Electroporation device

• US 7,625,729 B2
• Filed: 06/10/2003
• Issued: 12/01/2009
• Est. Priority Date: 06/07/2002
• Status: Active Grant

• Current Assignee: Igea Sri
• Original Assignee: IGEA S.p.A.
• Inventors: Mir, Lluis, Miklavcic, Damijan
• Primary Examiner(s): Beisner; William H
• Assistant Examiner(s): HOBBS, MICHAEL L
• Application Number:
  US10/517,038
• Publication Number:
  US 20060057706A1
• Time in Patent Office: 2,366 Days
• Field of Search: 435/285.2, 435/460, 435/173.6, 435/461, 435/470, 800/292
• US Class Current: 435/173.6
• 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 CGT 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 comprising a signal generator configured for generating a stimulating signal (S(t)) applied by means of electrodes to the substrate wherein an electric field (E(t)) permeabilizing the cells membranes is induced;

the device comprising;

an element configured for measuring, calculating and monitoring the instantaneous value of the ratio (GT) of current (ie) flowing between said electrodes and through the substrate and the voltage (Vp) of the stimulating signal (S(t)) applied to the substrate by means of said electrodes;

said device further comprising a controlling element configured for applying the stimulating signal in a controlled manner according to the waveform of only 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 element comprises a timing element configured 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 element is configured for calculating the slope of the waveform of curve C_GT after a minimum in curve C_GT has been reached.

4. Device as claimed in claim 1, claims, wherein said controlling element comprises a hazard detecting element configured for determining the instantaneous gradient (dG) of said ratio (GT) after a minimum has been reached in said curve C_GT;

said controlling element further comprises a first comparing element configured for comparing the calculated instantaneous gradient dG with at least a reference value (dG_ref1) and selecting a correcting element for performing an urgent correction to the stimulating signal S(t) in order to avoid lesions, damages or irreversible alterations in said substrate.

5. Device as claimed in claim 4, wherein said correcting element is configured to decrease 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 element comprises a slope determining element configured to calculate 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 element further comprising a second comparing element configured to compare 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 element 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) a continuing element is 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) an adjusting element is 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) a correcting element is selected.

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

said adjusting element subsequently selecting said element for calculating and monitoring the instantaneous value of the said ratio (GT) and said controlling element.

9. Device as claimed in claim 7, wherein said continuing element is configured to 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 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 element is configured to detect a minimum of said initial portion of said curve and determining the time Tm at which the minimum is reached.

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

said third comparing element is configured to perform the following operations;

if the detected Tm occurs before Ttmin (Tm<

Ttmin) then a correcting is selected;

if the detected Tm occurs after Ttmax (Tm>

Ttmax), then an adjusting is selected; and

if the detected Tm occurs between Ttmin and Ttmax, then a is selected.

12. A method for extracting molecules from living cells comprised in a substrate, comprising the step of using the device of claim 1 to generate a controlled stimulating signal applied to living cells by means of electrodes while extracting molecules from the living cells.

13. A method for introducing molecules into living cells comprising the step of using the device of claim 1 to generate a controlled stimulating signal applied to living cells by means of electrodes while introducing said molecules into the living cells.

14. The method for introducing molecules 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, phosphosulfates, or at the extremities, by addition 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, the antibiotic bleomycin or the cisplatinum;

a penicillin; and

other pharmacological agents.