Enhanced throughput mineral coatings for optimization of stem cell behavior
Disclosed are methods for cell transfection and regulating cellular behavior. More particularly, the present disclosure relates to methods of non-viral cell transfection and regulating cellular behavior using mineral coatings that allow for the enhanced transfection of cells. The mineral coatings bind polynucleotides and provide a source of calcium and phosphate ions to enhance transfection. The present disclosure also provides a high throughput platform for screening non-viral transfection of cells. The methods of the present disclosure also provide an advantageous polynucleotide delivery platform because the mineral coatings may be deposited on various medical device materials after being specifically developed using the high throughput screening platform.
1. A method of non-viral transfection comprising:
preparing a substrate comprising a mineral coating, wherein the mineral coating is formed by incubating the substrate in a simulated body fluid, wherein the simulated body fluid comprises from about 5 mM to about 12.5 mM calcium ions, from about 2 mM to about 12.5 mM phosphate ions, from about 4 mM to about 100 mM carbonate ions, and a pH of from about 4 to about 7.5;
contacting the substrate comprising the mineral coating with a polynucleotide-lipid complex, wherein the polynucleotide-lipid complex adsorbs to the mineral coating;
contacting a cell with the mineral coating, wherein the cell is transfected with the polynucleotide; and
culturing the cell.
2. The method of claim 1 , wherein the mineral coating comprises a calcium to phosphate ratio of from about 2.5:1 to about 1:1.
3. The method of claim 1 , wherein the substrate further comprises a poly(α-hydroxy ester).
4. The method of claim 3 , wherein the poly(α-hydroxy ester) is selected from the group consisting of poly(L-lactide), poly(lactide-co-glycolide), poly(ϵ-caprolactone), and combinations thereof.
5. The method of claim 1 , further comprising culturing the cell in a polynucleotide release medium, wherein the polynucleotide release medium comprises an ion selected from the group consisting of calcium, phosphate, and combinations thereof, wherein the calcium ion concentration comprises from about 0 mM to about 2.5 mM, and the phosphate ion concentration comprises from about 0 mM to about 1 mM.
6. The method of claim 1 , further comprising culturing the cell in a polynucleotide release medium, wherein the polynucleotide release medium comprises a pH of from about 6.2 to about 7.8.
7. The method of claim 1 , wherein the mineral coating comprises a morphology selected from the group consisting of a spherulitic microstructure, a plate-like microstructure, a net-like microstructure, and combinations thereof.
8. The method of claim 7 , wherein the spherulitic microstructure comprises an average spherulite diameter of from about 2 μm to about 4 μm.
9. The method of claim 1 , wherein the cell is selected from the group consisting of a pluripotent stem cell, a mesenchymal stem cell, and an umbilical vein endothelial cell.
10. The method of claim 1 , wherein the polynucleotide is contacted with the mineral coated substrate by a method selected from pipetting, pouring, and spraying the polynucleotide onto the mineral coated substrate.