MAGNETIC EXTRACELLULAR MATRIX

Abstract

Methods of making and using a magnetic ECM are disclosed. The ECM comprises positively and negatively charged nanoparticles, wherein one of said nanoparticles contains a magnetically responsive element. When the magnetic ECM is seeded with cells, the cells will be magnetized and can be levitated for 3-D cell culture.

Claims

1. A method of preparing tissue for implantation in a patient, said method comprising: a) combining cells with magnetic nanoparticles that are taken up by said cells; b) exposing said cells to a magnetic field such that said cells coalesce and levitate; c) growing said cells to form a 3D cell culture; d) decellularizing said 3D cell culture to make a magnetic ECM; e) seeding said magnetic ECM with allogeneic stem or progenitor cells from a patient; f) growing said seeded ECM in a magnetic field; and g) transplanting the seeded ECM from step f) into said patient.

2. The method of claim 1, wherein said magnetic nanoparticles further comprise: i) a negatively charged nanoparticle; ii) a positively charged nanoparticle; and iii) a support molecule; wherein one of said negatively charged nanoparticle or positively charged nanoparticle contains a magnetically responsive element, and wherein said support molecule holds said negatively charged nanoparticle and said positively charged nanoparticle in an intimate admixture.

3. The method of claim 1, wherein said magnetic nanoparticles are provided by NANOSHUTTLE™.

4. The method of claim 11, wherein said magnetic nanoparticles are provided by blasting, injection, electroporation, magnetic pressure, hydrogels, or cationic liposomes.

5. A method of making a magnetic ECM, said method comprising: a) placing magnetic nanoparticles inside a cell; b) levitating said cell in a magnetic field and growing said levitated cell in said magnetic field into a 3D culture of cells having an ECM until said magnetic nanoparticles are lost from said cells and retained by the ECM; and c) decellularizing said 3D culture of cells using a magnet to levitate and hold said ECM while washing away cell debris to produce a magnetic ECM.

6. A magnetic ECM made by the method of claim 1.

7. A magnetic ECM made by the method of claim 5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1. Schematic of decellularization process using a single cell type.

[0071] FIG. 2. Schematic of decellularization process using a two separate cell types in a sequential process, wherein Cell type A is used to make a 3D culture and thus generate ECM A, which is then used to treat Cell Type B. The final product will be magnetic ECM A further modified with ECM B. Of course, an ECM A & B mixture could also be prepared by co-culturing cells types A and in the initial 3D culture, and such method may be preferred.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0072] Generally speaking, the invention is a magnetic ECM, and methods of making and using same. A magnetic material is used that allows cells to uptake or adsorb magnetically responsive elements, and thus be levitable in cell culture when a magnetic field is applied.

[0073] In preferred embodiments, the magnetic 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 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.

[0074] After a period of incubation, the magnetic 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, allowing the preparation and use of a magnetic ECM. An intact 3D cell culture can be decellularized by any known method, or minced 3D culture fragments can be decellularized, depending on the ultimate use of the ECM. The magnetic ECM can then be seeded with desired cells, such as autologous stem cells, and cultured in a very natural 3D environment.

[0075] 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.

[0076] Preferably, the magnetic field used to levitate such cells or the magnetic ECM is about 300G-1000G. 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 ECM. Thus, the optimal field strength will vary, but is easily determined empirically.

[0077] The negatively charged nanoparticles include charge stabilized metals (e.g. silver, copper, platinum, palladium), but preferably is a gold nanoparticle.

[0078] 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.

[0079] One of the two nanoparticles must be magnetically responsive, but obviously either one (or both) could contain this feature.

[0080] The nanoparticles should have a nano-scale size, and thus are about 100 nm. Size can range, however, between about 5-250 nm, 50-200 nm, 75-150 nm, but they can be smaller or larger, provided only that the size is appropriate to allow entry or adsorption to the cell type in use. 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.

[0081] The “support molecule” is generally a polymer or other long molecule that serves to hold the nanoparticles and cells together in an intimate admixture. The support molecule can be positively charged, negatively charged, of mixed charge, or neutral, and can be combinations of more than one support molecule.

[0082] 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.

[0083] 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.

[0084] Generally, the nanoparticles are used in very low concentrations. Concentrations can range between 10.sup.−12-10.sup.−6 Molar, but are preferably in the nanomolar range, and the support molecule(s) 10.sup.−9-10.sup.−3 Molar, and are preferably in the micromolar range.

[0085] 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.

[0086] Coatings can also include protective or passivating coatings, particularly for the nanoparticles, such as PVP, dextran, BSA, PEG, 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.

[0087] In other embodiments, the compositions include the magnetic ECM alone or together with the cells that will be co-cultured therewith, including, but not limited to, stem or progenitor cells, cancer cells, primary cells, mammalian cells, human cells (particularly autologous cells), cells extracted directly from fresh tissue, bacteria, yeast, plant cells, or mixtures thereof.

[0088] If desired the magnetic nanoparticle assembly can be made free from biological molecules, such as phage or cell products because the 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 fibrous mat like structures allows for the incorporation of additional cell support molecules (such as extracellular matrix components) to be included into the nanoparticle magnetic assemblies.

[0089] Magnetizing cells with magnetic nanoparticle assemblies consists of only adding assembly to cells in 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. Cells can be cultured until a desired size is achieved, and then the 3D culture decellularized to produce magnetic ECM.

[0090] The 3D culture can be initiated with any cells, mixtures of cells or tissue fragments. Cell lines, heterologous or preferrably autologous cells can be used. The resulting magnetic ECM prepared from the 3D cultures can then be reseeded with the desired cell types, particularly with autologous cells from a patient, such that a few cells obtained from a patient can be amplified and grown in a very natural 3D cell culture on an existing ECM matrix. The final 3D culture can then be use in cell and tissue therapies, and perhaps even in organ transplantation in the future.

[0091] For example, ECM can be prepared by 3D culture of various cell lines originating from cardiac tissue and/or various allogenic cardiac cell types, and ECM prepared by the methods of the invention. The ECM can then be seeded with a patient's own fibroblasts, endothelial cells, stem cells, progenitor cells, adult-derived inducible pluripotent stem cells and the like, and treated with the appropriate growth factors to encourage differentiation into cardiomyocytes, vascular smooth muscle, and endothelial cells, etc. The resulting tissue can be implanted and used to repair damaged heart mucle. Because the ECM is grown from more readily available cell types, yet decellularized and seeded by a variety of autologous cells, including some stem and progenitor cells, the method has the advantages of providing readily available and natural ECM that can then be a scaffold for growing rare, but safe, patient-derived cells and thus regrow a viable, safe tissue for implant use.

[0092] In our test experiments, 0.5 ml of NANOSHUTTLE™ was combined with 5 ml of human primary fibroblasts and smooth muscle cells. This mixture was gently mixed by pipette action and allowed to incubate and settle for 5 minutes. The cells were then transferred to 3.5 cm petri dish and levitated with a ring shape neodymium magnet (20×(8.5×4.5)×7 mm; pull strength: 13 lbs). All cells levitated and coalesced into a 3D culture within minutes of applying a magnetic field.

[0093] The cells were then cultured for 4 days in DMEM medium with 10% FBS. After culture, the cells were decellurized according to known techniques (see starred methods below), but using a magnet to levitate and hold the ECM, whilst washing away debri. The final ECM was stained with DAPI, which showed the ECM lacked DNA (not shown), and positively stained for collagen and laminin (not shown).

[0094] A wide variety of ECM preparation techniques that can be used with the invention are listed below: [0095] 1. Freeze at −80° C. and thaw at RT. Repeat the cycle six times. Immerse in 25 mM NH.sub.4OH aqueous solution for 20 min and wash with MilliQ water. [0096] 2. Treat with 0.1% Triton-X100 and 1.5M KCl in 50 mM Tris buffer (pH 8.0) for 6 hr. Wash with 10 mM Tris buffer (pH 8.0) and MilliQ water [0097] 3. Freeze at −80° C. and thaw at RT. Repeat the cycle 6 times. Wash with MilliQ water. [0098] 4. Immerse in 25 mM NH.sub.4OH for 1 h and wash with MilliQ water. [0099] 5. Immerse in 0.1% TX100 in 50 mM Tris buffer (pH 8.0) for 6 h. Wash with 10 mM Tris buffer (pH 8.0) and MilliQ water. [0100] 6. Incubate in 1.5M KCl for 6 h and then in MilliQ water for 6 h. Repeat three times. Wash with MilliQ water. [0101] 7. 6 cycles of freezing at −80° C. and thawing at RT. Three cycles of osmotic shock with 3M NaCl to H.sub.2O. Wash with MilliQ water. [0102] 8. *5× washes with 8 mM CHAPS, 1M NaCl, and 25 mM EDTA in PBS (10 mM at pH 7.4), 10× washes with PBS, incubate overnight at 37° C. with PBS+10% FBS+10% penicillin/streptomycin; incubate tissue with benzonase (nuclease, 90 U/ml, Sigma-Aldritch) at 37° C. for 1 hour; 5× wash with PBS+10% FBS, 10% penicillin/streptomycin; store decellularized ECM at 4° C. in PBS+10% FBS, 10% penicillin/streptomycin [0103] 9. *Rinse tissue fragments 5× with PBS (10 mM pH 7.4), 5× washes in isotonic PBS+1% (w/v) Triton X-100; resuspend by gently pippeting 5×; 5× wash with isotonic PBS+2% (w/v) Triton X100; resuspend by gently pippeting 5×; rock sample overnight with isotonic PBS+3% (w/v) Triton X-100; 5× wash with isotonic PBS+3% (w/v) Triton X-100; 5 min. incubation in 2% Triton between washes; resuspend by using gently pippeting 5×; 10× wash with PBS containing 0.1% SDS (w/v); 10× wash PBS wash, Benzonase treatment as above, wash and store. [0104] 10. Freeze −80° C. 6 h; Thaw in deionized H.sub.2O 8 h; Perfuse deionized H.sub.2O; then PBS 37° C. 2 h; 0.02% Trypsin 0.05% EDTA 0.05% NaN.sub.3 2 h; 4% deoxycholate 2 h; 0.1% Peracetic acid; 4% EtOH 1 h [0105] 11. 10 μM adenosine 15 min; 1% SDS 12 h; 1% Triton X-100 30 min; PBS with antibiotics 124 h [0106] 12. 8 mM CHAPS, 1 M NaCl, 25 mM EDTA 2 h; PBS with antibiotics; 90 U/ml benzonase; PBS with 10% FBS [0107] 13. 0.1% SDS 120 min; 1% Triton X-100 10 min; PBS with antibiotics 72 h [0108] 14. Freeze −80° C. 24 h; 0.02% Trypsin 2 h; 3% Triton X 18-24 h; Peracetic acid 1 h [0109] 15. Freeze −80° C.; 4° C. PBS overnight; 0.01% SDS 24 h; 0.1% SDS 24 h; 1% SDS 24 h; 1% Triton X-100 30 min; 0.1% Peracetic acid 3 h [0110] 16. Wash—PBS & freeze 1 week; Freeze/thaw 24 h; Wash—DMEM 48 h; 11% SDS 5 weeks; DNAase 24 h

[0111] Seeding techniques currently employed in recellularization of ECM include injection of cells into the ECM, low dose seeding by perfusion, and multiple bolus seeding with perfusion. This can also be achieved by simply mixing cells with magnetized ECM. See FIG. 1 showing the magnetization of cells and the growth of a 3D culture, which eventually makes a magnetic ECM that can be decellularized according to known techniques. Although FIG. 1 shows a single cell type being used for 3D culture, a mixture of cell types could also be used herein.

[0112] The process can also be used to enrich or modify the purified magnetized ECM with cells of the same type or different type, respectively, as shown in FIG. 2. This would be achieved by recellularizing the magnetic ECM prepared as in FIG. 1 with cell type B, and then repeating the process to generate a mixed ECM made from two cell types.

[0113] A number of experiments were performed to show the usfeulness of the magnetic ECM and its value in assisting cells to grow in a more robust and natural way. One was a wound healing assay and the other a bone differentiation assay, wherein bone development was indicated by bone alkaline phosphatase level.

[0114] To make a decellularized matrix for the wound healing assay, T75 flasks of HPFs were treated with NanoShuttle™ (8 μL/cm.sup.2 surface area) to incubate overnight. The next day, the HPFs were detached from their substrate and levitated in 6-well plates at a concentration of 2.8 million cells/well in 2 mL of media (DMEM, with 10% fetal bovine serum, FBS, and 1% penicillin/streptomycin, P/S) and cultured in 3D for 2 days.

[0115] Afterwards, the cultures were gradually decellularized with increasing concentrations of Triton X-100 (1%, 2%, 3%) in PBS, with an overnight incubation with 3% Triton X-100. Next, the cultures were washed with 0.1% SDS in PBS, then PBS alone, before being mechanically broken up with pipette action and incubated overnight with 90 U/mL benzoase in PBS with 1% P/S at 37° C. The next day, the decellularized cultures were washed twice with PBS and resuspended in the same volume of PBS as the total volume of NanoShuttle added to the flasks. This provide magnetic ECM from HPF cells (mECM-HPF) for use in the following assays.

[0116] We then looked at the effect of the mECM-HPF on the wound healing ability of NNDFs. T75 flasks of NNDFs were incubated with NanoShuttle™ (8 μL/cm.sup.2 surface area). The next day, the cells were detached from substrate and counted. In a separate 96-well plate resting atop an array of 96 ring-shaped magnets, we added volumes of mECM-HPF in ratios between 0-2:1 with volumes corresponding to the amount of NanoShuttle™ in 200,000 cells. The mECM-HPF was allowed to pattern for 15 min at the bottom well. Next, 200,000 cells were added to each well and patterned for another 15 min. Afterwards, the magnetic field was removed, and the area of the ring was measured over 1400 minutes, then plotted against time and concentration. We found that with increasing concentrations of mECM-HPF, the ring of cells were able to close at a faster rate (Table 1). Thus, magnetic EMC from HPF cells improved the wound healing of NNDFs.

TABLE-US-00002 TABLE 1 Wound Healing of NNDFs Ratio mECM-HPF:NS Rate of ring/wound closure 2.00 1.17 1.67 0.82 1.33 1.59 1.00 0.75 0.67 0.78 0.33 0.58

[0117] Next, we studied whether the magnetic ECM from HPF helped to differentiate NNDFs into a bone phenotype. T75 flasks of NNDFs were treated with either NanoShuttle™ alone, mECM-HPF alone, or 1:1 combined NanoShuttle™: mECM-HPF (8 μL/cm.sup.2 surface area) to incubate overnight. The next day, NNDFs were detached from their substrate and levitated in 24-well plates at a concentration of 200,000 cells/well in 400 μL of media. The NNDFs were further split between cultures with DMEM media, and cultures with osteogenic media (DMEM with 10% FBS, 1% P/S, 10 mM β-glycerolphopshate, 100 nM dexamethasone, 50 μg/mL ascorbic acid).

[0118] Concomitantly, NNDFs treated with NanoShuttle™, and untreated NNDFs were plated in 2D with either media types. The cultures were cultured for 6 days, with media changes every 2 days. After 6 days, the 3D cultures were homogenized in 2% Triton X-100 in PBS, then centrifuged for 15 min at 10,000 g at 4° C. 2D cultures were similarly washed in 2% Triton X-100 in PBS, then centrifuged for 10 min at 2,500 g at 4° C. The supernatants were collected and analyzed using an bone alkaline phosphatase assay kit (SensoLyte®).

[0119] Bone alkaline phosphatase (BAP) is the bone-specific isoform of alkaline phosphatase, and the change of alkaline phosphatase activity is involved in a variety of physiological and pathological events, such as bone development, bone-related diseases, gestation-related diseases and inflammatory bowel diseases. Alkaline phosphatase is also a popular enzyme conjugated with secondary antibody for ELISA. p-Nitrophenyl phosphate (pNPP) is proven to be an effective chromogenic substrate for alkaline phosphate.

[0120] Samples of the supernatants were aliquotted and the alkaline phosphatase substrate, pNPP, was added at a 1:1 concentration. The solutions were mixed and the absorbances of the samples were then read on a spectrophotometer at an absorbance of 405 nm.

[0121] The results are shown in Table 2, and confirm that the addition of mECM-HPF to 3D cultures with osteogenic media increased BAP activity over those 3D cultures with no decellularized matrix, or a 1:1 ratio of NanoShuttle: mECM-HPF and osteogenic media. Thus, the addition of mECM-HPF increased bone development and improved the differentiation of NNDFs towards a bone phenotype in 3D culture.

TABLE-US-00003 TABLE 2 BAP Activity (relative to 2D untreated control without osteogenic media) Osteogenic Media − + 3D +mECM-HPF 0.265 0.402 +NS/mECM-HPF 0.239 0.315 +NS 0.222 0.274 2D +NS 0.805 1.511 untreated 1 1.229

[0122] In future test experiments, we will characterise and evaluate the performance of the magnetic ECM versus cells cultured with Nanoshuttle only. We will assess the efficacy of cells grown in our magnetic ECM against cells treated with Nanoshuttle. This experiment will include determining the yield of levitating cells versus non-levitated when treated with Magnetic ECM vs. Nanoshuttle. Next, we will evaluate the level of inflamatory cytokines, IL-6 and IL-8, in the supernatant of treated and levitated cultures versus the control system consisting of monolayer cultures and cells treated and levitated with Nanoshuttle only.

[0123] We will also investigate the effect of time of levitation on the composition of the magnetic ECM by probing ECM proteins with immunohistochemisty and/or western blotting. We will also characterize the magnetic ECM generated from a single cell type, more than one cell type levitated and co-cultured at the same time, and two cell types where one cell type generates the first batch of magnetic ECM (magnetic ECM A) that is then used to treat a second cell type (magnetic ECM B) which is levitated to further modify the original magnetic ECM (FIG. 2).

[0124] Finally, we will assess whether the magnetic ECM has any effect on regulating differentiation of pre-adipocyte stem cells (3T3 fibroblasts) into adipocytes versus cells treated/levitated with nanoshuttle only and in 2D. This will be accomplished by inducing differentiation into adipocytes of levitated cells (magnetic ECM and NANOSHUTTLE™ treated) and monolayers (magnetic ECM and NANOSHUTTLE™ treated and non-treated). We anticipate that our magnetic ECM will provide an easily obtainable and hospitable environment for the growth and correct differentiation of stem cells to make various tissue types.

[0125] The following reference are incorporated herein in their entirety for any and all purposes. [0126] Lu H., et al., Comparison of decellularization techniques for preparation of extracellular matrix scaffolds derived from three-dimensional cell culture, J Biomed. Mater. Res. 00A:000-000 (2012) (article preview available online). [0127] Reilly, G. C. & Engler, A. J., Intrinsic extracellular matrix properties regulate stem cell differentiation, J. Biomech. 43(1): 55-62(2010). [0128] WO2010036957 & WO2011038370 [0129] WO2006060171