The Oxystat 1000 System includes the following components:
• Oxystat Incubation Chamber with Stirrer 73-0117
• Power Supply for Stirrer 73-0118 or 73-0119
• pO2 Electrode 73-0120
• Control Unit Model 719 with amplifier and Pump 73-0122
• Stimulator 73-0124
• Thermocirculator 50-1932 or 50-1940
All of these components must be purchased separately. In addition to the above equipment, a chart recorder or data acquisition will permit recording of data for further analysis. The Oxystat chamber is an incubation chamber with a volume of 10 ml. Mechanical damage to the cells during stirring of cell suspensions was an important disadvantage of previous systems; special attention has therefore been paid here to a very gentle stirring action. The cells are introduced into the chamber and the chamber is filled with medium so as to be free from air bubbles (the venting cannula is used to remove air bubbles from the system). The pO2 electrode measures the fall in chamber pO2 due to the oxygen consumed by the cells. Since pO2 measurement is seriously affected by temperature, the chamber is provided with optimal thermostating.
The HSE-HA Oxystat can operate like conventional setups as a ‘closed system’. The oxygen consumption is calculated from the slope of the recorded pO2 reduction. Since the chamber is very well screened against oxygen diffusion it is possible to measure pO2 down to 0.1 mmHg. The really new application of the Oxystat is however an ‘open system’ with controlled addition of oxygen through the solution so that O2 consumption can be measured at constant pO2.
The pO2 signal is examined by the controller; if it differs from the preset value a pump is operated to introduce oxygen-rich solution into the chamber. This raises the pO2 in the chamber back to the set value and the pump is then stopped again. As a result a flow equilibrium is established through which, despite the oxygen consumption of the incubated cells, the chamber pO2 is maintained close to the set value (±5%). The volume in the chamber is maintained constant since the additional volume flows away through an outlet filter. The volume introduced into the Oxystat to maintain the set pO2 is used to calculate the oxygen consumption of the cells. A cannula permits withdrawal of samples for biochemical analysis without interfering with the measurement of oxygen consumption. As an option there is provision for fitting a fiber optic into the chamber for simultaneous measurement of additional parameters such as intracellular Ca++ concentration or redox potential. Two platinum mesh fittings inside the chamber can be used for electrical stimulation of muscle cells to experimentally increase the O2-consumption. A separate stimulator is required for this application.
This system includes the Oxystat control unit and the pump. It consists of a titrator connected to the pO2 meter. The volume is displayed and can be recorded.
Square Wave Stimulator G-270 has been developed for the stimulation of isolated cardiomyocytes in suspension. The main specifications are:
• Output voltage 50 to 250 V
• Maximum output current 7 A
• Square wave biphasic stimulation pattern
• Stimulation width 10 to 255 µsec
• Stimulation frequency 0.1 to 20.0 Hz
An example of this system: Isolated cardiomyocytes were incubated at a pO2 of 90 mm Hg and seven samples were withdrawn consecutively for biochemical analysis. The electrode was calibrated with aerated solution (155 mm Hg). After the cells were added, the pO2 in the chamber drops steeply. When the pO2 falls below 90 mm Hg the controller starts the pump and aerated solution is added until the pO2 has returned to its set value. The pump is operated more or less frequently depending on the O2 consumption of the cells. From the solution volume added in unit time it is possible to calculate the oxygen consumption of the isolated cardiomyocytes. After taking the samples (0.8 ml each, arrows) the total quantity of cells in the chamber is reduced and less solution is required in order to maintain the chamber pO2 at 90 mm Hg.
The relationship between available oxygen and oxygen consumption of isolated cardiomyocytes has been investigated under different forms of stimulation. Resting cardiomyocytes have a constant oxygen consumption over a wide pO2 range (1 to 120 mm Hg). It is only below a so-called ‘critical pO2’, at 1 mm Hg, that the oxygen consumption decreases with decreasing available oxygen. Electrical stimulation of the cells at a frequency of 9 Hz produces a 3-fold increase in oxygen consumption. Here again the oxygen consumption remains constant over a wide pO2 range. The critical pO2 is raised to 10 mm Hg. Below this pO2 the oxygen consumption decreases with decreasing available oxygen. Using the Oxystat system it could thus be shown that stimulation not only produces an increase in oxygen consumption but also that the critical oxygen value, below which the cells are in oxygen deficit, is displaced towards higher partial O2 pressures.