Glucose and oxygen concentrations are monitored, and glucose concentration and pH are adjusted as needed.
Figure 1 is a simplified schematic diagram of a system that automatically regulates the concentration of glucose or pH in a liquid culture medium that is circulated through a rotating-wall perfused bioreactor. Another system, shown in Figure 2, monitors the concentration of oxygen in the culture medium.
The glucose-regulating system includes an electrochemical sensor that measures the concentration of glucose in the medium flowing out of the bioreactor, a reservoir containing a concentrated glucose stock solution, and a peristaltic pump. The sensor reading is digitized and monitored by a computer, which generates commands to open and close valves, as described below, to adjust the concentration of glucose. This allows the system to maintain an optimal concentration of glucose without using large volumes of cell culture medium. The glucose control system operates in one of the following three modes (see Figure 1): perfusion, where the medium is simply circulated without adding or removing any liquid; infusion, where fresh cell medium is introduced into circulation, replacing the spent medium in the bioreactor; injection, where a small amount of concentrated glucose solution is added to the bioreactor when the glucose concentration falls below 75 mg/dL.
The electrochemical glucose sensor includes a membrane that is covered with a thin (10 to 200 mm thick) layer of immobilized glucose oxidase enzyme and is coupled to a three-electrode amperometric probe and a flow cell. Glucose diffuses through the membrane and, in the presence of the glucose oxidase, is converted to hydrogen peroxide and gluconic acid. The rate of generation of hydrogen peroxide is measured amperometrically on a platinum working electrode at a potential of 0.7 V with respect to an Ag/AgCl reference electrode. The sensor reading is correlated with the concentration of glucose by the Michaelis-Menten equation,
I=ImaxS/(K + S),
where I is the sensor current, Imax is the maximum sensor current for the amperometric reaction, K is a constant, and S is the concentration of glucose. The constants Imax and K are determined by means of a two-point calibration, using a commercial glucose analyzer to determine the glucose concentrations. This sensor system has been tested in cell culture experiments for over 50 days. The control of glucose in this manner reduces the amount of culture medium required to optimally grow cells.
The pH sensor, described in “Optoelectronic Instrument Monitors pH in a Culture Medium” (MSC-23107), NASA Tech Briefs, Vol. 28, No. 9 (September 2004), page 4a, was coupled to the above control system containing a small volume of a mixture of sodium and potassium hydroxide and bicarbonate solution to form a pH control system. When the sensor pH output fell below a preset level, a small amount of buffer was injected to the bioreactor to bring the pH to the desired level. This system was tested in a 120-day cell run, where the pH was controlled at 7.1±0.1 pH unit. The volume of buffer used was negligible compared to the volume of cell culture medium required for the same level of pH control. By controlling the culture pH in this manner, the cells produced less biofilm.