Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-07-27
2004-05-04
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S287100, C435S287500, C435S288700
Reexamination Certificate
active
06730471
ABSTRACT:
The invention relates to a method for monitoring metabolic activity of cells in liquid media, to a vessel particularly appropriate for this, and to a respective device for implementing said method.
In the vessels cells to be cultivated as well as a liquid medium are contained wherein the latter concerns with a conventional nutritive solution corresponding to the used cells, if necessary. The solution according to the invention can be employed for the most different cells and the most different studies, in particular in the pharmacological field wherein the metabolic activity of the cells can be monitored over a longer period. For example, monitoring the action with cytotoxic and biocompatibility tests, and optimizing the culture conditions for the production of biological molecules can be carried out.
Commonly, the major nutrient source for cell cultures is glucose which can be converted into lactate by means of aerobic glycolysis or oxidatively with the oxygen consumption and formation of carbon dioxide. Then, many influences of the physiology of a cell have an affect on its metabolic activity such that oxygen consumption is accordingly allowed to vary as well. Starting from the connection between the metabolic activity of cells with respect to the oxygen consumption, and e.g. the glucose consumption and L-glutamine consumption or the generation of lactate it is allowed to conclude the condition of the monitored cells, and as a result the influence of the respective culture conditions.
Based upon these findings, int. al., in “Noninvasive Oxygen Measurements and Mass Transfer Considerations in Tissue Culture Flasks” published in Biotechnology and Bioengineering, Vol. 51, pp. 466 to 478, it has been described by Lisa Randers-Eichhorn, how the oxygen consumption of cells cultivated in T-flasks can be determined by means of an optical measurement. Therein, it is suggested to arrange sensor membranes containing fluorescent indicators immediately on the flask bottom, and in the gas space above the nutritive solution within such a T-flask. During the measurement of oxygen concentration with such sensor membranes, the well known physical phenomenon of fluorescent erasure of known fluorescent dyes such as e.g. complexes of Ruthenium (II) is employed due to the influence of oxygen wherein the respective fluorescent intensity changes with the continuous excitation according to the oxygen concentration and the partial pressure of oxygen, respectively. For the determination of oxygen concentration the respective fluorescent intensity immediately, but also the fluorescence life can be measured, and the oxygen concentration can be determined according to a known calibration.
The set-up of measuring instruments described in this document, in particular the arrangement of the sensor membrane on the bottom of the T-flasks, and the neglected determination of some important influence quantities is not suitable to conclude the metabolic activity of the culture cells from oxygen consumption.
The charge of oxygen into the nutritive solution occurs for the most part through the interface of the nutritive solution toward the gas space, therefore here toward the gas space in the T-flask, and the consumption occurs by the cells being present on the bottom of the T-flask. The maximum enabled oxygen concentration which can be achieved within the nutritive solution is the saturation concentration of oxygen C
Sat
(of mg/1) which, according to the equation,
C
sat
=([
p−&ggr;*p
w
(
T
)]/p
0
*&agr;(
T
)*
X
02
is a function of the total gas pressure p (mbar), the relative humidity of air &ggr; (in values from 0 to 1, wherein 1 corresponds to a value of 100%, and 0 corresponds to a value of 0%), the partial pressure of water vapour p
w
(T) (mbar) as a function of the temperature T, the mole fraction of oxygen X
02
within the gas space of the T-flasks, the Bunsen absorption coefficient &agr;(T) (mg/1) as a function of the temperature T and the normal pressure p
0
=1013 mbar. Then, it is assumed that the gas space and the nutritive solution have the same temperature, and the gas space has been filled with atmospheric air which chemical composition thereof is sufficiently known. These preconditions are commonly present in breeding chambers in which cells will be cultivated. This saturation concentration of oxygen appears directly below the top surface within the nutritive solution. Therefrom, the oxygen is transported by means of different effects such as diffusion and/or convection toward the cells being present on the bottom. In such a system two quantities are significant. On the one hand, this is the consumption rate k
v
at which oxygen is consumed by the cells, and on the other hand, the transport rate k
T
at which oxygen is transported toward the cells. The two quantities are responsible together for that an oxygen gradient results from the top surface of the nutritive solution toward the bottom including cells. If the consumption rate is now slightly smaller than or equal to the transport rate, thus an oxygen concentration comprising a value of 0 is measured with the oxygen membrane on the bottom below the cells. In this case, the cells do not suffer from an oxygen supply since still sufficient oxygen is transported toward the cells, but which does not arrive toward the sensor membrane below the cells, and which, accordingly, cannot be measured any longer. If the consumption rate further increases, e.g. by spreading out the cells, and the consumption rate becomes greater than the transport rate thus this cannot be monitored any longer with the oxygen membrane located on the bottom below the cells. Furthermore, the saturation concentration of oxygen is a very important quantity in addition to the consumption and transport rates as a function of the total gas pressure, the humidity, the temperature and the mole fraction of oxygen and the partial pressure of oxygen, respectively, within the gas space of the T-flask. A change is causing a change of the oxygen gradient, and thus a change of oxygen concentration at any place between the cells and the top surface of the nutritive solution. Since the parameters of total pressure, humidity and temperature have not been determined or checked in the mentioned documentation, it cannot be excluded that measuring results became falsified due to variations of these parameters.
Therefore, it is the object of the invention to predetermine ways wherein monitoring the oxygen consumption and thus the metabolic activity of culture cells can be achieved in a cost effective manner and with an increased accuracy.
According to the invention this object is achieved with the features of claim
1
for a method, and with the features of claim
11
for an appropriate vessel. Advantageous embodiments and improvements of the invention result from the features mentioned in the subordinate claims.
The solution according to the invention is now assuming from that said cells will be cultivated in vessels using a liquid medium wherein as a rule here it concerns with a respective nutritive solution, and that the metabolic activity thereof takes place through the measurement of oxygen concentration at a location within the liquid medium between the cells consuming oxygen and the part being dominant for the oxygen charge into the liquid medium, which is here the top surface of the nutritive solution. Then, the saturation concentration of oxygen in the liquid medium will be determined according to comparison measurements in a vessel of cell cultures without any cells and/or by means of the determination of the parameters of pressure, humidity, temperature and with a chemical composition being well known and constant, of the ambient gas space, which are here the mole fractions of the gas components and the partial pressures thereof, respectively, in the ambient atmosphere. From the comparison between the saturation concentration of oxygen as a set value and the saturation concentration of oxygen at a location of the oxygen gradient within the vessel including the
Chemnitius Gabriele
Katerkamp Andreas
Key Göran
Institut fur Chemo-und Biosensorik Munster e.V.
Leydig , Voit & Mayer, Ltd.
Weber Jon P.
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