Electroporation device

• US 20060057706A1
• Filed: 06/10/2003
• Published: 03/16/2006
• Est. Priority Date: 06/07/2002
• Status: Active Grant

• Current Assignee: Igea Sri
• Original Assignee: Igea Sri
• Inventors: Mir, Lluis, Miklavcic, Damijan
• Granted Patent: US 7,625,729 B2
• Time in Patent Office: Days
• US Class Current: 435/285.200
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

Electro-poration device for the permeabilization of cells contained in a substrate comprising a signal generator for generating a stimulating signal applied by means of electrodes to the substrate wherein an electric field permeabilizing the cells membranes is induced. The device calculates and monitors the instantaneous value or the ratio GT between the current flowing through the substrate and the voltage of the stimulating signal applied to the substrate; in particular, the stimulating signal is applied in a modified manner according to the shape of an initial portion of the waveform of a curve C_GT representing the value of ratio GT in successive instants after the application of the stimulating signal. The system will achieve permeabilization of cells in substrate with no or minimum damage, allowing to introduce or to extract molecules in or from the electropermeabilized living cells.

Claims

1. Electroporation device for the permeabilization of cells (C) contained in a substrate (12) comprising signal generating means (3) for generating a stimulating signal (S(t)) applied by means of electrodes (6,7) to the substrate (12) wherein an electric field (E(t)) permeabilizing the cells membranes is induced;

the device being characterized by comprising;

means for measuring, calculating and monitoring (15,16,23) the instantaneous value of the ration (GT) of current (ie) flowing between said electrodes (6,7) and through the substrate (12) and the voltage (Vp) of the stimulating signal (S(t)) applied to the substrate (12) by means of said electrodes (6,7);

said device further comprising controlling means (100-170) for applying the stimulating signal in a controlled manner according to the waveform of an initial portion of the curve C_GT representing the ratio (GT) in successive instants after the beginning of the application of the stimulating signal (S(t)).

2. Device as claimed in claim 1, wherein said controlling means (100-170) comprise timing means (110) for applying said stimulating signal for a predetermined period of time Td and analysing the initial portion of the waveform of curve C_GT to detect a minimum value of the curve C_GT within the interval t=0 and t=Td.

3. Device as claimed in claim 1, wherein said controlling means (100-170) calculate the slope of the waveform of curve C_GT after that a minimum in curve C_GT has been reached.

4. Device as claimed in claim 1, claims, wherein said controlling means (100-170) comprise hazard detecting means (120) determining the instantaneous gradient (dG) of said ratio (GT) after a minimum has been reached in said curve C_GT;

said controlling means further comprise first comparing means (130) for comparing the calculated instaneous gradient dG with at least a reference value (dG_ref1) and selecting correcting means (140,145) for performing an urgent correction to the stimulating signal S(t) in order to avoid lesions, damages or irreversible alterations in said substrate (12).

5. Device as claimed in claim 4, wherein said correcting means (140,145) decreases the voltage of the stimulating signal S(t) in order to prevent deterioration in the cells (C).

6. Device as claimed in claim 1, wherein said controlling means (100-170) comprise slope determining means (150) calculating the average variation Δ

G of said ratio (GT) in a time interval that is successive to the instant Tm wherein a minimum in the curve (C_GT) has been reached and that has a pre-determined time width;

said controlling means further comprising second comparing means (160) comparing the calculated average variation Δ

G of said ratio (GT) with a reference interval of Δ

G values.

7. Device as claimed in claim 6, wherein said second comparing means (160) performs the following functions:

if the calculated average variation Δ

G of said ratio (GT) falls within the reference interval (0<

Δ

G<

Δ

G_obb) continuing means (170) are selected;

if the calculated average variation Δ

G of said ratio (GT) falls outside the reference interval and it is smaller than both limits delimiting the interval (Δ

G<

0<

Δ

G_obb) adjusting means (180) are selected; and

if the calculated average variation Δ

G of said ratio (GT) falls outside the reference interval and it is greater than both limits delimiting the interval (Δ

G>

Δ

G_obb>

0) correcting means (140) are selected.

8. Device as claimed in claim 7, wherein said adjusting means (180) increase the voltage of the stimulating signal in order to increase the value of the electric field E(t) applied to the substrate (12);

said adjusting means (180) subsequently selecting said means for calculating and monitoring (15,16,23) the instantaneous value of the said ratio (GT) and said controlling means.

9. Device as claimed in claim 7, wherein said continuing means (170) increase the voltage of the stimulating signal to an objective voltage V_opt in order to increase the value of the electric field E(t) applied to the substrate (12) so that the value of said average variation Δ

G tends to an expected value Δ

G_obb.

10. Device as claimed in claim 1, wherein said controlling means (100-170) detects (125) a minimum of said initial portion of said curve and determining (126) the time Tm at which the minimum is reached.

11. Device as claimed in claim 10, wherein third comparing means (127) are provided to compare the detected Tm with threshold values Ttmin and Ttmax;

said third comparing means (127) performing the following operations;

if the detected Tm occurs before Ttmin (Tm<

Ttmin) then correcting means (140) are selected;

if the detected Tm occurs after Ttmax (Tm>

Ttmax), then adjusting means (180) are selected; and

if the detected Tm occurs between Ttmin and Ttmax, then continuing means (170) are selected.

12. Use of a device as described in claim 1, to extract molecules from the living cells comprised in the substrate.

13. Use of a device as described in claim 1, to introduce molecules into living cells.

14. Use of the device as claimed in claim 13, wherein said molecules comprise one of the following:

a DNA or a RNA molecule containing regulatory sequences and sequence coding for therapeutic genes or genes of interest for biomedical or biotechnological purposes;

an oligonucleotide, (ribo- or deoxyribo-nucleotide, single or double strand, including the SiRNA), whether natural (phosphodiesters) or modified (inside the backbone of the oligonucleotide, such as phosphosulfates, or at the extremities, by addtion of groups to protect the oligonucleotides from digestion of nucleases;

a protein or peptide, whether natural or genetically or chemically modified, extracted from natural sources or obtained by synthesis, or a molecule simulating the structure of a protein or peptide, whatever its structure;

a cytotoxic agent, in particular, the antibiotic bleomycin or the cisplatinum;

a penicillin; and

other pharmacological agents.