AU2007202057B2 - Buffer solution for electroporation - Google Patents

Description

Pool Section 29 Regulation 3.2(2) AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: Buffer solution for electroporation The following statement is a full description of this invention, including the best method of performing it known to us: 1 BUFFER SOLUTION FOR ELECTROPORATION The invention relates to 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. 5 BACKGROUND OF THE INVENTION The introduction of biologically active molecules, such as for example, DNA, RNA or proteins, into living cells is an important instrument for studying biological functions of these molecules. A preferred method for introducing foreign molecules into the cells is electroporation which, unlike other methods, only 10 causes slight permanent changes to the biological structure of the target cell by the transfer reagents. During electroporation the foreign molecules are introduced into the cells from an aqueous solution by a brief current flow wherein the cell membrane is made permeable for the foreign molecules by the action of short electrical pulses. As a result of the "pores" briefly formed in the cell membrane, 15 the biologically active molecules initially enter the cytoplasm in which they can already exert their function to be studied if necessary. In cases where DNA is introduced into eukaryotic cells, this must enter the cell nucleus however, so that it is possible for the genetic information to be expressed. In the case of dividing cells, this can take place during the cell division wherein the DNA passively 20 enters the cell nucleus after the temporary dissolution of the nuclear membrane. In studies of quiescent or weakly dividing cells, for example, primary animal cells, however, the DNA does not enter into the cell nucleus in this way so that corresponding methods cannot be used here or at least are very tedious. Moreover, especially when DNA is introduced into animal cells, so-called 25 transfection, particular problems frequently occur as a result of the lability of the cells, since the survival rate of the cells influences the efficiency of the transfection as an important parameter. PRIOR ART In the past cell culture media (Anderson et al. (1991), J. Biochem. Biophys. 30 Meth. 22, 207) or salt solutions buffered with phosphate or HEPES (Potter et al. (1984), Proc. Nati. Acad. Sci. USA 81, 7161; Fromm et al. (1985), Nature 319, 791) were frequently used to take up the animal cells and DNA molecules. However, non-buffered or weakly buffered mannitol or saccharose solutions were 2 also used during the electroporation of animal cells (Shimizu et al. (1986), Med. Immunol. 11, 105; Riggs et al. (1986), Proc. Nati. Acad. Sci. USA 83, 5602). Such non-buffered or weakly buffered solutions have the disadvantage that their use leads to an increased cell mortality and significantly reduced transfection 5 efficiency (Yan et al. (1998), BioTechniques 24, 590). Yan et al. (1998) thus used a buffer solution for electroporation consisting of 100 mM HEPES, 137 mM NaCl, 4 mM Na 2

HPO

4 and 6 mM dextrose. Smooth muscle cells from human aorta could certainly be successfully transfected in this buffer but the transfection efficiency was only 15% with a survival rate of only 10 10 to 20%. Furthermore, the buffer used by Yan et al., is only optimised for voltage pulses up to 500 V .so that no indication is given as to whether this buffer can also be used for higher voltage pulses such as are required for the direct transfection into the cell nucleus. In any case, however, the transfection efficiencies achieved in this buffer are too low to meet higher demands. 15 In many cases, buffers having a low ionic strength and therefore low conductivity were used in order to avoid cell damage as a result of high currents which was observed when using buffer solutions having high conductivity, especially during the application of longer high-voltage pulses. Friedrich et al., 1998 (Bioelectrochem. and Bioenerg. 47, 103) showed that 20 the current flow during electroporation leads to a change in the pH in the vicinity of the electrodes as a result of electrolysis of the water. This change in the pH causes the release of cytotoxic A13+ ions from the aluminium electrodes of the cuvettes and therefore causes increased cell mortality. The authors here propose a shortening of the pulse duration to increase the transfection efficiency. No 25 information is given on any change to the buffer used. Divalent cations such as Mg 2 + for example, are frequently added to the electroporation buffer. Magnesium ions facilitate the binding of DNA to the surface of the cells and thereby bring about an increased transfection rate. However, this only seems to apply for Mg 2 + concentrations up to 10 mM since at 30 higher concentrations negative effects predominate, such as for example a reduction in the electrophoresis as a result of neutralisation of the charges of the DNA molecules or heating of the buffer as a result of an increase in the 3 conductivity (Xie and Tsong (1993), Biophys. J. 65, 1684); Neumann et al. (1982), EMBO J. 1, 841; Klenchin et al. (1991), Biophys. J. 60, 804). Furthermore, it was proposed that the buffer should be matched with regard to its composition to the intracellular conditions in order to increase the 5 survival rate of the cells. Thus, a buffer having high potassium and low sodium concentrations corresponding to the cytoplasm should be used so as to prevent any collapse of the intracellular Na*/K* ratio as a result of substance exchange via the pores formed in the cell membrane during the electroporation (van den Hoff et al. (1995), Methods in Mol. Biol. 48, Chapter 15, 185-197). However, a 10 reduction in the transfection efficiency was detected for pulses having a field strength higher than 1300 V/cm. All the buffer solutions described so far however have the disadvantage that the transfection efficiencies achieved when using them are relatively low and/or the buffers are not suitable for application during the electroporation of 15 quiescent or weakly dividing cells. DESCRIPTION OF THE INVENTION The object of the invention is thus to provide a buffer solution for electroporation which makes it possible to achieve higher transfection efficiencies with a low cell mortality rate. 20 The object is solved by the buffer solution according to the invention, comprising at least HEPES as buffer substance and having a buffer capacity of at least 20 mmol * 1-1 * pH-' and an ionic strength of at least 200 mmol * 1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25 0C, and further comprising at least 10 mmol * 1-1 magnesium ions (Mg 2 +). Contrary to the findings 25 so far, it has surprisingly been found that Mg 2 + in the buffer according to the invention, even in fairly high concentrations, can bring about an increase in the transfection rate and to a significantly greater extent than could be explained by the simultaneous slight increase in the ionic strength. Preferably, the buffer contains magnesium chloride (MgCl 2 ) and/or magnesium sulphate (MgSO 4 ). With 30 such a buffer solution it is possible to introduce biologically active molecules into animal or human cells by means of electroporation with transfection efficiencies up to 93% and at the same time with low cell mortality. Various cell types can be successfully transfected in this buffer, especially quiescent and weakly dividing 4 cells. Moreover, by using the buffer solution it is possible to use high-voltage pulses having a field strength of at least 2000 V/cm, which allows DNA molecules to be transfected directly into the cell nucleus of primary animal and human cells. A decisive factor for the advantageous properties of the buffer according to 5 the invention is the combination of a high buffer capacity with a high ionic strength. The buffer capacity P describes the quantity of a proteolyte required to change the pH of a buffer solution by one pH unit (P = dC * dpH 1 where dC = added amount of acid or base and dpH = change in the pH). The ionic strength I of a solution can be calculated using the formula I = 0.5 Eci * z2 (ci = ion 10 concentration in mol/l, z = ion charge). The "Java Buffer Calculator" program (Twigger & Beynon, 1998) was used to calculate the ionic strengths of the buffer substances given in this application. In this framework the buffer can be optimised in terms of its composition for the different cell types and requirements. In a preferred embodiment it is provided that the buffer solution has a 15 buffer capacity between 22 and 80 mmol * 1 * pH', preferably between 40 and 70 mmol * 1-1 * pH-. With regard to the ionic strength a range between 200 and 500 mmol * I, preferably between 250 and 400 mmol * 1 has been found to be particularly advantageous. In general, there is thus an optimum in relation to buffer capacity and Ionic strength, depending on the cell type and the other 20 experimental conditions, there being an interaction between these two parameters. In another preferred embodiment it is provided that the buffer solution comprises at most 20 mmol * [' magnesium ions. HEPES may be provided in a concentration of at least 5 mmol * 1'. 25 In an especially advantageous embodiment it is provided that the buffer according to the invention has a lower concentration of potassium ions (K*), preferably 2 to 6 mmol * 1-1 K*, and a higher concentration of sodium ions (Na'), preferably 100 to 150 mmol * 1- Na*, if compared with the cytoplasm of the cells. Thus, the buffer according to the invention is not matched to the intracellular 30 Na*/K* ratio but rather "extracellular" ratios are present in this respect. Surprisingly, however, this has no negative effects on the cells but nevertheless results in a significant increase in the transfection and cell survival rate.

5 It is advantageously provided that the buffer according to the invention contains HEPES and Na 2 HPOSNaH 2

PO

4 as buffer substances. However, Trs/HCI or K 2

HPO

4

KH

2

PO

4 can also be used as additional buffer substances. Phosphate buffer may be provided in a concentration of at least 30 mmol * r 1 , 5 preferably 30 to 200 mmol * 1-', in particular 30 to 160 mmol * 1-1. Furthermore, the buffer may also contain additional components, for example, sodium chloride, sodium succinate, mannitol, glucose, sodium lactobionate and/or peptides. Three groups of buffer systems, each optimised to different cell types, are mentioned below as examples for especially advantageous compositions of the 10 buffer solutions according to the invention. However, other compositions are also possible within the scope of the invention so that these examples should not be understood as a restriction. 1) 4 - 6 mM KCI, 10 - 20 mM MgCl 2 , 5 - 25 mM HEPES and 120 160 mM Na 2 HPOSNaH 2

PO

4 (pH 7.2) 15 2) 4 - 6 mM KC, 10 - 20 mM MgCl 2 , 5 - 25 mM HEPES, 50 - 160 mM Na 2 HPOSNaH 2

PO

4 (pH 7.2) and 5 - 100 mM sodium lactobionate or 5 - 100 mM mannitol or 5 - 100 mM sodium succinate or 5 - 100 mM sodium chloride. 3) 4 - 6 mM KC, 10 - 20 mM MgCl 2 , 80 - 100 mM NaCl, 8 - 12 mM glucose, 0.3 - 0.5 mM Ca(N0 3

)

2 , 20 - 25 mM HEPES and 50 - 100 mM trs/HCI 20 or 30 - 50 mM Na 2

HPO

4 /NaH 2

PO

4 (pH 7.2) The buffer solution according to the invention is especially suitable for carrying out an electroporation method in which biologically active molecules are introduced into the cells by a voltage pulse having a field strength of up to 2 to 10 kV*cm- and a duration of 10 to 200 pis and a current density of at least 2 A*cm- 2 . 25 As a result of the high voltage pulse, it is possible for DNA to be transfected directly into the cell nucleus of animal and human cells with irreversible membrane damage being avoided by the shortness of the pulse. As a result of the shortness of the high-voltage pulse, the high ionic strength or conductivity of the buffer also does not result in any disadvantageous heating of the solution so 30 that in addition to dividing cells, quiescent or weakly dividing cells can also be transfected with high efficiency and low mortality. The interaction of the high ionic strength of the buffer and short high-voltage pulses rather brings about efficient 6 pore opening, wherein the strong buffer effect can also compensate for extreme fluctuations of the pH. A current flow following said high-voltage pulse without interruption, having a current density of 2 to 14 A*cm- 2 , preferably up to 5 A*cm-2, and a duration of 1 5 to 100 ms, preferably up to 50 ms, can also be applied in a particularly advantageous fashion by using the buffer according to the invention in the method. The high buffer capacity of the buffer solution according to the invention reduces any change in the pH in the vicinity of the electrodes during the long lasting second pulse, which contributes to the electrophoresis of the DNA, so that 10 the cell mortality can be effectively reduced. As a result of the method according to the invention, the transfection of biologically active molecules is thus optimised by means of the electric current in the cell nucleus of animal or human cells. In this case, nucleic acids, proteins or peptides can be introduced into quiescent or dividing animal or human cells with a 15 high efficiency. The buffer solution according to the invention also favours the introduction of nucleic acids, proteins or peptides into primary cells by supporting high-voltage pulses. The nucleic acids can also be present in the buffer solution in complexes or in compounds with peptides, proteins or other biologically active molecules. 20 The method according to the invention is especially suitable for the transfection of primary human blood cells, pluripotent precursor cells of human blood, primary human fibroblasts and endothelial cells, as well as muscle cells or melanocytes. The cells produced by the method according to the invention are suitable 25 in an especially advantageous fashion for diagnostic and/or analytical methods and for the production of pharmaceutical products for ex-vivo gene therapy. Table 1 gives specific compositions of buffer solutions according to the invention which are each optimised for different applications or cell types and have proved to be especially advantageous with reference to high transfection 30 efficiencies and reducing cell mortality. However, other compositions may also be . understood within the scope of the invention so that these examples also should not be understood as restrictive.

7 Examples of application for individual buffer solutions are described in detail below with reference to the drawings. DESCRIPTION OF THE DRAWINGS The invention is described in detail below with reference to the drawings. 5 In the figures: Figure 1 is a graphical representation of the transfection efficiency and the survival rate for primary human endothelial cells as a function of the buffer capacity and the ionic strength of the buffer solution; Figure 2 is a graphical representation of the transfection efficiency and the 10 survival rate for primary human lymphocytes as a function of the buffer capacity and ionic strength of the buffer solution; Figure 3 shows a flow-cytometric analysis of primary human fibroblasts transfected with H-2Kk expression vector during which the cells were incubated with a CyS-coupled anti-H-2Kk antibody and analysed using a flow cytometer 15 (FACScalibur, Becton Dickinson) (FL-1, FL-2, FL-3 = fluorescence channel 1, 2, 3; SSC = sideward scatter, FSC = forward scatter); Figure 4 shows a flow-cytometric analysis of human smooth muscle cells transfected with H-2Kk expression vector during which the cells were incubated with a Cy5-coupled anti-H-2Kk antibody and anaysed using a flow cytometer 20 (FACScalibur, Becton Dickinson) (FL-1, FL-2, FL-3 = fluorescence channel 1, 2, 3; SSC = sideward scatter, FSC = forward scatter); Figure 5 shows a flow cytometric analysis of primary human melanocytes transfected with the GFP expression vector during which the cells were analysed using a flow cytometer (FACScalibur, Becton Dickinson) (FL-2, FL-3, FL-4 = 25 fluorescence channel 2, 3, 4; SSC = sideward scatter, FSC = forward scatter); and Figure 6 shows a flow cytometric analysis of the human CML cell line K562 transfected with the GFP expression vector during which the cells were analysed using a flow cytometer (FACScalibur, Becton Dickinson) (FL-2, FL-3, FL-4 = 30 fluorescence channel 2, 3, 4; SSC = sideward scatter, FSC = forward scatter). EXAMPLES The following examples illustrate the invention but should not be regarded as restrictive.

8 Example 1 Transfection efficiency and survival rate of primary human endothelial cells. In order to study the transfection and survival rate of cells during 5 electrotransfection as a function of the buffer capacity and the ionic strength, primary human endothelial cells were transfected with a vector coding for the heavy chain of the mouse MHC class 1 protein H-2K . Respectively 7 x 105 cells with respectively 5 pg vector DNA and the respective electrotransfection buffer were placed at room temperature in a 10 cuvette having an interelectrode gap of 2 mm and transfected by a 1000 V pulse of 100 ps duration. In addition, comparative values were determined using PBS (Phosphate Buffered Saline: 10 mM sodium phosphate, pH 7.2 +-137 mM NaCl, ionic strength = 161.5 mM, buffer capacity = 4.8 mM/pH). Directly after the electrotransfection the cells were washed out of the cuvette using 400 pl of 15 culture medium, incubated for 10 minutes at 37*C and then transferred to a culture dish with pre-heated medium. After incubating for 6 h, the cells were incubated with a Cy5-coupled anti-H-2Kk-antibody and analysed using a flow cytometer (FACScalibur, Becton Dickinson). As can be seen from Figure 1, both the transfection efficiencies and the 20 survival rates increase with increasing buffer capacity and ionic strength. In each case significantly better values could be achieved compared with the PBS comparative solution in which an extremely high mortality rate occurred. In some buffer solutions the mortality rate was so high that these values could not be evaluated statistically. 25 Example 2 Transfection efficiency and survival rate of primary human lymphocytes. Furthermore, the transfection and survival rate of primary human lymphocytes during electrotransfection was studied as a function of the buffer capacity and the ionic strength. 30 For this purpose respectively 5 x 100 cells with respectively 5 pg H-2Kk expression vector-DNA in the respective buffers were placed at room temperature in a cuvette having a 2 mm interelectrode gap and electrotransfected by a 1000 V, 100 ps pulse followed by a current flow having a current density of 4 A*cm-2 9 and 40 ms. In addition, comparative values were determined using PBS (ionic strength = 161.5 mM, buffer capacity = 4.8 mM/PH). The analysis was made after 16 hours. As can be seen from Figure 2, it is clear that the survival rates of the cells 5 increase as the buffer capacity and ionic strength of the buffer solutions used increases. In some cases significantly better values could be achieved compared with the comparative solution (PBS: 40.1% transfected cells 38.3% living cells). Here also the transfection efficiency can thus be increased significantly by the choice of suitable buffer solution according to the invention compared with 10 conventional solutions, such as medium or PBS for example. Example 3 Transfection of primary human fibroblasts. Figure 3 shows an FACScan analysis of primary human fibroblasts transfected with H-2Kk expression vector. 5 x 105 cells with 5 pg vector DNA in 15 buffer 6 from Table 1 were placed at room temperature in a cuvette having a 2 mm interelectrode gap and transfected by a 1000 V, 100 ps pulse followed by a current flow of 6 A*cm 2 and 33 ms. After incubating for 5 h, the cells were incubated with a Cy5-coupled anti-H-2Kk antibody and analysed using a flow cytometer (FACScalibur, Becton Dickinson). The number of dead cells was 20 determined by staining with propidium iodide. 93% of the living cells expressed the H-2Kk antigen which corresponds to a very high transfection efficiency. Example 4 Transfection of human smooth muscle cells. Figure 4 shows an FACScan analysis of human smooth muscle cells 25 transfected with H-2K'1 expression vector. 5 x 10 5 cells with 5 pg vector DNA in buffer 6 from Table 1 were placed at room temperature in a cuvette having a 2 mm interelectrode gap and transfected by a 1000 V, 100 ps pulse followed by a current flow having a current density of 5,6 A*cm 2 and 40 ms. After incubating for 13.5 h the cells were incubated using a Cy5-coupled anti-H-2Kk antibody and 30 analysed using a flow cytometer (FACScalibur, Becton Dickinson). The number of dead cells was determined by staining with propidium iodide. 53.8% of the living cells expressed the H-2Kk antigen which also corresponds to a high transfection efficiency.

10 Example 5 fransfection of primary human melanocytes igure 5 shows an FACScan analysis of primary human melanocytes which were transfected with GFP expression vector. 5 x 105 cells with 5 pg 5 pEGFF -Cl vector (Clontech Lab.) in buffer 40 from Table 1 were placed at room temperature in a cuvette having a 2 mm interelectrode gap and transfected by a 1000 \, 100 ps pulse followed by a current flow having a current density of 6 A*cm 2 and 33 ms. After incubating for 24 h the cells were analysed using a flow cytome ter (FACScalibur, Becton Dickinson). The number of dead cells was 10 determined by staining with propidium iodide. 56.2% of the living cells expressed the GFP which also corresponds to a very high transfection efficiency. Example 6 fransfection of human CML cell line K562 igure 6 shows an FACScan analysis of the human CML cell line K562 15 which was transfected with GFP expression vector. 5 x 105 cells with 5 pg pEGFF -Cl vector (Clontech Lab.) in buffer 42 from Table 1 were placed at room temperature in a cuvette having a 2 mm interelectrode gap and transfected by a 1000 V, 70 ps pulse followed by a current flow having a current density of 4 A*cm 2 and 10 ms. After incubating for 24 h the cells were analysed using a flow 20 cytometer (FACScalibur, Becton Dickinson). The number of dead cells was determined by staining with propidium iodide. 77.7% of the living cells expressed the GFP which also corresponds to a very high transfection efficiency. Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, 25 integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

11 LIST OF ABBREVIATIONS USED The following abbreviations were used in addition to those commonly used in Duden: A Ampere 5 APC Allphycocyanin CD Cluster of differentiation cm Centimetre DNA Deoxyribonucleic acid FACScan Fluorescence activated cell scanning 10 FCS Foetal calf serum FL Fluorescence FSC Forward scatter HEPES N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulphonic acid) ml Millilitre 15 mM Millimolar msec Millisecond PBS Phosphate buffered saline PE Phycoerythrin PerCP Peridinin chlorophyll protein 20 pH negative common logarithm of the hydrogen ion concentration RNA Ribonucleic acid RPMI Rosewell Park Memorial Institute SSC Sideward scatter 25 Tris Tris-(hydroxymethyl)-aminoethane-hydrochloride pg Microgram pl Microlitre psec Microsecond V Volt

Claims (8)

1. 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, comprising at least HEPES as 5 buffer substance and having a buffer capacity of at least 20 mmol * 1- * pH 1 and an ionic strength of at least 200 mmol * 1-1 at a change in the pH from pH 7 to pH 8 and at a temperature of 25 "C, and further comprising at least 10 mmol * 1 magnesium ions (Mg 2 +).

2. The buffer solution according to claim 1 having a buffer capacity (pH 7-8, 10 25 0 C) between 22 and 80 mmol * 1- * p-.

3. The buffer solution according to claim 1 or 2 having a buffer capacity (pH

7-8, 25 0 C) between 40 and 70 mmol * [- * pH~ 1 . 4. The buffer solution according to claim 1, 2 or 3 having an ionic strength between 200 and 500 mmol * 1-. 15 5. The buffer solution according to claim 1, 2, 3 or 4 having an ionic strength between 250 and 400 mmol * [-. 6. The buffer solution according to any one of the claims 1 to 5 comprising at most 20 mmol * 1- magnesium ions. 7. The buffer solution according to any one of the claims 1 to 6 wherein 20 HEPES is provided in a concentration of at least 5 mmol * r 1 .

8. The buffer solution according to any one of the claims 1 to 7 further comprising a phosphate buffer, preferably Na 2 HPO 4 /NaH 2 PO 4 and/or K 2 HPO4/KH 2 PO 4 as buffer substance. 13

9. The buffer solution according to claim 8 wherein the phosphate buffer is provided in a concentration of at least 30 mmol * 1-1, preferably 30 to 200 mmol * [ , in particular 30 to 160 mmol * 1-1.

10. The buffer solution according to any one of the claims 1 to 9 further 5 comprising a concentration of potassium ions (K*) between 2 to 6 mmol * r 1 K*, and a concentration of sodium ions (Na') between 100 to 150 mmol * 1-1 Na*.

11. The buffer solution according to any one of the claims 1 to 10 further comprising additional components, for example, sodium chloride, sodium succinate, mannitol, glucose, sodium lactobionate and/or peptides. 10 12. 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 having a composition substantially as hereinbefore described with reference to any of the examples in Table 1. 15 AMAXA GMBH WATERMARK PATENT & TRADE MARK ATTORNEYS P23334AU01 14 v. p.i p. Ir -Q00~ - 0 000 81100 0 0000 0000o 0 0 0 0 0 wo gist 00 PS 199 2 S1 S 111 S! 000 A 0