Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Fusion of cells
Reexamination Certificate
2001-10-15
2003-09-23
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Fusion of cells
C435S461000, C435S470000, C435S471000, C435S173600, C435S173700, C435S285200, C536S025410, C424S094100
Reexamination Certificate
active
06623964
ABSTRACT:
This is a continuation of co-pending international application No. PCT/FR00/00983, filed on Apr. 14, 2000, which designated the United States of America.
The present invention relates to a method for treating an aqueous flow colonised by cells by applying an electric field parallel to the flow direction, to a flow and electropulsing chamber and to its application to cell treatment, in particular cell destruction, transmembrane transfer of molecules, membrane fusion and insertion of membrane proteins.
The application of an electric field to cells is known: when a cell is placed in an electric field, it distorts the field lines, causing an accumulation of charge on the cell surface. This results in an induced transmembrane potential difference &Dgr;V which is superimposed on the native difference &Dgr;&PSgr;
0
[Bernhardt J. and Pauly H. (1973): (1)].
The most complete formula used in the case of a field with square wave kinetics and a spherical cell in suspension is as follows [Kinosita and Tsong (1979) (2)]:
&Dgr;
V
(
t
)=
fg
(&lgr;)
r E
(
t
)cos &thgr;(1−
e
−t/&tgr;p
) eq 1
The expression for this potential difference induced at a point M at time t is a fraction of:
E: the intensity of the applied electric field;
f: the form factor for the cell (1.5 in the case of a sphere);
g(&lgr;): factor (of the membrane permeability &lgr;) linked to the conductivities of the external and internal media and to that of the membrane;
r: the cell radius;
&thgr;: the angle between the macroscopic electric field vector and the normal to the plane of the membrane at the point considered, M;
&tgr;
p
: the charge time for the membrane capacity (of the order of one microsecond);
t: time of application of field.
When the pulse duration is much longer than the time to charge the membrane (t>>&tgr;
p
), the term (1−e
−t/&tgr;p
) tends towards 1 to give the stationary state of the conventional formula:
&Dgr;
V
(
t
)=
fg
(&lgr;)
rE
(
t
)cos &thgr; eq 2
The term in cos &thgr; indicates that for a given field, the amplitude of this potential difference is not identical at every point of the cell. It is a maximum at points facing the electrodes (poles) and reduces along the cell surface to become zero at the equator.
This potential difference generated by the field is added to the standing potential difference &Dgr;&PSgr;
0
. This produces a resultant potential difference &Dgr;V
&tgr;
.
&Dgr;
Vr=&Dgr;&PSgr;
0
+&Dgr;V
eq 3
For the cellular hemisphere facing the anode, the numerical values of &Dgr;&PSgr;
0
and &Dgr;V add to take into account the vector of the field effect, causing membrane hyperpolarisation. In contrast, for the hemisphere facing the cathode, the numerical values of &Dgr;&PSgr;
0
and &Dgr;V subtract and the membrane undergoes depolarisation.
When this resulting membrane potential difference exceeds a threshold value estimated to be 200-250 mV [Teissié and Tsong (1981): (3)], a permeabilisation phenomenon is induced [Ho and Mittal (1996): (4)].
The membrane structure responsible for this membrane permeability is unknown at the present time, and the term “transient permeabilisation structure” (TSP) is preferentially used, which is usually expressed by the term “pores”.
If the electropermeablisation conditions are controlled, this permeabilisation phenomenon is transient and reversible, and has little or no effect on cellular viability. This property induced by the field can provide direct access to the cytoplasmic contents [Mir et al., (1988): (5); Tsong (1991): (6); Hapala, (1997): (7)]. This allows foreign molecules that are naturally non permeating to penetrate and thus modifies the contents either transiently or permanently (electrocharging, electrotransformation, electroinsertion).
In contrast, under particularly drastic electropulsing conditions, electropertneabilisation is an irreversible phenomenon that leads to cell death, or electromortality [Sale and Hamilton (1967): (8); Sale and Hamilton (1967): (9), (1968): (10), Hulsheger et al., (1981): (11), (1983): (12); Mizuno and Hori (1988): (13); Kekez et al., (1996): (14), Grahl and Märkl (1996): (15)]. This property has been used either to lyse cells to recover a metabolite of interest, not naturally excreted by the cell, or to eradicate cells from the environment (disinfecting) or from alimentary fluids (non thermal sterilisation) [Jayaram et al., (1992): (16), Knorr et al., (1994): (17); Qin et al., (1996): (18); Qin et al., (1998): (19)].
The prior art discloses two systems for applying a pulsed electric field to a liquid medium, and the choice depends primarily on the volume of liquid to be treated. Fixed bed, or batch, pulse systems have been described. Such units (chambers) and methods can only treat small volumes, however, of the order of a fraction of a milliliter. The technical limit is linked to the power available from the electric pulse generators at a reasonable cost. In addition to research work, such an approach can produce genetically modified organisms (GMO) on an industrial scale.
Further, the application of a pulsed electric field to a flow has been described, which allows a flowing cell suspension to be treated. For the flow method, two strategies have been described: continuous flow and sequential flow.
In the second model, sequential flow, the pulse chamber is filled, the flow is stopped, the field is applied, the chamber is then emptied then refilled. The cells are immobile during application of the field. Thus, there are no hydrodynamic stresses. The operating conditions are thus identical to those described for fixed bed experiments. The flow rate is limited by the need to stop the flow to apply the pulses. However, large volumes can be used, for long periods.
The advantage of a flow system is that large volumes can be treated. The flow consists of an uninterrupted flow through the chamber and synchronising a series of pulses with the flow. Thus, it is possible to apply a defined number of pulses to the cells during their residence time in the pulse chamber. The cells are then moving and subjected to hydrodynamic stresses of deformation and orientation. The flow rate can be very high, being only limited by the frequency of the pulses. This approach, therefore, means that large volumes can be treated quickly.
To carry out certain sequential and/or continuous treatments of flows and in particular to treat certain colonised flows, it is known to use flow systems that apply a field perpendicular to the direction of flow [Teissié and Conte (1988): (20); Teissié and Rols (1988): (21); Sixou and Teissié (1990): (22), Teissié et al., (1992): (23), Rols et al., (1992): (24); Bruggeman et al., (1995): (25); Qin et al., (1996): (18)].
Systems in which the flow and the electrodes are coaxial have also been proposed, also systems in which a non uniform field is applied that is always perpendicular to the flow [Qin et al., (1996): (18); Qin et al., (1998): (19)]. In all of the prior descriptions, the applied field is perpendicular to the direction of flow.
According to Bruggeman et al., (1995): (25), for a given value of the electric field, the flow technique results in lower efficiency than that obtained with a batch system, as demonstrated when electrocharging inositol hexaphosphate onto red corpuscles. According to that method, an increase in the electric field intensity by 10% is necessary to obtain similar results to those obtained with a batch system in a continuous flow system. A more intense field intensity has to be used, and thus the costs are higher. In terms of charging efficacy, the flow approach enables a much larger volume to be treated.
It has now been shown that applying an electric field in a manner that is substantially parallel to the flow can result in higher efficiency for continuous flow treatment methods.
With certain species, in the case of electromortality, the method of the invention can completely eradicate the popula
Cabanes Pierre-André René
Teissie Justin
Vernhes Marie-Christine
Centre National de la Recherche Scientifique
Ketter James
Young & Thompson
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