BUFFER SOLUTION FOR ELECTROPORATION AND A METHOD COMPRISING THE USE OF THE SAME
A buffer solution for suspending animal or human cells and for dissolving biologically active molecules in order to introduce the biologically active molecules into the cells using electric current. The buffer solution includes HEPES and at least 10 mmol*1 −1 magnesium ions (Mg 2+ ). The buffer solution has a buffer capacity of at least 20 mmol*1 −1 *pH −1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25° C. The buffer solution also has an ionic strength of at least 200 mmol*1 −1 .
1 - 43 . (canceled)
44 . A buffer solution for suspending animal or human cells and for dissolving biologically active molecules in order to introduce said biologically active molecules into the cells using electric current, the buffer solution comprising HEPES and at least 10 mmol*1 −1 magnesium ions (Mg 2+ ),
wherein the buffer solution has a buffer capacity of at least 20 mmol*1 −1 *pH −1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25° C., and an ionic strength of at least 200 mmol*1 −1 .
45 . The buffer solution as recited in claim 44 having a buffer capacity between 22 and 80 mmol*1 −1 *pH −1 .
46 . The buffer solution as recited in claim 44 having a buffer capacity between 40 and 70 mmol*1 −1 *pH −1 .
47 . The buffer solution as recited in claim 44 having an ionic strength between 200 and 500 mmol*1 −1 .
48 . The buffer solution as recited in claim 44 having an ionic strength between 250 and 400 mmol*1 −1 .
49 . The buffer solution as recited in claim 44 comprising at most 20 mmol*1 −1 magnesium ions.
50 . The buffer solution as recited in claim 44 wherein HEPES is provided in a concentration of at least 5 mmol*1 −1 .
51 . The buffer solution as recited in claim 44 further comprising a phosphate buffer.
52 . The buffer solution as recited in claim 51 , wherein the phosphate buffer comprises at least one of Na 2 HPO 4 /NaH 2 PO 4 and K 2 HPO 4 /KH 2 PO 4 .
53 . The buffer solution as recited in claim 51 wherein the phosphate buffer has a concentration of at least 30 mmol*1 −1 .
54 . The buffer solution as recited in claim 44 further comprising at least one of sodium chloride, sodium succinate, mannitol, glucose, sodium lactobionate and peptides.
55 . The buffer solution as recited in claim 44 consisting essentially of:
4-6 mM KCl,
10-20 mM MgCl 2 ,
5-25 mM HEPES, and
120-160 mM Na 2 HPO 4 /NaH 2 PO 4 (at a pH of about 7.2).
56 . The buffer solution as recited in claim 44 consisting essentially of:
4-6 mM KCl,
10-20 mM MgCl 2 ,
5-25 mM HEPES,
50-160 mM Na 2 HPO 4 /NaH 2 PO 4 (at a pH of about 7.2), and
one of 5-100 mM sodium lactobionate, 5-100 mM mannitol, 5-100 mM sodium succinate, and 5-100 mM sodium chloride.
57 . The buffer solution as recited in claim 44 consisting essentially of:
4-6 mM KCl,
10-20 mM MgCl 2 ,
80-100 mM NaCl, 8-12 mM glucose,
0.3-0.5 mM Ca(NO 3 ) 2 ,
20-25 mM HEPES, and
one of 50-100 mM tris/HCl and 30-50 mM Na 2 HPO 4 /NaH 2 PO 4 (at a pH of about 7.2).
58 . A method for introducing biologically active molecules into animal or human cells using an electric current, comprising:
i. suspending the cells and dissolving the biologically active molecules in the buffer solution as recited in claim 44 , and
ii. applying an electric voltage to the suspension.
59 . The method as recited in claim 58 , wherein the applying an electric voltage to the suspension comprises a voltage pulse having a field strength between 2 and 10 kV*cm −1 , a duration of 10 to 200 μs, and a current density of at least 2 A*cm −2 .
60 . The method as recited in claim 58 , wherein the applying an electric voltage comprises a current flow following a high-voltage pulse without interruption, the current flow having a current density of 2 to 14 A*cm −2 , and a duration of 1 to 100 ms.
61 . The method as recited in claim 58 , wherein the biologically active molecules are transfected into a cell nucleus of the animal or human cells.
62 . The method as recited in claim 58 , wherein the biologically active molecules comprise at least one of nucleic acids, proteins and peptides and the animal or human cells include quiescent or dividing cells.
63 . The method as recited in claim 58 , wherein the biologically active molecules comprise at least one of nucleic acids, proteins and peptides and the animal or human cells include primary cells.
64 . The method as recited in claim 62 , wherein the nucleic acids are included in complexes or compounds comprising peptides, proteins or other biologically active molecules.
65 . The method as recited in claim 63 , wherein the nucleic acids are included in complexes or compounds comprising peptides, proteins or other biologically active molecules.
66 . The method as recited in claim 58 , wherein the cells comprise at least one of primary human blood cells, pluripotent precursor cells of human blood, primary human fibroblasts, endothelial cells, muscle cells and melanocytes.