An electroporation apparatus comprising an elongated hollow member in order to provide a uniform electric field during electroporation, wherein specifically, electroporation is carried out by applying electric pulses through a couple of electrodes from both end parts of the elongated hollow member, after the hollow member is charged with fluid specimen including cells and material which would be injected into the cells.

PTO Wrapper PDF
Dossier Espace Google
Patent
   RE50241
Priority
Jun 12 2004
Filed
Jul 13 2023
Issued
Dec 24 2024
Expiry
Jun 13 2025
Inventors
Chang, Jun…
Assg.orig
Life Techn…
Assg.curr
Life Techn…
Entity
Large
Referenced by
0
References
56
Maint.:
currently ok
MODE FOR INVENTION
Preferred Embodiment
1. An electroporation apparatus for applying an electric pulse or electric pulses to a specimen including cells to thereby electroporate cell membranes and infuse foreign materials into the cells, comprising:
a long an elongated hollow specimen-stuffing member composed of non-conductive material;
a pressure maintaining means connected to a distal first end of the elongated hollow specimen-stuffing member for fluid communication;
a reservoir connected to the other distal a second end of the elongated hollow specimen-stuffing member for fluid communication and disposed with an electrode for electrically contacting the a specimen or an electrolytic solution disposed in the elongated hollow specimen-stuffing member; and
a reservoir holder including a fixing unit for fixing the pressure maintaining means, an a first electrode terminal for electrically connecting the fixing unit to the pressure maintaining means and another a second electrode terminal for electrically connecting the electrode disposed at the reservoir;
wherein the pressure maintaining means is a pipette in which a conductive contact is disposed at part of the a pipette body thereof of the pipette and a movable electrode is inserted into the conductive contact for communication with a piston, the movable electrode also being inserted into the specimen stuffing elongated hollow specimen-stuffing member for fluid communication and for electrically contacting the specimen or an the electrolytic solution disposed in the specimen stuffing elongated hollow specimen-stuffing member, the pippette pipette being in electrical connection with the movable electrode through the conductive contact, and
wherein the elongated hollow specimen-stuffing member is directly attached and detached to a tip mounting shaft of the pipette.
2. The electroporation apparatus according to claim 1, wherein the elongated hollow specimen-stuffing member is a capillary or a tubing.
3. The electroporation apparatus according to claim 1, wherein the elongated hollow specimen-stuffing member has a ratio (R, cm−1) of a longitudinal length (L, cm) to horizontal cross-sectional area (A, cm2) in the range of 50 to 10,000.
4. The electroporation apparatus according to claim 1, wherein the movable electrode is a plastic of which a surface is coated with conductive material.
5. An electroporation system for introducing foreign materials into cells by eletroporating electroporating cell membranes by way of applying an electric pulse or electric pulses to a specimen including the cells, comprising:
the electroporation apparatus according to claim 1; and
a pulse generator for generating an electric pulse,
wherein the electrode disposed in the reservoir is disposed with an electrode contacting is in electrical contact with the specimen or an the electrolytic solution, the elongated hollow specimen-stuffing member is filled with the specimen by the pressure maintaining means, the specimen or the electrolytic solution filled in the reservoir is connected to a distal the second end of the elongated hollow specimen-stuffing member for fluid communication, and an electric pulse or electric pulses are applied to an the second electrode contacting the specimen or the electrolytic solution filled in the reservoir and another the first electrode being inserted into an electrode insertion unit of the of a connector to thereby electroporate the cells in the specimen filled in the elongated hollow specimen-stuffing member.
6. An electroporation system for introducing foreign materials into cells by eletroporating electroporating cell membranes by way of applying an electric pulse or electric pulses to a specimen including the cells, comprising:
the electroporation apparatus according to claim 1; and
a pulse generator for generating an electric pulse,
wherein the pressure maintaining means is a pipette disposed at part of the body thereof with a conductive contact, and a the movable electrode is disposed inside the elongated hollow specimen-stuffing member is inserted for communication with a the piston, and
wherein the hollow specimen-stuffing member is directly detached and attached to a tip mounted shaft of the pipette, the movable electrode is raised or lowered to a distal the first end or the second end of the elongated hollow specimen-stuffing member by a depression button of the pipette to fill the specimen in the elongated hollow specimen-stuffing member or retrieve it, the pipette is inserted and fixed to a reservoir holder inner pipe wall including the fixing unit, a the conductive contact of the pipette body is electrically connected to the first electrode terminal via the fixing unit of the reservoir holder inner pipe wall, the elongated hollow specimen-stuffing member is so positioned as to fluidly communicate with the specimen or the electrolytic solution stored in the reservoir, and an electric pulse or electric pulses are applied to the electrode contacting the specimen or the electrolytic solution stored in the reservoir to thereby electroporate the cells in the specimen filled in the elongated hollow specimen-stuffing member.
0. 7. The electroporation apparatus according to claim 1, wherein the reservoir is composed of non-conductive material.
0. 8. The electroporation apparatus according to claim 7, wherein the reservoir is transparent.
0. 9. The electroporation apparatus according to claim 8, wherein the reservoir is composed of polystyrene.
0. 10. The electroporation apparatus according to claim 1, wherein the elongated hollow specimen-stuffing member is transparent.
0. 11. The electroporation apparatus according to claim 10, wherein the elongated hollow specimen-stuffing member is composed of polypropylene.
0. 12. The electroporation apparatus according to claim 1, the electrode comprises platinum, gold, silver, or copper.
0. 13. The electroporation apparatus according to claim 1, wherein the reservoir holder comprises an upper lid and a body.
0. 14. The electroporation apparatus according to claim 13, wherein the upper lid and the body are separable.
0. 15. The electroporation apparatus according to claim 13, wherein the upper lid and the body are integrally formed.

wherein,
E is the applied intensity of the electric field,
V is the voltage difference between both ends of the electrodes, and
L is the channel length.

As a result, even if the same voltage is applied to both ends of the micro channel, mutually different electric fields can be obtained because the channel length varies.

The electroporation apparatus having the micro channel specimen-stuffing member may be integrally manufactured or may be manufactured by coupling glass substrates or plastic substrates. In case the electroporation apparatus is manufactured by coupling the plastic substrates, it is preferred that the electroporation apparatus should include an upper substrate (350a) and a lower substrate (350b), wherein the upper plate is formed with holes forming the wells, and the upper or the lower plate is formed with depressed channels.

Preferably, the electroporation apparatus according to the present invention is manufactured with a specimen-stuffing member whose length is 1 mm˜10 cm. More preferably, the length of the specimen-stuffing member is 1 cm˜5 cm. Preferably, the height of a channel, if the specimen-stuffing member has a channel structure, is 2 μm˜2 mm, and the width thereof is 10 μm˜10 mm. More preferably, the height of the channel is 20 μm˜200 μm, and the width is 100 μm˜5 mm. The electroporation apparatus having a channel structure according to the present invention can be manufactured by MEMS technique.

FIGS. 16 (a), (b), (c) and (d) illustrate various structures of an electroporation apparatus according to the present invention. FIGS. 16(a) to (c) illustrate structures wherein several pairs of wells for inserting electrodes of the pulse generator are formed at both sides, and each channel forming a space for connecting the plural pairs of wells and filling the specimen is formed for each pair of wells. Particularly, the electroporation apparatus illustrated in FIG. 16(c) is arranged with wells in such a manner that the distance of each pair of wells is different.

The electroporation apparatus illustrated in FIG. 16(d) is formed at both sides thereof with a pair of wells for inserting the electrodes of the pulse generator, and is also formed with a plurality of channels for connecting the pair of wells and filling the specimens.

An electroporation apparatus illustrated in FIG. 17 is formed at both sides thereof with a pair of wells for inserting the electrodes of the pulse generator, and each pair of wells is formed with a channel, wherein each channel width is different from each other. If the channel length of each channel of the specimen-stuffing member is different, each channel can be applied with different electric field.

An electroporation apparatus illustrated in FIGS. 18(a) and (b) is such that each channel length and channel width connecting the corresponding wells are all different. The electroporation apparatus according to the present invention is such that a far less amount of current flows in the same electric field, compared with the conventional electroporation apparatus because the current flows only through the hollow specimen-stuffing member. As a result, power consumption can be minimized and if necessary, it can be manufactured for portable purpose using battery as a power source.

Hereinafter, an electroporation experiments and biological results using the electiOporation apparatus and electroporation system according to the present invention will be described.

MODE FOR INVENTION
Preferred Embodiment

Preferred Embodiment 1: Electroporating Experiment of HEK-293 Cell Line using Pipette Type Electroporation Apparatus

1-1. Preparation of Cells

HEK-293 cell line (ATCC, CRL-1573) was stored in a medium supplemented with 10% FBS in a 25 cm2 culturing flask, cultured in CO2 incubator, and cultured up to 70% confluency. Next, the medium was removed, and the cell was washed using PBS buffer solution, and treated with trypsin. It was added by medium supplemented with FBS and centrifuged. Next, the cell was washed by PBS buffer solution, and suspended again in medium supplimented with 10% FBS to prepare a cell specimen.

1-2. Electroporation

Approximately 100 μl of HEK-293 cell specimen thus prepared at 1-1 was introduced into a reservoir at room temperature. The specimen of 100 βl was inserted with 5 μg of plasmid DNA pEGFP (obtained from: GenBank Accession: U55762; CLONTECH Lab.) as transfection material and mixed. A distal end of the specimen-stuffing member of the electroporation apparatus (see FIG. 8) according to the present invention was inserted into the mixed solution in the reservoir. A pipette-type pressure maintaining means was used to fill an interior of the specimen-stuffing member with specimens while the distal end of the specimen-stuffing member was so connected as to fluidly communicate with the mixed solution stored in the reservoir. The reservoir was replaced by a reservoir containing only the electrolytic solution and the distal end of the specimen-stuffing member was made to dip in the reservoir containing the electrolytic solution for fluid communication. In addition, electric field applying conditions such as pulse voltage, pulse duration and pulse repetition frequency and the like were set up. In the present experiment, it was so set up that an electric field of 0.57 kV/cm was once applied for a pulse duration of 30 ms. Next, an electric pulse was applied to the electrodes inserted into a connector out of the electroporation apparatus and the reservoir containing only the electrolytic solution under the electric condition.

1-3. Retrieve of Electroporated Cells

The specimen in the specimen-stuffing member was moved to a culture plate using a pipette and applied with medium. Cells were cultured in CO2 incubator for 24˜48 hours. The cells were counted and transfection rate thereof was measured.

1-4. Results

FIGS. 19 and 20 are microscopic photographs of HEK-293 cells into which plasmid DNA pEGFP was inserted by the electroporation apparatus according to the present invention. FIG. 19 is a photograph via bright field and FIG. 20 is a photograph observed in fluorescence. As a result of the experiment, the transfection rate (number of fluorescently expressed cells/number of surviving cells) was in the range of 90 to 95%, as shown in the photographs, and the survival rate of the cells was over 90%. FIGS. 21 and 22 are microscopic photographs of experimental results under the same condition as was done by using the conventional electroporation apparatus illustrated in FIG. 1 in which HEK-293 cells were transfected by plasmid DNA pEGFP. FIG. 21 is a photograph via bright field and FIG. 22 is a photograph observed in fluorescence. As a result of the experiment, the transfection rate was approximately 50% and the cell survival rate was observed to be less than that of the present invention. It could be noticed that dead or less-grown cells frequently observed in the prior arts (round-shape cells in FIG. 21) were drastically reduced in the result of the present invention (FIGS. 19 and 20). Therefore, it can be noted that the transfection rate and survival rate were much improved when an electroporation apparatus according to the present invention was employed. Furthermore, if the electroporation apparatus according to the present invention was used, it is easy to retrieve cells introduced with particular materials.

1-5. Effect Analysis Based on Geometrical Structure Changes of Specimen-Stuffing Member

HEK-293 cell specimen of approximately 100 μl prepared in 1-1 was infused into a reservoir at room temperature. Plasmid DNA pEGFP of 5 βg is added to the specimen of 100 μl as transfection material and mixed, and an experiment was conducted using the electroporation apparatus of FIG. 8. The specimen was picked up by a pipette-type pressure maintaining means, and reservoir was replaced by a reservoir containing only the electrolytic solution. A distal end of the specimen-stuffing member is dipped into the reservoir containing the electrolytic solution for fluid communication. The electric condition was set up in such a manner that an electric field of 425 V/cm was applied three times for a pulse duration of 10 ms. The electroporation was implemented in such a manner that the specimen-stuffing member of a capillary is fixed with a cross-sectional diameter of 0.135 cm while the lengths between the distal ends change from 0.4 cm to 4 cm. Table 1 shows the geometric conditions and experimental conditions.

TABLE 1
Trans-
cell fection
L (cm) D (cm) A (cm2) R (cm−1) voltage counting rate
4 0.135 0.014307 279.6 2500 160/163 98.0
3.6 0.135 0.014307 251.6 2250 138/142 97.0
3.2 0.135 0.014307 223.7 2000 122/127 96.0
2.8 0.135 0.014307 195.7 1750 158/165 96.0
2.4 0.135 0.014307 167.8 1500 117/124 94.0
2 0.135 0.014307 139.8 1250 107/117 91.0
1.6 0.135 0.014307 111.8 1000 104/117 89.0
1.2 0.135 0.014307 83.9 750 46/63 73.0
0.8 0.135 0.014307 55.9 500 43/62 69.0
0.4 0.135 0.014307 28.0 250 18/68 26.0

In the above table, L denotes a longitudinal length (cm) of the specimen-stuffing member, D denotes a diameter (cm) of cross-section, A denotes an area (cm2) of the cross-section, and R (cm−1)=L/A.

Following the electroporation under the condition thus described, the specimens in the specimen-stuffing member were moved to a culturing plate and cultured for 24 hours. The cells were counted and the transfection rate was measured. FIG. 22 shows a result thereof

FIG. 23 illustrates a graph in which transfection rate was greatly reduced when R is below 50. FIGS. 24 to 33 are microscopic photographs of HEK-293 cells into which plasmid DNA pEGFP was inserted by the electroporation apparatus according to the present invention. Left side of each figure is a photograph observed via bright field, and right side is a photograph observed in fluorescence.

1-6. Electroporation Experiments using Various Cell Lines

The electroporations were conducted in the same conditions with various cell lines. The experimental results, as shown in FIG. 34, are such that all the cells described in Table 2 showed an excellent transfection rate by the electroporation apparatus according to the present invention. FIG. 34 shows a transfection rate given in a graph, and FIG. 35 illustrates a microscopic photographic result relative to GFP expression.

TABLE 2
ACC. No. origin Tissue
HEK293 ATCC: CRL-1573 Human Embryonic kidney
CHO-K1 ATCC: CRL-9618 Hamster Ovarian
NIH3T3 ATCC: CRL-1658 Mouse Fibroblast
3T3-L1 ATCC. CL-173 ™ Mouse Pre-adipocyte
MDA-MB-231 ATCC: HTB-26 Human Breast
Raw264.7 ATCC: TIB-71 Mouse Macrophage
Cos07 ATCC: CRL-1651 Monkey Kidney
C2C12 ATCC: CRL-1772 Mouse Myoblast
RKO ATCC: CRL-2577 Human Colon
MCF-ADR ATCC: HTB-22 Human Breast
PA317 ATCC: CRL-9078 ™ Human Embryonic firoblast
ChangX31 ATCC CCL-13 ™ Human Liver
BJ ATCC: CRL-2522 Human Foreskin Primary
culture

1-7. Transfection of siRNA

CHO cell line (ATCC:CRL-9618), HeLa cell line (ATCC, CCL-2) and SK-OV-3 cell line (ATCC, HTB-77) were employed for experiments. The electroporation was conducted in the same ways as in those of 1-1 to 1-4 to observe the GFP expression except that GFP siRNA (Ambion, NO. 4626, USA) of 0.25 nmol and pEGFP 5 of μg as transfection materials were mixed with a specimen of 100 μl. As illustrated in FIG. 36, GFP expression was barely observed when employed with a mixed solution of GFP siRNA and pEGFP as transfection materials, which suggests that pEGFP and siRNA were all effectively transmitted within the cells and thereby the GFP expression was inhibited by the siRNA in the cells.

Preferred Embodiment 2: Electroporating Experiment of SK-OV-3 Cell using a Channel-Structured Electroporation Apparatus

2-1. Manufacturing of Micro Channel Structure

In the preferred embodiment 2, a biological experiment was conducted employing an electroporation apparatus having a specimen-stuffing member of a micro channel structure. An electroporation apparatus disposed with wells for inserting electrodes and channels as hollow specimen-stuffing members for connecting the wells was manufactured by a method such as molding or the like. The channel structured specimen-stuffing members were variably manufactured with 20 μm in height, 2 cm in length and 100 to 500 μm in width of the channel. However, it should be apparent that the channel pattern was formed by photolithographic method using photomasks. For example, first of all, negative photoresist (SU-8, MicroChem, Massachusetts, USA) is spin-coated on a silicon wafer to form a mold master of 20 μm thickness. The soft baking is performed to make the mask pattern on the SU-8 coated silicon wafer by the mask aligner (MA-6, Karl Suss GmbH, Germany). SU-8 pattern is exposed to light, and post-exposure bake, development and hard baking process are performed. Then, mixture (Sylgard 184, DOW Corning Co., USA) of PDMS and cure agent is poured on the pattern. The curing condition is 90° C. for 30 minutes. The PDMS layer processed by 25 W oxygen plasma is coupled to a glass substrate to form a micro channel.

2-2. Cell Preparation and Culture

SK-OV-3 cell (ATCC, HTB-77) was cultured in an CO2 incubator of 37° C., humidity 5% using DMEM (Dulbecco's modified Eagle's Medium) supplied with heat inactivated Fetal Bovine Serum(FBS, Sigma), penicillin (100 unit/ml), streptomycin (100 μg/ml) and L-glutamine (4 mM). Trypsin-EDTA was used to separate cells from 25 cm2 tissue culturing flask. The final cell suspension concentration was adjusted to 1×107 cells/ml. The survival rate of cells following the application of pulse was used as a direct proof of viability. Before the electric pulse is applied, PI (propidium iodide) was added to cell medium. PI is a conventionally used fluorescent marker. The PI is an indicator of cell membrane introduction in a living cell and is inserted into nucleic acid. If the cell membrane is permeable, the PI enters the cell, and is combined with nucleic acid to emit a red fluorescence. As the intensity of the red fluorescence is determined by the amount of PI combined with the nucleic acid, it is possible to perform quantitative analysis. In the present experiment, PI 1.0 mg/ml was applied to cell medium in the ratio of 1:20 (v/v).

Because GFP(green fluorescent protein) extracted from Aequorea Victoria has a higher visibility and emission of effective inner fluorophore, it is variably used in the fields of biochemistry and cell biology. The GFP is used as a gene expression marker of protein targeting in cells and organs. In the present experiment, plasmid isolation kit (Promega, USA) was used for extracting and refining pEGFP-NI plasmid for transmitting GFP of colitis germs E. coli. The extracted plasmid DNA was checked on an agarose gel by way of electrophoresis. The concentration of the plasmid was determined by measuring the absorbance at 260 nm with a spectrophotometer. Before the pulse was applied, plasmid pEGFP-NI was applied to a specimen in the concentration of 0.1 βg/μL. A reporter gene expression was used as evaluation of successful transfection. In order to inspect the expression, cells exposed to electric pulse were cultured. After the pulse was applied, the channel structured electroporation apparatus was dipped in the DMEM medium, and placed in an incubator for 24 hours before EGFP expression inspection. For cell culture, no prior process was conducted except for O2 plasma to the micro channel device.

2-3. Electroporation

A system for electroporation comprises the aforementioned 2-1 electroporation apparatus having a channel structured specimen-stuffing member, home-made high voltage pulse generator, Pt electrodes and an electrode holder. The cell specimens prepared in the aforementioned 2-2 were introduced into one well to allow the channel type specimen-stuffing members to be filled with cell specimens or to allow an excess quantity of specimen to be filled in other wells by capillary or water head pressure action, or to allow the wells and specimen-stuffing members to be infused by pumping. By fixing the electrode holder on the microscope, the electro-permeating process could be observed under application of electric pulse. The high voltage pulse generator was connected to a computer via an analogue output board (COMI-CP301, Comizoa, Korea), and was controlled by LabVIEW ver 6.1 (National Instrument, USA) program. In order to verify the performance of the electroporation apparatus according to the present invention, our experimental results were compared with those of square wave electroporation apparatus (ECM 830, BTX, USA, see FIG. 2) and cuvette of 2 mm gap equipped with parallel aluminum electrodes (see FIG. 1). In order to analyze the performance of said two systems under the same electric field (1 kV/cm), the cuvette was applied with an voltage of 200 V, and the micro channel device according to the present invention was applied with an voltage of 2 kV. The channel width was changed as 100 μm, 200 μm, 300 μm, 400 μm, and 500 μm, and experiments were conducted using PI relative to the five cases. For GFP transfection and expression, experiments were carried out for 10 ms under various pulse conditions from 0.75 kV/cm to 0.25 kV/cm. In order to observe PI absorption, a reverse phase fluorescent microscope (LX790, Olympus, USA) equipped with 100 W mercury lamp and ×20 object lens (0.4 NA) was used. The light was optically filtered by a 530+20 nm band pass filter, and the fluorescence induced from the electroporated cells was filtered by 590 nm long pass filter. Image of resolution 640×80 pixels were obtained with a rate of 15 frames/sec using 12-bit CCD camera (PCO, Kelheim, Germany). The exposed time of 10 ms was given for all the cases. In order to observe the fluorescence relative to cell viability and GFP transfection, the excited light was filtered by 475±5 nm band pass filter, and the induced fluorescence was filtered by 520 nm long pass filter. Images of resolution 640×80 pixel were obtained using a color 3IT CCD camera (AW-E300, Panasonic, USA).

2-4. Results

When an electric pulse was applied in the cuvette using Al electrodes (see FIG. 1), air bubbles were electrochemically generated on the surfaces of the electrodes to form two layers of liquid and gas phases. FIG. 37 illustrates an Al electrode surface before (a) and after (b) the application of electric pulse when a cuvette equipped with a conventional Al electrode is used to perform an electroporation. FIG. 37(b) shows air bubbles formed on the electrode surface. The air bubbles are created very fast and generate a complicated liquid movement. The said air bubble movement, in cooperation with the electrophoresis during pulse application, results in an uneven state of bulk media and cells. Furthermore, aluminum is a material easily formed with an oxide layer (Al2O3), which acts as a high resistance layer. In the electroporation apparatus according to the present invention, the air bubble formation or the complicated medium movement was not found, which explains that the electrodes positioned only on both the distal ends and the chemical stability of the electrode material (Pt) used in the present invention prevent the air bubble formation from directly influencing on the specimens. As a result, although the bulk medium movement in the cuvette wherein the air bubbles are created is strong, the medium in the electroporation apparatus having a channel structure according to the present invention can maintain a stable condition because of less air bubble influence. The micro channel structure according to the present invention is excellent in visualization and is not visually affected by electrodes, such that mechanism of the electroporation can be visually studied using microscopes and the like.

2-5. Intercalation Rate and Electro-Permeability Process Inspection

A local introduction of PI during the milli-sec (ms) unit in the channel was observed after a pulse was applied to the electroporation apparatus having a micro channel specimen-stuffing member according to the present invention. If the same scope of electric field is applied in the conventional system, the PI permeability process was detected from almost all the cells within the micro channel. FIG. 38 illustrates an infusion process of PI in a 100 μm width micro channel. Right after the application of pulse, PI was infused only from an anode direction. As time goes by, the fluorescence was dissipated all over the interiors of the cells (c to d), and after 10 seconds, the nucleic acid started to radiate fluorescence (e to h).Observation directly reflects the PI characteristics coupling to the nucleic acid. The function of observing in real time in a single living cell is very advantageous because it can provide an important information about the basic cell process. For example, by way of detecting FRET (fluorescence resonance energy transfer), the oligo DNA pair in the cytoplasm can be observed in being combined with c-fos mRNA. Because it can be directly observed in real time, the micro channel device according to the present invention is very useful.

2-6. Electroporation Effect Based on Channel Width Changes in Electroporation Apparatus having a Micro Channel Specimen-Stuffing Member

In the electroporation apparatus, the intensity of fluorescence relative to dye absorption is differently observed according to the channel width. If the same electric pulse is applied, the intensity of grey scale unit relative to the cell region decreased as the channel width increased. FIG. 39 is a microscopic photograph of cells infused by PI via electroporation in two micro channels each having a different channel width of 100 μm(a) and 500 μm(b) respectively after 30 seconds of pulse application, wherein the electric field was 1 kV/cm, and pulse duration was 10 ms. It was confirmed that PI absorption of cells in a micro channel having a narrow channel width (100 μm) is much greater than that of cells in a micro channel having a broader channel width (500 μm). In order to compare the PI absorption relative to five micro channels each having a different channel width, images were photographed at 15 frames/sec during the experiments. Image process was conducted at every 50 frame. By using graphic software (Paint Shop Pro 7.0, Jasc Software, USA) and MATLAB program (MathWorks, Inc., USA), an average intensity of the grey scale unit for the background was subtracted from the grey scale unit for the cell region. A comparative data relative to the PI intensity is illustrated in FIG. 40. It can be noticed that the channel width affects the PI absorption. Because the geometric parameters except the electrode gap in the cuvette based system were not seriously considered, the said phenomenon in the micro channel specimen-stuffing members should be given a special attention. The effect of the electric pulse to the cells was analyzed in bright field. The bright field analysis method was conducted under two conditions of 150 μm and 500 μm widths. The pulse conditions are the same as those of other experiments (10 ms in 1 kV/cm electric field). Images following the exposure to the electric pulse were obtained 25 seconds after the pulse application. Exposed to the electric pulse, the cells were immediately swelled. FIG. 41 illustrates a cell size change before and after an electroporation in two micro channels, each having a different channel width of 150 μm(a) and 500 μm(b), respectively. By using AutoCAD 2002 (Autodesk, Inc., USA), the cell diameter increase was measured and increased rate relative to diameter before the pulse application was calculated. Although the cell diameter at 150 μm channel width increased approximately by 23%, the cell diameter at 500 μm channel width increased approximately by 10%. The said difference relative to the channel width seems to be resulted from the different degrees in electroporation. From these results, it was confirmed that geometrical shapes of channel cross-sections such as channel widths or heights during the electroporation of micro channel specimen-stuffing members should be considered.

2-7. Cell Culture in PDMS Channel Specimen-Stuffing Member

PDMS is a material appropriate for the cell culture system of channel device due to its biological suitability and permeability. Because it usually takes 24 hours to express in cells following the electroporation in EGFP transfection experiment, it is necessary to have a cell culture function in the EGFP transfection experiment in the channel specimen-stuffing member according to the present invention. Inspection was made as to whether the channel specimen-stuffing member could be used as a reservoir for cell culture. Cells were infused into the channel and the entire PDMS channel device was dipped into the cell medium (DMEM), and stored in an incubator for 7 days. FIG. 42 illustrates a culture result thereof. Only the O2 plasma process for coupling PDMS to the glass was conducted in the PDMS channel device. Following 7 days, as a result of observation on the wells and cells at the distal ends of the channel, it could be noticed that the cells were well dispersed on the floor surface and maintained an excellent condition. The cells in the central channel of 50 μm channel width still survived, but conditions thereof were not good (see FIG. 42(a)). It seems that the cell culture was ill affected by lack of fresh culture media and CO2, and cramped physical space caused by narrow channel width relative to the channel length. FIGS.42 (b), (c) and (d) also illustrate a result wherein cells are cultured for seven days in a micro channels each having a different channel width of 150 βm, 200 μm and 250 βm respectively. It could be noted that the cells were attached, dissipated and successfully moved in a broader micro channel. As a result of the aforementioned experiment, it was confirmed that the cell culture was possible in the electroporation apparatus according to the present invention. This shows that many advantages could be provided along with the real time visualization function in the study of various cells for a long time. Furthermore, the electroporation apparatus according to the present invention could be expectedly used in simultaneously tracing the routes of multiple proteins within a living cell for a long time by using nano-sized quantum dot semiconductor.

2-8. EGFP Expression in SK-OV-3 Cell

A biological experiment was carried out by EGFP which is widely used as gene expression marker. First of all, an electric pulse of 1.5 kV was applied to induce an electric field of 0.75 kV/cm for 10 ms. This is an adequate condition for infecting SK-OV-3 cells using currently marketed BTX electroporation apparatus. The said electric field condition was too harsh for cells in the channel structured specimen-stuffing members. The cells were inspected after 24 hours and a result thereof is illustrated in FIG. 43 (a). It could be noticed that the cells are not under an excellent state to be dissipated on the floor surface. The fluorescence was not detected. The electric field adequate for the currently marketed electroporation apparatus is too strong to be adopted for the channel structured electroporation apparatus according to the present invention, so the electric field was changed in the range of 0.25 kV/cm to 0.75 kV/cm in the present experiment. As a result thereof, the cells were successfully infected in the range of 0.4 kV/cm to 0.5 kV/cm, and it was confirmed that green fluorescence was expressed. The most preferable condition was 0.4 kV/cm. FIGS.43 (b) and (c) show the result thereof. From this result, it was verified that the energy efficiency for electroporation in the electroporation apparatus according to the present invention is far more excellent than that of using the cuvette-type electroporation apparatus.

As mentioned earlier, the infusing process can be visualized in real time using the same electroporation apparatus according to the present invention. In the electroporation according to the method of the present invention, the generation of air bubbles and complicated movement of cell media and cells were not observed either. Unlike the cuvette, the long, thin and hollow specimen-stuffing member restricts the current direction due to its geometrical structure, an even electric field is formed on the entire specimen-stuffing member. The uniform environment in the said specimen-stuffing member enhances the material absorption rate in the cells.

Industrial Applicability

As earlier mentioned, cells can be easily electroporated using the electroporation apparatus according to the present invention. Furthermore, because the cells are electroporated in a capillary, pipe including tubing or micro channel, the electroporated cells can be effectively retrieved and used. The thin, long and hollow structured specimen-stuffing member enables the current to flow only through the narrow piping, such that an even electric field can be provided in the specimen-stuffing member compared with the conventional broad and short cuvette. Therefore, it is possible to reduce errors resulting from experimental conditions. The electroporation apparatus according to the present invention has the electrodes and the specimen-stuffing members, which are attachable and detachable therefrom, to allow the eternal use of platinum electrodes of excellent performance, or cheaper disposable electrodes, such that the specimen-stuffing members can be conveniently disposed for one time use. As electrodes of excellent performance are used, the generation of oxygen due to decomposed water or formation of metal ions can be reduced. Furthermore, there is little loss of specimens. In addition, experiments can be conducted with only a small amount of specimens because the small amount of specimens can be filled in and retrieved from the specimen-stuffing member and retrieved by eletroporation. Furthermore, by properly controlling the pressure maintaining means, a large amount of specimen can be automatically experimented, and by using a plurality of electroporation apparatuses in parallel, optimum experimental conditions can be easily created, enabling to process several specimens at the same time.

INVENTORS:

Chang, Jun-Keun, Cho, Keun-Chang, Chung, Chan-Il, Jung, Neon-Cheol, Shin, Young-Shik, Kim, Jeong-Ah

THIS PATENT IS REFERENCED BY THESE PATENTS:
Patent Priority Assignee Title
THIS PATENT REFERENCES THESE PATENTS:
Patent Priority Assignee Title
4099548, Aug 25 1976 Sherwood Medical Company Hand-held pipette for repetitively dispensing precise volumes of liquid
4959321, Mar 26 1988 Cell fusion apparatus
5007995, May 11 1989 Olympus Optical Co., Ltd. Device for electrofusion of cells
6001617, Jun 07 1989 Queen's University at Kingston Electroporation device and method of use
6074605, Mar 10 1995 MaxCyte, Inc Flow electroporation chamber and method
6241701, Aug 01 1997 OncoSec Medical Incorporated Apparatus for electroporation mediated delivery of drugs and genes
6287776, Feb 02 1998 MDS Sciex Method for detecting and classifying nucleic acid hybridization
6293749, Nov 21 1997 ASM America, Inc Substrate transfer system for semiconductor processing equipment
6352853, Dec 07 1998 Rosetta Inpharmatics LLC Multi-channel electrode arrays
6356173, May 29 1998 Kyocera Corporation High-frequency module coupled via aperture in a ground plane
6627421, Apr 13 1999 CEREVAST THERAPEUTICS, INC Methods and systems for applying multi-mode energy to biological samples
6628382, Aug 20 1999 Nanodrop Technologies, LLC Liquid photometer using surface tension to contain sample
6677114, Apr 20 1999 TARGET DISCOVERY, INC Polypeptide fingerprinting methods and bioinformatics database system
6897069, Jun 08 2004 Applied Biosystems, LLC System and method for electroporating a sample
6936462, Jun 12 1998 SOPHION BIOSCIENCE A S High throughput screen
7384781, Sep 21 2005 Sensors for biomolecular detection and cell classification
7393681, May 17 2005 Applied Biosystems, LLC System and method for electroporating a sample
7456012, Nov 06 1998 Cellectricon AB Method and apparatus for spatially confined electroporation
7678564, Feb 20 2002 Lonza Cologne AG Container with at least one electrode
8017399, Jun 08 2004 Applied Biosystems, LLC System and method for electroporating a sample
8101401, Mar 15 2004 Lonza Cologne AG Container and device for generating electric fields in different chambers
8932850, Jun 12 2004 LIFE TECHNOLOGIES HOLDINGS PTE LTD Electroporation apparatus having an elongated hollow member
20020090649,
20030009148,
20030022153,
20030070923,
20030104588,
20040029101,
20050118705,
20050164161,
20070275454,
20080064511,
20080213854,
20100196998,
20110263005,
EP338667,
EP338667,
JP11290058,
JP2002504232,
JP2007167006,
JP2008051169,
JP2008502356,
JP63049070,
JP63160574,
JP6349070,
KR101084528,
WO63408,
WO233066,
WO3057819,
WO2004031353,
WO2005123931,
WO2006001614,
WO2008051169,
WO2009129327,
WO9858251,
WO9924110,
ASSIGNMENT RECORDS    Assignment records on the USPTO
/
Executed onAssignorAssigneeConveyanceFrameReelDoc
Jul 13 2023Life Technologies Holdings PTE Ltd.(assignment on the face of the patent)
MAINTENANCE FEES AND DATES:    Maintenance records on the USPTO
Date Maintenance Fee Events
Jul 13 2023BIG: Entity status set to Undiscounted (note the period is included in the code).


Date Maintenance Schedule
Dec 24 20274 years fee payment window open
Jun 24 20286 months grace period start (w surcharge)
Dec 24 2028patent expiry (for year 4)
Dec 24 20302 years to revive unintentionally abandoned end. (for year 4)
Dec 24 20318 years fee payment window open
Jun 24 20326 months grace period start (w surcharge)
Dec 24 2032patent expiry (for year 8)
Dec 24 20342 years to revive unintentionally abandoned end. (for year 8)
Dec 24 203512 years fee payment window open
Jun 24 20366 months grace period start (w surcharge)
Dec 24 2036patent expiry (for year 12)
Dec 24 20382 years to revive unintentionally abandoned end. (for year 12)