# US 2021/0340520 A1 · Methods for stimulating the proliferation and differentiation of eukaryotic cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2021/0340520 A1 - Document ID: US20210340520A1 - Status: Published application - Kind: A1 - Application number: 17/369,271 - Filed: 2021-07-07 - Published: 2021-11-04 - Earliest claimed: 2019-01-10 - Assignees: Nanovis, LLC; Purdue Research Foundation - Applicants: Nanovis, LLC; Purdue Research Foundation - Inventors: Allen L. GARNER; Ram Anand VADLAMANI; David Alan DETWILER; Aginiprakash DHANABAL - Web page: https://patentgrep.com/patent/US20210340520A1 ## CPC - C12N 13/00: Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves - A61K 35/32: Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane - A61K 35/34: Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44) - A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca - A61N 1/326: {for promoting growth of cells, e.g. bone cells} - A61N 1/3616: {Voltage density or current density} - A61N 1/36171: {Frequency} - C12M 35/02: {Electrical or electromagnetic means, e.g. for electroporation or for cell fusion} - C12N 5/0654: {Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth} - C12N 5/0658: {Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts} - A61N 1/327: {for enhancing the absorption properties of tissue, e.g. by electroporation} - A61N 1/36: for stimulation - A61N 1/36034: {specified by the stimulation parameters} - C12N 2529/00: Culture process characterised by the use of electromagnetic stimulation - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61N: ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY (measurement of bioelectric currents A61B; surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body A61B18/00; anaesthetic apparatus in general A61M; incandescent lamps H01K; infrared radiators for heating H05B) - 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} ## Abstract The present disclosure relates to methods of stimulating cell proliferation, promoting differentiation of cells, regenerating cells, promoting nodule formation, and promoting myotube formation. The methods include applying one or more pulses of electricity to cells, each pulse of electricity having a duration of between about 10 nanoseconds and about 1,000 nanoseconds, wherein said pulses of electricity are applied under conditions effective to stimulate cell proliferation, promote differentiation of cells, regenerate cells, promote nodule formation, and promote myotube formation. ## Claims ### Claim 1 A method of stimulating cell proliferation, said method comprising: applying one or more pulses of electricity to cells, each pulse of electricity having a duration of between about 10 nanoseconds and about 1,000 nanoseconds, wherein said pulses of electricity are applied under conditions effective to stimulate cell proliferation. ### Claim 2 (depends on 1) The method of claim 1, wherein each pulse of electricity has a duration of between about 10 nanoseconds and about 300 nanoseconds. ### Claim 3 (depends on 1) The method of claim 1, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.01 Hz to about 1,000 Hz. ### Claim 4 (depends on 3) The method of claim 3, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.1 Hz to about 300 Hz. ### Claim 5 (depends on 4) The method of claim 4, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.5 Hz to about 10 Hz. ### Claim 6 (depends on 1) The method of claim 1, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm to about 30.0 kV/cm. ### Claim 7 (depends on 1) The method of claim 1, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm and about 25.0 kV/cm. ### Claim 8 (depends on 7) The method of claim 7, wherein each pulse of electricity has an intensity peak in a range of about 5.0 kV/cm and about 10.0 kV/cm. ### Claim 9 (depends on 1) The method of claim 1, wherein each pulse of electricity has an intensity peak of about 1.0 kV/cm. ### Claim 10 (depends on 1) The method of claim 1, wherein each pulse of electricity has an intensity peak in a range of about 2.5 kV/cm to about 25.0 kV/cm. ### Claim 11 (depends on 1) The method of claim 1, wherein a 30 nanosecond rise and fall time is present between a peak intensity and a baseline intensity. ### Claim 12 (depends on 1) The method of claim 1, wherein a time between rise and fall times of a peak intensity and a baseline intensity is less than about 10 nanoseconds. ### Claim 13 (depends on 1) The method of claim 1, wherein said electrical pulses are applied to said cells in vivo. ### Claim 14 (depends on 1) The method of claim 1, wherein said electrical pulses are applied to said cells in vitro. ### Claim 15 (depends on 14) The method of claim 14 further comprising: inserting said cells into a subject following applying of said one or more electrical pulses. ### Claim 16 (depends on 1) The method of claim 1, wherein said cells are selected from the group consisting of stem cells, satellite cells, myoblasts, osteoblasts, chondrocytes, fibroblasts, tenocytes, precursor cells, embryological cells, progenitor cells, mesenchymal stem cells, neural stem cells, glial progenitor cells, angioblast hematopoietic stem cells, induced pluripotent stem cells, allograft stem cells, and xenograft stem cells. ### Claim 17 (depends on 1) The method of claim 1, wherein said cells are subject to up to 150 pulses of electricity. ### Claim 18 (depends on 17) The method of claim 17, wherein said cells are subject to five or fewer pulses of electricity. ### Claim 19 (depends on 1) The method of claim 1 further comprising: administering an additional agent. ### Claim 20 (depends on 19) The method of claim 19, wherein said additional agent is selected from group consisting of an antibiotic compound, an antimicrobial compound, an antibody, a biocidal agent, nanoparticles, self-assembling nanoparticles, viral particles, bacteriophage particles, bacteriophage DNA, genetic material, chemotherapy agent, growth factor, synthetic scaffold, natural scaffold, electrode, drug, a microbe, and a bacteria. ### Claim 21 A method of promoting differentiation of cells, said method comprising: applying one or more pulses of electricity to cells, each pulse of electricity having a duration of between about 10 nanoseconds and about 1,000 nanoseconds, wherein said pulses of electricity are applied under conditions effective to promote differentiation of cells. ### Claim 22 (depends on 21) The method of claim 21, wherein each pulse of electricity has a duration of between about 10 nanoseconds and about 300 nanoseconds. ### Claim 23 (depends on 21) The method of claim 21, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.01 Hz to about 1,000 Hz. ### Claim 24 (depends on 23) The method of claim 23, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.1 Hz to about 300 Hz. ### Claim 25 (depends on 24) The method of claim 24, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.5 Hz to about 10 Hz. ### Claim 26 (depends on 21) The method of claim 21, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm to about 30.0 kV/cm. ### Claim 27 (depends on 21) The method of claim 21, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm and about 25.0 kV/cm. ### Claim 28 (depends on 27) The method of claim 27, wherein each pulse of electricity has an intensity peak in a range of about 5.0 kV/cm and about 10.0 kV/cm. ### Claim 29 (depends on 21) The method of claim 21, wherein each pulse of electricity has an intensity peak of about 1.0 kV/cm. ### Claim 30 (depends on 21) The method of claim 21, wherein each pulse of electricity has an intensity peak in a range of about 2.5 kV/cm to about 25.0 kV/cm. ### Claim 31 (depends on 21) The method of claim 21, wherein a 30 nanosecond rise and fall time is present between a peak intensity and a baseline intensity. ### Claim 32 (depends on 21) The method of claim 21, wherein a time between rise and fall times of a peak intensity and a baseline intensity is less than about 10 nanoseconds. ### Claim 33 (depends on 21) The method of claim 21, wherein said electric pulses are applied to said cells in vivo. ### Claim 34 (depends on 21) The method of claim 21, wherein said electrical pulses are applied to said cells in vitro. ### Claim 35 (depends on 34) The method of claim 34 further comprising: inserting said cells into a subject following applying of said one or more electrical pulses. ### Claim 36 (depends on 21) The method of claim 21, wherein said cells are selected from the group consisting of stem cells, satellite cells, myoblasts, osteoblasts, chondrocytes, fibroblasts, tenocytes, precursor cells, embryological cells, progenitor cells, mesenchymal stem cells, neural stem cells, glial progenitor cells, angioblast hematopoietic stem cells, induced pluripotent stem cells, allograft stem cells, and xenograft stem cells. ### Claim 37 (depends on 21) The method of claim 21, wherein said cells are subject to up to 150 pulses of electricity. ### Claim 38 (depends on 37) The method of claim 37, wherein said cells are subject to five or fewer pulses of electricity. ### Claim 39 (depends on 21) The method of claim 21 further comprising: administering an additional agent. ### Claim 40 (depends on 39) The method of claim 39, wherein said additional agent is selected from group consisting of an antibiotic compound, an antimicrobial compound, an antibody, a biocidal agent, nanoparticles, self-assembling nanoparticles, viral particles, bacteriophage particles, bacteriophage DNA, genetic material, chemotherapy agent, growth factor, synthetic scaffold, natural scaffold, electrode, drug, a microbe, and a bacteria. ### Claim 41 A method of regenerating cells, said method comprising: applying one or more pulses of electricity to cells, each pulse of electricity having a duration of between about 10 nanoseconds and about 1,000 nanoseconds, wherein said pulses of electricity are applied under conditions effective to promote cell regeneration. ### Claim 42 (depends on 41) The method of claim 41, wherein each pulse of electricity has a duration of between about 10 nanoseconds and about 300 nanoseconds. ### Claim 43 (depends on 41) The method of claim 41, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.01 Hz to about 1,000 Hz. ### Claim 44 (depends on 43) The method of claim 43, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.1 Hz to about 300 Hz. ### Claim 45 (depends on 44) The method of claim 44, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.5 Hz to about 10 Hz. ### Claim 46 (depends on 41) The method of claim 41, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm to about 30.0 kV/cm. ### Claim 47 (depends on 44) The method of claim 44, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm and about 25.0 kV/cm. ### Claim 48 (depends on 47) The method of claim 47, wherein each pulse of electricity has an intensity peak in a range of about 5.0 kV/cm and about 10.0 kV/cm. ### Claim 49 (depends on 41) The method of claim 41, wherein each pulse of electricity has an intensity peak of about 1.0 kV/cm. ### Claim 50 (depends on 41) The method of claim 41, wherein each pulse of electricity has an intensity peak in a range of about 2.5 kV/cm to about 25.0 kV/cm. ### Claim 51 (depends on 41) The method of claim 41, wherein a 30 nanosecond rise and fall time is present between a peak intensity and a baseline intensity. ### Claim 52 (depends on 41) The method of claim 41, wherein a time between rise and fall times of a peak intensity and a baseline intensity is less than about 10 nanoseconds. ### Claim 53 (depends on 41) The method of claim 41, wherein said electric pulses are applied to said cells in vivo. ### Claim 54 (depends on 41) The method of claim 41, wherein said electrical pulses are applied to said cells in vitro. ### Claim 55 (depends on 54) The method of claim 54 further comprising: inserting said cells into said subject following applying of said one or more electrical pulses. ### Claim 56 (depends on 41) The method of claim 41, wherein said cells are selected from the group consisting of stem cells, satellite cells, myoblasts, osteoblasts, chondrocytes, fibroblasts, tenocytes, precursor cells, embryological cells, progenitor cells, mesenchymal stem cells, neural stem cells, glial progenitor cells, angioblast hematopoietic stem cells, induced pluripotent stem cells, allograft stem cells, and xenograft stem cells. ### Claim 57 (depends on 41) The method of claim 41, wherein said cells are subject to up to 150 pulses of electricity. ### Claim 58 (depends on 57) The method of claim 57, wherein said cells are subject to five or fewer pulses of electricity. ### Claim 59 (depends on 41) The method of claim 41 further comprising: administering an additional agent. ### Claim 60 (depends on 59) The method of claim 59, wherein said additional agent is selected from group consisting of an antibiotic compound, an antimicrobial compound, an antibody, a biocidal agent, nanoparticles, self-assembling nanoparticles, viral particles, bacteriophage particles, bacteriophage DNA, genetic material, chemotherapy agent, growth factor, synthetic scaffold, natural scaffold, electrode, drug, a microbe, and a bacteria. ### Claim 61 A method of promoting nodule formation, said method comprising: applying one or more pulses of electricity to cells, each pulse of electricity having a duration of between about 10 nanoseconds and about 1,000 nanoseconds, wherein said pulses of electricity are applied under conditions effective to promote nodule formation. ### Claim 62 (depends on 61) The method of claim 61, wherein each pulse of electricity has a duration of between about 10 nanoseconds and about 300 nanoseconds. ### Claim 63 (depends on 61) The method of claim 61, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.01 Hz to about 1,000 Hz. ### Claim 64 (depends on 63) The method of claim 63, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.1 Hz to about 300 Hz. ### Claim 65 (depends on 64) The method of claim 64, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.5 Hz to about 10 Hz. ### Claim 66 (depends on 61) The method of claim 61, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm to about 30.0 kV/cm. ### Claim 67 (depends on 61) The method of claim 61, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm and about 25.0 kV/cm. ### Claim 68 (depends on 67) The method of claim 67, wherein each pulse of electricity has an intensity peak in a range of about 5.0 kV/cm and about 10.0 kV/cm. ### Claim 69 (depends on 61) The method of claim 61, wherein each pulse of electricity has an intensity peak of about 1.0 kV/cm. ### Claim 70 (depends on 61) The method of claim 61, wherein each pulse of electricity has an intensity peak in a range of about 2.5 kV/cm to about 25.0 kV/cm. ### Claim 71 (depends on 61) The method of claim 61, wherein a 30 nanosecond rise and fall time is present between a peak intensity and a baseline intensity. ### Claim 72 (depends on 61) The method of claim 61, wherein a time between rise and fall times of a peak intensity and a baseline intensity is less than about 10 nanoseconds. ### Claim 73 (depends on 61) The method of claim 61, wherein said electric pulses are applied to said cells in vivo. ### Claim 74 (depends on 61) The method of claim 61, wherein said electrical pulses are applied to said cells in vitro. ### Claim 75 (depends on 74) The method of claim 74 further comprising: inserting said cells into a subject following applying of said one or more electrical pulses. ### Claim 76 (depends on 61) The method of claim 61, wherein said cells are osteoblasts. ### Claim 77 (depends on 61) The method of claim 61, wherein said cells are subject to up to 150 pulses of electricity. ### Claim 78 (depends on 77) The method of claim 77, wherein said cells are subject to five or fewer pulses of electricity. ### Claim 79 (depends on 61) The method of claim 61 further comprising: administering an additional agent. ### Claim 80 (depends on 79) The method of claim 79, wherein said additional agent is selected from group consisting of an antibiotic compound, an antimicrobial compound, an antibody, a biocidal agent, nanoparticles, self-assembling nanoparticles, viral particles, bacteriophage particles, bacteriophage DNA, genetic material, chemotherapy agent, growth factor, synthetic scaffold, natural scaffold, electrode, drug, a microbe, and a bacteria. ### Claim 81 (depends on 61) The method of claim 61, wherein promoting nodule formation comprises bone formation. ### Claim 82 A method of promoting myotube formation, said method comprising: applying one or more pulses of electricity to cells, each pulse of electricity having a duration of between about 10 nanoseconds and about 1,000 nanoseconds, wherein said pulses of electricity are applied under conditions effective to promote myotube formation. ### Claim 83 (depends on 82) The method of claim 82, wherein each pulse of electricity has a duration of between about 10 nanoseconds and about 300 nanoseconds. ### Claim 84 (depends on 82) The method of claim 82, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.01 Hz to about 1,000 Hz. ### Claim 85 (depends on 84) The method of claim 84, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.1 Hz to about 300 Hz. ### Claim 86 (depends on 85) The method of claim 85, wherein each pulse of electricity has a frequency of repetition in a range of between about 0.5 Hz to about 10 Hz. ### Claim 87 (depends on 82) The method of claim 82, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm to about 30.0 kV/cm. ### Claim 88 (depends on 82) The method of claim 82, wherein each pulse of electricity has an intensity peak in a range of about 1.0 kV/cm and about 25.0 kV/cm. ### Claim 89 (depends on 88) The method of claim 88, wherein each pulse of electricity has an intensity peak in a range of about 5.0 kV/cm and about 10.0 kV/cm. ### Claim 90 (depends on 82) The method of claim 82, wherein each pulse of electricity has an intensity peak of about 1.0 kV/cm. ### Claim 91 (depends on 82) The method of claim 82, wherein each pulse of electricity has an intensity peak in a range of about 2.5 kV/cm to about 25.0 kV/cm. ### Claim 92 (depends on 82) The method of claim 82, wherein a 30 nanosecond rise and fall time is present between a peak intensity and a baseline intensity. ### Claim 93 (depends on 82) The method of claim 82, wherein a time between rise and fall times of a peak intensity and a baseline intensity is less than about 10 nanoseconds. ### Claim 94 (depends on 82) The method of claim 82, wherein said electric pulses are applied to said cells in vivo. ### Claim 95 (depends on 82) The method of claim 82, wherein said electrical pulses are applied to said cells in vitro. ### Claim 96 (depends on 95) The method of claim 95 further comprising: inserting said cells into a subject following applying of said one or more electrical pulses. ### Claim 97 (depends on 82) The method of claim 82, wherein said cells are myoblasts. ### Claim 98 (depends on 82) The method of claim 82, wherein said cells are subject to up to 150 pulses of electricity. ### Claim 99 (depends on 98) The method of claim 98, wherein said cells are subject to five or fewer pulses of electricity. ### Claim 100 (depends on 82) The method of claim 82 further comprising: administering an additional agent. ### Claim 101 (depends on 100) The method of claim 100, wherein said additional agent is selected from group consisting of an antibiotic compound, an antimicrobial compound, an antibody, a biocidal agent, nanoparticles, self-assembling nanoparticles, viral particles, bacteriophage particles, bacteriophage DNA, genetic material, chemotherapy agent, growth factor, synthetic scaffold, natural scaffold, electrode, drug, a microbe, and a bacteria.