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    • 专利摘要:
    MATERIALS FOR CELL MAGNETIZATION AND MAGNETIC HANDLING This is a material that comprises negatively charged nanoparticles, in which one of the said nanoparticles contains a magnetically responsive element, which are combined with a support molecule, which is a molecule or a long natural polymer. or synthetic to produce a magnetic nanoparticle assembly. When the magnetic nanoparticle assembly is combined with cells, it will magnetize those cells. The magnetized cells can then be washed to remove the magnetic nanoparticle assembly and the magnetized cells manipulated in a magnetic field.
    公开号:BR112012006558B1
    申请号:R112012006558-0
    申请日:2010-09-27
    公开日:2021-01-26
    发明作者:Glauco R. Souza
    申请人:Greiner Bio-One North America, Inc.；
    IPC主号:
    专利说明:

    [0001] [0001] This order claims priority for the provisional order under number US61 / 245846, which was filed on September 25, 2009 and is incorporated by reference in its entirety. FIELD OF THE INVENTION
    [0002] [0002] The invention relates to the fields of nanotechnology, materials, biosynthesis, medicine, cell biology and tissue engineering. More particularly, the compositions and methods of the present disclosure refer to methods of cell magnetization and cell culture in 3D, cell manipulation and cell patterning with the use of magnetic fields. BACKGROUND OF THE INVENTION
    [0003] [0003] The manipulation of cells, the control of their environment and the promotion of conditions that mimic or prohibit tissue or cellular responses in vivo or natural is an area of intense research. In the area of stem cells and regenerative medicine, there is a particular need for methods and materials that replicate the native conditions in which cells grow in vivo. The conditions that cells experience when removed from their native environment promote homeostasis, where cells change to adapt to their new environment, thereby inducing cellular changes. Many of these processes are not elastic or reversible, so cells cannot return to their native state. There is a strong need for materials and methods that promote natural cellular environments and minimize or control adverse cellular changes before cell and tissue engineering can reach its full potential.
    [0004] [0004] Currently, materials are being developed that can withstand three-dimensional (3D) cell culture conditions. Most 3D cell culture techniques involve rotating the flasks, using an outer framework to which cells can adhere, using magnetic fields to suspend cells, or some combination of these approaches.
    [0005] [0005] For example, Felder in US2005054101, WO2005010162 describes a hydrogel substrate that forms an outer framework in which cells can grow and be supported in a 3D environment. This introduces an artificial substrate with which cells interact, rather than rapidly promoting cell-cell interactions, and although an improvement over 2D culture, the framework is prone to disturbing cells and remains in the finished product. Additionally, cells can grow on or in microcarriers, but cells cannot be levitated in a way that everything around cell-cell contact / interaction is possible.
    [0006] [0006] Nationally, there is a significant level of complexity involved in the manufacture of Felder microcarriers, which includes difficult chemistry and the need for complex equipment. Additionally, algimatrix, one of the main reagents in the production of microcarriers, can be a source of endotoxins. The buoyancy control also seems to be relevant to facilitate levitation and is controlled by the infusion of glass bubbles in the microcarriers, again contributing to the complexity and difficulty. Finally, specialized hardware is needed for agitation, which is necessary to achieve gas exchange and to prevent agglutination of microcarriers, and impellers are often used to agitate cells. However, the shear stress resulting from agitation is known to cause cellular damage. In addition, agitation gives any control of the magnetic field shape of 3D cultures.
    [0007] [0007] Becker in US2009137018, WO2005003332 uses a coating of bioactive magnetized core particles, thus initiating the adhesion of biological cells to the magnetized core particles and allowing their suspension in a magnetic field. The coating remains with the cells during culture, thus introducing an unnatural element into the culture and probably disturbing the cells. The inventors contemplate the use of a biodegradable coating that could eventually be eliminated, but none of them is revealed, so it is not known whether this approach would be successful. Additionally, due to the fact that the cells are grown in the nucleus of the microcarriers, the levitation of individual cells in which they can be joined by magnetic levitation for the purpose of promoting cell-cell interaction is unlikely to occur. Therefore, it is not obvious that the rapid assembly (hours) of 3D multicellular structures due to cell-cell contact can be demonstrated when using microcarriers. Furthermore, through the growth of cells in the microcarriers, the co-culture of different types of cells, especially through the levitation of individual cells and, therefore, their mechanical union, is not demonstrated. Finally, this system is inconvenient and not suitable for scaling and high performance applications.
    [0008] [0008] A better approach may be to temporarily magnetize the cells, allowing their 3D culture. For example, Akira in US2006063252, WO2004083412, WO2004083416 uses magnetic cationic liposomes (MCL) to magnetize cells by absorbing the liposomes. The magnetized cells are then grown on a slide at the bottom of a plate that uses magnetic attraction and then released for use. However, although they are capable of producing sheets of cells, the cells are still grown at the bottom of a plate and, therefore, this is not the case for 3D culture by magnetic levitation. Additionally, Shimizu and Akira et. al. In a recent publication entitled "Effective Cell-Seeding Technique Using Magnetite Nanoparticles and Magnetic Power onto Decellularized Blood Vessels for Vascular Tissue Engineering" they use a magnetic guide to seed cells over a decellularized blood vessel.1 Their study shows encouraging results, but they do not use the magnetized cells as the tissue source to be decellularized. Magnetized cells are only used to recellularize decellularized blood vessels.
    [0009] [0009] In the patent application WO2010036957 de Souza, the cells are levitated in a magnetic field through the contact of the cells with a "hydrogel" that comprises a bacteriophage with nanoparticles that are responsive to a magnetic field. In particular, filamentous phage, such as bacteriophage fd, fl or Ml 3, are used. Bearing in mind that the method's work is not completely clear, however, it is theorized that the phage provides a gel-like structure or assembly that lines the cells, and somehow assists the cells in the absorption or adsorption of magnetically responsive nanoparticles. In this way, even when the hydrogel is washed away, the cells remain magnetically responsive, and can be levitated in an appropriate magnetic field. However, although the hydrogel is mostly removed by washing, the potential for phage infectivity or transfer of genetic material remains and, therefore, it is desired to provide a material that allows cellular absorption or adsorption without the use of phage.
    [0010] [0010] The present disclosure overcomes the existing deficiencies in the technique by providing improved materials and methods that promote native cellular environments. These include using compositions and methods to generate nanoparticle-based materials and preparing the cells to allow 3D cell culture, cell standardization and cell imaging. SUMMARY OF THE INVENTION
    [0011] [0011] As used in the present invention, "positively charged nanoparticle" or "positive nanoparticle" is defined as any particle less than 200 nm, preferably 100 nm or less, that has a total positive charge. Preferably, the particle is non-toxic, but this is not essential, since the particles do not remain with the cells.
    [0012] [0012] As used in the present invention, "negatively charged nanoparticle" or "negative nanoparticle" is defined as any particle less than 200 nm, preferably 100 nm or less, and most preferably, about 2 to 25 nm, which has a total negative charge. Preferably, the particle is non-toxic, but this is not essential, since the particles do not remain with the cells for a long period of time.
    [0013] [0013] As used in the present invention, a "magnetically responsive element" can be any element or molecule that will respond to a magnetic field. As detailed below, one of the nanoparticles must contain or be a magnetically responsive element.
    [0014] [0014] As used in the present invention, "support molecule" refers to any long molecule that will interact with the nanoparticles to create a fibrous structure like a blanket or gel and thereby retain the magnetic nanoparticle very close to the cell for absorption.
    [0015] [0015] The following abbreviations are used in this document:
    [0016] [0016] In general terms, the invention is a new material that allows cells to absorb or adsorb the magnetically responsive elements and, thus, to be levitable in cell culture when a magnetic field is applied. The materials include negatively and positively charged nanoparticles, one of which must contain one or more magnetically responsive elements, such as iron oxide. These nanoparticles are additionally combined with a polymer, preferably a cell-derived or natural polymer or other long molecule that acts as a support (referred to in this document as a "support molecule") for the charged nanoparticles and the cells, which it retains the nanoparticles in place for their absorption or adsorption by the cells.The inclusion of both negative and positive nanoparticles allows mixing by intense addition of the nanoparticles and triggers the assembly of the three components, thus ensuring uniform distribution and good absorption. It intensively combines all three components with cells in a fibrous blanket type structure that allows cells to absorb the magnetically responsive element.
    [0017] [0017] After an incubation period, the material can be removed by washing, allowing the cells to be manipulated in a magnetic field. An alternative step is to optimize or tune the absorption of magnetic material by increasing the ratio between the number of cells and the amount of magnetic nanoparticle. If a large number of cells are present, they will absorb most of the magnetic nanoparticles, and the step of washing out any material left behind may not be necessary, particularly if the remaining support molecules and / or nanoparticles are non-toxic. and / or beneficial to the cell. This is particularly true where support molecules comprise one or more extracellular matrix proteins, glycoproteins or polysaccharides. The magnetic particles are eventually lost from the cells, leaving them in a completely natural state.
    [0018] [0018] In addition to simple 3D culture, the magnetic field can be used to manipulate the shape, patterns and movement of the cell. For example, the use of a toroidal magnet (in the shape of a washer) can promote the assembly of cells in a similar shape or an inclined field can make the 3D cell culture thicker on one side. Strong dense sheets of cells were also created by placing a strong magnet on the bottom of a culture plate for a period of growth. The simple reversal of the field allows the blade to then be levitated and the blade can then continue to grow in a 3D culture. It is also possible to combine several formats and continue the culture in 3D and, thus, create more complex formats in a 3D culture.
    [0019] [0019] The magnetically responsive element can be any element or molecule that will respond to a magnetic field, for example, rare earth magnets (for example, samarium-cobalt (SmCo) and neodymium-iron-boron (NdFeB)), ceramic magnet materials (for example, strontium ferrite), magnetic elements (for example, iron, cobalt and nickel and their alloys and oxides). Paramagnetic materials that react to a magnetic field are particularly preferred, but they are not the magnets themselves, since this allows for easier assembly of the materials.
    [0020] [0020] Preferably, the magnetic field used to levitate such cells is about 300 G to 1000 G. However, the field resistance varies both with the distance of the cell and with the amount and type of magnetic response element absorbed or adsorbed by cells. In this way, the ideal field resistance will vary, but it is easily determined empirically.
    [0021] [0021] Negatively charged nanoparticles include charge-stabilized metals (eg silver, copper, platinum, palladium), but are preferably a gold nanoparticle.
    [0022] [0022] The positively charged nanoparticles include oxides and / or alloys coated or stabilized with a surfactant or polymer (for example, elemental iron, cobalt iron, nickel oxide) and, preferably, is an iron oxide nanoparticle.
    [0023] [0023] One of the two nanoparticles needs to be magnetically responsive, but obviously one could contain this feature.
    [0024] [0024] The nanoparticles must have a nanoscale size and, therefore, are about 100 nm. The size can be in the range, however, between about 5 to 250 nm, 50 to 200 nm, 75 to 150 nm, but it can be smaller or larger, provided that only the size is appropriate to allow entry or adsorption to the type. cell in use. It has been shown in this document that there is an upper limit on the effective size of the magnetic nanoparticle, and the micrometer size is too large for effectiveness, although some functionality was still observed.
    [0025] [0025] The "support molecule" is generally a polymer or other long molecule that serves to retain nanoparticles and cells together in a mixture by intense addition. The support molecule can be positively charged, negatively charged, mixed charge or neutral, and can be combinations of more than one support molecule.
    [0026] [0026] Examples of such support molecules include natural polymers, such as peptides, polysaccharides, nucleic acids and the like, but synthetic polymers can also be used. Particularly preferred support molecules include polylysine, fibronectin, collagen, laminin, BSA, hyaluronan, glycosaminoglycan, anionic unsulfated glycosaminoglycan, gelatin, nucleic acid, extracellular matrix protein mixtures, matrigel, antibodies, and mixtures and derivatives thereof.
    [0027] [0027] In general terms, the concentration of the support molecule is substantially higher than the concentration of the positively and negatively charged nanoparticles, in the range of 1 to 1000 times greater, 10 to 500 or 20 to 200 times greater. However, larger or smaller amounts are possible, depending on what type of cell is being used and which support molecule and nanoparticles are being used. The longer the polymer, the less it may be necessary to form enough structure to retain the nanoparticles at the site for absorption.
    [0028] [0028] In general, nanoparticles are used in very low concentrations. Concentrations can be in the range of 10-12 to 10-6 Molar, but are preferably in the nanomolar range, and the support molecule (s) 10-9 to 10-3 Molar, and , are preferably in the micromolar range.
    [0029] [0029] The three components come together through electrostatic interaction and, therefore, charged or mixed charge support molecules, such as polylysine, are preferred. However, any of the three components can be functionalized, derivatized or coated in order to further promote the interaction of the components and / or cells. In this way, one or more members can be functionalized, derivatized or coated with an antibody that, for example, binds to a cell surface antigen. That way, the interactions between the components and / or cells would be further promoted. Other binding pairs included receptor-ligands, biotin-strepavidin, complementary nucleic acids, wheat germ agglutinin (WGA), molecules containing sialic acid and the like.
    [0030] [0030] Coatings may also include protective or passivate coatings, particularly for nanoparticles, such as PVP, dextran, BSA, PEG and the like. Nanoparticles, especially the nanoparticle that comprises the magnetically responsive element, can be identified for viewing, for example, with a fluorophore, radiolabel or the like, particularly during the development and in vitro testing of magnetized cells and tissues. However, for therapeutic uses, it may be preferable to omit such markings.
    [0031] [0031] In other embodiments, the compositions include cells that will be levitated or manipulated in a magnetic field, including, but not limited to, stem cells, cancer cells, primary cells, mammalian cells, human cells, cells extracted directly of fresh tissue, bacteria, yeast, plant cells or mixtures thereof.
    [0032] [0032] The present disclosure provides methods of cell culture, cell standardization and cell imaging in three dimensions, which comprises mixing the cells with one or more of the compositions presently disclosed and culturing, standardizing or manipulating the mixture in the presence of a magnetic field. The magnetic field can be above or below the culture vessel, closer or farther (for example, stronger or weaker), tilted or to the side, or the shape of the field can be varied or combinations of one or more of the they can be applied. In this way, cells can be standardized or moved to achieve particular goals.
    [0033] [0033] The present extensive test of the system described above has shown that there are a large number of improvements available now over the prior art methods. First, a self-assembling manufacturing chemistry makes the method simple and reproducible, and no specialized equipment is needed to manufacture the magnetic nanoparticle assembly or for subsequent magnetization, culture or cell manipulation. The only requirements were a magnetic field, pipettes, containers and a hot plate. Thus, the method is compatible with large scale and high throughput.
    [0034] [0034] If desired, the magnetic nanoparticle assembly can become free of biological molecules, such as phage or cell products, due to the fact that support molecules, such as polylysine, can be easily produced synthetically. Yet all components are generally non-toxic, inexpensive, or easy to produce. In addition, fibrous mat-like structures allow the incorporation of additional cell support molecules (such as extracellular matrix components) to be included in the magnetic nanoparticle assemblies.
    [0035] [0035] The magnetization of cells with magnetic nanoparticle assemblies consists only of adding assembly to the cells in regular cell culture media. The cells can be magnetized within minutes from the magnetic nanoparticle treatment (5 minutes) and the fixed or suspended cells can be treated with magnetic nanoparticle assemblies. The cells can be cryopreserved before or after treatment with magnetic nanoparticles and the method still works. If desired, the magnetic nanoparticle assembly can be removed by washing the magnetized cells prior to use, and the remaining cells will still levitate.
    [0036] [0036] Levitation and cell culture in 3D through magnetic levitation does not require any specialized or costly equipment or methods (such as to agitate or maintain buoyancy) beyond the standard 2D cell culture requirements, and format control of magnetically levitated 3D cell culture can be achieved by varying the shape of the magnetic field. Finally, and perhaps most importantly, the invention promotes rapid cell-cell interaction (scale of seconds and minutes) with cell levitation and assembly in 3D multicellular clusters within minutes, and these complex culture structures can be produced by manipulating the magnetic field and / or through the magnetic contact of different cell types. BRIEF DESCRIPTION OF THE DRAWINGS
    [0037] [0037] Figure 1 is a schematic view for a direct assembly method for generating nanoparticle assemblies. In this diagram, 1 is the negatively charged nanoparticle, 3 is the positively charged nanoparticle and 5 is the support molecule and the completed magnetic nanoparticle assembly is 7. The three components can be combined in any order, but the nanoparticles are shown here first combined and then added to the support molecule.
    [0038] [0038] Figure 2A is a schematic view showing a magnetic nanoparticle assembly variant 19. The positive nanoparticle is 15, the negative nanoparticle is 17, and 21 and 23 refer to two different support molecules, for example, polylysine ( charge +) and a peptide rich in glutamate and / or aspartate (charge -) or, as another example, laminin and fibronectin.
    [0039] [0039] Figure 3 is the absorbance spectra of real nanoparticle assemblies composed of magnetic nanoparticles of iron and gold oxide and various support molecules as mentioned on the right.
    [0040] [0040] Figure 4 shows the image formation in the dark field (a) and the hyperspectres (b) of an exemplary FeO2-Au-PL nanomount.
    [0041] [0041] Figure 5a-d shows fluorescent and bright field images at 10x magnification, a is a bright field image with support molecule IgG-AlexaFluor 488, b is a green fluorescent image of the same field, c is the bright field photo with mouse IgG and anti-mouse IgG support molecules AlexaFluor 555 ed is the red fluorescent image of the same field.
    [0042] [0042] Figure 6a-d is photographs of HEK293 cells levitating after treatment with magnetic nanoparticle assemblies of different compositions, including a = PL, b = COL, c = LAM, d = NT.
    [0043] [0043] Figure 7 is a schematic view indicating the use of cells or fluid from an animal in the method of the invention.
    [0044] [0044] Figure 8 is a schematic view showing the cyclic use of cells or extracts of the invention to be added to already growing samples.
    [0045] [0045] Figure 9 shows photographs of several examples of manipulation of cell shape and density using the methods of the invention.
    [0046] [0046] Figure 10 shows a change in thickness of 3D culture by tilting the magnetic field.
    [0047] [0047] Figure 11 is a photograph showing an example of the method in Figure 10.
    [0048] [0048] Figure 12 is a schematic view that illustrates the use of a magnetic film for magnetized cells, in which the cells are not intensively mixed by addition with the magnetic nanoparticle assembly, but only seated on the surface thereof. DESCRIPTION OF MODALITIES OF THE INVENTION
    [0049] [0049] The present disclosure provides compositions comprising negatively charged nanoparticles, positively charged nanoparticles and a support molecule comprising a polymer or long molecule, or metal-binding polymer or molecule. The following examples are illustrative only and are not intended to improperly limit the invention. EXAMPLE 1: NANOPARTICLE MAGNETIC ASSEMBLIES
    [0050] [0050] Figure 1 shows a general scheme for preparing magnetic nanoparticle assemblies (7) by combining negative nanoparticles (1) and positive nanoparticles (3) (at least one nanoparticle is magnetic in nature) and support molecules (5 ).
    [0051] [0051] Nanoparticle solutions are prepared by mixing the nanoparticles separately in water or low ionic resistance buffer (salt concentration <10 mM) at a desired pH. The surface particle charge can be adjusted by choosing the appropriate pH, where the low pH buffer (such as citrate or carbonate buffer) can generally increase the total charge on the nanoparticles. Adversely, the high pH buffer (such as borate buffer) can generally decrease the total charge on the particles. Ideally, the pH of choice for each solution should result in opposite charges between the two particles. This can be achieved due to the fact that nanoparticles of different composition usually have different isoelectric points.
    [0052] [0052] For example, Au nanoparticles are negatively charged at most pH values, often due to the presence of adsorbed citrate or chloride ions, in contrast to iron oxide nanoparticles, which can vary from pH = 3, 3 to 8, depending on the type of iron oxide. Therefore, at pH 4, it is expected that Au nanoparticles are negatively charged and iron oxide nanoparticles should be positively charged. The nanoparticles can also be coated with molecules, such as dextran, polyethylene glycol or thiools, which can also dictate the charge as a whole of the nanoparticles. It is desirable that a mismatch of charges ensures that the assembly occurs with support molecules that are positively or negatively charged, mixed or neutral.
    [0053] [0053] Solutions of support molecules (5) are generally prepared by solubilizing or mixing them with water or buffered solutions (such as citrate, phosphate, borate) of a desired pH and, preferably, low ionic resistance (of preferably, salt concentration <10 mM, but the salt concentration could be higher if necessary to ensure solubilization of the molecule). Low ionic resistance is generally desirable to reduce the charge rating and, therefore, promotes charge interaction between nanoparticles and oppositely charged polymers. The concentrations of support molecules (5) should generally be greater than the concentration of the nanoparticle (range from 10 nM to 1 mM, but other concentrations can be used), usually above 10X in relation to the molar concentrations of nanoparticles (1) and (3).
    [0054] [0054] In additional details, the support molecule can also provide functionality for assembly, such as: improving the adhesion of magnetic nanoparticles to cells (such as polylysine); improve the cell culture environment (such as through the use of extracellular matrix proteins, such as collagen and laminin); allow delivery of a specific molecule (such as DNA, drugs, ligands, tags, etc.) to cells; provide function as signal reporters (such as fluorescence marking); and / or enhance cell / tissue biocompatibility of magnetic nanoparticle assemblies (for example, by providing nutritional support or a compatible immunosurface and the like).
    [0055] [0055] Figure 2 shows a magnetic nanoparticle union (19) in which the negatively charged nanoparticle (17) and the positively charged nanoparticle (15) are retained can two support molecules (21, 23). An example of the pairing of support molecules could be laminin and fibronectin, another could be antibody and antigen.
    [0056] [0056] A wide variety of magnetic nanoparticle assemblies were prepared, and their functionality was tested with various cells in various media. Table 3 shows the range of tested components:
    [0057] [0057] Figure 3 shows the absorption spectra of several joints. The variations in extinction spectra between different solutions are a result of varying levels of electrostatic crosslinking between nanoparticles and the various support molecules. With the exception of spectra for laminin (5), collagen (7) and FuGENE-GFP (21), all had a broad centralized support at approximately 560 nm, resulting from the characteristic absorption of Au nanoparticles. The nature of the flat spectra in relation to the Au nanoparticle spectrum is a result of the presence and broad extinction spectra of the polydispersed iron oxide nanoparticle (generally less than 100 nm). The spectra for laminin (5) and collagen (7) are nanoparticle assemblies prepared with extracellular matrix components. The spectra designated as FuGENE-GFP (21) are from nanoparticle assemblies prepared with FuGENE (a liposome) loaded with a DNA plasmid encoding a GFP reporting molecule. The presence of the DNA plasmid is indicated by the presence of the UV support at 260 nm (DNA absorbs at 260 nm) in the trace for (21).
    [0058] [0058] Figure 4 shows the image formation in the dark field (a) and the hyperspectres (b) of an exemplary FeO2-Au-PL nanomount. Hyperspectral imaging is an optical technology that combines microscopy based on a dark field with resolved spectra with a wavelength of diffused light (400 to 1000 nm) from an imaged sample. This technology allows the identification of nanomaterials based on their characteristics of optical diffusion. A peak of monodisperse nanoparticles is expected, but here, the expected expansion of the spectra is due to the presence of iron oxide nanoparticles and their assembly. Although there is broadening of the spectra, this still provides sufficient spectral resolution to identify these samples within a tissue. Thus, this is a method for monitoring assembly in vitro or in vivo.
    [0059] [0059] Referring now to Figure 5, a fluorescence signal of magnetic nanomounts (a, c) prepared with fluorescent protein is conjugated as the support molecule (b, d). The nanoparticle assembly shown in the aec images was generated using the scheme in Figure 1A, with the use of iron oxide and gold nanoparticles, and AlexaFluor 555 monkey anti-mouse IgG and protein mouse IgG as duel support molecules . First, the iron oxide and gold were mixed, then the protein mouse IgG was added to form a magnetic nanoparticle assembly. Then, in a second / separate step, the assembly was incubated with AlexaFluor anti-mouse IgG fluorescence-reporting antibody. In this way, the magnetic nanoparticle assembly can be marked and monitored in use.
    [0060] [0060] Figure 6a-d shows photographs of cells that levitate after treatment with a magnetic nanoparticle assembly of different compositions generated with the method described in Figure 1. The procedure used was as follows: an iron oxide-Au assembly was first generated by mixing 6 ml of a X mg / ml solution with an Au nanoparticle solution with a diameter of approximately 74 nm (generated by citrate reduction, with 4.1 extinction at wavelength 548 nm) with 3 ml of 1.0 mg / ml of polydispersed iron oxide nanoparticle solution in picopure water. Then, 1.0 ml of the iron oxide-Au mixture was immediately added to separate 1.0 ml of polylysine (0.0001%), collagen (at 30 μg / ml), laminin (at 10 μg / ml) ml) or oligonucleotide (at 1 μg / ml) all dissolved in picopure water.
    [0061] [0061] After mixing with the support molecules, the solutions were allowed to incubate overnight. A fraction of the supernatant was removed with the use of magnetic separation, pulling the magnetic nanoparticle assembly to the bottom with the use of a magnet and the discarded supernatant. To remove excess unreacted reagents, the magnetic nanoparticle assembly was washed twice with 5 ml of picopure water each time. This was then stored in 5 ml of picopure water at 4 ° C.
    [0062] [0062] Finally, 5 ml of HEK293 cells (50,000 cells / ml) were carefully pipetted into a 15 ml conical tube with 0.5 ml of the nanoparticle assembly. This mixture was slowly mixed through the action of the pipette and allowed to incubate and set for 5 minutes. Then, the cells were allowed to settle in the conical tube for 5 minutes and then the supernatant was removed (placed in a conical tube) and replaced with culture media. This washing procedure was repeated twice more.
    [0063] [0063] Due to the fact that the cells attached to the nanoparticle assemblies settle much faster than the non-adherent nanoparticles, the non-adherent nanoparticles were removed with the supernatant. Non-adherent nanoparticles can be visually detected in the supernatant by placing a magnet at the bottom of the conical tube with the supernatant, and the nanoparticles visualized due to magnetic sedimentation. After the third wash, the supernatant appeared to be depleted of free magnetic nanoparticle assemblies. The cells were then transferred to a 3.5 cm Petri dish and levitated with a ring-shaped neodymium magnet (20 x (8.5 x 4.5) x 7 mm; pull resistance: 13 lbs). All cells levitated and coalesced in a 3D culture within minutes of applying a magnetic field, regardless of which support molecule was used. Many other support materials have been tested on the present invention and all have worked, but only a few exemplary results are shown. EXAMPLE: COMPONENT MOLECULES
    [0064] [0064] The importance of the various components of the magnetic nanoparticle assembly of the present invention has also been demonstrated through the testing of Fe2O3 nanoparticles alone, (<50 nm particle size), Au- (Fe2O3) nanoparticles and magnetic assemblies of complete nanoparticles of PL-AU- (Fe2O3).
    [0065] [0065] The cells were treated with samples that carry the same amount of magnetic iron oxide nanoparticles or approximately 1.0 μ! per 5,000 cells, and the microphotographs were taken directly after the levitation started with a 500G magnet, as well as after the cells had been cultured by magnetic levitation for 7 days with a 300G magnet or a 500G magnet (data not shown ).
    [0066] [0066] All cells were able to levitate in one day, however, these samples treated with PL-AU- (Fe203) showed larger and more cohesive 3D structures, especially when cultured with the 500G magnet. Therefore, all three components are necessary for effective cell levitation and coalescence in a 3D structure. Although the nanoparticles alone showed poor performance, there is still value in the ability to levitate and grow cells that are treated with the nanoparticles that require less manipulation than the modalities in Figure 1. EXAMPLE: DELIVERY OF MATERIAL FOR CELLS
    [0067] [0067] The ability of magnetic nanoparticle assemblies to carry and deliver materials to cells has also been demonstrated. As proof-of-principle, HEK293 cells were treated and levitated with the magnetic nanomounts that carry GFP DNA, and then the GFP fluorescence signal was detected inside the cells of the levitated 3D cell culture.
    [0068] [0068] In additional details, the procedure for preparing the magnetic nanomounts that carry DNA was as follows: first, the FuGENE solutions were prepared according to the manufacturer's instructions (Roche Applied Science, FuGENE HD Transfection Reagent), where 2 μg of GFP plasmid DNA (DNA) were dissolved in 200 μl of serum-free DMEM medium.
    [0069] [0069] Then, 6 μl of FuGENE solution was added directly to the 200 μl solution of the diluted DNA (FuGENE-DNA). This mixture was allowed to settle for 15 minutes at room temperature. Two other samples were prepared: one with 6 μl of FuGENE added directly to 200 μl of serum-free DMEM medium (without DNA, negative control, called FuGENE in this document), and another with 4 μl of DNA in 200 μl of serum-free DMEM medium (referred to in this document as DNA). Then, 3 ml 2: 1 (v / v) ratio of the nanoparticle solution of iron oxide and Au (1 mg / ml of iron oxide before mixing with Au) was combined and mixed thoroughly and then added directly to 100 μl of FuGENE-DNA, 100 μl of FuGENE and 100 μl of DNA.
    [0070] [0070] The solution was mixed well and allowed to incubate overnight at 4 ° C. After an overnight incubation period, the nanoparticles settled. The extinction spectra taken from the nanoparticle assemblies were composed of iron and gold oxide magnetic nanoparticles and their nanoparticle-free supernatant (collected after the nanoparticles settle), which indicated the incorporation of DNA and / or FuGENE of its high absorption of UV. The supernatant spectra show lower absorbance than the mixed samples, indicating the incorporation of DNA and FuGENE in the nanoparticle assembly.
    [0071] [0071] Magnetic nanoparticle assemblies were added to the cells, and the cells levitated as above, and the cells in levitation were then photographed. The presence of GFP fluorescence signal in the cells of both systems that carry DNA (not shown), shows the ability of these magnetic nanoparticle assemblies to carry and deliver DNA to cells while magnetizing and cultivating these cells in 3D through magnetic levitation. This result proves that the magnetic nanoparticle assembly can be used both to magnetize cells and to deliver functional materials to the cells, such as DNA or drugs. EXAMPLE: PRIMARY CELL LEVITATION
    [0072] [0072] Primary cells are often difficult to grow in vitro, but primary cells have been shown to be successfully cultured in 3D by magnetic levitation after being magnetized with iron oxide-Au-PL.
    [0073] [0073] Several magnetic nanoparticle assemblies were made, including Au, PL nanoparticles and different iron oxide nanoparticles. The various iron oxides were Ferridex-PL, iron oxide-Au-PL (<50 nm), iron oxide-Au-PL (~ 5 nm Au) and iron oxide-Au-PL (<5 μl, a example of a microparticle). Ferridex is made up of dextran-coated superparanan magnetic iron oxide particles (SPIONs). The isoelectric point (pi) or pH at which a particular molecule or surface carries no network electrical charge for the following is: SPION, 7.3-5 magnetite (y-Fe2O3), 3.3 to 6.7, ' 2 and magnetite (Fe3O4), 6.5 to 6.8.2
    [0074] [0074] For the different nanoparticle assemblies, the following were used: 11.2 mg / ml Feridex IV (Advanced Magnetics, Inc., Cambridge, MA, USA), iron (III) oxide (Sigma Aldrich, 544884, <50 nm particle size, Fe2O3 (This product mainly consists of magnetite in the gamma form, but both the alpha and gamma forms are present in the mineral form naturally). Iron oxide (II, III) oxide (Sigma Aldrich, 310069, 310069, Fe3O4, magnetite) was also used. All three iron samples were diluted to 1 mg / ml working solutions in water. Au ~ 5 nm colloid was prepared according to Duff et al.6
    [0075] [0075] The preparation of iron oxide-Au-PL consisted of six serial dilutions of aqueous poly-L-lysine (PL) with concentrations ranging from 1.00x10-3 to 3.12x10-5%, which were prepared separately. A 1: 1 ratio of equal part (v / v) of the nanoparticle solution of iron oxide and Au was added to each dilution. After an overnight incubation period, the nanoparticles settle to the bottom of each flask and half of the supernatant was removed. Starting with the lowest dilution, mixtures of serial dilutions were combined and mixed with the next lowest dilution in series until the entire solution remained in a flask. The total PL concentration was 1.24x10-3%.
    [0076] [0076] Mixing the reagents as serially diluted samples allows unbound reagents at higher dilutions the chance of being incorporated into the assembly. In addition, different dilutions often have distinct optical, structural, adhesive and other physical characteristics; by mixing the different dilutions, these properties can be integrated. Often, the integration of such properties may be desirable. On the other hand, this process can be a good visual diagnosis to determine and choose the ideal conditions depending on the application. For example, one or several dilutions could be sticky on the plastic or pipettes.
    [0077] [0077] Four types of primary human lung cells (SCIENCELL RESEARCH LABORATORIES ™, Carlsbad, CA, USA) were treated with iron oxide-Au-PL assemblies and then cultured in 3D. This procedure consisted of culturing these cells as monolayers for -80% confluence in a 2D cell culture flask. Then, the iron oxide-Au-PL solution was added to the cell flask (with concentrations in the range of 1.56 to 13.00 μl / cm2) and incubation with the cells was allowed. The incubation time was approximately overnight (or -12 hours). The primary cells were then washed with PBS (non-adhered iron oxide-Au-PL removed). Finally, the cells were separated by trypsin digestion, transferred to 3.5 cm Petri dishes and, finally, levitated with a ring-shaped neodymium magnet (20 x (8.5 x 4.5) x 7 mm pull resistance: 13 lbs).
    [0078] [0078] All four types of nanomounts were prepared with different shapes and sizes of adhered iron oxide and were able to promptly levitate the cells (data not shown), even where the original cell contact employed adhered cells. However, assemblies prepared with larger iron oxide (<5 μm, Fe3O4, magnetite) do not form cell structures as large and cohesive as the others, indicating that there is an upper limit to the size of the magnetically responsive element that can be absorbed by cells. Here, smaller clusters of cells are likely to result from the presence of larger iron oxide particles that do not readily enter cells, so cells are less magnetized and such larger size particles can also coat the cell surface and hinder or prevent cell-cell interaction. EXAMPLE: EX VIVO CELL LEVITATION
    [0079] [0079] Referring to Figure 7, any cells taken from animals or humans (41), including, but not limited to, blood, serum, plasma or disaggregated tissue cells (43), are mixed with nanoparticles (45) and then incubated together for 30 seconds to 48 hours. During incubation, the sample (43) and the nanoparticles (43) interact electrostatically together with any proteins, DNA or polysaccharides that may be contained therein to form the nanoparticle assembly (47). Then, the magnetic nanoparticle assembly (47) is separated by magnetic force, centrifugation and / or sedimentation, where the supernatant is separated from the mixture, and a magnet used to manipulate the remaining magnetized cells.
    [0080] [0080] Still referring to Figure 7, the nanoparticle assembly is generated from the interaction between proteins, DNA or polysaccharide present in the blood or another sample (43). The types of interactions may include, but are not limited to, interactions of an electrostatic, covalent (thiol functional groups and / or other crosslinkers), short-range and / or hydrophobic (usually through a bridged molecule) interactions of a specific nature and not specific. For example, electrostatic interactions can be allowed and / or controlled through pH manipulation, in which proteins from different isoelectric points interact with two corresponding nanoparticle surface charges.
    [0081] [0081] Additionally, additional support molecules can also be added to the assembly to support cell growth or additionally bridge the various components of the assembly. This type of bridging can be achieved by modifying the original sample (43) or by assembling a magnetic nanoparticle (47) with antibodies or other molecules (for example, peptide or protein labels), then the specific molecules are enriched / captured in the assembly.
    [0082] [0082] The modality described in this document may be of value for preparing nanoparticle assemblies with native proteins in order to reduce any immune response when the final cells are to be used therapeutically. This could be significant when it comes to cells in culture for autologous cell procedures, since serum, blood, or other body fluids or tissues could be used to prepare the nanoparticle assemblies to mask the effects of a foreign body. EXAMPLE: FROZEN MAGNETIZED CELLS
    [0083] [0083] It has been shown in this document that cells can be frozen when magnetized and then later thawed and used for 3D culture. The cells are mixed with the magnetic nanoparticle assembly by the procedures above. The excess assembly of the magnetic nanoparticle is then removed, and the cells are washed and cryopreserved according to standard techniques. Subsequently, they are thawed and cultivated in a 3D culture system. This is very convenient, given that it allows preparing and commercializing magnetized cells for use in research and therapy.
    [0084] [0084] The following procedure was used. A vial of Human Embryonic Kidney T-25 cells (HEK293) (ATCC CRL-1573) was grown to 80% confluence and treated with 200 µl of iron oxide-Au-PL. After an incubation period of 12 hours. Then, the cells were washed with PBS (iron oxide-Au-PL removed removed), trypsinized, divided into two flasks, placed in media containing DMSO and then frozen at -80 ° C. The next day, they were transferred to a liquid nitrogen Dewar for long-term storage.
    [0085] [0085] Eighty-one days later, 1 vial was thawed in a 37 ° C water bath for 2 minutes. After resuspending the flask in 7.5 ml of media, the cells were divided between three Petri dishes of 35 mm diameter Petri and 1000G magnets were placed on top of each dish. The cells were recovered from cryopreservation and were cultured for days in 3D by magnetic levitation. It was possible to grow satisfactory 3D levitated cultures with these cells. This procedure was also successfully performed with primary smooth muscle cells, primary fibroblast cells, glioblastoma cells (LN299) and hepatoma (H4IIE cancer cells, hepatocarcinoma). Therefore, magnetized cells can be prepared in advance for later use. EXAMPLE: CYCLIC TREATMENT
    [0086] [0086] Multiple cell levitation cycles can also be performed, adding cell or cell extracts to existing 3D cultures. In this way, cultures can be enriched for various cells or cellular products as the 3D culture is grown.
    [0087] [0087] In Figure 8, cells (51) are added to a magnetic nanoparticle assembly (53) and the cells and magnetic nanoparticle assembly incubated together (55) to allow the cells to magnetize. Then, the magnetized cells are levitated (59) with the use of a magnet (61). Subsequently, the cells are levitated for 4 hours, overnight, or days (depending on the type of cell), the cells were removed from levitation, the media were replaced with picopure water (73), and the cells were lysed by freeze / thaw action (75) (the cells were placed in liquid nitrogen for at least 10 minutes). Alternatively, the cells can be pulled to the bottom of the culture plate (65) with the use of a magnet (61), where this step can be repeated 1 to 4 times. Then, the cells (67), a cell extract (69) or different cells (71) can be fed back into another sample and the 3D culture process continued. Photographs are not shown, but these procedures have been successfully demonstrated. EXAMPLE: SIZED CULTURES
    [0088] [0088] It has also been shown that the shape of the 3D cell culture can be varied by modifying the magnetic field. The use of a strong magnet that pulls cells to the bottom of the plate creates a well-formed, clearly dense sheet of cells (not shown). This can then be levitated by reversing the magnetic field. 3D cultures have also been created that are thicker on one side by tilting the magnet. The donut-shaped cultures were created using toroidal magnets.
    [0089] [0089] Figure 9a-e, for example, shows few exemplary cell cultures where cells can be compressed more or less by varying the resistance of the magnetic field, or the distance of the magnet from the plate, and where the toroidal shape is achieved with the use of a ring magnet. Figures 10 and 11 show a 3D culture made thicker on one side by tilting the magnetic field.
    [0090] [0090] It was also foreseen that stacking such cell cultures in a blade or screw shape, in this way, eventually creating more complex structures. For example, a tube-like structure could be created by stacking disks. A compartment can be created by stacking disks and then layered blades at one or both ends, and the compartment could be filled with the same or different cell type. In this way, the most complex tissue engineering can be performed.
    [0091] [0091] It is also possible to move the cells using the magnetic field and this movement can affect the properties and composition of the 3D culture. When cells are seeded with the hydrogel (also known as magnetic nanoparticle assembly), the vertical and / or horizontal movement of the magnetic fields, through the movement of permanent magnets or through the variation of the magnetic fields generated from electromagnets, prevents the cells attach directly to the cell culture plate. The frequency of this movement can vary, for example, from 1 Hz (60 times per minute) to 0.001 Hz. In addition, different cell types could interact very differently with a moving gel. Through the combination of vertical and / or horizontal movements, the rigidity of the moving material will additionally dictate the type of cells that would differentially adhere to the moving gel. A variation of this approach could also be achieved by applying an oscillating motion to the magnet. This method is a valuable alternative for preparing, separating and classifying cells for magnetic manipulation, including, but not limited to, 3D cell culture by magnetic levitation. EXAMPLE: SURFACE CONTACT
    [0092] [0092] In the above modalities, the magnetic nanoparticle assembly was mixed with the cells, thus providing a mixture by intense addition of the components. However, it has also been shown that it is not necessary, and that simply being adjacent to the magnetic nanoparticle assembly, the cells will absorb the magnetic nanoparticles. This is the benefit when cells need to be free of materials from the magnetic nanoparticle assembly.
    [0093] [0093] Figure 12 illustrates the method by which the magnetic nanoparticle assembly (200) is exposed to a magnetic field. This has the effect of concentrating or compressing the magnetic nanoparticle assembly to form a denser magnetic film. The cells (400) can be added above the film and will settle naturally through gravity on the film, and their proximity to the film will allow the cells to be magnetized. Then, the cells can be easily levitated and separated from the film, levitated and grown in 3D culture. This concept has been tested with a wide variety of support molecules and has been shown to work.
    [0094] [0094] All compositions and / or methods disclosed and claimed in this document may be made and performed without undue experimentation in the light of the present disclosure. Although the compositions and methods of that invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods and in the steps or sequence of steps of the method described in this document without that departs from the concept, spirit and scope of the invention. More specifically, it will be evident that certain agents that are both chemically and physiologically related can be replaced by the agents described in this document, although the same or similar results are achieved. All such substituents and similar modifications evident to those skilled in the art are intended to be included in the spirit, scope and concept of the invention as defined by the appended claims.
    [0095] (1) Shimizu, K.; Ito, A.; Arinobe, M; Murase, Y.; Iwata, Y.; Narita, Y.; Kagami, H.; Ueda, M.; Honda, H. J Biosci. Bioeng. 2007, 103, 472 a 8. (2) Kosmulski, M; Marcel Dekker: Chemical Propriedades of Material Superfícies: New York, 2001. (3) Mahmoudi, M.; Simchi, A.; Imani, M. J. Iran. Chem. Soc. 2010, 7, S1 a S27. (4) Bacri, J.-C; Perzynski, R.; Salin, D.; Cabuil, V.; Massart, R. J Magn. Magn. Mater. 1990, 55, 27 a 32. (5) Douziech-Eyrolles, L.; Marchais, H.; Herve, K.; Munnier, E.; Souce, M.; Linassier, C; Dubois, P.; Chorapa, I. Int. J. Nanomed. 2007, 2, 541 a 550. (6) Duff, D. G.; Baiker, A.; Edwards, P. P. Langmuir 1993, 9, 2301 a 2309. US2005054101, WO2005010162US2009137018, WO2005003332US2006063252, WO2004083412, WO2004083416WO2010036957[0095] The following references are incorporated by reference in their entirety: (1) Shimizu, K .; Ito, A .; Arinobe, M; Murase, Y .; Iwata, Y .; Narita, Y .; Kagami, H .; Ueda, M .; Honda, H. J Biosci. Bioeng. 2007, 103, 472 to 8. (2) Kosmulski, M; Marcel Dekker: Chemical Properties of Material Surfaces: New York, 2001. (3) Mahmoudi, M .; Simchi, A .; Imani, MJ Iran. Chem. Soc. 2010, 7, S1 to S27. (4) Bacri, J.-C; Perzynski, R .; Salin, D .; Cabuil, V .; Massart, R. J Magn. Magn. Mater. 1990, 55, 27 to 32. (5) Douziech-Eyrolles, L .; Marchais, H .; Herve, K .; Munnier, E .; Souce, M .; Linassier, C; Dubois, P .; Chorapa, I. Int. J. Nanomed. 2007, 2, 541 to 550. (6) Duff, DG; Baiker, A .; Edwards, PP Langmuir 1993, 9, 2301 to 2309. US2005054101, WO2005010162 US2009137018, WO2005003332 US2006063252, WO2004083412, WO2004083416 WO2010036957
    权利要求:
    Claims (9)
    [0001]
    Composition (7) for magnetizing cells, said composition comprising: a) a negatively charged nanoparticle (1); b) a positively charged nanoparticle (3); and c) a support molecule (5), wherein the positively charged nanoparticle (3) is a magnetically responsive element, and wherein said support molecule (5) retains said negatively charged nanoparticle (1) and said positively charged nanoparticle (3) in a mixture by intense addition that forms a fibrous blanket type structure, characterized by the fact that a negatively charged nanoparticle (1) is a gold nanoparticle.
    [0002]
    Composition according to claim 1, characterized by the fact that said positively charged nanoparticle (3) is an iron oxide nanoparticle.
    [0003]
    Composition according to claim 1, characterized by the fact that the support molecule (5) comprises peptides, polysaccharides, nucleic acids, polymers or combinations thereof; and / or in which the support molecule (5) comprises polylysine, fibronectin, collagen, laminin, BSA, hyaluronan, glycosaminoglycan, anionic unsulfated glycosaminoglycan, gelatin, nucleic acid, extracellular matrix proteins, cell extract, antibody, or mixtures or derived therefrom.
    [0004]
    Composition according to claim 1, characterized in that said cells are obtained from animal fluids or tissue, and said support molecule (5) is provided by said fluid tissue.
    [0005]
    Composition, according to claim 1, characterized by the fact that a) the support molecule (5) comprises peptides, polysaccharides, nucleic acids, polymers, polylysine, fibronectin, collagen, laminin, BSA, hyaluronan, glycosaminoglycan, anionic unsulfated glycosaminoglycan, gelatin, nucleic acid, extracellular matrix protein mixtures, antibody , or mixtures or derivatives thereof, and b) said positively charged nanoparticle (3) is an iron oxide nanoparticle.
    [0006]
    Composition characterized by the fact that it comprises magnetized cells that are produced by incubation with the composition as defined in claim 1, wherein said cells are frozen.
    [0007]
    Method for moving cells characterized by the fact that it comprises contacting the cells with the composition as defined in claim 1 and incubating for 1 to 12 hours until the cells become magnetized, and subjecting said magnetized cells to a magnetic field sufficient to move said cells.
    [0008]
    Method according to claim 7, characterized by the fact that said magnetic field is asymmetric, and / or in said cells are in suspension or adhered.
    [0009]
    Method according to claim 7, characterized in that it further comprises washing said cells to remove said composition before subjecting said magnetized cells to a magnetic field sufficient to move said cells.
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    同族专利:
    公开号 | 公开日
    US20150104844A1|2015-04-16|
    ES2657242T3|2018-03-02|
    US8883471B2|2014-11-11|
    BR112012006558A2|2016-04-26|
    JP2013505728A|2013-02-21|
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    WO2011038370A1|2011-03-31|
    EP2480343A1|2012-08-01|
    US9688955B2|2017-06-27|
    EP2480343B1|2017-12-20|
    EP2480343A4|2016-11-30|
    JP5769717B2|2015-08-26|
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    法律状态:
    2016-07-26| B08F| Application fees: dismissal - article 86 of industrial property law|Free format text: REFERENTE A 5A ANUIDADE. |
    2016-09-06| B08G| Application fees: restoration|
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-06-04| B25A| Requested transfer of rights approved|Owner name: GREINER BIO-ONE NORTH AMERICA, INC. (US) |
    2020-12-01| B09A| Decision: intention to grant|
    2021-01-26| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 26/01/2021, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US24584609P| true| 2009-09-25|2009-09-25|
    US61/245,846|2009-09-25|
    PCT/US2010/050445|WO2011038370A1|2009-09-25|2010-09-27|Materials for magnetizing cells and magnetic manipulation|
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