USE OF MAGNETIC CELLS TO MANIPULATE NON-MAGNETIC CELLS

Abstract

Methods of using magnetic display cells to replace secondary antibodies and magnetic beads in any cell manipulation methods. Cells displaying ligands for target cells are magnetized, and then used to bind the target cells. The complex can then be collected in a magnetic field, and thereby manipulated according to the application needs.

Claims

1) A method of isolating target cells from a mixture of cells, comprising: a) combining a mixture of cells comprising target cells and non-target cells with magnetized display cells that display one or more ligands on their surfaces, said ligands specific for a binding partner on a surface of said target cells; b) allowing said ligand to bind to said binding partner to produce magnetized display cells bound to said target cells; c) applying a magnetic field to collect said magnetized display cells bound to said target cells; and d) washing away non-target cells, thereby isolating said target cells.

2) The method of claim 1, wherein said ligands comprising antigen binding sites and said binding partner comprises an antigen that is recognized by said antigen binding sites.

3) The method of claim 1, wherein said magnetized display cells are fibroblasts.

4) The method of claim 1, wherein said magnetized display cells are from a fibroblast derived cell line.

5) The method of claim 1, wherein said target cells and said magnetized display cells are human.

6) The method of claim 1, comprising the further step of 3D culturing said magnetized display cells bound to said target cells.

7) The method of claim 1, comprising the further step of 3D culturing said magnetized display cells bound to said target cells in a magnetic field such that said magnetized display cells bound to said target cells are levitated.

8) The method of claim 1, wherein said magnetized display cells are provided by treating cells displaying one or more ligands with magnetic nanoparticles.

9) The method of claim 8, wherein said magnetic nanoparticles are introduced into said cells displaying one or more ligands by blasting, injection, electroporation, magnetic pressure, hydrogels, or cationic liposomes.

10) The method of claim 8, wherein said magnetic nanoparticles are introduced into said cells displaying one or more ligands with a composition comprising: a) a negatively charged nanoparticle; b) a positively charged nanoparticle; and c) a support molecule, d) wherein one of said negatively charged nanoparticle or positively charged nanoparticle is a magnetically responsive element or compound, and wherein said support molecule holds said negatively charged nanoparticle and said positively charged nanoparticle in an intimate admixture forming a fibrous mat-like structure.

11) A method of isolating target cells from a mixture of cells, comprising: a) combining cells displaying one or more antigen binding site(s) on their surfaces with a composition to form magnetized display cells, said composition comprising: i) a negatively charged nanoparticle; ii) a positively charged nanoparticle; and iii) a support molecule; iv) wherein one of said negatively charged nanoparticle or positively charged nanoparticle is a magnetically responsive element or compound, and wherein said support molecule holds said negatively charged nanoparticle and said positively charged nanoparticle in an intimate admixture forming a fibrous mat-like structure; b) combining a mixture of cells comprising target cells and non-target cells with said magnetized display cells; c) allowing said antigen binding site(s) to bind to one or more target cell(s) displaying an antigen that is recognized by said antigen binding site(s); d) applying a magnetic field to collect magnetized display cells bound to said target cells; and e) washing away non-target cells, thereby isolating magnetized display cells bound to said target cells.

12) The method of claim 11, comprising the further step of separating the magnetized display cells from the target cells.

13) The method of claim 11, comprising the further step of 3D culturing said isolating magnetized display cells bound to said target cells in a magnetic field sufficient to levitate said isolating magnetized cells bound to said target cells.

14) The method of claim 11, a) wherein the support molecule comprises peptides, polysaccharides, nucleic acids, polymers, poly-lysine, fibronectin, collagen, laminin, BSA, hyaluronan, glycosaminoglycan, anionic, non-sulfated glycosaminoglycan, gelatin, nucleic acid, extracellular matrix protein mixtures, antibody, or mixtures or derivatives thereof, b) wherein said negatively charged nanoparticle is a gold nanoparticle, and c) wherein said positively charged nanoparticle is an iron oxide nanoparticle.

15) A method of manipulating target cells, comprising: a) combining target cells with magnetized display cells that display one or more ligands on their surfaces, said ligands for specifically binding a target molecule on a surface of said target cells; b) allowing said ligands to bind to said target molecule to produce magnetized display cells bound to said target cells; c) applying a magnetic field to collect said magnetized display cells bound to said target cells, thereby allowing manipulation of said target cells.

16) A magnetic cell complex comprising: a) at least one magnetized display cell displaying at least one ligand on a surface of the at least one magnetized display cell; and b) at least one target cell having at least one binding partner on a surface of the at least one target cell, wherein said at least one ligand on the surfaces of the at least one magnetized display cell binds said binding partner on the surface of the at least one target cell so as to form the magnetic cell complex.

17) The magnetic cell complex of claim 16, wherein said at least one ligand comprises antigen binding site(s) and said at least one binding partner comprises an antigen that is recognized by said antigen binding site(s).

18) The magnetic cell complex of claim 16, wherein the at least one magnetized display cell comprises magnetic nanoparticles.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0117] FIG. 1. Diagram of an expression plasmid for display of scFv on mammalian cells. PCMV cytomegalovirus promoter; Ig SP, murine Ig chain signal peptide; VH, heavy chain variable region; VL, light chain variable region; Linker, a flexible synthetic linker between VH and VL; myc, an epitope tag to measure the scFv expression level; PDGFR, the transmembrane domain of human platelet-derived growth factor receptor; PSV40/ori, SV40 promoter and origin facilitating episomal replication in mammalian cells expressing SV40 large T antigen; Neo/KanR, neomycin- and kanamycin-resistance gene. From Ho, 2009.

[0118] FIG. 2. Schematic illustration of surface display on mammalian cells. An additional 10-amino acid epitope tag (c-myc) was fused to the C-terminus of the scFv (based on the anti-CD22 RFB4 Fv structural model), allowing quantitation of fusion display with mAb 9E10 independent of antigen (CD22) binding. Fusion to the N-terminal portion of the PDGFR transmembrane domain was used to anchor scFv on the mammalian (HEK-293T) cell surface. Ho, 2009.

[0119] FIG. 3. Schematic illustration of general interactions between magnetized display cells and target cells.

[0120] FIG. 3A. Interaction, including protein-protein, lipid-lipid, lipid-protein, electrostatic, Van der Walls. In the case of collecting and assembling cells that exist in low numbers in probed samples, such blood with circulating tumor cells (CTCs) or small tissue biopsy samples. Magnetized display cells can function as supporting or feeding layer to support growth of CTCs or biopsy cells.

[0121] FIG. 3B. Interaction between proteins at cell surface, such antibody-antigen or protein-protein interactions.

[0122] FIG. 3C. Interaction mediated or triggered by adding a coupling or bridging ligand (molecule or protein or metal ion) which will interact with proteins or antibodies present on the surface of both cell types. These molecules could also be protein, DNA in the form of aptamers and/or metal if a chelating polypeptide, such as polyhistidine, mediates the cell-cell interaction.

[0123] FIG. 3D. Similar to C, but here magnetized display cells are incubated with molecules that will be captured by surface protein/antibody, free/unbound molecules are removed, then the magnetized is combined with target cell.

[0124] FIG. 3E. Similar to D, but here target cells are incubated with molecule to be captured by its surface protein/antibody, free/unbound molecules are removed, then magnetized display cells are combined with target cell.

[0125] FIG. 3F. Magnetized display cells are modified with cell surface modifying molecules or polymers, such as extracellular matrix proteins, poly-L-lysine, fibronectin, collagen, laminin, and the like. The added molecules promote interaction between the magnetized display cell with the target cell.

[0126] FIG. 4A-E. Schematic illustration of how to dissociate interaction between magnetized display cells and target cells. Dissociation action includes: A. enzymatic digestion, change in ionic strength, change in media composition, change in pH, change in temperature, surfactant action. B., dissociation by one or more dissociation actions. C. Addition of excess ligand. D. Excess ligand or chelating molecule such as EDTA. E. Dissociation by dissociation action.

[0127] FIG. 5. Experimental layout, where primary tracheal smooth muscle cells (SMC) were magnetized (magnetized display cell) and lung cancer adenocarcinoma A549 cells (target cell) were not magnetized. Tracheal SMC are part of pulmonary/lung tissue system, therefore affinity between SMC and lung cancer adenocarcinoma target cell is expected. Number of cells used were: 50K, 40K, 30K, 20K, 10K, and 0 or no cells, as indicated in the plate array labels.

[0128] FIG. 6. Photomicrographs (2.5 magnification) of cells according to experimental layout shown in FIG. 5 at time 0. Time zero is immediately after the two cell types, one magnetized and the other not, are magnetically aggregated or bioprinted at the bottom of a cell repellent 384-well plate after the two cell types were allowed to interact for 15 to 30 minutes.

[0129] FIG. 7. Photomicrographs (2.5 magnification) of cells according to experimental layout listed in FIG. 5 and shown in FIG. 6 after 24 hours of culturing these cells.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0130] Generally speaking, the invention is a method of using a magnetic display cell in any of the ways that magnetic beads were previously used. The use of cells, instead of artificial beads, means that no polymers or crosslinking agents or activators need ever be used. Additionally, the use of linkers and associated chemistry is no longer needed, since the magnetized cells already display the relevant antigen binding sites on the cell surface. Further, the cells can be retained, and if fibroblasts, including irradiated fibroblasts, can serve as feeder cells for subsequent cultures.

[0131] The targeting sequences preferrably include antigen binding sites, but any cell targeting protein or reagent could be used. As another option, cells that naturally bind to the target cells could be used, e.g., immune cells, cancer cells, smooth muscle cells, edothelial cells, bone cells, epithelial cells, and the like.

[0132] In preferred embodiments, the magnetizing materials include positively and negatively charged nanoparticles, one of which must contain one or more magnetically responsive elements, such as nanosized iron oxide. These nanoparticles are further combined with a polymer, preferably a cellular or synthetic polymer, or other long molecule that acts as a support (herein called a support molecule) for the charged nanoparticles and the cells, holding the nanoparticles in place for their uptake or adsorption by the cells. The inclusion of both positive and negative nanoparticles allows intimate admixing of the nanoparticles and drives the assembly of the three components, thus ensuring even distribution and good uptake. The support molecule intimately combines all three components with the cells in fibrous mat-like structure that allows the cells to take up the magnetically responsive element.

[0133] After a period of incubation, the magnetizing material can be washed away, allowing the cells to be manipulated in a magnetic field. The magnetic nanoparticles are eventually lost from the cells by 8 days, but we now know they are retained by the ECM for the duration of the culture.

[0134] The magnetically responsive element can be any element or molecule that will respond to a magnetic field, e.g., rare earth magnets (e.g., samarium cobalt (SmCo) and neodymium iron boron (NdFeB)), ceramic magnet materials (e.g., strontium ferrite), the magnetic elements (e.g., iron, cobalt, and nickel and their alloys and oxides). Particularly preferred are paramagnetic materials that react to a magnetic field, but are not magnets themselves, as this allows for easier assembly of the materials.

[0135] Preferably, the magnetic field used to levitate such cells or the magnetic ECM is about 300 G-1000 G. However, the field strength varies with both distance from the culture, and with the amount and type of magnetic response element taken up or adsorbed by the cells. Thus, the optimal field strength will vary, but is easily determined empirically.

[0136] The negatively charged nanoparticles include charge stabilized metals (e.g. silver, copper, platinum, palladium), but preferably is a gold nanoparticle.

[0137] The positively charged nanoparticles include surfactant or polymer stabilized or coated alloys and/or oxides (e.g. elementary iron, iron-cobalt, nickel oxide), and preferably is an iron oxide nanoparticle.

[0138] One of the two nanoparticles must be magnetically responsive, but obviously either one (or both) could contain this feature.

[0139] The nanoparticles should have a nano-scale size, and thus are about 50 nm. Size can range, however, between about 5-250 nm, 50-200 nm, 75-150 nm, but they can be smaller or larger or an assembly of nanoparticles, provided only that the size is appropriate to allow entry or adsorption to the cell type in use. Larger particles are less efficient at cell entry. We have shown in other work that there is an upper limit on the effective size of the magnetic nanoparticle, and micrometer size is too big for effectiveness, although some functionality was still observed.

[0140] The support molecule is generally a polymer or other long molecule that serves to hold the nanoparticles and cells together in an intimate admixture, like a tangled felt mat. The support molecule can be positively charged, negatively charged, of mixed charge, or neutral, and can be combinations of more than one support molecule.

[0141] Examples of such support molecules include the natural polymers, such as peptides, polysaccharides, nucleic acids, and the like, but synthetic polymers can also be employed. Particularly preferred support molecules include poly-lysine, fibronectin, collagen, laminin, BSA, hyaluronan, glycosaminoglycan, anionic, non-sulfated glycosaminoglycan, gelatin, nucleic acid, extracellular matrix protein mixtures, matrigel, antibodies, and mixtures and derivatives thereof.

[0142] Generally speaking, the concentration of the support molecule is substantially greater than the concentration of the negatively and positively charged nanoparticles, ranging from 10-1000 fold greater, 20-500, or 50-200 fold greater. However, greater or lesser amounts are possible, depending on what cell type is being used and which support molecule and nanoparticles are being used. The longer the polymer, the less may be needed to form sufficient structure to hold the nanoparticles in place for uptake.

[0143] Generally, the nanoparticles are used in very low concentrations. Concentrations can range between 10.sup.6-10.sup.12 Molar, but are preferably in the nanomolar range, and the support molecule(s) 10.sup.3-10.sup.9 Molar, and are preferably in the micromolar range. At least 1 magnetic nanoparticle is needed per cell, but preferably there are hundreds or thousands as more nanoparticles means a lesser field strength in needed.

[0144] The three components assemble by electrostatic interaction, and thus charged or mixed charge support molecules, such as poly-lysine, are preferred. However, any of the three components can be functionalized, derivatized, or coated so as to further promote interaction of the components and/or the cells. Thus, one or more members can be functionalized, derivatized, or coated with an antibody that e.g., binds to a cell surface antigen. Thus, interactions between the components and/or the cells would be further promoted. Other binding pairs included receptors-ligands, biotin-strepavidin, complementary nucleic acids, wheat germ agglutinin (WGA), sialic acid containing molecules, and the like.

[0145] Coatings can also include protective or passivating coatings, particularly for the nanoparticles, such as PVP, dextran, BSA, PEG, poly-L-lysine and the like. The nanoparticles, especially the nanoparticle that comprises the magnetically responsive element, can be labeled for visualization, e.g., 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 preferred to omit such labels.

[0146] If desired the magnetic nanoparticle assembly can be made free from biological molecules, such as phage or cell products, because support molecules, such as poly-lysine, can easily be made synthetically. Yet all of the components are generally non-toxic, inexpensive or easy to make. Further, the tangled, fibrous, mat- or felt-like structures allows for the incorporation of additional cell support molecules (such as extracellular matrix components) to be included into the nanoparticle magnetic assemblies.

[0147] Magnetizing cells with magnetic nanoparticle assemblies consists of only adding assembly to cells in e.g., regular cell culture media. Cells can be magnetized within minutes from magnetic nanoparticle treatment (5 minutes) and either attached or suspended cells can be treated with magnetic nanoparticle assemblies.

[0148] The magnetized antibody display cells are then incubated with the target cells, such as blood or a tissue homogenate, and the target cell will then bind to the antigen binding sites of the magnetized cells, allowing their collection in a strong magnetic field. These can be washed one or more times if desired. The collected cells can be used as is or can be further cultured before use.

[0149] If it is desired to separate the magnetized display cells from the target cells, some methods are shown in FIG. 4. One could use excess of unbound ligand for competitive binding with cell-cell interactions. Another methods uses enzymes that cleave specific sites of one member of the binding pair, and cell, and such sites could even be engineered into the display molecule. We could also use trypsinization, or general enzymatic digestion with enzyme mixture, such as papain, dispase, collagenase, hyaluronidase, and trypsin, then remove magnetic cells with magnet. A brief temperature shock can also dissociate some interactions. Finally, EDTA could also be added if the cell-cell interaction is mediated by chelating proteins or molecule, such as histidine polypeptide.

[0150] In a preferred embodiment, the magnetized cells are fibroblast cells or cells from a target organ, and a 3D culture is initiated, using the fibroblasts as feeder cells or cells from a target organ. The 3D culture can be initiated with by the application of a magnetic field, which levitates or bioprints the cells, which quickly coalesce into a 3D structure. The 3D culture shape can also be changed by changing the shape of the magnetic field. The final 3D culture can then be use in cell and tissue therapies, and perhaps even in organ transplantation in the future.

[0151] FIG. 5 shows the experimental or plate layout, while FIG. 6 shows the results. FIG. 6 demonstrates that cell type associated with the tissue in which a particular type of cancer grow in vivo, when magnetized can be used to collect and culture the target cancer cells.

[0152] The top four rows and six columns (top-left quadrant) were a mixture of the two cell types, where the total number of cells was kept constant (at 50K cells), but the ratio between the two cell types are varied as indicated. The top four rows were replicas of the conditions set for each column to show reproducibility. Columns seven to twelve and top four rows (top-right quadrant) are the controls for the top-left quadrant, where no target cell (lung adenocarcinoma) is present. The bottom four rows and seven to twelve right columns (bottom-right quadrant) the number for magnetized display cell (SMC) is kept constant at 25K cells per well and the number of target cell (A549 adenocarcinoma) varied from 0 to 50K cells, as indicated. The bottom four rows and first six left columns (bottom-left quadrant) there were no magnetized cells (SMC) and the number of target cell (A549 adenocarcinoma) varied from 0 to 50K cells.

[0153] More specifically, primary tracheal smooth muscle cells (SMC) are magnetized (magnetized display cell) and allowed to interact with target lung cancer adenocarcinoma A549 (target cell) which is not magnetized. Of the displayed culture wells, only the wells where magnetized display cell were presented generated the magnetically bioprinted circular structures (top-left, top-right, and bottom-right quadrants). The wells in bottom left quadrant (bottom four rows and first six left columns), which only target cells were added, cells were dispersed in the well, a circular pattern was not generated because these cells were not magnetized, therefore were not guided into the magnetic circular pattern.

[0154] The protocol for mixing magnetized display cells and nonmagnetized cells is as follows:

[0155] 1. Detach and count both cells types, one to be magnetized (magnetized display cell) and the other non-magnetized (target cell).

[0156] 2. Bring both cells types to a concentration of 1 million per milliliter.

[0157] 3. For the cells to be magnetized, add NanoShuttle at a concentration of 1 L per 10 thousand cells and centrifuge the mixture 3 at 100 G for 5 minutes. Cells are gently resuspended after each centrifugation method. Alternatively, cells can be magnetized overnight incubation of NanoShuttle while in a monolayer culture.

[0158] 4. Add the desired or unknown amount of the nonmagnetized cells to the well.

[0159] 5. Add the desired amount of magnetized cells to the well.

[0160] 6. Add media to the well to bring the total volume to 300 L.

[0161] 7. Let cells interact for 15 minutes with gentle swirling of microwell plate every 3 minuteskeep cells in incubator at 5% CO.sub.2, 37 C.

[0162] 8. Place the plate on the magnet drive for 15 minutes to assemble collected cells together, magnetized and target cells.

[0163] 9. Remove the plate from the magnet drive and incubate at 5% CO.sub.2, 37 C.

[0164] FIG. 7 shows morphological differences between all four quadrants as a result of the different number of cells per well and ratio between magnetized display cells and target cells (non-magnetized). More specifically, top-right quadrant (magnetized display cells only) show distinct morphology, shape, and size relative to the left-top quadrant with the target cell present. More specifically, in the top-left quadrant, the overall sized is kept nearly the same as the number of magnetized display cells decrease from 50K (left wells) to 0K or no cells (right wells), but size remained nearly the same, with the exception of the wells with no magnetized display cells (0K SMC, right wells). This indicates that the magnetized display cell is collecting and facilitating the growth of the target cell.

[0165] Next, in the bottom-right quadrant, where the number of magnetized display cells was kept constant at 25K and the number of target cells (non-magnetized) increased, the 3D structures increased in size as the number of target cells increased. Again, this shows target cells are being collected and facilitated to grow by the magnetized display cells.

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