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Method for combined sequential agent delivery and electroporation for cell structures and use thereof

a cell structure and sequential agent technology, applied in the field of combined sequential agent delivery and cell structure electroporation, can solve the problems of difficult transfer of all these compounds and particles

Inactive Publication Date: 2005-01-27
CELLECTRICON
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention is a method for quickly delivering multiple agents into cells using a process called electroporation. This technology allows for the rapid screening of drugs and the identification of intracellular proteins and enzymes. The method can be used for profiling, screening, and probing of e.g. intracellular proteins or drug actions inside living cells. The invention also provides a way to alter the biochemical content of cell structures and to detect the presence of certain enzymes or proteins in biosensor-chemical separation formats. The method can be used in conjunction with fluorescent markers and fluorescence microscopy for the detection of proteins and enzymes in cells. Overall, the invention provides a reliable and efficient way to study the effects of drugs and other molecules on intracellular processes."

Problems solved by technology

Although these probes and indicators are available, the main challenge so far in applying these probes and indicators is to introduce them into the cellular interior.
It is, however, difficult to transfer all these compounds and particles to the cytosol of a cell owing to the presence of a cell plasma membrane barrier, which acts as a physical boundary to the external solution that prevents the entrance of exogenous compounds and particles.

Method used

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  • Method for combined sequential agent delivery and electroporation for cell structures and use thereof
  • Method for combined sequential agent delivery and electroporation for cell structures and use thereof
  • Method for combined sequential agent delivery and electroporation for cell structures and use thereof

Examples

Experimental program
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Effect test

third embodiment

sequential delivery of multiple cell-loading agents is achieved by introducing a separation step while pumping the fluid through the EFC. When using electrophoretic pumping for delivery of reagents, all species present in the electrolyte solution is separated based on their charge-to-frictional drag ratio and will be delivered in a sequential manner at the cellular target. Analogously, sequential reagent delivery can be achieved by incorporating any separation technique applicable to the EFC format, for example, it is feasible to utilize chromatographic separation techniques.

The sequential delivery of cell-loading agents can be performed together with parallel delivery of cell-loading agents in a combinatorial fashion. Such combinatorial delivery may be achieved by selection of cell-loading agents to each EFC from a plurality of containers containing said cell-loading agents as shown in FIG. 3b. It may also be achieved by activating different EFCs in an array of EFCs where each EFC...

example 1

Detection of the Intracellular Receptor Ryanodine Type II

Fluo-3 AM ester was from Molecular Probes (Leiden, The Netherlands). Cyclic ADP ribose and the chemicals used for buffer solutions, were all of analytical grade and purchased from Sigma (St. Louis, Mo., USA). All solutions were made in distilled water from a Milli-Q system (Millipore).

NG108-15 cells were plated on no. 1 cover slips or in a Petri dish and allowed to grow for 1-3 days. Cell dishes were mounted in a circular polycarbonate holder and transferred to the stage on the microscope. Prior to experiments the culture medium was replaced by a HEPES buffer (NaCl 140 mM, KCl 5.0 mM, MgCl2 1.0 mM, CaCl2 1.0 mM, D-glucose 10 mM, HEPES 10 mM, pH was adjusted to 7.4 with NaOH).

The cells were stained with fluo-3 AM ester by incubating the cells for 30 minutes in dye solution (10 μM fluo-3 AM ester in HEPES buffer) at room temperature. To remove excess uncaptured dye, the cells were washed three times in HEPES buffer and sto...

example 2

Detection of Intracellular Enzymes I. Detection of Proteases

Casein BODIPY FL was obtained from Molecular Probes (Leiden, The Netherlands). The chemicals used for buffer solutions were all of analytical grade and purchased from Sigma (St. Louis, Mo., USA). All solutions were made in distilled water from a Milli-Q system (Millipore).

Cell culturing and preparations were made according to methods used in example 1 above, and apparatus and instrumentation was the same as in example 1.

Electroporation was performed as in example 1 and an EFC (30 cm long, 50 μm id., 375 μm o.d.) was used. Casein BODIPY FL was used in a concentration of 100 μg / ml and electroporated into cells using a 10 second pulse at 10 kV.

The results of detection of intracellular proteases using casein-BODIPY-FL is shown in FIG. 9. Specifically, the intracellular protease activity was investigated using a protein, casein, which was heavily loaded with the green-fluorescent molecule BODIPY FL, as enzyme substrate. ...

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Abstract

Disclosed is a method for sequential delivery of agents to and / or into a cell structure, wherein an electrolyte-filled tube is provided together with a counter electrode, said tube is connected to a voltage or current generator, at least two agents are introduced in a discrete mode into the electrolyte solution contained in the tube, which is placed close to the cell structure, one agent at the time being transported through the tube to and / or into said cell structure in which a pore has been formed by application of an electric field focused on the cell structure, resulting in electroporation of the cell structure. Also different applications of the method is disclosed, e.g. us of the method in order to transfer cell-impermeant solutes, such as drugs or genes, into the cell structure or out of the cell structure.

Description

FIELD OF THE INVENTION The present invention relates to a highly spatially resolved method for the electroporation and sequential delivery of one or several different compounds into cell structures, such as cells, cell-like structures, or a population of cells. Preferably, an electrolyte-filled capillary (EFC), a linear array of EFCs, or a two-dimensional matrix array of EFCs coupled to a voltage generator is used as a combined electroporation and delivery tool of, for example, multiple dyes, drugs, DNA, RNA, antisense oligonucleotides and biomolecules into the cytoplasm of single cells or populations of cells in a sequential or parallel manner or combinations thereof. The invention also relates to the use of these methods. In particular, it relates to methods for rapid screening of drugs that affect intracellular chemistry, and for the identification and detection of intra-cellular proteins. BACKGROUND OF THE INVENTION Fast and reliable methods for investigating drug actions on i...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/50A61K31/74A61K33/00A61K35/12A61K38/00A61K38/43A61K39/00A61K39/395A61K45/00A61K48/00A61N1/30A61P43/00C12N13/00C12N15/09C12N15/87G01N21/78G01N33/15G01N33/487G01N33/86G01N35/10
CPCA61N1/30G01N2035/1039G01N35/1074C12N15/87A61P43/00
Inventor ORWAR, OWE E.KARLSSON, MATTIASNOLKRANTZ, KERSTINFARRE, CECILIA
Owner CELLECTRICON
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