibrium between the gas and liquid phase. Therefore, 100% oxygen concentration (or level or pO 2 ) means that the gas phase is at 21% oxygen and is in equilibrium with the liquid. Note that the actual oxygen available to the cells in absolute terms is much lower and is determined by the solubility of oxygen in the medium, which is approximately 8 mg/l or a mere 250 μM in distilled water at 35° C. In practice, solutes decrease this even more and this is why oxygen supply to cells growing in liquids is such a challenge. 
     Every one of these studies cited below where oxygen levels are referred to used gas phase equilibration to achieve the stated oxygen level in the medium. However, the actual oxygen level experienced by the cells has NOT been continuously measured. Nevertheless, as the studies show, growth and differentiation are significantly affected by pO 2 , and thus its accurate measurement and control are critical. 
     Hyperoxia has been long known to have deleterious effects on all types of cells due to oxidative stress caused by increased oxygen free radical production ( 33 ). More recently, hypoxia also appears to play a critical role in cell migration, growth and regulation largely via induction of Hypoxia Inducible Factor (HIF) ( 28 ,  34 ,  35 ). Hypoxia&#39;s role in stem cells is also drawing more scrutiny and appears to be important both in normal as well as in cancer stem cells ( 12 - 15 ,  36 ). 
     Stem cells are regarded to occupy niches, which have been compared to ecological niches ( 9 ,  11 ). It would appear that oxygen plays a major role in this niche, as recent evidence points to the stem cells residing in a hypoxic environment ( 12 ,  13 ). It has been shown that when some cells are grown under hypoxic (10-15%) compared to normoxic (100%) conditions, they proliferated more and displayed phenotypic changes. In addition, differentiation ability has been found to be significantly affected. For example, Malladi et al. show that adipose derived MSCs have severely diminished osteogenesis capability when cultured at 10% oxygen versus 100% ( 37 ). 
     Different types of cells have been grown in the presence of low oxygen. For example maintenance of cord blood progenitor cells under low oxygen condition was comparable to growing them on irradiated stromal cells ( 15 ). Adult and Neonatal fibroblasts seeded as single cells did not proliferate under 100% oxygen but had been shown to expand and form colonies when grown under low oxygen culture condition ( 38 ). In the case of CD34+ bone marrow cells, low oxygen culture condition increased their proliferation rate significantly and increased the number of colony forming units ( 39 ). Murine mesenchymal stem cells (MSCs) under hypoxic culture conditions showed greater migration and higher secretion of VEGF and tube formation ( 40 ). 
     It has been demonstrated that culturing rat neural stem cells in more physiological oxygen condition (10-15% O2) increases cell proliferation and enhances clonal expansion and reduces apoptosis ( 41 ). These studies also showed an effect of oxygen level in culture on differentiation of NSPCs. It was observed that lowered oxygen enhanced dopaminergic neuronal phenotype upon differentiation ( 41 ). 
     In another study, ASCs were suspended in alginate beads and cultured in control or chondrogenic media in either hypoxic (25%) or normoxic oxygen tension (100%) for up to 14 days ( 42 ). Under chondrogenic conditions, low oxygen tension greatly inhibited the proliferation of ASC cells, but was found to induce a two-fold increase in the rate of protein synthesis and a three-fold increase in total collagen synthesis. Hypoxia was found to increase glycosaminoglycan synthesis at certain timepoints. These findings suggest oxygen tension plays an important role in regulating the proliferation and metabolism of ASC cells as they undergo chondrogenesis, and provide evidence that the exogenous control of oxygen tension may provide a means of increasing the overall accumulation of matrix macromolecules in tissue-engineered cartilage ( 42 ). 
     Ultimately, if stem cells are to ever become successful in therapy, their in vivo niches need to be recreated in vitro ( 11 ). As the brief literature survey presented above indicates, oxygen levels are a critical factor in stem cell physiology and it is important to measure and control these levels. A recent report has raised the possibility of using hypoxia to arrest stem cell differentiation ( 12 ). This report may give rise to a strategy of expanding stem cells under controlled hypoxia before the induction of normoxic differentiation. For such a strategy to be implemented, oxygen levels would need to be reliably measured and controlled under in vitro culture conditions. 
     As their therapeutic use increases, stem cells will need to be manufactured under Food and Drug Administration (FDA) oversight and licensing. A critical aspect of obtaining approval for therapeutic use will require that the manufacturing conditions follow Good Manufacturing practices (GMP). The FDA is actively encouraging industry to implement Process Analytical Technologies (PAT) as a part of this effort in order to obtain consistent product quality. As a parallel, one can look at the biopharmaceutical industry where protein-based therapeutics are typically produced by mammalian cells grown in bioreactors. In these systems, oxygen, pH and temperature levels are controlled in order to achieve consistent scale-up and product quality as measured by amino acid sequence, glycosylation profiles, purity and potency. Similar metrics for stem cells will also be required and the present invention can help validate the degree to which oxygen level control can be used to make a consistent product as measured by markers and differentiation assays. 
     Given the clear evidence that oxygen levels impact the fate and proliferation of cells, vendors of incubators have made available systems which allow the investigator to blend gas mixtures to achieve desired pO 2  levels in culture. It has been believed that the cells in the incubator quickly achieve equilibrium with the gas environment. This premise has been used in the references cited above to achieve hypoxic and normoxic conditions. Based on the observed results, it appears that elaborate conditioning protocols may be effective in preparing cells for different applications, such as expansion, preconditioning for implantation, differentiation, wound priming etc.. 
       FIG. 1  illustrates several hypothetical profiles that can be achieved by varying the gas environment in the incubator. Again, the critical assumption underlying all these approaches is that the cells growing in liquid media are in equilibrium with the gas phase. We have tested this basic and key assumption because, if it is correct, protocols that are currently in use are appropriate. However, if it is not correct, a major rethinking of how cells are cultured becomes necessary. 
     The present invention allows for feedback control for individual T-flasks, which is not achievable with prior art shaker incubators. One could achieve quasi-control by manipulating the rocking rate of a shaker incubator, but shaker incubators do not provide the means to achieve feedback control by using the output of an oxygen sensor that measures the liquid phase pO 2 . In order to truly replicate an in vivo stem cell niche, the present invention controls pO 2  based on feedback from an actual liquid pO 2 . The problem with currently published studies where oxygen levels have been manipulated using the gas phase is that the precise oxygen levels experienced by the cells are not known in those studies. The cells would have been normoxic or hypoxic, but the exact degree to which they were and the reproducibility of the experiments are questionable without actual measurements. 
     Commonly assigned and related U.S. Pat. Nos. 7,041,493 and 6,673,532, both by Govind Rao at al., describe a system and method for non-invasively measuring cultivation parameters in culture vessels. One of the parameters that can be measured is dissolved oxygen. 
     The optical oxygen sensors work on equilibrium principles.  FIG. 2  illustrates their principle of operation. A sterilizable oxygen sensor patch  140  with chemistry unique to oxygen is affixed inside the vessel  20  where measurements are to be made. The oxygen sensor patch  140  is illuminated at an appropriate wavelength from the outside using, for example, an LED  30  and the resulting fluorescence signal is measured using a photodetector  40 . The oxygen concentration in the vessel is deduced from a previously made calibration curve. The major advantages of this approach are as follow:
     In situ measurement—no sampling needed   Non-invasive—no penetration into the vessel thus avoiding possible contamination   Low-cost light sources, semiconductor detectors can be used   Measures through the material (any transparent vessel)   Simple calibration—patches are pre-calibrated   Miniaturization is possible   

     Other technologies, such as blood gas analyzers, are available. However, their main drawback is the necessity of a sample from the culture vessel, which is inconvenient and labor intensive. Other approaches require invasive insertion of electrodes/optrodes into the culture vessel, requiring modification of the culture vessel and increasing the risk of contamination. The electrodes also require individual calibration, precluding their use in large numbers of culture vessels. 
     In order to determine whether cells that are growing in liquid media in static culture flasks in an incubator are in equilibrium with the gas phase, an experiment was performed where hybridoma cells were cultured in a CO 2  incubator where the headspace gas concentration was 5% CO 2  and the balance was air. A T-flask was set up with two oxygen sensor patches inside it. One of the oxygen sensors was located in the headspace above the liquid and the other one was located in the liquid ( 44 ). Continuous measurements of oxygen were made for the duration of the culture, with samples withdrawn once a day to determine cell count. 
     As can be seen in the plot of  FIG. 3 , the headspace oxygen level remained close to 100%, indicating that it was in equilibrium with the incubator atmosphere. The liquid oxygen level shows a very different profile. As the cells grew (indicated by the viable cell counts), the oxygen consumption rate became greater than the oxygen diffusion rate into the medium (as evidenced by the decreasing oxygen level in the liquid phase) until it reached zero. The cells stayed in this hypoxic regime and the liquid phase oxygen levels only started increasing gradually as the viable cell counts started decreasing as the cells started dying. The spikes in oxygen levels were caused by the mixing of the flask contents that took place when a sample was withdrawn. However, the increased oxygen was temporary as it was quickly consumed once the flasks were static again. 
     These data clearly demonstrate that, in static cultures, oxygen diffusion into the medium is rate limiting and that cells experience widely varying oxygen levels as a result. This is not a surprising result if one considers that the diffusion coefficient for oxygen in nitrogen is 0.240 cm 2 s −1 , but for oxygen in water it is only 0.0000324 cm 2 s −1 ( 56 ). Therefore, a strategy for gently agitating the culture vessel to promote better mixing should result in better equilibration of the gas and liquid phases and promote more uniform oxygen levels. 
     This approach was tested in the following experiment. An aquarium pump was placed on the top shelf of the CO 2  incubator and turned on for the duration of the experiment. The steady vibrations emitted by the pump were sufficient to gently mix the liquid medium and allowed for much greater oxygen diffusion from the headspace into the liquid. As seen in the plot of  FIG. 4 , and in contrast with the static culture results shown in  FIG. 3 , the liquid phase oxygen levels were observed to be above 50% throughout the duration of the culture. 
     The vibration intensity was constant, however, had we been able to control its intensity, we should have been able to obtain a different oxygen profile in the liquid. These data provide proof-of-principle that agitation can be used to provide feedback control of pO 2  levels in cell culture vessels in an incubator. 
       FIG. 5  is a schematic diagram of a system  100  for non-invasive measurement and control of pO 2  in a culture vessel, in accordance with one embodiment of the present invention. The system  100  is designed for use in an incubator (not shown). 
     The system includes a culture vessel  110  that holds cells  120  and a cell culture medium  130 . An oxygen (pO 2 ) sensor patch  140  is affixed inside the culture vessel  110 , and is monitored optically (non-invasively) from the outside using an illuminating light source  150  and a photodetector  160  for measuring the optical signal emitted by the oxygen sensor patch  140 . An agitator, suitably a vibratory mixer  170 , is attached to the culture vessel  110  for providing agitation and promote rapid equilibration between the culture medium  130  and the gas phase. The vibratory mixer  170  is preferably under the control of a controller  180 , such as a computer. pO 2  control of gas phase concentrations is controlled by the controller  180 . The mixer driven equilibration will result in precise control of the liquid medium pO 2 . 
     The culture vessel  110  is suitably a T-flask with a 0.2 m pore size filter vented cap  190 . These minimize diffusive resistance between the C-chamber atmosphere and the headspace in the flask and provide a tight and sterile seal. However, the culture vessel  110  can be any type of vessel that can be used to culture cells such as, for example, a Petri dish, a spin tube, a spinner flask or a shaker flask. The oxygen sensor  140  is not affected by the size of vessel  110  used. 
     The oxygen sensor patch  140  contains an oxygen-sensitive luminescent dye that is a complex of Ruthenium, Platinum, or any other metal ligand complex. The compound is preferably immobilized in silicone rubber to provide an inert, steam, ethanol or radiation sterilizable patch. The oxygen sensor patch  140  is then calibrated. One advantage of this type of oxygen sensor patch  140  is that calibration is required on only one of the patches when a batch of patches is made. All patches from a common lot will behave identically, so individual calibration of each patch is not required. The measurement of the dissolved oxygen is preferably based on Stern-Volmer quenching of emission, as described below. 
     The excited state of the luminescent dye in the oxygen sensor patch  140  is quenched proportionately to the oxygen concentrations. Intensity-modulated light at a frequency (w=2p×Hz) generated from the light source  150 , preferably light emitting diodes (LED&#39;s), serves as the excitation source. The phase shift (f) of the resulting emission from the oxygen sensor patch  140  is related to the decay time (t) by 
       tan f w =wt   (1)
 
     Collisional quenching by oxygen is described by the Stern-Volmer equation, 
         t   0   /t= tan  f   0 /tan  f= 1+ Ksv[Q]   (2)
 
     where t 0  and f 0  are the decay time and phase in the absence of oxygen, respectively, Ksv is the Stern-Volmer constant and is the oxygen concentration. 
     For calibration, the phase angle is measured as a function of various mixtures of oxygen in nitrogen and a curve fit is performed. This is then entered into software on the controller  180  and, when an oxygen measurement is needed, the software automatically calculates the oxygen concentration from the calibration file. The typical calibration curve behaves like a saturation binding curve. Consequently, the oxygen sensor patch  140  becomes more sensitive as the oxygen tension decreases and has greater accuracy at lower oxygen levels. 
     The oxygen sensor patch  140  is preferably steam sterilized and inserted into the culture vessel  110  under sterile operating conditions in a laminar flow hood. The oxygen sensor patch  140  preferably have a biocompatible adhesive that is designed as a “peel-and-stick” unit. 
     The accuracy of the preferred oxygen sensor patch described above has been extensively valiated ( 45 , 47 , 51 , 52 , 55 ).  FIG. 6  is a plot comparing the preferred oxygen sensor patch  140  with conventional electrochemical sensors (a Clark electrode) during a cell culture experiment ( 45 ). As the data show, the optical sensor data agree well with the Clark oxygen electrode. Furthermore, the preferred oxygen sensor patch  140  has been extensively validated using microarray analysis to ensure that the sensor patch chemistry and/or optical energy used for the measurement do not affect the cells or product adversely ( 47 ). 
     The plot of  FIG. 9  also demonstrates pO 2  control in a 24 well plate system where 3 wells were equipped with oxygen sensor patches  140  and computer control was set at a setpoint of 20% ( 55 ). The cells were  E. coli,  which have a very high oxygen demand compared to mammalian cells and are much harder to control. 
     Initially, the oxygen levels were high until the cells grew to a point where oxygen supply was less than the consumption rate of the respiring cells. Once the oxygen levels reached the setpoint, control was achieved by increasing the stirring speed in the wells whenever oxygen dropped below the setpoint. The increased mixing had the effect of increased oxygen transport into the liquid phase and raised its level. If the oxygen level crossed the setpoint, then the stirring speed was decreased. This control algorithm relies on cells consuming the oxygen to maintain a steady oxygen level and was done by feedback control using the controller, preferably a computer. 
     Towards the end (six hours onwards) after the cells ran out of nutrient and respiration slowed down, the oxygen levels again rose and reached equilibrium with the supplied air. Reasonable accuracy was obtained despite no attempt being made to tune the control algorithm. 
     The light source  150  and the photodetector  140  are preferably packaged together to form an oxygen sensor module  200 , as shown in the photograph of  FIG. 7 . The oxygen sensor module  200  is mounted such that the light from the light source  150  illuminates the oxygen sensor patch  140 . The emission from the oxygen sensor patch  140  is then detected by photodetector  160 . The oxygen sensor module  200  is preferably connected to an analog-to-digital converter (ADC)  175 , that communicates with the controller  180 . The ADC  175  preferably has a variable voltage output that is used to drive the vibratory mixer  170  under the control of the controller  180 . 
     The vibratory mixer  170  is preferably a miniature vibration motor  250 , such as the one shown in the photograph of  FIG. 8 , that is typically used in cell phones and pagers. This type of vibrator motor body is typically only 0.44″ L×0.18″ Dia., and has a weight attached to its shaft to provide inertial damping. It has 2 flexible terminals (an important feature for a vibrating device so that the power leads do not fall off) for power connection and can operate from 1VDC up to 9VDC. The resistance of the motor is 10 ohms. 
     The vibration motor  250  is preferably affixed to the culture vessel  110  with double-sided tape. The optimum location on the culture vessel  110  to attach the vibration motor  250  is experimentally determined based on simple mixing studies conducted by dropping a colored dye into water and observing the mixing patterns, as has been done with minibioreactors ( 58 ). This allows one to choose the vibration motor location that results in the shortest mixing times. It is important to avoid a location that matches the natural resonant frequency of the system, as that will result in standing waves and poor mixing. 
     Although the agitator shown in  FIG. 5  is a vibratory mixer  170 , any other type of agitator known in the art may be used while still falling within the scope of the present invention.  FIGS. 9A-9D  illustrate examples of other types of agitators that may be used.  FIG. 9A  shows the use of an ultrasonic transducer  172  to induce ultrasonic waves in the culture medium  130 .  FIG. 9B  illustrates the use of a rocker  300 . The oxygen sensor module  200  is attached to the bottom of the culture vessel  110 , and the culture vessel/oxygen sensor module combination sits on top of the rocker  300 . The rocker  300  tilts the culture vessel/oxygen sensor module combination back and forth in a “teeter-totter” motion to agitate the culture medium  130 . 
       FIG. 9C  illustrates the use of a rotator  310  to agitate the culture medium  130 . The culture vessel/oxygen sensor module combination sits on top of the rotator, which gyrates in a back-and-forth circular motion. 
       FIG. 9D  illustrates the use of a turntable  320  to agitate the culture medium  130 . The culture vessel/oxygen sensor module combination sits on top of the turntable, which rotates to swirl the culture vessel/oxygen sensor module combination. 
     The agitator, whichever type is used, is preferably powered with a feedback loop from the sensor software run by the controller  180  to agitate when oxygen levels drop below a user entered set point. When this happens, the diffusion of oxygen into the liquid phase will increase until the set point is reached, at which point the controller will turn the motor off. 
     All of the instrumentation is preferably contained in water-proof enclosures. This allows them to be sprayed with 70% ethanol and wiped down prior to placement in the incubator chamber to maintain sterility. It also prevents fogging of the optical windows internally during use in a humidified incubator. 
     The controller suitably runs a LabVIEW based data acquisition software. When a vibratory mixer  170  is used as the agitator, dissolved oxygen is controlled by changing the vibration rate of the vibratory mixer  170 . The vibration rate is changed by controlling the voltage supply to the vibratory mixer  170 . pO 2  is preferably controlled by the following well-known PID (proportional-integral-derivative) algorithm for sampled systems: 
     
       
         
           
             
               
                 
                   
                     
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     where A is the controller output (vibration rate), E is the difference between the measured and desired pO 2,  and K c , t, and t d  are the gain, integral time constant, and derivative time constant, respectively. T is the time between successive measurements. 
     The design of the control software assumes that cells are actively consuming oxygen. Control is achieved by varying the oxygen supply rate, which is a function of the agitation rate provided by the vibratory mixer  170 . This is significant, as it means that the upper limit for control will be determined by the gas phase oxygen concentration. At equilibrium, this will be the maximum pO 2  achievable in the liquid. The lower ranges will be determined by the agitation rate. In practice, for studies on normoxic or hypoxic respiring cells, we hypothesize that it may be possible to eliminate expensive gas phase environment controls. With respiring cells, one could simply run all incubators with inexpensive house air and achieve control at any desired oxygen level with the systems and methods of the present invention. This could be achieved at a fraction of the cost of an incubator controller. 
     As discussed above, the system  100  is designed to be placed inside any type of incubator.  FIG. 10  schematically shows an incubator  400  that is supplied with a blend of O 2 , N 2  and CO 2  in any desired combination using a commercially available oxygen controller  410 , such as the OxyCycler from Biospherix. The system  100  would preferably placed inside a cell culture C-Chamber that resides inside the temperature controlled incubator  400 . 
     The foregoing embodiments and advantages are merely exemplary, and are not to be construed as limiting the present invention. The present teaching can be readily applied to other types of apparatuses. The description of the present invention is intended to be illustrative, and not to limit the scope of the claims. Many alternatives, modifications, and variations will be apparent to those skilled in the art. Various changes may be made without departing from the spirit and scope of the invention, as defined in the following claims (after the Appendix below). 
     APPENDIX  
     
         
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