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WO2010110767A1 - Bioréacteur, kit et son procédé d'utilisation - Google Patents

Bioréacteur, kit et son procédé d'utilisation Download PDF

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Publication number
WO2010110767A1
WO2010110767A1 PCT/US2009/001798 US2009001798W WO2010110767A1 WO 2010110767 A1 WO2010110767 A1 WO 2010110767A1 US 2009001798 W US2009001798 W US 2009001798W WO 2010110767 A1 WO2010110767 A1 WO 2010110767A1
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WO
WIPO (PCT)
Prior art keywords
magnetic field
bioreactor
controlling circuit
field emitter
cell
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.)
Ceased
Application number
PCT/US2009/001798
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English (en)
Inventor
Robert G. Dennis
David A. Wolf
Donnie Rudd
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.)
Regenetech Inc
Original Assignee
Regenetech Inc
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 Regenetech Inc filed Critical Regenetech Inc
Priority to PCT/US2009/001798 priority Critical patent/WO2010110767A1/fr
Publication of WO2010110767A1 publication Critical patent/WO2010110767A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C12M35/06—Magnetic means
    • 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 to cell and tissue culture systems, more particularly to a bioreactor, a kit and a method of using the bioreactor for use in regenerating mammalian cells, and a method of using the bioreactor for growing and/or culturing a cell.
  • Some methods involve culturing cells in two-dimensional cultures utilizing such culture chambers as flasks and petri dishes, and others involve culturing cells in three-dimensional cultures utilizing such culture chambers as bioreactors.
  • Methods of optimally culturing cells include adding molecules to cells in a culture such as growth factors, hormones, and others that, for instance, up or down regulate expansion of cells.
  • Some methods are optimized for culturing individual cells and others are optimized for tissue culture.
  • cell cultures are performed under various conditions.
  • This invention relates to a new and novel device, kit and method of using the device to improve the regeneration of cells, cell aggregates, tissue, and tissue like structures, by the induction of a sequence of electromagnetic pulses with a mandatory relaxation period between the pulses.
  • the period of time during which the electromagnetic energy is induced, or present, in the area of interest, usually an area in need of regeneration and growth is herein referred to as the "active" period.
  • the period of time between the active periods is referred to as the “relaxation period” also called an “inactive period.”
  • There may be a transition period between the active and relaxation periods usually required because the apparatus and real target system does not have an instantaneous response nor is it always advisable to induce such rapid response into the target area.
  • the preferred embodiment utilizes an extremely short active period, 200 microseconds, during which an electromagnetic field is induced over the area of a broken bone or other tissue to be regenerated.
  • the relaxation period is preferably 100 milliseconds. The relaxation period thus can occupy 99.8% of the time. It is thought that this long relaxation period is important because during this time the tissues, surrounding interstitial components, fluids, soluble ions, and other species are able to function in a normal manner not under the influence of the electromagnetic field or pulse.
  • Pulse-relaxation events presumably act upon molecules and charged species directly, and the time scale of the change in the electromagnetic field is such that it corresponds to the time constants for molecular events such as ion diffusion across membranes, ligand binding and release events, altering molecule associations, and protein folding (nano-seconds to micro-seconds).
  • the present bioreactor, kit and method of using, according to the principles of the present invention overcomes a number of the shortcomings of the prior art by providing a novel bioreactor, kit and method for use in promoting the growth and/or culture.
  • the bioreactor includes a controlling circuit coupled to magnetic field emitter that emits relatively steep and short-lived magnetic field pulses during these an active ephemeral period, and a culture container located within the magnetic field generated by the magnetic field emitter.
  • the bioreactor also provides a relatively long-term inactive phase in which no magnetic field pulses are imposed. It is thought that both these steep short-lived magnetic field pulses and the inactive periods play important roles in the growth and/or culture of cells.
  • the kit includes the unassembled components of the bioreactor.
  • the method of growing and/or culturing cells includes the step of applying a time variant magnetic field through the cell to promote growth and/or culture.
  • the present invention essentially comprises a controlling circuit coupled to magnetic field emitter that emits relatively steep and short-lived magnetic field pulses during these an active ephemeral period in which the bioreactor also provides a relatively long-term inactive phase in which no magnetic field pulses are imposed.
  • the invention may also include an optional power supply.
  • An even further aspect of the present invention is to provide a bioreactor that has a low cost of manufacture with regard to both materials and labor, and which accordingly is then susceptible of low prices of sale to the consuming public, thereby making the bioreactor economically available to the buying public.
  • FIG. 1 depicts a schematic view of a preferred embodiment of the bioreactor constructed in accordance with the principles of the present invention
  • FIG. 2 depicts a perspective view of a preferred embodiment of the bioreactor
  • FIGS. 3A, 3B, 3C and 3D depict a number of different embodiment configurations of the bioreactor
  • FIGS. 4A, 4B, 4C, 4D, 4E and 4F depict a number of different electronic schemes of how the bioreactor can be configured
  • FIGS. 5A, 5B, and 5C depict a number of electromagnetic physical characteristics experienced by the magnetic field emitter during active and inactive periods
  • FIGS. 6A, 6B, 6C, 6D, 6E, 6F, and 6G depict a number of embodiments of showing different magnetic field output patterns as a function of time;
  • FIGS. 8 depicts the bioreactor internally mounted to promote growing and/or culturing of a cell
  • FIG. 9 is a schematic of the electronic circuit utilized to drive the time variant magnetic field generated by the coil magnetic field emitter.
  • a bioreactor 10 for promoting the growth and/or culture of a cell comprises a controlling circuit 14, a magnetic field emitter 18, and a culture containment device 17.
  • the controlling circuit 14 is configured to be powered by a power source 16 and is configured to output an electric pulse train.
  • the electric pulse train outputted from the controlling circuit 14 comprises an output current, an electrical cycle period, an electrical active and inactive period, a peak voltage amplitude, and a peak current amplitude.
  • the magnetic field emitter 18 electrically coupled to the controlling circuit 14 is configured to provide a time variant magnetic field when driven by the electric pulse train of the controlling circuit 14.
  • the magnetic field emitter 18 electrically coupled to the controlling circuit 14 is configured to provide a time variant magnetic field when driven by the electric pulse train of the controlling circuit 14.
  • the magnetic field emitter 18 that is electrically coupled to the controlling circuit 14 is configured to provide a time variant magnetic field comprising a magnetic (B) field exhibiting a magnetic slew rate of at least about 10 kiloGauss/sec when driven by the electric pulse train from the controlling circuit 14.
  • the bioreactor 10 is subject to almost any infinite number of design variations as long as the bioreactor 10 can produce a magnetic slew rate (either rising or falling, or both rising and falling) of at least about 10 kiloGauss/sec.
  • a magnetic slew rate either rising or falling, or both rising and falling
  • the magnetic field of the time variant magnetic field can be configured to exhibit a slew rate (either rising or falling, or both rising and falling) being between about 25 to about 1000 kiloGauss/sec.
  • the magnetic field of the time variant magnetic field can be configured to exhibit a magnetic cycle period between about 0.01-1000 Hertz.
  • the magnetic field of the time variant magnetic field can be configured to exhibit a magnetic field active duty between about 0.01 to 50 (preferably 0.01 to 2) percent of the cycle period wherein the magnetic active field duty defined as when the magnetic field emitter 18 emits the magnetic field. Still yet another variation is that the magnetic field of the time variant magnetic field can be configured to exhibit a magnetic inactive duty being between about 50 to 99.99 (preferably 98 to 99.99) percent of the cycle period wherein the magnetic field inactive duty defined as when the magnetic field emitter 18 does not emit the magnetic field. Even yet another variation is that the magnetic field of the time variant magnetic field can be configured to exhibit a peak magnetic amplitude being between about -20 to +20 Gauss.
  • controlling circuit 14 is configured to exhibit an electrical current slew rate (either rising or falling, or both rising and falling) between about 10 to about 1000 Amperes/sec.
  • the output current of the electric pulse train outputted from the controlling circuit 14 can be configured to exhibit a falling slew rate being between about 10 to about 1000 Amperes/sec.
  • Yet another variation of the controlling circuit 14 is that it can be configured to the output the electric pulse train to exhibit an electrical cycle period being between about 0.01-100 Hertz.
  • the output current of the electric pulse train outputted from the controlling circuit 14 can be configured to exhibit an electrical active period between about 0.01 to 50 (preferably 0.01 to 2) percent of the electrical cycle period wherein the electrical active period defined as when the output current is outputted.
  • the output current of the electric pulse train outputted from the controlling circuit 14 can be configured to exhibit an electrical inactive period between 50 to 99.99 (preferably 98 to 99.99) percent of the electrical cycle period wherein the electrical inactive period defined as when the output current is not outputted.
  • the output current of the electric pulse train outputted from the controlling circuit 14 can be configured to exhibit a peak voltage amplitude being between about -5 to +5 Volts and to exhibit a peak current amplitude being between about -5 to +5 kiloAmps.
  • the electric pulse train of the controlling circuit 14 is that the output current can be configured to exhibit a rising electrical current slew rate between about 10 to about 1000 Amperes/sec and to exhibit a falling electrical slew rate being between about 10 to about 1000 Amperes/sec.
  • the electrical cycle period can be configured to be between about 0.01-100 Hertz.
  • controlling circuit 14 Yet another variation of the controlling circuit 14 is that the electrical active period can be configured to be between about 0.01 to 2 percent of the electrical cycle period and that the electrical inactive period can be configured to be between 98 to 99.99 percent of the electrical cycle period. Even yet another variation of the controlling circuit 14 is that the peak voltage amplitude can be configured to be between about 1 to 10 Volts. Still yet another variation of the controlling circuit 14 is that the peak current amplitude can be configured to be being between about 1 to 10 Amps.
  • the magnetic field emitter 18 of the bioreactor 10 may be made of any known is selected from the group consisting of a coil magnetic field emitter 18, a plurality of coil magnetic field emitters 18, an antenna magnetic field emitter, and a plurality of loop magnetic field emitters 18.
  • the magnetic field emitter 18 may exhibit any known inductance value.
  • the controlling circuit 14 of the bioreactor 10 may have an optional current switch 20 may be added to the controlling circuit 14 of the bioreactor 10 in which the optional current switch 20 is configured to control the output current of the electric pulse train outputted from the controlling circuit 14.
  • the controlling circuit 14 of the bioreactor 10 may have an optional cycle length switch 22 may be added to the controlling circuit 14 of the bioreactor 10 in which the optional cycle length switch 22 is configured to control the electrical cycle period of the electric pulse train outputted from the controlling circuit 14.
  • the controlling circuit 14 of the bioreactor 10 may even have an optional pulse direction switch 24 may be added to the controlling circuit 14 of the bioreactor 10 in which the optional pulse direction switch 24 is configured to control the peak voltage and current amplitudes of the electric pulse train outputted from the controlling circuit 14.
  • the controlling circuit 14 of the bioreactor 10 may also have an optional output mode switch 26 may be added to the controlling circuit 14 of the bioreactor 10 in which the output mode switch 26 is configured to control various patterns of the electric pulse train outputted from the controlling circuit 14. Alternating polarity, or other sequences with low net DC values over time may yield the advantage of not introducing or accumulating long term net electric or magnetic motive forces. In cases where such accumulated forces are desirable, the instrumentation may be adjusted to produce such, and in a degree found to optimize the tissue response.
  • controlling circuit 14 of the bioreactor 10 may also have an optional rising slew rate switch 28 may be added to the controlling circuit 14 of the bioreactor 10 in which the rising slew rate switch 28 is configured to control the output current rising slew rate of the electric pulse train outputted from the controlling circuit 14.
  • the controlling circuit 14 of the bioreactor 10 may have an optional falling slew rate switch 30 may be added to the controlling circuit 14 of the bioreactor 10 in which the falling slew rate switch 30 configured to control the output current falling slew rate of the electric pulse train outputted from the controlling circuit 14.
  • the "slew" or rate of change of the energizing signal may be constant or variable, and in practical terms, variability is accepted in most practically realizable implementations. Variability, such as "tapering" or "wave shaping” at inflection points and sharp signal transition points are introduced or accepted as advantage in target cell response or for practical circuitry implementation.
  • the bioreactor 10 may optionally comprise the power source 16 electrically coupled to the controlling circuit 14.
  • the optional power source 16 may be selected from the group consisting of a battery power source 16, a high capacity capacitor power source 16, and an electrical outlet power source 16.
  • the culture containment device 17 of the bioreactor 10 is in relation to the magnetic field emitter 18 such that, when in use a magnetic field is generated by the magnetic field emitter 18, the magnetic field affects the cells in the culture container.
  • the culture containment device 17 is located a preferred distance from the magnetic field emitter, including, but not limited to, within the magnetic field emitter which would be a distance of zero, such that in use, the magnetic field generated by the magnetic field emitter 18 reaches the interior portion of the culture containment device 17 which is adapted to receive cells.
  • a "culture containment device” it is intended to comprise any and all vessels adapted to receive and supporting a cell culture.
  • the culture containment device 17 may be a single culture container or more than one culture container, as preferred.
  • the culture containment device 17 may be any preferred configuration that can be functionally located within the magnetic field generated by the magnetic field emitter 18 when the bioreactor 10 is in use.
  • kits for a bioreactor 10 comprises a magnetic field emitter 18 coupled to a controlling circuit 14.
  • the magnetic field emitter 18 is configured to be electrically coupled to the controlling circuit 14 in which the magnetic field emitter 18 is configured to provide a time variant magnetic field when driven by the electric pulse train of the controlling circuit 14.
  • the time variant magnetic field comprises a magnetic (B) field exhibiting a magnetic slew rate of at least about 10 kiloGauss/sec.
  • the controlling circuit 14 may be configured to be powered by a power source 16 and is also configured to output an electric pulse train.
  • the controlling circuit 14 of the kit of the bioreactor 10 may optionally have a current switch 20 which is configured to control the electrical cycle period of the electric pulse train to output from the controlling circuit 14.
  • the controlling circuit 14 of the kit of the bioreactor 10 may also optionally have a cycle length switch 22 configured to control the electrical cycle period of the electric pulse train outputted from the controlling circuit 14.
  • the controlling circuit 14 of the kit of the bioreactor 10 may also optionally have a pulse direction switch 24 configured to control the peak voltage and current amplitudes of the electric pulse train outputted from the controlling circuit 14.
  • the controlling circuit 14 of the kit of the bioreactor 10 may also optionally have an output mode switch 26 configured to control various patterns of the electric pulse train outputted from the controlling circuit 14.
  • the controlling circuit 14 of the kit of the bioreactor 10 may also optionally have a rising slew rate switch 28 configured to control the output current rising slew rate of the electric pulse train outputted from the controlling circuit 14.
  • the controlling circuit 14 of the kit of the bioreactor 10 may also optionally have a falling slew rate switch 30 configured to control the output current falling slew rate of the electric pulse train outputted from the controlling circuit 14.
  • the magnetic field emitter 18 of the kit of the bioreactor 10 may be any known commercially available magnetic field emitter 18. Some preferred magnetic field emitters 18 may be selected from the group consisting of a coil magnetic field emitter 18, a plurality of coil magnetic field emitters 18, a plurality of loop magnetic field emitters 18, and an antenna magnetic field emitter 18.
  • the kit of the bioreactor 10 also comprises a culture containment device 17.
  • the culture containment device 17 is preferably configured to the intended test, result, and/or cell to be grown and/or cultured.
  • a preferred configuration of the culture containment device 17 is a rotating bioreactor, rotatable about a substantially horizontal longitudinal central axis.
  • Another preferred configuration of the culture containment device 17 is a petri dish, more preferably a flask, and most preferably a plate.
  • An optional power source 16 may be added to the kit of the bioreactor 10 in which the optional power source is configured to be electrically coupled to the controlling circuit 14.
  • the power source 16 of the kit of the bioreactor 10 may be any known power source 16 in which some preferred power sources 16 may be selected from the group consisting of a battery, a high capacity capacitor, and an electrical outlet.
  • An optional stabilizing agent 32 may be added to the kit of the bioreactor 10 in which the stabilizing agent 32 may be any known and commercially available stabilizing agent 32.
  • the stabilizing agent 32 is made from a non-conductive material.
  • the stabilizing agent provides support and positioning to the magnetic field emitter 18 such that, in use, the bioreactor 10 can function to provide a magnetic field in the interior portion of the culture containment device 17 and to the cells contained therein.
  • One preferred method for growing and/or culturing a cell comprises the step of applying a time variant magnetic field through the cell to promote the growth and/or culture of the cell.
  • growing and/or culturing and similar terms, it is intended that throughout this paper, either a cell is increased in number to more than one or more than the number that was present in the culture containment device before the bioreactor was in use (growth) and/or the cell is merely exposed to the time variant magnetic field generated by the magnetic field emitter 18, in which case the number of cells in the culture containment device may increase, decrease, or remain unchanged.
  • a combination of the growth and culture may also be preferred.
  • the desired outcome and final use of the cells will dictate the preferred method whether it be growing the cells in number, culturing the cells, or growing and/or culturing the cells. Therefore, by the term "desired level" it is intended that the cell, cell aggregate, tissue, and/or tissue like structure, by the method of the present invention grow and/or culture to a level that is most beneficial for the object of the culture. For instance, if in a preferred embodiment the object of the culture is to achieve a greater number cells Y than the original number before growing and/or culturing X, then as soon as the cells reach Y, then the desired level is reached.
  • the object of the culture is merely culturing the cells until certain membrane proteins are up regulated and others down regulated by a factor of X or Y, then once the membrane proteins are detected at that level, regardless of the number of cell, then the desired level has been reached.
  • RNA phenotype transcribed RNA patterns
  • Cell structural proteins may include cell membrane components and cell cytoskeletal components, and cell functional proteins may, among others, effect cell metabolism.
  • genes may be up regulated by the time variant magnetic field, and others may be down regulated.
  • cells cultured by this method may have heightened functionality.
  • cells cultured by this method produce significantly different cells with significantly distinct expressions from cells grown by any other method.
  • the growing and/or culturing a cell comprises the step of applying a time variant magnetic field through the cell
  • the applying step comprises a magnetic field having a slew rate, either a rising or a falling, or both a rising and falling) of at least 10 kiloGauss/sec.
  • an experiment was conducted with two cultures: a rotating bioreactor culture containing non-adherent peripheral blood CD34+ stem cells (already shown to produce expanded cells that are significantly different from cells grown under non-rotating conditions); and a rotating bioreactor culture containing cells exposed to a time variant magnetic field wherein the cells are from the same lot of non-adherent peripheral blood CD34+ stem cells as the culture without exposure to a time variant magnetic field.
  • AU conditions were the same except for the application of a time variant magnetic field to the latter culture.
  • the gauss range of this particular experiment was between about 1.0 gauss to about 1.2 gauss. This experiment is an example of the results that are expected across the entire range of the time variant magnetic field disclosed by the methods and embodiments of this invention.
  • metallothioneins are significantly upregulated in the time variant magnetic field expanded cells as compared to the non time variant magnetic field expanded cells, through their role in zinc metabolism play a role in controlling gene transcription levels, and some of those target genes include genes involved in proliferation and differentiation.
  • the data provides that the expression of certain HOX genes (homeobox containing genes) are also significantly upregulated under the time variant magnetic field conditions as compared to non- time variant magnetic field conditions.
  • the ability of certain HOX genes to enhance hematopoietic stem cell self-renewal, and to increase the number of engraftable hematopoietic stem cells is well known in the art.
  • the time variant magnetic field expanded cells may have enhanced stem cell renewal and engraftability compared to control cultures.
  • BNIP3 and BNIP3-like genes are also significantly upregulated in the time variant magnetic field expanded cells. It is well known in the art that BNIP3 and related genes are induced under conditions of hypoxia. Hypoxia can enhance the quantity and quality of certain stem cells from blood and other tissues. For instance, the clonogenic capacity of the cells can increase as well as the number of stem cells.
  • the time variant magnetic field expanded cells are further characterized by significantly decreased expression of genes involved in chromatin remodeling, including, for instance, HDAC3.
  • HDAC inhibitors like valproic acid and trichostatin A strongly enhance the expansion of primitive blood stem cells and improve the cell engraftment in animal models. Since time variant magnetic field exposed cultures appear to naturally down-regulate HDAC3, which is a biologically similar outcome to the addition of IIADC inhibitors into a culture, this suggests that the time variant magnetic field in this type of cell is inducing the beneficial effects of histone deacetylation on stem cell numbers and quality, but in a more targeted and specific manner. Modulating chromatin structure via HDACs and other genes should allow the time variant magnetic field expanded cells to modify stem cell fate and enhance the time variant magnetic field expanded stem cell expansion.
  • Angiopoeitin type 1 expression is also significantly elevated in the time variant magnetic field expanded cells versus non- time variant magnetic field expanded cells. It is known in the art that Angptl is a key component of the blood stem cell niche, namely of the binding between the bone and blood cells. It is also thought that Angiopoietin 1 helps blood stem cells maintain their stem-like character by controlling cell division and promoting survival. This suggests that the effect of increased expression of Angiopoietin 1 in the time variant magnetic field cultures may affect the ability of these cells to bind to the hematopoietic stem cell niche in the bone.
  • the differences between the two culture conditions includes transcription levels for RNA's coding for differently expressed proteins such as cell surface proteins, proteins involved in the cell replication process, growth factors and components of the cellular transcriptional control machinery.
  • the applying step may last for any known length of time in which one preferred embodiment is that the applying step is applied for a duration of at least one week without interruption. Another preferred embodiment is that the applying step lasts for a duration of at least one week and is performed at least 8 hours in each day during the duration of the applying step.
  • the time variant magnetic field may be applied in any known direction.
  • One preferred embodiment is that the time variant magnetic field is always applied along a substantially identical direction during the applying step, whereby the time variant magnetic field being a unidirectional time variant magnetic field.
  • Another preferred embodiment is that the time variant magnetic field is alternately applied along substantially alternate opposite directions during the applying step, whereby the time variant magnetic field being an alternating bi-directional time variant magnetic field.
  • time variant magnetic field is applied using a current pulse train through a magnetic field emitter generated by a circuit.
  • the time variant magnetic field comprises the active duty is between about 0.1 to about 1 percent of the cycle period; the rising edge magnetic slew of at least about 10 kiloGauss/sec; and the falling edge magnetic slew rate being of at least about 10 kiloGauss/sec.
  • One preferred embodiment of the electric pulse train comprises an output current exhibiting a rising slew rate between of at least 1 Amperes/sec; the output current exhibiting a falling slew rate of at least about 1 Amperes/sec; an electrical cycle period being at least about 0.01 Hertz; an electrical active periodicity may be any function, such as being between about 0.01 to 2 percent of the electrical cycle period wherein the electrical active period defined as when the output current is outputted; an electrical inactive period between 98 to 99.99 percent of the electrical cycle period wherein the electrical inactive period defined as when the output current is not outputted; a peak voltage amplitude being between about -5 to +5 Volts; and a peak current amplitude being between about -5 to +5 kiloAmps.
  • the present method is suitable for promoting the growing and/or culturing of a cell, preferably an animal cell, more preferably a mammalian cell.
  • the mammalian cell is selected from the group consisting of human, goat, sheep, domesticated dog, domesticated cat, a rat, mouse, guinea pig, rabbit, horse, cow, llama, alpaca, mule, donkey, gorilla, gibbon, orangutan, chimpanzee, lemur, rhinoceros, monkey, bat, bison, camel, wolf, coyote, fox, jackal, tiger, oryx, water buffalo, elephant, giraffe, antelope, deer, elk, lion, cheetah, panda, leopard, puma, serval, opossum, kangaroo, platypus, armadillo, lemur, muskox, baboon, zebra, pig
  • the present method is suitable for regenerating and/or improving the function(s) of cells in mammals selected from the group consisting of a human, a goat, a sheep, a domesticated dog, a domesticated cat, a rat, a mouse, a guinea pig, a rabbit, a horse, a cow, a llama, an alpaca, a mule, a donkey, a gorilla, a chimpanzee, a lemur, a rhinoceros, a monkey, a bat, a bison, a camel, a wolf, a coyote, a fox, a jackal, tiger, an oryx, a water buffalo, a elephant, a giraffe, an antelope, a deer, an elk, a lion, a cheetah, a panda, a leopard, a puma, a serval, an
  • Regenerating and/or improving the function of mammalian cells can be done either by introduction of these cells to an autologous or allogeneic source.
  • This invention also contemplates the regeneration and/or improvement of the function of mammalian cells ex vivo or in vitro.
  • cells that are grown and/or cultured by the methods of the present invention are used to prepare a medicament for regenerating and/or improving the function of mammalian cells, whether in vivo or ex vivo.
  • cells that are grown and/or cultured by the methods of the present invention are used for the treatment of mammalian disease whereby mammalian cells are regenerated or improved in function, either in vivo or ex vivo (including in vitro).
  • An optional aligning step may be added to the method in which the aligning step is used to align the cell in a desired orientation.
  • An optional stabilizing step may be added to the method in which the stabilizing step is used to stabilize the magnetic field emitter 18 by providing support with a stabilizing agent 32.
  • An optional mounting step may be added to the method in which the mounting step is used to mount a magnetic field emitter 18 near a culture containment device 17.
  • the mounting step of the magnetic field emitter 18 may be performed in any known manner such as being mounted external relative to the culture containment device 17, or may be mounted such that the culture containment device 17 in contained within the magnetic field emitter 18. If the magnetic field emitter 18 is mounted external to the culture containment device 17 it is considered to be "adjacent to" the culture containment device 17. By the term “adjacent” it is meant that, when in use, the magnetic field emitter 18 applies a magnetic field to the cells in the culture containment device 17.
  • the magnetic field emitter 18 contains the culture containment device 17 within, it is referred to by the term "around" wherein it is intended that the magnetic field emitter 18 encompass the culture containment device 17, so that in use, the cells in the culture containment device 17 are exposed to a magnetic field generated by the magnetic field emitter, preferably a uniform magnetic field, preferably uniformly exposed.
  • An optional turning off step may be added to the method in which the turning off step is used to turn off the time variant magnetic field after a substantial amount of growing and/or culturing has occurred.
  • An optional withdrawing step may be added to the method in which the withdrawing step is used to withdraw the magnetic field emitter 18 away from the culture containment device 17 subsequent to when the growing and/or culturing of the cells has occurred.
  • the stabilizing agent 32 may be any known stabilizing agent 32.
  • the stabilizing agent 32 may preferably be a device for supporting the magnetic field emitter in a preferred configuration.
  • the stabilizing agent 32 may preferably provide support to the magnetic field emitter, and may also preferably position the magnetic field emitter 18 a distance from the cell to be grown and/or cultured, preferably in a culture containment device 17.
  • the distance may preferably be zero if the culture containment device 17 is within the magnetic field emitter 18.
  • the distance from the culture containment device 17 is such that when in use, the culture containment device 17 is functionally located within the magnetic field generated by the magnetic field emitter 18.
  • the culture containment device 17 is located adjacent to the magnetic field emitter 18.
  • the magnetic field emitter 18 is functionally located adjacent to the culture containment device 17, wherein in use, the magnetic field generated by the magnetic field emitter 18 is applied to the cell in the culture containment device 17.
  • FIG. 1 depicts a schematic view of a preferred embodiment of the bioreactor 10 showing the optional power supply 16 electrically coupled to the controlling circuit 14, and the culture containment device 17.
  • the controlling circuit 14 is shown having the optional current switch 20, the cycle length switch 22, the pulse direction switch 24, the output mode switch 26, the rising slew rate switch 28, the falling slew rate switch 30. Also shown is the magnetic field emitter 18 electrically coupled to the controlling circuit 14.
  • FIG. 2 depicts a perspective view of a preferred embodiment of the bioreactor 10 showing the assembly of an optional power supply 16 and the magnetic field emitter 18 electrically coupled to the controlling circuit 14, to a culture containment device 17 which, upon assembly, will be functionally located within the magnetic field emitter 18.
  • Figure 2 also depicts a stabilizing agent 32.
  • the stabilizing agent 32 provides support to the magnetic field emitter 18 such that, in use, it remains in relation to the culture containment device 17 so that a magnetic field is generated within the culture containment device 17 and applied to the cell therein.
  • FIGS. 3A, 3B, and 3C depict a number of different embodiments of the bioreactor 10.
  • the bioreactor 10 is shown having any number of different designs or configurations.
  • FIG. 3A illustrates one preferred configuration of the controlling circuit 14 which is coupled to only one coil magnetic field emitter 18 and is powered by only one power supply 16, and a culture containment device 17, preferably a flask.
  • FIG. 3B illustrates another preferred configuration of the controlling circuit 14 which is coupled to a plurality of loop magnetic field emitters 18 and is powered by only one power supply 16, and a culture containment device 17 is preferably a rotatable bioreactor.
  • FIG. 3B also depicts a stabilizing agent 32.
  • FIG. 3C illustrates yet another preferred configuration of the controlling circuit 14 which is coupled to a plurality of coil magnetic field emitters 18 and is powered by only one power supply 16, and a culture containment device 17, preferably a petri dish.
  • FIG. 3D illustrates still yet another preferred configuration of the controlling circuit 14 which is coupled to a plurality of coil magnetic field emitters 18 and is coupled to a plurality of power supplies 16, and a culture containment device 17, preferably a plate.
  • each power supply 16 is shown configured via the controlling circuit 14 to individually drive only a single corresponding coil magnetic field emitters 18.
  • a bioreactor 10 that is preferably a rotatable bioreactor is rotatable about a substantially horizontal axis, 360° in one direction, so that in use, the cells are suspended in an essentially quiescent three-dimensional environment that provides for low shear stress and turbulence.
  • Such a three-dimensional environment also provides that cells grown and/or cultured therein have a unique phenotypic expression due to the cells adaptation to the environment in the rotatable bioreactor.
  • the rotation of a rotatable bioreactor provides a stabilized culture environment into which cells may be introduced, suspended, maintained, grown, cultured and/or expanded with improved retention of delicate three-dimensional structural integrity by simultaneously minimizing the fluid shear stress, providing three-dimensional freedom for cell and substrate spatial orientation, and increasing localization of cells in a particular spatial region for the duration of the expansion. It is expected that rotation along with exposure to a time variant magnetic field provides the cells that are grown and/or cultured in the bioreactor 10 of the present invention with a genetic expression that is unique.
  • FIGS. 4A, 4B, 4C, 4D, 4E, and 4F depict a number of different electronic schemes of how the bioreactor 10 can be configured. These electronic schemes are depicted to illustrate just a few of the infinite number of electronic configurations of the bioreactor 10 as long as each can realize the invention as described in the claims.
  • FIGS. 5A, 5B, and 5C depict a number of electromagnetic physical characteristics experienced by the magnetic field emitter 18 during active and inactive periods.
  • FIG. 5 A depicts a voltage step function across the magnetic field emitter 18 showing an almost instantaneous potential jump between two potential states (i.e., on state and off state).
  • FIG 5B depicts a current step function across the magnetic field emitter 18 showing an out of phase or delayed current, relative to the voltage step function, through the magnetic field emitter 18.
  • FIG. 5C depicts a magnetic field step function emitted from the magnetic field emitter 18 showing an out of phase or delayed magnetic field, relative to the voltage step function, in which the magnetic field step function is approximately in phase with the current step function.
  • FIGS. 6 A, 6B, 6C, 6D, 6E, 6F, and 6G depict a number of embodiments of showing different magnetic field output patterns as a function of time.
  • the magnetic field emitter 18 is envisioned to be capable of producing any number of different patterns or modes of the resultant magnetic field along with being capable of producing alternating directional magnetic fields.
  • one embodiment of the bioreactor 10 provides that the magnetic field emitter 18 driven by the controlling circuit 14 can be configured to produce a unidirectional magnetic field for a short time period (i.e., during the active mode) and afterwards remain quiescent(i.e., the inactive mode) until the end of the cycle period.
  • FIG 6B another embodiment of the bioreactor 10 provides that the magnetic field emitter 18 driven by the controlling circuit 14 can be configured to produce alternately produce magnetic field pulses in opposite directions.
  • the bioreactor 10 is envisioned to be capable of producing the various magnetic field pulse patterns as depicted in FIGS. 6A, 6B, 6C, 6D, 6E, 6F, and 6G. which are illustrative and not limited to the infinite number of magnetic field pulse patterns that the bioreactor 10 is envisioned to be capable of producing.
  • FIGS. 7A, 7B, 7C, 7D, 7E, and 7F depict various ways the bioreactor 10 can be externally mounted to promote growing and/or culturing of a cell.
  • Each figure, (i.e., FIGS. 7A, 7B, 7C, 7D, 7E, and 7F) shows the controlling circuit 14 operationally coupled to the optional power source 16 and operationally coupled to at least one magnetic field emitter 18.
  • FIG. 7B depicts that the magnetic field emitter 18 can be mounted within a stabilizing agent 32.
  • FIG 8 shows the controlling circuit 14 operationally coupled to the optional power source 16 and operationally coupled to a coil magnetic field emitter 18 with a culture containment device 17 functionally located within the magnetic field emitter 18. Also shown in FIG. 8 is the stabilizing agent 32 .

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Abstract

L'invention concerne un bioréacteur, un kit et un procédé d'utilisation du dispositif permettant de favoriser la croissance et/ou la culture d'une cellule, et un procédé permettant de régénérer et/ou améliorer la fonction des cellules de mammifères. Le bioréacteur inclut un circuit de commande couplé à un émetteur de champ magnétique qui émet des impulsions de champ magnétique de durée de vie courte et relativement abruptes pendant une phase active éphémère. Le bioréacteur assure également une phase inactive de durée relativement longue dans laquelle aucune impulsion de champ magnétique n'est imposée. Le kit inclut les composants démontés du bioréacteur. Le procédé d'utilisation du bioréacteur et de régénération et/ou d'amélioration de la fonction des cellules de mammifères incluent les étapes consistant à appliquer un champ magnétique variable dans le temps à travers la cellule afin de favoriser sa croissance et/ou sa culture, puis à introduire les cellules dans le corps d'un mammifère pour régénérer les cellules.
PCT/US2009/001798 2009-03-23 2009-03-23 Bioréacteur, kit et son procédé d'utilisation Ceased WO2010110767A1 (fr)

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PCT/US2009/001798 WO2010110767A1 (fr) 2009-03-23 2009-03-23 Bioréacteur, kit et son procédé d'utilisation

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PCT/US2009/001798 WO2010110767A1 (fr) 2009-03-23 2009-03-23 Bioréacteur, kit et son procédé d'utilisation

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020009797A1 (en) * 2000-06-02 2002-01-24 National Aeronautics & Space Administration Growth stimulation of biological cells and tissue by electromagnetic fields and uses thereof
DE10034750A1 (de) * 2000-07-18 2002-02-07 Graviton Gmbh Verfahren und Vorrichtung zur computergesteuerten elektromagnetischen Beeinflussung der Kinetik von organischen und anorganischen Prozessen in Flüssigkeiten
WO2004011631A2 (fr) * 2002-07-26 2004-02-05 Ebi, L.P. Procedes et compositions pour traiter des anomalies tissulaires au moyen d'une excitation par champ electromagnetique pulse
US20050009161A1 (en) * 2002-11-01 2005-01-13 Jackson Streeter Enhancement of in vitro culture or vaccine production using electromagnetic energy treatment
US20060024822A1 (en) * 2004-07-27 2006-02-02 Chang Walter H System and method for cultivating cells
WO2009025722A1 (fr) * 2007-08-20 2009-02-26 Regenetech, Inc. Procédé et composition pour réparer un tissu cardiaque

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020009797A1 (en) * 2000-06-02 2002-01-24 National Aeronautics & Space Administration Growth stimulation of biological cells and tissue by electromagnetic fields and uses thereof
DE10034750A1 (de) * 2000-07-18 2002-02-07 Graviton Gmbh Verfahren und Vorrichtung zur computergesteuerten elektromagnetischen Beeinflussung der Kinetik von organischen und anorganischen Prozessen in Flüssigkeiten
WO2004011631A2 (fr) * 2002-07-26 2004-02-05 Ebi, L.P. Procedes et compositions pour traiter des anomalies tissulaires au moyen d'une excitation par champ electromagnetique pulse
US20050009161A1 (en) * 2002-11-01 2005-01-13 Jackson Streeter Enhancement of in vitro culture or vaccine production using electromagnetic energy treatment
US20060024822A1 (en) * 2004-07-27 2006-02-02 Chang Walter H System and method for cultivating cells
WO2009025722A1 (fr) * 2007-08-20 2009-02-26 Regenetech, Inc. Procédé et composition pour réparer un tissu cardiaque

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