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Improving Electrotransfection Efficiency by Post-Pulse Centrifugation

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Improving Electrotransfection Efficiency by Post-Pulse Centrifugation Gene Therapy (1999) 6, 364–372 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt Improving electrotransfection efficiency by post-pulse centrifugation LH Li1,2, P Ross1 and SW Hui1 1Membrane Biophysics Laboratory, Roswell Park Cancer Institute, Buffalo, NY, USA We have demonstrated that the viability of electro- dorf desktop centrifuge. Pelleting improves the cell viability transfected adherent CHO and suspended NK-L, K-562, over the whole range of the NK-L, K-562, L1210 and MC2 L1210 and MC2 cells is improved if pelleting by centrifug- cell concentrations studied. When this pelleting method is ation is performed immediately after pulsing. The protec- applied to load CHO cells with FITC-dextran (41 000 MW), tion effect on cell viability is cell line- and pellet thickness- not only is the success rate close to 100%, but the growth dependent. For forming CHO cell pellets, centrifugation rate is similar to the control, which is far better than the force (300–13 000 g) and duration are not crucial; about conventional electroporation method. Furthermore, the five to 10 cell layers in the pellet provide the optimal protec- transfection efficiency of the five cell lines in pellet is sig- tion effect. NK-L, K-562, L1210 and MC2 cell pellets are nificantly higher than that in suspension. optimally formed by centrifugation at 13 000 g in an Eppen- Keywords: centrifugation pellet; viability; electroporation; electroloading; electrotransfection Introduction method that can improve cell viability is likely to improve electrotransfection efficiency. Electroporation has become a popular method in biotech- Pelleting by centrifugation effectively reduces extra- nology for transferring genetic materials, as well as other cellular volume between cells and leads to reduced biochemicals, such as foreign proteins and drugs, into erythrocyte electrohemolysis by restricting post-pulse cell 1–3 cells. The advantage of this method is that it can be swelling.10,15 This feature may be used to reduce post- 4,5 applied to almost all cell types. Also, the parameters in pulse cell mortality in cultured cells. Furthermore, post- electroporation are easier to control and the efficiency in pulse pelleting does not interfere with the pulsing pro- 6–9 some cases is higher than those by other methods. cedure. The combination of the post-pulse pelleting tech- Moreover, it avoids biological contamination that poten- nique and the conventional electroporation method may tially exists in virus-induced methods. It offers an attract- improve cell viability, thereby increase electrotransfec- ive way for ex vivo gene delivery if the transfection tion and electroloading efficiency. efficiency can be significantly improved. In this article, we demonstrated that the post-pulse pel- Electroporation includes two major steps. The first step leting technique could improve cell viability of pulsed is the temporary breakdown of the membrane barrier to cultured cells. Five cell lines were studied, an adherent allow uptake of exogenous molecules, such as DNA or cultured cell line CHO cells and four suspension cultured 10,11 other materials, by diffusion or electrophoresis. The cell line, NK-L, K-562, L1210 and MC2 cells. We deter- second step is to allow cell membranes to recover to their mined that the post-pulse pelleting technique improves 7 original impermeable state. Initially, in the reversible the electroloading and electrotransfection efficiency by breakdown step, the higher the pulse energy (including improving cell viability in these five cell lines. pulse duration and pulse strength), the higher the intake by electroloading.12 If the pulse electrical field is too strong, an irreversible breakdown occurs, and the Results efficiency of electrotransfection and electroloading suffers due to the drop of cell viability.13,14 It is found that the Effects of electroporation and pelleting on post-pulse best electrotransfection and electroloading occur when cell viability 8,12 the cell viability is approximately 50%. This viability Our goal is two-fold. The first is to determine if post- problem is especially serious in the application of elec- pulse pelleting can improve the viability of pulsed cul- troporation for gene delivery in some cell lines, for tured cells; if the result is positive, the second goal is to example, natural killer cells and lymphomas. Any apply this technique to improve the efficiency of electro- transfection and electroloading. It is known that post- pulse pelleting can decrease the electrolysis of pulsed Correspondence: SW Hui, Roswell Park Cancer Institute, Elm & Carlton erythrocytes due to the inhibition of colloidal osmotic Streets, Buffalo, NY 14263, USA 10,15 2On leave from: Biomedical Engineering Department, Hunan Medical swelling. The thicker the post-pulse pellet, the lower University, Changsha, PR China the electrolysis. However, the thick pellet also poses a Received 13 March 1998; accepted 18 September 1998 hindrance to the supply of nutrients. Therefore, one may Improving electroporation efficiency by pellet LH Li et al 365 expect that there is an optimal thickness of the pellet for post-pulse viability of cultured cells. To check this idea, the dependence of the viability of the pulsed CHO cells on cell density was examined, as shown in Figure 1a. For cells incubated in pellet, the opti- mal viability occurs in the concentration range of 8 × 106/ml–16 × 106/ml (120 000–240 000 total cells). The viability is very low at cell densities less than 8 × 106/ml, and decreases gradually when cell density exceeds 16 × 106/ml. The decrease of cell viability in pellet with the increase of cell density shows the adverse effect of pellet thickness on cell viability once it exceeds the optimal thickness. For cells incubated in suspension, the viability increases with the increase of the cell density. When the cell density reaches 40 × 106/ml (corresponding to 600 000 total cells), there is little difference in viability between cells incubated in pellet or in suspension. When cell density is low (Ͻ16 × 106/ml), the viability of cells incu- bated in pellet is much higher than that incubated in suspension. To illustrate the advantage of pelleting at low cell den- sity further, we pulsed CHO cells at the highest cell den- sity (80 × 106/ml, corresponding to 1 200 000 cells) and resuspended the pulsed cells in 115 ␮l of culture medium (about eight times dilution, 10 × 106/ml). The diluted sample was divided into two parts, one with 120 000 cells and pelleted, the other with the remaining cells in sus- pension. The resultant viability is shown in Figure 1b. Again, the viability of cells incubated in the post-pulse pellet is much higher than that incubated in suspension. There is no pulse treatment for cells in the control sample. The effect of the pellet thickness on post-pulse cell viability is studied by changing the bottom surface area of the centrifuge chambers. Cylindrical chambers with different flattened bottom surface areas were used to allow the estimation of relative pellet thickness, using the same number of cells. Cells were centrifuged by a table- top centrifuge at 2200 g for 0.5 min (IEC HN-S Centrifuge, Needham Heights, MA, USA). In Figure 1c, the depen- dence of cell viability on relative pellet thickness, defined as cell number per unit bottom surface area, is shown. The shape of the curves in Figure 1c is similar to that in Figure 1a (solid circles), though a different centrifugation force was used. Also, the thickness of the pellet for opti- mal viability occurs at the same value for three chambers with different bottom diameters, 2, 3 and 4.3 mm, respectively. The effect of post-pulse manipulation on cell viability Figure 1 (a) The dependence of CHO cell viability on cell density. CHO was examined. Figure 2 shows the dependence of the cells were pulsed by four 2.5 kV/cm, 400 ␮s pulses. Solid circles represent pulsed cell viability on the delay time between electro- the viability of cells incubated in pellet, while the empty circles represent poration and centrifugation. When cells were centrifuged those incubated in suspension. (b) The dependence of CHO cell viability at a delay time of 5 min or more after pulses, the cell on incubation methods. CHO cells were pulsed by four 2.5 kV/cm, 400 ␮s × 6 viability decreases to almost zero. It is clear that cells pulses in a cell density of 80 10 /ml (1 200 000 total cells), diluted eight times and divided into two parts. Pellet column represents the viability have to be pelleted immediately (usually within 0.5 min) of one part with 120 000 CHO cells incubated in pellet; suspension column after pulses to achieve high cell viability. represents the viability of the other part with 1 000 000 CHO cells incu- Centrifugation time is another concern of how post- bated in suspension. Control column is the viability without pulse appli- pulse centrifugation affects cell viability. As shown in cation. (c) The dependence of CHO cell viability on relative pellet thickness Figure 3, the centrifugation (13 000 g) times, within a 3 to (cell number per unit bottom surface area). CHO cells were pulsed by ␮ 60 s range, does not affect the CHO cell viability if cells four 2.5 kV/ml, 400 s pulses. Circles, squares and triangles represent the cell viability incubated as pellets in cylindrical chambers with 4.3, 3 and are centrifuged immediately after pulses. 2 mm bottom diameter, respectively. Suspension NK-L, K-562, L1210 and MC2 cells were used to test how generally this pelleting method could be applied. Similar advantages in the viability were found for cells incubated in pellet as against those incu- Improving electroporation efficiency by pellet LH Li et al 366 Figure 2 The dependence of CHO cell viability on the delay time between electroporation and centrifugation.
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