EP1137765A1 - Appareil pour l'ecoulement tangentiel, la concentration et la fusion des cellules - Google Patents

Appareil pour l'ecoulement tangentiel, la concentration et la fusion des cellules

Info

Publication number
EP1137765A1
EP1137765A1 EP00919388A EP00919388A EP1137765A1 EP 1137765 A1 EP1137765 A1 EP 1137765A1 EP 00919388 A EP00919388 A EP 00919388A EP 00919388 A EP00919388 A EP 00919388A EP 1137765 A1 EP1137765 A1 EP 1137765A1
Authority
EP
European Patent Office
Prior art keywords
cell
bladder
cells
fusion
liquid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00919388A
Other languages
German (de)
English (en)
Other versions
EP1137765A4 (fr
Inventor
Richard E. Walters
Alan D. King
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cyto Pulse Sciences Inc
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP1137765A1 publication Critical patent/EP1137765A1/fr
Publication of EP1137765A4 publication Critical patent/EP1137765A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/18—Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/20—Accessories; Auxiliary operations
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
    • B03C5/02—Separators
    • B03C5/022—Non-uniform field separators
    • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

Definitions

  • the present invention relates generally to methods and apparatus for fusing biological cells together. More specifically, the present invention provides methods and apparatus for fusing biological cells using the application of electrical fields to the biological cells to be fused together.
  • An important number for any cell fusion system is the percentage of cells that are fused.
  • the probability of getting fusion products in a system which has the objective of fusing two and only two cell types, that have equal cell populations, are:
  • Cell Type l to Cell Type 1 25% of no use Cell Type l to Cell Type 2 25% desired Cell Type 2 to Cell Type 1 25% desired Cell Type 2 to Cell Type 2 25% of no use
  • the chemical method usually involves concentrating cells in the presence of polyethylene glycol . This method is toxic to cells and the maximum yields of desirably fused cells are approximately only 0.1 %.
  • the electrical method for fusing cells requires first contact between the cell membranes of two or more cells, and then the application of a high voltage electrical pulse.
  • the high voltage electrical pulse induces cellular pores (openings) which lead to cell fusion in the touching cells.
  • the contact between the cell membranes prior to induction of cell fusion is a critical part of the process.
  • the method of bringing the cells into contact highly influences the percent of different-cell-type fused cell pairs (desired) , the percent of same-cell-type cells fused (not desired) , and the viability of the cells fused.
  • the method of applying the electrical pulses also has an effect on the efficiency. To date ordinary pulses have been used, exponential decay pulses (see Sowers, A.E., 1986, J.
  • PulseAgile (Registered in U. S. Patent and Trademark Office), Model PA-4000 Electroporation System made by Cyto Pulse Sciences, Inc., P. 0. Box 609, Columbia, MD 21045. It is noted that the Model PA-4000 delivers rectangular waves of various amplitudes (voltages), width, and intervals. It is expected that use of the PulseAgile System may improve efficiency.
  • Dielectrophoresis A well-known electrical method for bringing cells into contact is called dielectrophoresis, which was developed by Zimmerman in 1982 (see Zimmerman, U., 1982, Biochm Biophys Acta, 694: 227-277). Dielectrophoresis is the induction of dipoles in cells through the application of a low voltage potential. This is usually applied as a very high frequency sine wave, but direct current can also be used. The dipoles in the cells cause the cells to line-up in a chain called a pearl chain. A brief high voltage electrical pulse is applied to the cells while they are lined up.
  • the high voltage pulses are delivered after centrifugation.
  • the methods in which the biological cells are concentrated prior to electrofusion are most desirable.
  • the liquid flow flows in a direction perpendicular to the surface of the porous filter.
  • the liquid flow is obstructed by the porous filter.
  • a cell fusion apparatus were provided which employs a concentrating porous filter which is not oriented perpendicular to the liquid flow through the apparatus.
  • a cell fusion apparatus provides both unobstructed liquid flow and concentration of biological cells that are to undergo electrofusion.
  • a cell fusion apparatus could be contained in a sealed, sterile system.
  • a cell fusion apparatus were modular in form that could be used, disposed of, and replaced in a simple and easy manner.
  • a cell fusion module could be placed in a module manipulator that interacts with and operates a cell fusion module from outside the module without entering the module so that the sterility inside the module is not disturbed.
  • a cell fusion apparatus which has the following combination of desirable features: (1) employs a concentrating porous filter which is not oriented perpendicular to the direction of liquid flow through the apparatus; (2) provides both unobstructed liquid flow and concentration of biological cells that are to undergo electrofusion; (3) provides an electroporation or electrofusion apparatus which employs tangential flow filtration; (4) can be contained in a sealed, sterile system; (5) can be modular in form and can be used, disposed of, and replaced in a simple and easy manner; and (6) can employ a programmable module manipulator that interacts with and operates a cell fusion module from outside the module without entering the module so that the sterility inside the module is not disturbed.
  • the foregoing desired characteristics are provided by the unique tangential flow, cell concentration and fusion apparatus of the present invention as will be made apparent from the following description thereof.
  • a cell concentration and fusion apparatus includes a housing.
  • a first input channel is contained in the housing.
  • a concentration chamber includes a proximal end connected to the first input channel and is contained in the housing.
  • the concentration chamber extends along a longitudinal axis through the housing.
  • a first output channel is contained in the housing and is connected to a distal end of the concentration chamber.
  • a filter serves as a wall of the concentration chamber. The filter is located between the first input channel and the first output channel. The filter extends in a direction parallel to the longitudinal axis.
  • An effluent chamber is contained in the housing.
  • the filter serves as a wall of the effluent chamber.
  • a second output channel is connected to the effluent chamber.
  • Electrodes are supported by the housing, and the electrodes are located between the first input channel and the first output channel.
  • the housing is made of transparent material.
  • the electrodes are located adjacent to the first output channel, and, in one configuration, the electrodes are oriented parallel to the longitudinal axis.
  • the filter is a porous membrane and has a porosity which prevents biological cells from passing through but permits liquids to pass through.
  • a second input channel is contained in the housing, and the second input channel is connected to the effluent chamber.
  • the second input channel serves as a vent channel .
  • a cell mixture bladder is connected to the first input channel.
  • a liquid bladder is connected to the first input channel.
  • a fused cell bladder is connected to the first output channel, and an exhaust bladder is connected to the second output channel .
  • a first valve is located between the cell mixture bladder and the first input channel .
  • a second valve is located between the liquid bladder and the first input channel, and a third valve is located between the fused cell bladder and the first output channel.
  • An input port is connected to the cell mixture bladder.
  • An output port is connected to the fused cell bladder, and an electrical connection jack is electrically connected to the wires .
  • the input port and the output port can be made from self-sealing elastomeric material, which is suitable for receiving a hypodermic needle.
  • a bag envelopes the cell concentration and fusion apparatus, the cell mixture bladder, the liquid bladder, the fused cell bladder, the exhaust bladder, and the wires for providing a cell fusion module.
  • the bag leaves access to the input port, the output port, and the electrical connection jack.
  • the bag is hermetically sealed.
  • the interior of the cell fusion module is maintained in a sterile status by the bag.
  • the bag is made from flexible material .
  • a module manipulator unit which can be programmable, is provided for receiving and operating the cell fusion module.
  • the module manipulator unit includes a manipulator housing.
  • a first bladder operation assembly is supported by the manipulator housing for operating the cell mixture bladder.
  • a second bladder operation assembly is supported by the manipulator housing for operating the liquid bladder, and a bladder assemblies controller is provided for controlling the first bladder operation assembly and the second bladder operation assembly.
  • a third bladder operation assembly can be supported by the manipulator housing for operating the fused cell bladder, and a fourth bladder operation assembly, supported by the manipulator housing, can be provided for operating the exhaust bladder.
  • the third bladder operation assembly and the fourth bladder operation assembly are also controlled by the bladder assemblies controller.
  • Each of the bladder operation assemblies can include a respective shaft drive unit driven by the bladder assemblies controller. A respective shaft is driven by the shaft drive unit, and a respective ram is moved by the shaft.
  • the cell concentration and fusion apparatus includes a base block and a front block that is supported by the base block.
  • the front block includes a milled front slot, a vent channel, front fastener- eception channels, and a drain channel.
  • a back block is supported by the base block and includes an input channel, a transverse hole in communication with the input channel, a milled back slot in communication with the transverse hole, an electrode, an output channel in communication with the back slot, a pair of electrical jacks connected to the electrode, and back fastener-reception channels.
  • a gasket is located adjacent to the front block between the front block and the back block.
  • the gasket includes gasket fastener-reception channels and a filter access channel.
  • a filter is located between the gasket and the back block.
  • Fasteners are received in the back fastener- reception channels, the gasket fastener-reception channels, and the front fastener-reception channels when the back fastener-reception channels, the gasket fastener- reception channels, and the front fastener-reception channels are placed in registration.
  • the electrodes are supported by the housing and are oriented perpendicular to the longitudinal axis .
  • a method for concentrating and electrically treating biological cells. The method includes the steps of : obtaining a mixture of the biological cells in a liquid carrier, moving a portion of the cell/liquid mixture into a solid/liquid separation chamber in a first direction towards an electrical treatment region, separating a portion of the liquid in the solid/liquid separation chamber from the cell/liquid mixture in a second direction, which is perpendicular to the first direction, as the cell/liquid mixture moves toward the electrical treatment region in the first direction, whereby the biological cells are concentrated in the liquid remaining in the solid/liquid separation chamber, applying an electrical field to the concentrated biological cells, collecting the biological cells that have been concentrated and treated with the electrical field.
  • the method of the invention includes the step of applying an electrical field to the concentrated biological cells.
  • the method of the invention can include applying an electrical field to bring about dielectrophoresis in the concentrated biological cells, can include applying an electrical field to bring about cell fusion in the concentrated biological cells, and can include applying an electrical field to bring about dielectrophoresis in the fused cells to result in post-fusion maturation of the fused cells.
  • the electrical field that is applied to the concentrated cells can bring about cell fusion.
  • the electrical field that is applied to the concentrated cells can bring about electroporation.
  • a method of concentrating and electrically treating biological cells includes the steps of: obtaining a cell fusion module which includes a cell concentration and fusion apparatus, placing the cell fusion module in a programmable module manipulator unit, obtaining a mixture of the biological cells in a liquid carrier, introducing a portion of the cell/liquid mixture into the cell concentration and fusion apparatus, under control of the module manipulator unit, moving a portion of the cell/liquid mixture into a solid/liquid separation chamber in the cell concentration and fusion apparatus, in a first direction, towards an electrical treatment region in the cell concentration and fusion apparatus, separating a portion of the liquid in the solid/liquid separation chamber from the cell/liquid mixture in a second direction, which is perpendicular to the first direction, as the cell/liquid mixture moves toward the electrical treatment region in the first direction, whereby the biological cells are concentrated in the liquid remaining in the solid/liquid separation chamber, applying an electrical field to the concentrated biological cells
  • the electrial-field-treated cells can be moved either to a collection region outside the cell fusion module or to a collection region inside the cell fusion module. If moved to a collection region inside the cell fusion module, then, the electrial-field-treated cells are moved to a location outside the cell fusion module.
  • the cell fusion module can be considered disposable and can be disposed of in its entirety after the fused cells have been collected.
  • It is therefore an object of the present invention is to provide a new and improved tangential flow, cell concentration and fusion apparatus which employs a concentrating porous filter which is not oriented perpendicular to the direction of liquid flow through the apparatus .
  • Still another object of the present invention is to provide a new and improved tangential flow, cell concentration and fusion apparatus that provides both unobstructed liquid flow and concentration of biological cells that are to undergo electrofusion.
  • Yet another object of the present invention is to provide a new and improved tangential flow, cell concentration and fusion apparatus which provides an electroporation or electrofusion apparatus which employs tangential flow filtration.
  • Yet another object of the present invention is to provide a new and improved tangential flow, cell concentration and fusion apparatus that can be contained in a sealed, sterile system.
  • Still a further object of the present invention is to provide a new and improved tangential flow, cell concentration and fusion apparatus which can be modular in form and can be used, disposed of, and replaced in a simple and easy manner.
  • Yet another object of the present invention is to provide a new and improved tangential flow, cell concentration and fusion apparatus that can employ a module manipulator that interacts with and operates a cell fusion module from outside the module without entering the module so that the sterility inside the module is not disturbed.
  • Fig. 1 is a cross-sectional view of a first embodiment of the tangential flow, cell concentration and fusion apparatus of the invention which employs electrodes oriented parallel to the liquid flow path.
  • Fig. 2 is a schematic diagram of a second embodiment of the invention in which the embodiment of the invention shown in Fig. 1 is contained in a sterile cell fusion module .
  • Fig. 3 is a schematic diagram of a third embodiment of the invention in which the cell fusion module shown in Fig. 2 is placed in a module manipulator unit.
  • Fig. 4 is a schematic close-up diagram of a ram in the embodiment of the invention shown in Fig. 3 in contact with a bulging surface of a bladder.
  • Fig. 5 is a schematic close-up diagram of a portion of another embodiment for applying a squeezing pressure onto the bulging surface of a bladder.
  • Fig. 6 is an exploded side view of another embodiment of the cell concentration and fusion apparatus of the invention.
  • Fig. 7 is a rear view of the embodiment of the invention shown in Fig. 6 that is fully assembled.
  • Fig. 8 is a top view of a filter used in the embodiment of the invention shown in Figs. 6 and 7.
  • Fig. 9 is a top view of a gasket used in the embodiment of the invention shown in Figs. 6-8.
  • Fig. 10 is a top view of a back block used in the embodiment of the invention shown in Figs. 6-9.
  • Fig. 11 is an enlarged schematic diagram of a portion of a cell concentration and fusion apparatus of the invention in which electrodes are oriented parallel to the flow path of liquid past the electrodes.
  • Fig. 12 is an enlarged schematic diagram of a portion of a cell concentration and fusion apparatus of the invention in which electrodes are oriented perpendicular to the flow path of liquid past the electrodes .
  • the cell concentration and fusion apparatus 12 includes a housing 24, and a first input channel 1 is contained in the housing 24.
  • a concentration chamber 26 includes a proximal end connected to the first input channel 1 and is contained in the housing 24.
  • the concentration chamber 26 extends along a longitudinal axis 28 through the housing 24.
  • a first output channel 5 is contained in the housing 24 and is connected to a distal end of the concentration chamber 26.
  • a filter 3 serves as a wall of the concentration chamber 26. The filter 3 is located between the first input channel 1 and the first output channel 5. The filter 3 extends in a direction parallel to the longitudinal axis 28.
  • An effluent chamber 34 is contained in the housing 24.
  • the filter 3 serves as a wall of the effluent chamber 34.
  • a second output channel 4 is connected to the effluent chamber 34.
  • Electrodes 6 are supported by the housing 24, the electrodes 6 are located between the first input channel 1 and the first output channel 5.
  • the housing 24 is made of transparent material.
  • the electrodes 6 are located adjacent to the first output channel 5, and, in one configuration, the electrodes 6 are oriented parallel to the longitudinal axis 28.
  • the filter 3 is a porous membrane.
  • the filter 3 has a porosity which prevents biological cells from passing through but permits liquids to pass through.
  • a second input channel 2 is contained in the housing 24, and the second input channel 2 is connected to the effluent chamber 34.
  • the second input channel 2 serves as a vent channel .
  • FIG. 1 Operation of the embodiment of the invention shown in Fig. 1 is as follows. A mixture of biological cells in a liquid carrier is introduced into the first input channel 1. The mixture passes through the first input channel 1 into the concentration chamber 26 in the direction shown by directional arrow 7. Once in the concentration chamber 26, a portion of the liquid carrier passes through the filter 3 into the effluent chamber 34. Since the biological cells are retained by the filter 3 and some of the carrier liquid is separated from the mixture, the concentration of the biological cells in the mixture increases. As the biological cells flow toward the first output channel 5, more liquid carrier is separated through the filter 3, and the biological cells become more concentrated when they reach the electrodes 6. At the electrodes 6, the concentrated cells are subjected to the application of electric fields from the voltage waveform generator 36 through wires 8. As a result of the application of the electric fields, the concentrated biological cells undergo cell fusion.
  • tangential flow past a filter is moved in a first direction towards an electrical treatment region.
  • a portion of the liquid in the solid/liquid separation chamber is separated from the cell/liquid mixture through the filter in a second direction, which is perpendicular to the first direction.
  • the filter is oriented parallel to the direction of flow of the cell/liquid mixture, and the cell/liquid mixture flows tangentially along the surface of the filter.
  • the liquid which separates from the cell/liquid mixture through the filter moves away from the cell/liquid mixture in a direction perpendicular to the direction of flow of the cell/liquid mixture.
  • the tangential filtration and concentration system of the invention is designed for cells to flow along the surface of the filter membrane.
  • This apparatus and method insure that there are fewer tendencies for the filter membrane to be blocked by the biological cells.
  • the apparatus and method also prevent excessive packing of the cells on the filter membrane.
  • the outflow from the concentration chamber 26 is restricted or blocked at the first output channel 5 to encourage accumulation of cells in the region of the electrodes 6.
  • introducing varying amounts of fluid into the first input channel 1 will increase and control cell packing density. The introduction of extra fluid will encourage further tangential flow of cells toward the electrodes 6.
  • the cell concentration and fusion apparatus 12 of the invention can be used in a wide variety of protocols for carrying out electrofusion and/or electroporation of biological cells. More specifically with respect to electrofusion, electrofusion is the fusion of two or more cells using pulsed electric fields. The membrane effects resulting from applied pulsed electrical fields in electrofusion are similar to those in electroporation. The principle difference is that membranes are in close contact permitting them to fuse together in the process of pore formation. For this reason, electric field densities used in electrofusion are similar to those used in electroporation .
  • Electrofusion is done in three major steps. First, cells are brought into contact with other cells. This is done for the devices herein using cell concentration and/or dielectrophoresis. Dielectrophoresis is a force on cells created in non-homogeneous (divergent) electric fields. The divergent electric field induces movement toward higher field densities, in the direction of the electrodes. The movement is independent of polarity and can occur in alternating current fields.
  • the second step in electrofusion is to apply one or more high voltage pulses to the cells, inducing membrane fusion. The voltage required must be above a threshold to induce membrane breakdown and below a maximum voltage that would cause cell death. Threshold voltage is approximately one volt across the cell membrane or two volts across two cell ' s membranes.
  • the voltage across a cell is equal to 1.5 times the cell radius times the electric field strength times the cosine of the angle of the membrane in relation of the direction of the field. This is the same formula used for electroporation.
  • Multiple fusion pulses may be more efficient than a single pulse.
  • the last step in the electrofusion process (when using dielectrophoresis as an alignment tool) is post fusion alignment. Electrofusion is a process that continues to occur over some time after the fusion pulse is applied. Re-applying dielectrophoresis after the fusion pulse allows maturation of the fusion process by holding cells in optimal alignment and contact.
  • the cell concentration and fusion apparatus 12 can be used in a number of different modes.
  • the cell/liquid mixture can flow continuously into the cell concentration and fusion apparatus 12, and electrial-field-treated cells can flow continuously out from the cell concentration and fusion apparatus 12.
  • the cell/liquid mixture can be introduced into the cell concentration and fusion apparatus 12 by a series of batches, and the electrial- field-treated cells can flow out from the cell concentration and fusion apparatus 12 in a batchwise manner.
  • the following steps comprise a protocol for electrofusion which involves dielectrophoresis and intermittent flow through the cell concentration and fusion apparatus 12, wherein fused hybridoma cells are produced.
  • An alternative to the wash is medium replacement using a modified tangential flow apparatus. Two filters would be used. One for introducing new medium and another for collecting filtrate.
  • the following steps presented below comprise a protocol for electrofusion which involves using intermittent flow through the cell concentration and fusion apparatus 12, without using dielectrophoresis, wherein fused hybridoma cells are produced.
  • An alternative to the wash is medium replacement using a modified tangential flow apparatus of the invention.
  • Two filters would be used. One for introducing new medium and another for collecting filtrate. 8. Count the cells before the last centrifugation. Re-suspend the cells at 1,000,000 cells/ml after the last centrifugation.
  • a second embodiment of the invention is shown.
  • Cell mixture bladder 40 is connected to the first input channel 1.
  • a liquid bladder 42 is connected to the first input channel 1.
  • a fused cell bladder 44 is connected to the first output channel 5, and an exhaust bladder 46 is connected to the second output channel 4.
  • a first valve 41 is located between the cell mixture bladder 40 and the first input channel 1.
  • a second valve 43 is located between the liquid bladder 42 and the first input channel 1, and a third valve 45 is located between the fused cell bladder 44 and the first output channel 5.
  • the first valve 41, second valve 43, and the third valve 45 can be embodied in different forms. For example, they can be simply in the form of flexible tubes that are pinched appropriately by an electromechanical clamp controlled by the bladder assemblies controller 49. Alternatively, the valves can be automatically operating one-way check valves.
  • An input port 9 is connected to the cell mixture bladder 40.
  • An injection output port 10 is connected to the fused cell bladder 44, and an electrical connection jack 14 is electrically connected to the wires 8.
  • the input port 9 and the injection output port 10 are made from self-sealing elastomeric material.
  • the input port 9 is an injection input port 9, and the output port 10 is an injection output port 10.
  • the input port 9 and the injection output port 10 are suitable for receiving a hypodermic needle.
  • the input port 9 and the output port 10 can be equipped with valves so that a continuous input of cell/liquid mixture can be introduced through the input port 9 and so that a continuous output of fused biological cells can be produced from the output port 10.
  • valves at the input port 9 and the output port 10 cell/liquid mixtures can be processed through the cell fusion module 20 in a series of batches.
  • a bag 13 envelopes the cell concentration and fusion apparatus 12, the cell mixture bladder 40, the liquid bladder 42, the fused cell bladder 44, the exhaust bladder 46, and the wires 8 for providing a cell fusion module 20.
  • the bag 13 leaves access to the input port 9, the injection output port 10, and the electrical connection jack 14.
  • the bag 13 is hermetically sealed.
  • the interior of the cell fusion module 20 is maintained in a sterile status by the bag 13.
  • the bag 13 is made from flexible material .
  • a module manipulator unit 22 is provided for receiving and operating the cell fusion module 20.
  • the module manipulator unit 22 includes a manipulator housing 48.
  • a first bladder operation assembly 50 is supported by the manipulator housing 48 for operating the cell mixture bladder 40.
  • a second bladder operation assembly 52 is supported by the manipulator housing 48 for operating the liquid bladder 42, and a bladder assemblies controller 49 is provided for controlling the first bladder operation assembly 50 and the second bladder operation assembly 52.
  • the bladder assemblies controller 49 can be programmable.
  • a third bladder operation assembly 54 is supported by the manipulator housing 48 for operating the fused cell bladder 44, and a fourth bladder operation assembly 56, supported by the manipulator housing 48, is provided for operating the exhaust bladder 46.
  • the third bladder operation assembly 54 and the fourth bladder operation assembly 56 are controlled by the bladder assemblies controller 49. It is noted that the third bladder operation assembly 54 and the fourth bladder operation assembly 56 may be considered optional. With certain operating conditions of the cell fusion module 20, the fused cell bladder 44 and the exhaust bladder 46 may be filled simply as a result of pressure applied to the cell mixture bladder 40 by the first bladder operation assembly 50 and/or pressure applied to the liquid bladder 42 by the second bladder operation assembly 52.
  • the third bladder operation assembly 54 would be employed to do just that.
  • Each of the bladder operation assemblies includes a respective shaft drive unit 60 driven by the bladder assemblies controller 49.
  • a respective shaft 62 is driven by the shaft drive unit 60, and a respective ram 64 is moved by the shaft 62.
  • the third embodiment of the invention, shown in Fig. 3, is suitable for clinical applications. Briefly, the self-contained cell fusion module 20 is placed in the module manipulator unit 22, and the voltage waveform generator 36 is connected to the electrical connection jack 14. A mixture containing biological cells and a liquid are injected through the injection input port 9 into the cell mixture bladder 40 using a hypodermic needle and syringe. Detailed operation of the module manipulator unit 22 is controlled by the bladder assemblies controller 49.
  • the bladder assemblies controller 49 first controls the first bladder operation assembly 50 to push a portion of the cell/liquid out of the cell mixture bladder 40, through the first valve 41 and into the cell concentration and fusion apparatus 12. To do so, the shaft drive unit 60 of the first bladder operation assembly 50 pushes the associated shaft 62 to push the associated ram 64 against the flexible bag 13 which pushes against the cell mixture bladder 40. A portion of the cell/liquid mixture is pushed through the first input channel 1 and into the concentration chamber 26. Some of the liquid passes through the filter 3, whereby the biological cells in the cell/liquid mixture are concentrated, and whereby the filtered liquid passes into the effluent chamber 34. The concentrated cells are pulsed at the electrodes 6 by the voltage waveform generator 36 which is electrically connected to the electrical connection jack 14.
  • the bladder assemblies controller 49 then operates the second bladder operation assembly 52.
  • the liquid bladder 42 contains a phosphate-buffered saline solution (PBS)
  • PBS phosphate-buffered saline solution
  • the second bladder operation assembly 52 forces a quantity of the PBS through the second valve 43 and into the concentration chamber 26 so that the fused cells are forced through the third valve 45 and into fused cell bladder 44.
  • a quantity of fused cells can be removed from the fused cell bladder 44 by inserting a hypodermic needle into the injection output port 10.
  • the close-up diagram shows a ram 64 in the embodiment of the invention shown if Fig. 3 in contact with a bulging surface 65 of a bladder. The bladder is squeezed by mechanical compresion when the ram 64 compresses the bulging bladder surface 65.
  • the close-up diagram shows a portion of another embodiment for applying a squeezing pressure onto the bulging surface 65 of a bladder. More specifically, driving bladder 67 is placed over the bulging surface 65 of an underlying bladder. The driving bladder 67 contains a hydraulic fluid, such as water 69. Hydraulic pressure is applied to the driving bladder 67 through and intake tube 71. With this embodiment, it is understood that the respective bladder operation assembly includes means for applying hydraulic pressure, such as a pump or a piston/cylinder combination.
  • the following steps comprise a protocol for electrofusion which involves a clinical application using the cell fusion module 20 and the module manipulator unit 22 of the invention shown in Figs. 2 and 3. More specifically, this protocol carries out the fusing of human dendritic cells and tumor cells. l. Obtain and culture dendritic cells using published methods (Schuler et al and Romani et al) .
  • the cell concentration and fusion apparatus 12 includes a base block 21, which is made from clear polycarbonate and that has the following dimensions, 70 mm. X 40 mm. X 10 mm..
  • a front block 23 is made from clear polycarbonate, has the following dimensions, 60 mm. X 40 mm. X 10 mm., and is supported by the base block 21.
  • the front block 23 includes a milled front slot 25 which has the following dimensions, 40 mm. X 3 mm. X 3 mm., includes a vent channel 27 which is a drilled hole 0.8 mm. in diameter and 10 mm. deep, includes front fastener-reception channels 55, and includes a drain channel 29 which is a through hole 1 mm. in diameter.
  • a back block 31 is made from clear polycarbonate, has the following dimensions, 60 mm. X 40 mm. X 20 mm. , and is supported by the base block 21.
  • the back block 31 includes an input channel 33 which is a 0.8 mm. diameter hole 11 mm. deep and includes a transverse hole 35 in communication with the input channel 33.
  • the transverse hole 35 is 1 mm. in diameter and 1.8 mm. deep.
  • the back block 31 includes a milled back slot 37 in communication with the transverse hole 35.
  • the back slot 37 has the following dimensions, 40 mm. X 2 mm. X 1 mm..
  • the back block 31 includes an electrode 39 which is a 1 mm. X 1 mm.
  • platinum bar includes an output channel 51 which is a hole 0.8 mm. in diameter and 21 mm. deep from the side and is in communication with the back slot 37.
  • the back block 31 also includes a pair of electrical jacks 53 connected to the electrode 39 and includes back fastener- reception channels 57.
  • the gasket 72 is made from transparent silicone rubber, and has the following dimensions 0.5 mm. X 60 mm. X 40 mm..
  • the gasket 72 includes gasket fastener-reception channels 74, and includes a filter access channel 76 which has dimensions 2 mm. X 40 mm..
  • a filter 63 has dimensions 50 mm. X 10 mm. X 0.0008 mm. and is located between the gasket 65 and the back block 31. Fasteners 61 are received in the back fastener- reception channels 57, the gasket fastener-reception channels 74, and the front fastener-reception channels 55 when the back fastener-reception channels 57, the gasket fastener-reception channels 74, and the front fastener- reception channels 55 are placed in registration.
  • FIG. 11 an enlarged schematic diagram of a portion of a cell concentration and, fusion apparatus of the invention is shown in which electrodes are oriented parallel to the flow path of liquid past the electrodes.
  • This embodiment of the invention is shown in detail in Fig. 1.
  • FIG. 12 an enlarged schematic diagram of a portion of a cell concentration and fusion apparatus of the invention is shown in which electrodes are oriented perpendicular to the flow path 7 of liquid past the electrodes. That is, the electrodes 15 are supported by the housing 24 and are oriented perpendicular to the longitudinal axis 28. In Fig. 12, the electrodes 15 are shown as screen electrodes 15.
  • biological cells can be concentrated with the invention prior to subjecting the cells to electroporation for the uptake or delivery of DNA, RNA, peptide, or protein material so that smaller quantities of the DNA, RNA, peptide, or protein material can be employed. From the above description, it is evident that a novel method for the concentration and electrical treatment of biological cells is provided by the invention.
  • the method of concentrating and electrically treating biological cells includes the steps of: obtaining a mixture of the biological cells in a liquid carrier, moving a portion of the cell/liquid mixture into a solid/liquid separation chamber in a first direction towards an electrical treatment region, separating a portion of the liquid in the solid/liquid separation chamber from the cell/liquid mixture in a second direction, which is perpendicular to the first direction, as the cell/liquid mixture moves toward the electrical treatment region in the first direction, whereby the biological cells are concentrated in the liquid remaining in the solid/liquid separation chamber, applying an electrical field to the concentrated biological cells, and collecting the biological cells that have been concentrated and treated with the electrical field.
  • the method of the invention includes the step of applying an electrical field to the concentrated biological cells.
  • the method of the invention can include applying an electrical field to bring about dielectrophoresis in the concentrated biological cells, can include applying an electrical field to bring about cell fusion in the concentrated biological cells, and can include applying an electrical field to bring about dielectrophoresis in the fused cells to result in post-fusion maturation of the fused cells.
  • a method of concentrating and electrically treating biological cells includes the steps of : obtaining a cell fusion module which includes a cell concentration and fusion apparatus, placing the cell fusion module in a module manipulator unit, obtaining a mixture of the biological cells in a liquid carrier, introducing a portion of the cell/liquid mixture into the cell concentration and fusion apparatus 12, under control of the module manipulator unit 22, moving a portion of the cell/liquid mixture into a solid/liquid separation chamber in the cell concentration and fusion apparatus 12, in a first direction, towards an electrical treatment region in the cell concentration and fusion apparatus 12, separating a portion of the liquid in the solid/liquid separation chamber from the cell/liquid mixture in a second direction, which is perpendicular to the first direction, as the cell/liquid mixture moves toward the electrical treatment region in the first direction, where
  • the cell fusion module 20 can be considered disposable and can be disposed of in its entirety after the fused cells have been collected.
  • a tangential flow, cell concentration and fusion apparatus provides both unobstructed liquid flow and concentration of biological cells that are to undergo electrofusion.
  • a tangential flow, cell concentration and fusion apparatus provides an electroporation or electrofusion apparatus which employs tangential flow filtration.
  • a tangential flow, cell concentration and fusion apparatus is provided which can be contained in a sealed, sterile system.
  • a tangential flow, cell concentration and fusion apparatus which can be contained in a module which can be used, disposed of, and replaced in a simple and easy manner.
  • a tangential flow, cell concentration and fusion apparatus is provided which can employ a module manipulator that interacts with and operates a cell fusion module from outside the module without entering the module so that the sterility inside the module is not disturbed.

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  • Engineering & Computer Science (AREA)
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Abstract

L'invention porte sur un appareil (12) utilisé pour l'écoulement tangentiel, la concentration et la fusion des cellules et comprenant un corps (24) transparent constitué d'un premier canal (1) d'entrée. Dans le corps (24), une chambre (26) de concentration, comprenant une extrémité proximale raccordée au premier canal (1) d'entrée, s'étend le long d'un axe (28) longitudinal. Un premier canal (5) de sortie est raccordé à une extrémité distale de la chambre (26) de concentration. Un filtre (3), servant de paroi à la chambre (26) de concentration, est placé entre le premier canal (1) d'entrée et le premier canal (5) de sortie et s'étend dans un sens parallèle à l'axe (28) longitudinal.
EP00919388A 1999-03-22 2000-03-21 Appareil pour l'ecoulement tangentiel, la concentration et la fusion des cellules Withdrawn EP1137765A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US12558399P 1999-03-22 1999-03-22
US125583P 1999-03-22
PCT/US2000/006420 WO2000060065A1 (fr) 1999-03-22 2000-03-21 Appareil pour l'ecoulement tangentiel, la concentration et la fusion des cellules

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EP1137765A1 true EP1137765A1 (fr) 2001-10-04
EP1137765A4 EP1137765A4 (fr) 2002-09-18

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Publication number Priority date Publication date Assignee Title
EP1458846A4 (fr) * 2001-12-06 2005-02-09 Bio Rad Laboratories Insert de cuvette d'electroporation facilitant la fusion par membrane
EP1620936A2 (fr) * 2003-03-17 2006-02-01 Virginia Tech Intellectual Properties, Inc. Appareil et methode permettant d'eviter un inversement d'ecoulement dans la matiere de retour arriere de stator d'un srm biphase (tpsrm)
AU2003902363A0 (en) * 2003-05-15 2003-05-29 Life Therapeutics Limited Cell separation
EP2270130B1 (fr) 2005-06-13 2020-04-29 Tosoh Corporation Dispositif pour la fusion des cellules et procédé d'utilisation de celui-ci

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0208637A1 (fr) * 1985-06-19 1987-01-14 Centre National De La Recherche Scientifique (Cnrs) Procédé perfectionné de fusion cellulaire
US4832814A (en) * 1987-12-28 1989-05-23 E. I. Du Pont De Nemours And Company Electrofusion cell and method of making the same

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Publication number Priority date Publication date Assignee Title
US6010613A (en) * 1995-12-08 2000-01-04 Cyto Pulse Sciences, Inc. Method of treating materials with pulsed electrical fields

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0208637A1 (fr) * 1985-06-19 1987-01-14 Centre National De La Recherche Scientifique (Cnrs) Procédé perfectionné de fusion cellulaire
US4832814A (en) * 1987-12-28 1989-05-23 E. I. Du Pont De Nemours And Company Electrofusion cell and method of making the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO0060065A1 *

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WO2000060065A1 (fr) 2000-10-12
CA2374649A1 (fr) 2000-10-12

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