U.S. patent application number 11/836656 was filed with the patent office on 2009-01-22 for circuit arrangement for injecting nucleic acids and other biologically active molecules into the nucleus of higher eucaryontic cells using electrical current.
This patent application is currently assigned to AMAXA GMBH. Invention is credited to Ludger Altrogge, Rainer Christine, Juliana Helfrich, Elke Lorbach, Herbert Muller-Hartmann, Gudula Riemen, Kirsten Rothmann-Cosic, Gregor Siebenkotten, Corinna Thiel, Meike Weigel, Heike Wessendorf.
Application Number | 20090023131 11/836656 |
Document ID | / |
Family ID | 7682433 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090023131 |
Kind Code |
A1 |
Muller-Hartmann; Herbert ;
et al. |
January 22, 2009 |
CIRCUIT ARRANGEMENT FOR INJECTING NUCLEIC ACIDS AND OTHER
BIOLOGICALLY ACTIVE MOLECULES INTO THE NUCLEUS OF HIGHER
EUCARYONTIC CELLS USING ELECTRICAL CURRENT
Abstract
The invention relates to a novel circuit arrangement for
electrotransfection or electrofusion, which enables the
transportation of DNA and/or other biologically active molecules to
the nucleus of higher eukaryotic cells or the fusion of cells,
independent of cell division and with reduced cell mortality.
Inventors: |
Muller-Hartmann; Herbert;
(Koln, DE) ; Riemen; Gudula; (Langenfeld, DE)
; Rothmann-Cosic; Kirsten; (Berlin, DE) ; Thiel;
Corinna; (Koln, DE) ; Altrogge; Ludger;
(Pulheim, DE) ; Weigel; Meike; (Koln, DE) ;
Christine; Rainer; (Koln, DE) ; Lorbach; Elke;
(Koln, DE) ; Helfrich; Juliana; (Glauchau, DE)
; Wessendorf; Heike; (San Francisco, CA) ;
Siebenkotten; Gregor; (Frechen-Konigsdorf, DE) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 770, Church Street Station
New York
NY
10008-0770
US
|
Assignee: |
AMAXA GMBH
Koln
DE
|
Family ID: |
7682433 |
Appl. No.: |
11/836656 |
Filed: |
August 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10475840 |
Mar 15, 2004 |
|
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11836656 |
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Current U.S.
Class: |
435/3 |
Current CPC
Class: |
C12N 15/87 20130101;
A61N 1/0412 20130101; H03K 3/57 20130101; A61N 1/327 20130101 |
Class at
Publication: |
435/3 |
International
Class: |
C12Q 3/00 20060101
C12Q003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 23, 2001 |
DE |
101 19 901.5 |
Apr 23, 2002 |
DE |
PCT/DE02/01489 |
Claims
1. A method for introducing nucleic acids, peptides, proteins
and/or other biologically active molecules into the cell nucleus of
eukaryotic cells by means of electric current or for the treatment
of cells, cell derivatives, subcellular particles and/or vesicles
with electric current, said method comprising: supplying at least
one voltage pulse to the cells, cell derivatives, subcellular
particles and/or vesicles, measuring the quantity of charge
supplied by the voltage pulse in at least one selectable time
interval, wherein a preset desired quantity of charge is compared
with the actually supplied quantity of charge, and terminating the
voltage pulse on reaching or exceeding the desired quantity of
charge.
2. The method of claim 1, wherein the supplied quantity of charge
is determined from the difference between the original charge of a
corresponding storage device and the residual charge.
3. The method of claim 1, wherein a first pulse with the capacitor
voltage (U.sub.1) is supplied to the cells and subsequently without
interruption at least one second pulse with the capacitor voltage
(U.sub.2) is also supplied to the cells.
4. The method of claim 3, wherein a first pulse having a field
strength of 2-10 kV/cm, a duration of 10-100 .mu.s and a current
density of at least 2 Acm.sup.-2 is applied to the cells, and
subsequently without interruption a second pulse having a current
density of 2-14 Acm.sup.-2 and a maximum duration of 100 ms is also
supplied to the cells.
5. The method of claim 1, wherein the time interval for determining
the supplied charge is specified simultaneously with the supply of
the charge of a first and/or preferably a second or each further
pulse.
6. The method of claim 3, wherein the switch-on time (T.sub.2) of
the second pulse is specified by comparing the desired quantity of
charge with the actual quantity of charge supplied by the
measurement time and terminated when the desired quantity of charge
is reached.
7. The method of claim 1, wherein in order to determine the actual
quantity of charge a measuring cycle of 1 msec is selected, wherein
during the time (T.sub.2) the capacitor voltage decreases
exponentially and the pulse is terminated on reaching the specified
quantity of charge (Q.sub.2).
8. The method of claim 1, wherein after at least one pre-determined
time interval after triggering one first and/or second pulse the
flowing current is measured and, if said current exceeds or falls
below a desired value, the duration of the pulse is readjusted in
order to keep the supplied quantity of charge constant.
9. The method of claim 1, wherein after at least one pre-determined
time interval after triggering one first and/or second pulse the
flowing current is measured and, if said current exceeds or falls
below a desired value, an error message is given.
10. The method of claim 1, wherein after at least one
pre-determined time interval after triggering one first and/or
second pulse the flowing current is measured and, if said current
exceeds or falls below the desired value, the desired value is
readjusted.
11. The method of claim 1, wherein pre-selected setting parameters
of the pulse (U.sub.1, T.sub.1, I.sub.2, T.sub.2, K.sub.2) are
inputted manually or by entering a code.
12. The method of claim 3, wherein an overcurrrent cutoff for the
first and second pulse is accomplished.
13. The method of claim 3, wherein the resistance R of the cuvette
used to calculate (U.sub.2) is determined by a resistance
measurement before triggering a power semiconductor.
14. The method of claim 3, wherein the resistance R of the cuvette
used to calculate (U.sub.2) is predetermined.
15. The method of claim 3, wherein pre-selected setting parameters
of the pulse (U.sub.1, T.sub.1, I.sub.2, T.sub.2, K.sub.2, R) are
read in via a memory card.
16. The method of claim 1, wherein the method is used for the
transfection of quiescent or dividing eukaryotic cells.
17. The method of claim 1, wherein the method is used for the
transfection of primary cells.
18. The method of claim 1, wherein the method is used for the
transfection of human blood cells.
19. The method of claim 1, wherein the method is used for the
transfection of pluripotent precursor cells of human blood.
20. The method of claim 1, wherein the method is used for the
transfection of primary human fibroblasts, endothelial cells,
muscle cells or melanocytes.
21. The method of claim 1, wherein the method is used for the
fusion of cells, cell derivatives, subcellular particles and/or
vesicles.
22. The method of claim 1, wherein the method further includes
using eukaryotic cells transfected according to the method for
analytical or diagnostic purposes.
23. The method of claim 1, wherein the method further includes
using eukaryotic cells transfected according to the method for the
manufacture of a pharmaceutical product for ex vivo gene therapy.
Description
CROSS-REFERENCE TO PRIOR APPLICATION
[0001] This is a division of U.S. patent application Ser. No.
10/475,840, filed Oct. 21, 2003, which is the national stage of
PCT/DE02/01489.
BACKGROUND OF THE INVENTION
[0002] The invention relates to a circuit arrangement for
introducing nucleic acids, peptides, proteins and/or other
biologically active molecules into the cell nucleus of eukaryotic
cells by means of electric current, or for the treatment of cells,
cell derivatives, subcellular particles and/or vesicles with
electric current, consisting of at least two storage devices for
quantities of electric charge, each supplied by a high-voltage
power supply which each have at least one power semiconductor for
transferring the quantities of charge present in the storage
devices into a suspension in a cuvette and at least one monitoring
device for controlling the power semiconductor.
[0003] Since the place of action of eukaryotic DNA is the cell
nucleus, DNA supplied from outside must enter the nucleus in order
to be read out. Conventional transfection methods only bring about
transport of DNA through the cell membrane into the cytoplasm. It
is only because the nuclear membrane is temporarily dissolved
during the cell division of higher eukaryotes that the DNA can
passively enter the nucleus so that proteins encoded by it can be
expressed. Only very small DNA molecules (oligonucleotides) can
diffuse freely through the pores of the nuclear membrane. For the
effective transfection of quiescent or weakly dividing cells it is
thus necessary to create conditions which have the result that
larger DNA molecules enter the nucleus through the nuclear membrane
in sufficient quantity. The circuit arrangement described here
makes this possible in higher eukaryotic cells.
STATE OF THE ART
[0004] It has been known for some time that DNA from a buffer can
be introduced into cells with the aid of electric current. However,
the circuit arrangements for electroporation described so far are
based on the transport of DNA into the cytoplasm of higher
eukaryotic cells so that the expression of transfected DNA remains
dependent on the dissolution of the nuclear membrane during the
cell division. None of the circuit arrangements for electroporation
known so far is concerned with bringing DNA electrically
specifically into the nucleus of higher eukaryotic cells. Thus, a
circuit arrangement for electrotransfection optimised for
electrical nucleus transport is not known.
[0005] U.S. Pat. No. 4,750,100 from Bio-Rad Laboratories, Richmond,
USA (1986), describes a specific equipment structure which can
provide a maximum of 3000 V at a maximum of 125 A by capacitor
discharge.
[0006] U.S. Pat. No. 5,869,326 (Genetronics, Inc., San Diego, USA,
1996) describes a specific equipment structure by which means two,
three or a plurality of pulses can be generated using two separate
current sources. However it is not claimed or shown that these
pulses have an effect which goes beyond the transport of DNA into
the cytoplasm.
[0007] U.S. Pat. No. 6,008,038 and the European Patent Application
EP 0 866 123 A1 (Eppendorf-Netheler-Hinz GmbH, Hamburg, 1998)
describe a device with which short pulses of 10-500 .mu.s and a
maximum of 1.5 kV can be generated but again give no indication
that certain conditions could lead to conveying DNA into the
nucleus.
[0008] None of the circuit arrangements known so far is optimised
to make it possible for DNA and/or other biologically active
molecules to be effectively transported into the cell nucleus with
low cell mortality.
[0009] The object of the invention is to provide a circuit
arrangement which makes it possible for DNA and/or biologically
active molecules to be transported effectively into the cell
nucleus with low cell mortality.
DESCRIPTION OF THE INVENTION
[0010] In order to solve the object according to the invention it
is provided that the first storage device is charged with the
preset voltage (U.sub.1) as a parameter and the second storage
device is charged with a voltage
U.sub.2=R.times.I.sub.2.times.K.sub.2, wherein R is the resistance
of the cuvette and the suspension contained therein, I.sub.2 is the
desired current and K.sub.2 is a correction value which takes into
account the cuvette properties and wherein at least one first pulse
with the capacitor voltage (U.sub.1) of the storage device can be
transferred to the cell for a preset time (T.sub.1) by controlling
a power semiconductor.
[0011] In a development of the invention it is provided that
without interruption at least one second pulse with the capacitor
voltage (U.sub.2) of the storage device can also be applied to the
cuvette by controlling a power semiconductor, wherein the delivered
quantity of charge in at least one selectable time interval can be
measured by the monitoring device, wherein the preset desired
quantity of charge is compared with the actual delivered quantity
of charge and on reaching or exceeding the desired quantity of
charge, the power semiconductor is blocked.
[0012] In addition to the possibility of determining the delivered
quantity of charge using the current flowing from the storage
device, alternatively the preset desired quantity of charge is
compared with the actual delivered quantity of charge in an
interval of time and on reaching or exceeding the desired quantity
of charge, the power semiconductor is blocked. On this occasion,
depending on the pulse shape used and the number of pulses, the
time interval which can be selected for the determination can be
individually predefined in order, for example, to determine the
delivered quantity of charge during the first or each subsequent
pulse. The delivered quantity of charge can be determined by
determining the difference between the original charge at least of
one of the storage devices and the residual charge. In this case it
is possible that according to the number of pulses used, more than
one of the at least two storage devices is used in a circuit
fashion wherein each storage device is assigned at least one
high-voltage power supply, a monitoring device and a power
semiconductor to transfer the quantity of charge to the cuvette
containing the cell suspension. For the pulse transfer it is
provided that the first power semiconductor transfers a pulse of
2-10 kV/cm having a duration of 10-100 .mu.s and a current density
of at least 2 Acm.sup.-2 and, without interruption, the second
power semiconductor transfers a pulse having a current density of
2-14 Acm.sup.2 and a maximum duration of 100 ms. The time interval
for determining the delivered quantity of charge can consequently
be specified with the delivery of a first and/or preferably a
second or each further pulse.
[0013] The delivered quantity of charge of the second pulse is
preferably monitored wherein the switch-on time (T.sub.2) of the
second pulse can be specified by comparing the desired quantity of
charge with the actual quantity of charge delivered by the
measurement time and ends when the desired quantity of charge is
reached and wherein a measurement cycle of 1 msec is provided to
determine the actual quantity of charge, wherein during the time
(T.sub.2) the capacitor voltage decreases exponentially and the
power semiconductor can be blocked on reaching the specified
quantity of charge (Q.sub.2).
[0014] Alternatively it is possible that after at least one
predetermined time interval after triggering a first and/or second
pulse, the flowing current is measured and if this exceeds or falls
below a desired value, the pulse duration can be re-adjusted in
order to keep the delivered quantity of charge constant. In another
alternative it is possible that after at least one predetermined
time interval after triggering a first and/or second pulse, the
flowing current is measured and if this exceeds or falls below a
desired value, an error message is generated to give a warning to
the user of the device. It is furthermore possible that after at
least one predetermined time interval after triggering a first
and/or second pulse, the flowing current is measured and if this
exceeds or falls below a desired value, the desired value is
readjusted.
[0015] In order to determine any necessary constants, especially of
the cuvette used with the cell suspension, it can be provided that
a preliminary measurement of the resistance of the cuvette with the
cell suspension is made. The other necessary pulse parameters are
preferably pre-selected manually or if necessary specified by
entering a code. It is thus also possible to use retrievable data
via a card reader. The card reader can also be used at the same
time to store the time profile of the voltage applied to the
cuvette or the current flowing through the cuvette for
documentation purposes for one or a plurality of pulse delivery
processes on a commercially available memory card. This memory card
is preferably used at the same time for storing the pulse
parameters to be set.
[0016] As a result of the circuit regulation of the pulse delivery,
the transfer of the envisaged quantity of charge is thus monitored
in a reliable and advantageous fashion at least for one pulse and a
controlled and sample-dependent transfer of a preset quantity of
charge as well as a controlled monitoring to avoid any damage to
the cells located in the sample can be achieved.
[0017] For further safety of the user and the samples used it is
provided that an overcurrent cutoff is provided for the first and
each subsequent pulse. The overcurrent cutoff thus allows the
high-voltage pulse to be interrupted at any time in the event that
preset limiting values are exceeded.
[0018] The high-voltage pulse of 2-10 kV/cm described is suitable
for creating conditions such that DNA can enter the cell nucleus
independently of the cell division. In order to keep cell damage
low, this pulse is limited to between 10 and a maximum of 200
.mu.s, preferably 10-50 .mu.s. This is sufficient to achieve
transfection independent of cell division. For example, such a
short single high-voltage pulse was found to be optimum for the
transfection of endothelial cells from the human umbilical vein.
Another current pulse of lower field intensity or lower current
strength or current density but of longer duration, following
without interruption influences the efficiency of the transfection.
As a result of the significantly lower current density, this pulse
can persist significantly longer with little cell damage. An
optimum current density or duration of the second pulse is obtained
depending on the cell type and sensitivity of the cell. Such
combined pulses are found to be optimal, for example, for primary
human dermal fibroblasts or melanocytes or various human blood
cells. In experiments using different cell lines and expression
systems, the following was shown: the higher the current density of
the second pulse, the stronger its influence on the transfection
rate, i.e. the percentage of transfected cells. The lower the
current density, the more the second pulse causes pure DNA
transport into cells already transfected by the first pulse. The
expression level of the transfected cells increases with increasing
pulse duration but not the fraction of transfected cells. In order
to maintain a precise cell-specific control of the transfection
rate, the expression level and the cell vitality, the pulse
duration and current density of the second pulse must therefore be
controlled.
[0019] In order to achieve precise control of the pulse actually
delivered to the cell suspension, in a preferred embodiment the
delivered quantity of charge is controlled. In order to control the
current strength or current density by a selectable capacitor
voltage of the storage unit, the resistance of the cuvette and the
cell suspension contained therein must be predefined initially. It
was found that the resistance of the cuvettes when using aluminium
electrodes varies during the pulse as a result of electrochemical
processes. This variation is taken into account by a pulse-specific
predefined correction value. Thus, precise pulse shapes for the
second pulse can be predetermined using
U.sub.2=R.times.I.sub.2.times.K.sub.2 by controlling the charge,
where U.sub.2 is the capacitor voltage with which the storage
device is charged, R is the resistance of the cuvette and the cell
suspension contained therein, I.sub.2 is the desired current and
K.sub.2 is the pulse-specific correction value.
[0020] In one embodiment of the invention the ohmic cuvette
resistance R can be measured directly before the beginning of pulse
delivery by applying a test voltage and taken into account
accordingly in the calculation of the voltage U.sub.2. Since the
resistance measured before pulse delivery is subjected to larger
fluctuations than the resistance during pulse delivery, presumably
as a result of electrochemical processes, it is found to be
advantageous to fixedly predefine the resistance R to calculate the
capacitor voltage U.sub.2 as a parameter. In a preferred embodiment
of the invention the resistance of the cuvette is measured before
the commencement of pulse delivery regardless of this in order to
determine whether this lies within a predefined resistance window.
If the measured resistance lies outside this window, there is a
fault and the pulse delivery is not released.
[0021] For every cell type optimum conditions can be established
for transfection rate, transfection intensity and cell vitality. In
a preferred embodiment of the circuit arrangement the field
intensity and duration of the first pulse and initial current
intensity or current density and empirical duration of the second
pulse can be selected and optimum conditions can simply be
established for various cell types via a code.
[0022] The circuit arrangement can be used in an advantageous
fashion for the transfection of quiescent or dividing eukaryotic
cells. In the same way the circuit arrangement is also suitable for
the transfection of primary cells such as human blood cells,
pluripotent precursor cells from human blood, primary human
fibroblasts, endothelial cells, muscle cells or melanocytes and can
be used for diagnostic purposes or for the manufacture of a
medicinal product for ex-vivo gene therapy.
[0023] The circuit arrangement according to the invention is
furthermore also suitable, for example, for electrofusion, i.e.,
methods for the fusion of cells, cell derivatives, subcellular
particles and/or vesicles by means of electric current, wherein,
for examples the cells, cell derivatives, subcellular particles
and/or vesicles are initially suspended in a suitable density in an
aqueous solution, the suspension is then transferred to a cuvette
and finally an electric voltage is applied to the electrodes of the
cuvette and a current flow is generated through the suspension.
Alternatively, for example, adherent cells, cell derivatives,
subcellular particles and/or vesicles or however, also adherent
cells with suspended cells, cell derivatives, subcellular particles
or vesicles can be fused.
[0024] The circuit arrangement described here generates very high
field intensities of 2 to 10 kV/cm which have the effect that DNA
and/or biologically active molecules can enter the nucleus
independently of the cell division. These field intensities are far
above those normally used for electroporation and far beyond those
sufficient for efficient opening of the pores in the cell membrane
(on average 1 kV/cm according to Lurquin, 1997, Mol. Biotechnol. 7,
5).
[0025] The subject matter of the invention is thus a circuit
arrangement for implementing a method for introducing nucleic
acids, peptides, proteins and/or other biologically active
molecules into the cell nucleus of higher eukaryotic cells using
electric current wherein the introduction into the nucleus is
achieved by a pulse having a field intensity 2-10 times that
sufficient for opening the pores in the cell membrane and a
duration of at least 10 .mu.s and a current density of at least 2
Acm.sup.-2.
[0026] The introduction of nucleic acids, peptides, proteins and/or
other biologically active molecules into the cell nucleus can be
achieved by a pulse of 2-10 kV/cm, preferably 3-8 kV/cm, wherein
the pulse is a maximum of 200 .mu.s long.
[0027] The circuit arrangement is designed so that the first pulse
can be followed without interruption by a current flow having a
current density of 2 Acm.sup.-2 up to a maximum of 14 Acm.sup.-2,
preferably up to 5 Acm.sup.-2, of 1 ms up to a maximum of 100 ms,
preferably up to 50 ms in length.
[0028] Since the circuit arrangement makes transfection possible
regardless of the cell division, in addition to dividing cells,
quiescent or weakly dividing primary cells can also be
transfected.
[0029] In other preferred embodiments the higher eukaryotic cells
comprise primary human fibroblasts, endothelial cells and
melanocytes.
[0030] The eukaryotic cells transfected using the circuit
arrangement according to the invention can also be used for
diagnostic and analytic purposes to produce a pharmaceutical
product for ex-vivo gene therapy.
[0031] The circuit arrangement according to the invention makes it
possible to achieve transfection independent of cell division and
thus to considerably speed up transfection experiments. In
transfection experiments using expression vectors, an analysis
according to promoter and expressed protein can be made even a few
hours after the transfection.
[0032] The concept "biologically active molecules" means peptides,
proteins, polysaccharides, lipids or combinations or derivatives of
these molecules as long as they develop a biological affinity in
the cell.
[0033] Electroporation buffers having a high ionic strength and
high buffer capacity are especially suitable for use with the
circuit arrangement according to the invention.
[0034] The following protocol can be used to introduce nucleic
acids into the cell nucleus of eukaryotic cells:
1.times.10.sup.5-1.times.10.sup.7 cells and up to 10 .mu.g DNA are
incubated in 100 .mu.l electroporation buffer in a cuvette having a
2 mm interelectrode gap for 10 min at room temperature and then
transfected according to the conditions according to the invention.
Immediately afterwards the cells are washed out of the cuvette with
400 .mu.l of cell culture medium and incubated for 10 min at
37.degree. C. The cells are then plated out in 37.degree. C. warm
cell culture medium.
[0035] Suitable cuvettes are commercially available cuvettes for
the electroporation of prokaryotes having an interelectrode gap or
2 mm or 1 mm, for example.
[0036] Evidence that the nucleic acids enter the cell nucleus
independently of cell division can be furnished by analysing the
cells which have not divided between transfection and analysis.
This is achieved on the one hand by the transfection of
non-dividing cells, such as for example cells of peripheral human
blood and on the other hand for dividing cells by an analysis a few
hours after transfection at a time when at most a fraction of the
cells can have divided.
[0037] The following abbreviations are used in addition to those in
general use:
TABLE-US-00001 FACS Fluorescence activated cell sorting FCS Foetal
calf serum PBMC Peripheral blood mononuclear cells PE
Phycoerythrin
EXAMPLES
[0038] The following examples illustrate the invention but should
not be regarded as restrictive.
Example 1
Transfection of Cytotoxic T Cells from Human Blood
[0039] Freshly prepared unstimulated (non-dividing) mononuclear
cells from peripheral human blood (PBMC) were transfected with a
vector which codes for the heavy chain of the mouse MHC class 1
protein H-2K.sup.k. 5.times.10.sup.6 cells together with 5 .mu.g of
vector DNA in a buffer having a high buffer capacity (48
mM.times.pH.sup.-1) and high ionic strength (280 mM) were placed at
room temperature in a cuvette having a 2 mm interelectrode gap and
transfected by a 1000 V pulse of 100 .mu.s duration, followed by a
current flow having a current density of 5 Acm.sup.-2 and 40 ms.
Immediately afterwards, the cells were washed from the cuvette
using 400 .mu.l of culture medium, incubated for 10 minutes at
37.degree. C. and then transferred to a culture dish with
pre-heated medium. After incubating for 24 h, the cells were
successively incubated with digoxigenin-coupled
anti-H-2K.sup.k-antibody and Cy5-coupled anti-digoxigenin-antibody,
as well as with a PerCP-coupled anti-CD8-antibody to identify human
cytotoxic T cells and analysed using a flow cytometer (FACScalibur,
Becton Dickinson). The number of dead cells was determined by
staining with propidium iodide. As shown in FIG. 1, 74.3% of the
living cells express the H-2K.sup.k antigen which corresponds to a
very high transfection efficiency.
Example 2
Transfection of Human Haematopoeitic Stem Cells (CD34)
[0040] CD34-positive cells were pre-enriched from freshly prepared
PBMC described as in Example 1 by magnetic cell sorting.
Respectively 1.times.10.sup.4 CD34-positive cells were then mixed
with 1.times.10.sup.6 PBMCs, placed together with 5 .mu.g
H-2K.sup.k-expression vector DNA in a buffer having a high buffer
capacity (54 mM.times.pH.sup.-1) and high ionic strength (260 mM)
at room temperature in a cuvette having a 2 mm interelectrode gap
and transfected by a 1000 V pulse of 100 .mu.s duration, followed
by a current flow having a current density of 4 Acm.sup.-2 and 20
ms duration. Immediately afterwards, the cells were washed from the
cuvette using 400 .mu.l of culture medium, incubated for 10 minutes
at 37.degree. C. and then transferred to a culture dish with
pre-heated medium. After incubating for 16 h, the cells were
successively incubated with phycoerythrin-coupled
anti-H-2K.sup.k-antibody, as well as with an APC-coupled anti-CD34
antibody to identify human haematopoietic stem cells and analysed
using a flow cytometer (FACScalibur, Becton Dickinson). The number
of dead cells was determined by staining with propidium iodide. As
shown in FIG. 2, 66.7% of the cells express the H-2K.sup.k antigen
which corresponds to a high transfection efficiency.
Example 3
Transfection of Human Neonatal Dermal Fibroblasts (NHDF-Neo)
[0041] Human neonatal dermal fibroblasts (5.times.10.sup.5 cells)
together with 5 .mu.g H-2K.sup.k-expression vector DNA were placed
in a buffer having a high buffer capacity (67 mM.times.pH.sup.-1)
and high ionic strength (380 mM) at room temperature in a cuvette
having a 2 mm interelectrode gap and transfected by a 1000 V pulse
of 100 .mu.s duration, followed by a current flow having a current
density of 6 Acm.sup.-2 and of 33 ms duration. Immediately
afterwards, the cells were washed from the cuvette using 400 .mu.l
of culture medium, incubated for 10 minutes at 37.degree. C. and
then transferred to a culture dish with pre-heated medium. After
incubating for 5 h, the cells were incubated with a Cy5-coupled
anti-H-2K.sup.k-antibody and analysed using a flow cytometer
(FACScalibur, Becton Dickinson). The number of dead cells was
determined by staining with propidium iodide. As shown in FIG. 3,
93% of the cells express the H-2K.sup.k antigen which corresponds
to a very high transfection efficiency.
Example 4
Transfection of Human Neonatal Melanocytes
[0042] Human neonatal melanocytes (2.5.times.10.sup.5 cells)
together with 5 .mu.g H-2K.sup.k-expression vector DNA were placed
in a buffer having a high buffer capacity (54 mM.times.pH.sup.-1)
and high ionic strength (260 mM) at room temperature in a cuvette
having a 2 mm interelectrode gap and transfected by a 1000 V pulse
of 100 .mu.s duration, followed by a current flow having a current
density of 6 Acm.sup.-2 and 33 ms duration. Immediately afterwards,
the cells were washed from the cuvette using 400 .mu.l of culture
medium, incubated for 10 minutes at 37.degree. C. and then
transferred to a culture dish with pre-heated medium. After
incubating for 5 h, the cells were incubated with a Cy5-coupled
anti-H-2K.sup.k-antibody and analysed using a flow cytometer
(FACScalibur, Becton Dickinson). The number of dead cells was
determined by staining with propidium iodide. As shown in FIG. 4,
75.1% of the cells express the H-2K.sup.k antigen which corresponds
to a very high transfection efficiency.
Example 5
Transfection of Human Endothelial Cells from the Umbilical Vein
(HUVEC)
[0043] Endothelial cells from the human umbilical vein
(1.times.10.sup.6 cells) together with 5 .mu.g
H-2K.sup.k-expression vector DNA were placed in a buffer having a
high buffer capacity (67 mM.times.pH.sup.-1) and high ionic
strength (378 mM) at room temperature in a cuvette having a 2 mm
interelectrode gap and transfected by a 1000 V pulse of 100 .mu.s
duration. Immediately afterwards, the cells were washed from the
cuvette using 400 .mu.l of culture medium, incubated for 10 minutes
at 37.degree. C. and then transferred to a culture dish with
pre-heated medium. After incubating for 5 h, the cells were
incubated with a Cy5-coupled anti-H-2K.sup.k-antibody and analysed
using a flow cytometer (FACScalibur, Becton Dickinson). The number
of dead cells was determined by staining with propidium iodide. As
shown in FIG. 5, 49.7% of the cells express the H-2K.sup.k antigen
which corresponds to a high transfection efficiency.
Example 6
Transfection of the Human Cell Line K562
[0044] K562 cells (1.times.10.sup.6 cells) together with 5 .mu.g
H-2K.sup.k-expression vector DNA were placed in a buffer having a
high buffer capacity (24 mM.times.pH.sup.-1) and high ionic
strength (254 mM) at room temperature in a cuvette having a 2 mm
interelectrode gap and transfected by a 1000 V pulse of 100 .mu.s
duration, followed by a current flow having a current density of 8
Acm.sup.-2 and 10 ms duration. Immediately afterwards, the cells
were washed from the cuvette using 400 .mu.l of culture medium,
incubated for 10 minutes at 37.degree. C. and then transferred to a
culture dish with pre-heated medium. After incubating for 4 h, the
cells were incubated with a Cy5-coupled anti-H-2K.sup.k-antibody
and analysed using a flow cytometer (FACScalibur, Becton
Dickinson). The number of dead cells was determined by staining
with propidium iodide. As shown in FIG. 6, 69.5% of the cells
express the H-2K.sup.k antigen which corresponds to a very high
transfection efficiency.
Example 7
Transfection Efficiency and Average Fluorescence Intensity of
Cycle3-GFP-Transfected CHO Cells
[0045] In order to investigate the transfection efficiency and the
average fluorescence intensity of transfected cells as a function
of the quantity of charge flowing in the second pulse, respectively
7.times.10.sup.5 CHO cells together with 5 .mu.g
Cycle3-GFP-vector-DNA were placed in electroporation buffer in a
cuvette having an interelectrode gap of 2 mm and transfected by a
1000 V, 10 .mu.s pulse and subsequent second pulses differing in
the variation of the current intensity or current density and pulse
time. After cultivation for 5 hours, the cells were analysed using
a flow cytometer. FIG. 7 shows the transfection efficiency
determined as a function of the integral of the current over the
pulse time (the quantity of charge Q). It is found that the
transfection efficiency can be increased with increasing current
intensity (open circles). An increase in the pulse time for the
same current intensity on the other hand results in no appreciable
increase in efficiency (closed circles). The fluorescence intensity
(brightness) of the transfected cells increases with increasing
quantity of charge Q, with saturation being reached for high Q. No
major differences are found whether the increase in Q was achieved
by increasing the current intensity (open circles) or increasing
the pulse length (closed circles).
Example 8
Transfection Efficiency and Average Fluorescence Intensity of
Cycle3-GFP-Transfected Jurkat Cells
[0046] In order to investigate the transfection efficiency and the
average fluorescence intensity of transfected cells as a function
of the quantity of charge flowing in the second pulse, respectively
4.times.10.sup.5 Jurkat cells together with 5 .mu.g
Cycle3-GFP-vector-DNA were placed in electroporation buffer in a
cuvette having an interelectrode gap of 2 mm and transfected by a
1000 V, 10 .mu.s pulse and subsequent second pulses differing in
the variation of the current intensity or current density and pulse
time. After cultivation for 5 hours, the cells were analysed using
a flow cytometer. FIG. 8 shows the transfection efficiency
determined as a function of the integral of the current over the
pulse time (the quantity of charge Q). As when using CHO cells, it
is found that the transfection efficiency can be increased with
increasing current intensity (open circles). An increase in the
pulse time for the same current intensity on the other hand results
in no appreciable increase in efficiency (closed circles). The
fluorescence intensity (brightness) of the transfected cells
increases with increasing quantity of charge Q, with saturation
being reached for high Q. No major differences are found whether
the increase in Q was achieved by increasing the current intensity
(open circles) or increasing the pulse length (closed circles).
Example 9
Transfection Efficiency and Average Fluorescence Intensity of
H-2K.sup.k-Transfected Jurkat Cells
[0047] In order to investigate the transfection efficiency and the
average fluorescence intensity of transfected cells as a function
of the quantity of charge flowing in the second pulse, respectively
1.times.10.sup.6 Jurkat cells together with 2 .mu.g of
H2K.sup.k-expression vector DNA were placed in electroporation
buffer in a cuvette having an interelectrode gap of 2 mm and
transfected by a 1000 V, 10 .mu.s pulse and subsequent second
pulses differing in the variation of the current intensity or
current density and pulse time. After cultivation for 3.5 hours,
the cells were incubated with Cy5-coupled anti-H2K.sup.k and
analysed using a flow cytometer. FIG. 9 shows the transfection
efficiency determined as a function of the integral of the current
over the pulse time (the quantity of charge Q). It is found that
the transfection efficiency can be increased with increasing
current intensity (open circles). An increase in the pulse time for
the same current intensity on the other hand results in no
appreciable increase in efficiency (closed circles). The
fluorescence intensity (brightness) of the transfected cells
increases with increasing quantity of charge Q, with saturation
being reached for high Q. No major differences are found whether
the increase in Q was achieved by increasing the current intensity
(open circles) or increasing the pulse length (closed circles).
[0048] The invention is explained further with reference to the
following figures.
[0049] In the figures
[0050] FIG. 1 shows a transfection of cytotoxic T cells from human
blood,
[0051] FIG. 2 shows a transfection of pluripotent precursor cells
from human blood,
[0052] FIG. 3 shows a transfection of human neonatal dermal
fibroblasts,
[0053] FIG. 4 shows a transfection of human neonatal dermal
melanocytes,
[0054] FIG. 5 shows a transfection of human endothelial cells from
the umbilical cord,
[0055] FIG. 6 shows a transfection of the cell line K562 (analysis
4 h after transfection),
[0056] FIG. 7 shows an investigation of the transfection efficiency
as a function of the current intensity, pulse time and quantity of
charge and of the expression intensity as a function of the
quantity of charge, experiment using the CHO cell line,
[0057] FIG. 8 shows an investigation of the transfection efficiency
as a function of the current intensity, pulse time and quantity of
charge and of the expression intensity as a function of the
quantity of charge, experiment using the Jurkat cell line,
[0058] FIG. 9 shows an investigation of the transfection efficiency
as a function of the current intensity, pulse time and quantity of
charge and of the expression intensity as a function of the
quantity of charge, experiment using the Jurkat cell line and the
surface marker protein H-2K.sup.k,
[0059] FIG. 10 shows a block diagram of an electroporator
circuit,
[0060] FIG. 11 shows a circuit diagram of a control panel,
[0061] FIG. 12 shows a circuit diagram of a card reader,
[0062] FIG. 13 shows a circuit diagram of a supply unit,
[0063] FIG. 14 shows a circuit diagram of a HV power supply,
[0064] FIG. 15 shows a circuit diagram of an HV switch,
[0065] FIG. 16 shows a circuit diagram of a current regulating
system,
[0066] FIG. 17 shows a circuit diagram of a flash recognition
system,
[0067] FIG. 18 shows a circuit diagram of a control system, and
[0068] FIG. 19 is a flow diagram to explain the sequence of the
pulse delivery processes.
[0069] FIG. 1 shows the flow cytometric analysis of PBMC which had
been transfected with H-2K.sup.k-expression vector. The cells were
successively incubated with digoxigenin-coupled
anti-H-2K.sup.k-antibody and Cy5-coupled anti-digoxigenin-antibody,
as well as with a PerCP-coupled anti-CD8-antibody to identify human
cytotoxic T cells and were analysed using a flow cytometer
(FACScalibur, Becton Dickinson). (FL-2, FL-3=fluorescence channel
2, 3; SSC=sideward scatter, FSC=forward scatter, PerCP=peridinin
chlorophyll protein, CD=cluster of differentiation).
[0070] FIG. 2 shows the flow cytometric analysis of CD34-positive
stem cells enriched from PBMC which had been transfected with
H-2K.sup.k-expression vector. The cells were successively incubated
with phycoerythin-coupled anti-H-2K.sup.k-antibody, as well as with
a APC-coupled anti-CD34-antibody to identify human CD34 positive
haematopoietic stem cells and were analysed using a flow cytometer
(FACScalibur, Becton Dickinson). (FL-1, FL-3=fluorescence channel
1, 3; SSC=sideward scatter, FSC=forward scatter, PE=phycoerythrin,
APC=allophycocyanin, CD=cluster of differentiation).
[0071] FIG. 3 shows the flow cytometric analysis of human neonatal
dermal fibroblasts (NHDF-Neo), which had been transfected with
H-2K.sup.k-expression vector. The cells were incubated with
Cy5-coupled anti-H-2K.sup.k and analysed using a flow cytometer
(FACScalibur, Becton Dickinson). (FL-1, FL-2, FL-3=fluorescence
channel 1, 2, 3; SSC=sideward scatter, FSC=forward scatter).
[0072] FIG. 4 shows the flow cytometric analysis of human neonatal
melanocytes (NHEM-Neo), which had been transfected with
H-2K.sup.k-expression vector. The cells were incubated with
Cy5-coupled anti-H-2K.sup.k and analysed using a flow cytometer
(FACScalibur, Becton Dickinson). (FL-1, FL-2, FL-3=fluorescence
channel 1, 2, 3; SSC=sideward scatter, FSC=forward scatter).
[0073] FIG. 5 shows the flow cytometric analysis of endothelial
cells from human umbilical cord (HUVEC), which had been transfected
with H-2K.sup.k-expression vector. The cells were incubated with
Cy5-coupled anti-H-2K.sup.k and analysed using a flow cytometer
(FACScalibur, Becton Dickinson). (FL-1, FL-2, FL-3=fluorescence
channel 1, 2, 3; SSC=sideward scatter, FSC=forward scatter).
[0074] FIG. 6 shows the flow cytometric analysis of the human cell
line K562 which had been transfected with H-2K.sup.k-expression
vector. The cells were incubated with Cy5-coupled anti-H-2K.sup.k
and analysed using a flow cytometer (FACScalibur, Becton
Dickinson). (FL-1, FL-2, FL-3=fluorescence channel 1, 2, 3;
SSC=sideward scatter, FSC=forward scatter).
[0075] FIG. 7 shows a graphical representation of the transfection
efficiency of CHO cells and the average fluorescence intensity
(brightness) of the positive cells as a function of the quantity of
charge Q which has flowed. The CHO cells were transfected with
Cycle3-GFP expression vector and analysed after five hours using a
flow cytometer (FACScalibur, Becton Dickinson). Closed circles
correspond to a gradual increase in the pulse time for the same
current intensity (2 A) or current density (4 Acm.sup.-2), open
circles correspond to an increase in current intensity.
[0076] FIG. 8 shows a graphical representation of the transfection
efficiency of Jurkat cells and the average fluorescence intensity
(brightness) of the positive cells as a function of the quantity of
charge Q which has flowed. The Jurkat cells were transfected with
Cycle3-GFP expression vector and analysed after five hours using a
flow cytometer (FACScalibur, Becton Dickinson). Closed circles
correspond to a gradual increase in the pulse time for the same
current intensity (2 A) or current density (4 Acm.sup.-2), open
circles correspond to an increase in current intensity.
[0077] FIG. 9 shows a graphical representation of the transfection
efficiency of Jurkat cells and the average fluorescence intensity
(brightness) of the positive cells as a function of the quantity of
charge Q which has flowed. The Jurkat cells were transfected with
H-2K.sup.k expression vector and incubated after 3.5 hours with a
Cy5-coupled anti-H-2K.sup.k and analysed using a flow cytometer
(FACScalibur, Becton Dickinson). Closed circles correspond to a
gradual increase in the pulse time for the same current intensity
(2 A) or current density (4 Acm.sup.-2), open circles correspond to
an increase in current intensity.
[0078] FIG. 10 shows a block diagram of the electroporator 1 with
the necessary individual components. These comprise an adjusting
unit 2, a control unit 3 to which a voltage supply unit 4 is
connected as well as at least two HV power supplies 5, 6 with
following storage devices 7, 8 and two power semiconductors 9, 10
provided for pulse delivery. The power semiconductors 9, 10 are
controlled via a potential divider stage 11, 12 by the control unit
3 by means of an HV switch 13 and a regulating unit 14. The storage
devices 7, 8 are directly connected to the inputs of the power
semiconductors 9, 10, wherein the storage devices 7, 8 can consist
of one or a plurality of capacitors depending on the field strength
and the pulse duration. The power semiconductor 9 can for example
consist of an IGBT and the power semiconductor 10 can consist of a
MOSFET. However, the term "power semiconductor" should comprise all
other electronic components or component assemblies by which means
the voltages and currents to be switched within the scope of the
invention can be switched with the required switching times. The
output of the IGBT is directly connected to the cuvette connection
15 whereas the output of the MOSFET 10 is connected via a
resistance 16 and a diode 17 to the cuvette connection 15 so that
no pulse can flow back via the second power semiconductor 10 if
both power semiconductors 9, 10 are controlled simultaneously. For
this purpose the diode 17 is connected to the cuvette connection 15
on the cathode side. The second cuvette connection 18 is connected
to earth via a resistance 19. The resistance 19 comprises a
measuring shunt to measure the voltage drop and supply to an
overcurrent switching stage 20. The overcurrent switching stage 20
can interrupt the pulse delivery by means of a switch 21 via the
potential divider stage 11 and the HV switch 13 whereas a second
overcurrent switching stage 22 interrupts a control system of the
regulating unit 14 for the MOSFET 10 via a switch 23. The voltage
applied via the resistance 16 is fed to the overcurrent switching
stage 22 in order to bring about a current switchoff in the event
that the maximum current is exceeded. Since the resistance 16 is
located directly in the high-voltage circuit, the switch 23 is
located after the potential divider stage 12 so that no
high-voltage pulses can enter the control unit 3 and the operating
staff are not endangered. In the case of the overcurrent switching
stage 20, the low-resistance measuring resistance 19 lies behind
the cuvette connections 15, 18 and is connected to earth so that
the transmission of high-voltage pulses can be eliminated.
Depending on the intended usage of the electroporator 1, one or a
plurality of high-voltage power supplies 5, 6 with the relevant
storage devices 7, 8 and the necessary potential divider stages 11,
12 and HV switch 13 or regulating unit 14 to control the power
semiconductors 9, 10 can be used. The storage devices 7, 8 are
equipped with one or a plurality of capacitors of the required
capacity and breakdown voltage so that a suitably high quantity of
charge can be stored and transferred to the cuvette connection
15.
[0079] The following FIGS. 11 to 18 shows the circuit diagrams of
the individual components in the block diagram.
[0080] FIG. 11 shows a circuit diagram of the control panel for
entering the parameter signals to be set wherein these can be
preselected via a pushbutton switch 30 and checked visually using
display elements 31. LEDs 32 shows when the equipment is ready for
operation. The necessary parameters are prepared in the circuit and
transmitted via a connector 33 to the control system in accordance
with FIG. 14.
[0081] FIG. 12 shows a circuit diagram of a card reader 34 via
which preset parameters for certain biological substances are read
in and transmitted to the control unit as shown in FIG. 14.
[0082] FIG. 13 shows a circuit diagram of the supply unit which
substantially consists of a 150/230 Volt changeover switch 35, a
transformer stage 36 with primary-side wiring and voltage lead and
secondary regulating stages to produce the necessary operating
voltages. For this purpose a plurality of voltage regulators 38 are
inserted after the rectifier 37.
[0083] FIG. 14 shows a circuit diagram of the two HV power supplies
5, 6 which can be identified from the block diagram. Both HV power
supplies 5, 6 are acted upon by the voltage U.sub.1 from the supply
unit, wherein each regulating stage 39 receives a control signal
U3on, U5on from the control system and the applied voltage U.sub.1
charges the storage device 7, 8 consisting of a plurality of
capacitors, in pulsed mode via a transformer stage 40. The desired
voltage reached is transmitted via output signals U3sense, U5sense
of the control system as shown in FIG. 15. The voltage U.sub.5 of
the storage device 7 is fed to an HV switch 13 as shown in FIG. 15
and the voltage U.sub.3 of the storage device 8 is fed to a current
regulating stage as shown in FIG. 16.
[0084] FIG. 15 shows a circuit diagram of the HV switch 13. The HV
switch 13 receives the signal HIN generated by the pulse monitoring
stage as shown in FIG. 16 to control the first power semiconductor
9. This transmits the applied voltage U.sub.5 to a solder pad 41
for the HV cable for connecting the cuvette which is then connected
to earth via a second solder pad 42 via a low-resistance measuring
resistance. An overcurrent cutoff stage 20 delivers a control
signal for the control unit as shown in FIG. 18 for switching off
the power semiconductor 9 in the event of a preset maximum current
rise being exceeded. The first solder pad 41 is further connected
to the voltage output U.sub.4 of the current regulating stage from
FIG. 12 in order that a controlled current flow into the cuvette to
deliver a specific quantity of charge can be achieved following the
high-voltage pulse. The current regulating stage from FIG. 16
receives the control signals from the control unit from FIG. 18 via
a potential divider stage and regulates the voltage U.sub.3 applied
to the storage device 8 to the voltage U.sub.4 delivered via the
solder pad 41. In this case, according to the invention, Q
regulation or current regulation is used whereby the charge in the
storage device 8 is determined at predefined time intervals of, for
example, one millisecond and the delivered quantity of charge is
determined taking into account the original charge.
[0085] FIG. 18 shows a circuit diagram of the control unit 3 which
either takes into account the preset manual values or the values
entered via a card reader 34 and controls the current regulating
unit 14 as shown in FIG. 16 on the basis of further monitoring
signals. The HV switch 13 as shown in FIG. 15 however is controlled
after manually triggering the high-voltage pulse via a pulse
monitoring stage 43 as shown in FIG. 17 so that after the HV pulse
has been delivered, the quantity of charge can be monitored via the
current regulating unit 14 as shown in FIG. 16.
[0086] The pulse parameters can thus on the one hand be preset
manually and on the other hand via a card reader so that when a
pulse is triggered manually via the existing regulating
electronics, a high-voltage pulse with or without monitoring of the
flowing current and if necessary, a continuous current signal with
monitoring of the quantity of charge can be delivered via a second
HV power supply.
[0087] FIG. 19 shows a schematic flow diagram of the operating
sequence of a pulse delivery process controlled by the control unit
3 (see FIG. 10) according to a preferred embodiment of the
invention. First, the required pulse parameters are predefined
manually or by reading out a memory card (not shown). After
starting the process (e.g. by actuating a corresponding trigger
button), the ohmic resistance of the cuvette is first measured by
briefly applying a low voltage (e.g. 12 V) to the cuvette
connections 15, 18 and a subsequent current measurement (e.g. for 2
ms) in step 44. As part of the interrogation 45 it is checked
whether this resistance lies within a predefined window. If not,
the subsequent process is interrupted. The measured resistance is
not used subsequently to calculate the charging voltage U.sub.2 in
the present embodiment of the invention. If the resistance lies in
order within the predefined window, the storage devices 7, 8 are
charged to the predefined voltages U.sub.1 and U.sub.2 in step 46.
When the desired charging voltages are achieved, the charging by
the HV power supplies 5, 6 is switched off. During the following
pulse delivery, no recharging of the storage devices takes place.
The pulse delivery for the high-voltage pulse then begins in step
47 by closing the semiconductor switch 9. As a result, a relatively
high current flows through the cell. An excessively steep current
rise is recognised by the overcurrent cutoff stage 20 and results
in immediate opening of the switch 9 for safety reasons and
interrupts the routine. In the present embodiment the high-voltage
pulse is terminated after a predefined time of a few microseconds
whereupon the second pulse follows immediately and without
interruption. For this purpose in step 48 the second semiconductor
switch 10 is already closed a short time before opening the first
semiconductor switch 9 so that there is an interruption-free
transition between the two pulses. In the short time interval in
which both high-voltage switches 9, 10 are closed simultaneously,
the diode 17 prevents any higher voltage from being able to flow
from the storage device 7 into the storage device 8. The
semiconductor switch 10 then remains open (provided that the
maximum current is not exceeded by an overcurrent cutoff stage 22)
until the predefined charge Q has flowed through the cuvette. For
this purpose in step 49 the current flowing through the cuvette is
measured and integrated in predefined time intervals (e.g. 1 ms).
As soon as the predefined charge has been reached (see
interrogation 50), the switch 10 is opened and the routine is
terminated. The capacity of the storage device 8 is selected so
that the voltage decreases gradually or slowly during the duration
of the second pulse. If as a result of a fault, the predefined
desired charge is still not yet achieved even when the storage
device is almost completely discharged, the process will also be
interrupted after a suitably selected time limit has been
exceeded.
REFERENCE LIST
[0088] 1 Electroporator
[0089] 2 Adjusting unit
[0090] 3 Control unit
[0091] 4 Voltage supply unit
[0092] 5 HV power supply
[0093] 6 HV power supply
[0094] 7 Storage device
[0095] 8 Storage device
[0096] 9 Power semiconductor
[0097] 10 Power semiconductor
[0098] 11 Potential divider stage
[0099] 12 Potential divider stage
[0100] 13 HV switch
[0101] 14 Regulating unit
[0102] 15 Cuvette connection
[0103] 16 Resistance
[0104] 17 Diode
[0105] 18 Cuvette connection
[0106] 19 Resistance
[0107] 20 Overcurrent cutoff stage
[0108] 21 Switch
[0109] 22 Overcurrent cutoff stage
[0110] 23 Switch
[0111] 30 Push-button switch
[0112] 31 Display element
[0113] 32 LED
[0114] 33 Connector
[0115] 34 Card reader
[0116] 35 Changeover switch
[0117] 36 Transformer
[0118] 37 Rectifier
[0119] 38 Voltage regulator
[0120] 39 Regulating stage
[0121] 40 Transformer stage
[0122] 41 Solder pad
[0123] 42 Solder pad
[0124] 43 Pulse monitoring stage
[0125] 44 to 51 steps
* * * * *