E¡ect of serum on in vitro electrically mediated gene delivery andexpression in mammalian cells

Christine Delteil, Justin Teissie, Marie-Pierre Rols *IPBS-CNRS (UMR 5089), 205 route de Narbonne, 31077 Toulouse Cedex, France

Received 3 February 2000; received in revised form 11 May 2000; accepted 12 May 2000

Abstract

In many cell systems, electric pulses can efficiently mediate gene transfer with a high level of expression in vitro. In vivoresults have been reported where decrease in efficiency was obtained. The mechanisms involved in the process are unknown.Since, in vivo, the efficiency of non-viral methods of gene transfer is generally limited by the presence of serum, we reporthere the effect of serum on in vitro electrically mediated chinese hamster ovary cell membrane permeabilization, viability,gene transfer and expression. The results indicate that permeabilization and gene transfer are not inhibited by serum. Byacting as a protector of cell viability, serum indeed increases gene transfer and expression. ß 2000 Elsevier Science B.V. Allrights reserved.

Keywords: Electropermeabilization; Electroporation; Gene transfer; Serum; Chinese hamster ovary cell

1. Introduction

The use of in vivo gene delivery methods makes itpossible to deliver a gene in the living host. Despitehigh transfection e¤ciencies, viruses present di¡erentdrawbacks such as toxic^in£ammatory and inducehost^immune responses, cytopathicity or scaling-updi¤culties that limit their therapeutic applications[1]. Optimization of in vivo non-viral delivery meth-ods of DNA therefore represents a challenge. Syn-thetic cationic lipid complexes with DNA have beendeveloped. However, the presence of serum preventsthe use of such complexes [1,2] even though new

conditions could maintain the transfection e¤ciencywhen up to 8% serum is present [3]. Particle bom-bardment is an e¤cient method but it su¡ers from itslimited penetration [4].

An alternative method for gene delivery is the useof electric ¢elds. Since the ¢rst experiments of Neu-mann and collaborators in 1982, electropulsation ofcells in the presence of plasmid DNA has been re-ported to be an e¤cient method to induce in vitrogene transfer and expression [5]. In vivo applicationsof electric ¢elds have been developed, primarily tointroduce anti-tumor drugs into cancer cells on pa-tients, a method called electrochemotherapy [6], andmore recently to get gene expression. Electro-medi-ated gene transfer experiments have been performedin skin [7], in rat liver [8], murine melanoma [9], aswell as in muscle and cardiac tissues [10^12]. Thismethod is of great interest since it allows the target-ing of gene transfer in tissues. Electric pulses are

0005-2736 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 3 6 ( 0 0 ) 0 0 2 3 5 - 2

* Corresponding author. Fax: +33-5-61-17-59-94;E-mail : rols@ipbs.fr

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delivered directly at the site of DNA injection by adirect contact between the tissue and the electrodes,but the molecular processes involved in such phe-nomena remain poorly understood. While the levelof safety is very high, the in vivo use appears re-stricted by their relative low e¤ciency in comparisonwith viral vectors. The e¤ciency of gene transfer andexpression indeed decreases by at least one order ofmagnitude from in vitro to in vivo studies [7^9]. Thisdecrease in transfection e¤ciency can have di¡erentorigins. In particular, the presence of serum has beenshown to strongly a¡ect cationic lipid-mediated genetransfer [1]. Serum could either prevent the free ex-change of macromolecules across the permeabilizedmembrane, a¡ect cell viability or, by a direct inter-action with DNA, a¡ect gene transfer as has beenshown with chemical vectors.

To go further into the mechanism, we examined inthe present study the in£uence of serum on in vitrocell membrane permeabilization, cell viability andtransfection.

2. Materials and methods

2.1. Cell culture and plasmid

Chinese hamster ovary (CHO) cells were used. TheWTT clone which is not strictly anchorage dependentwas selected. It grows at 37³C in monolayers on Petridishes (35 mm in diameter, Nunc, Denmark), but hasbeen adapted for suspension culture under gentle ag-itation (100 rpm) in Eagle's minimum essential me-dium (MEM0111, Eurobio, France) supplementedwith glucose (3.5 g/l), tryptose phosphate (2.95 g/l),sodium bicarbonate (3.5 g/l), vitamins and 8% new-born calf serum. Antibiotics, penicillin (100 U/l) andstreptomycin (100 Wg/ml), and glutamine (0.585 ng/ml) are added extemporally. Cells are maintained inthe exponential growth phase (4U105^1.2U106 cells/ml) by daily dilution of the suspension. Cells in sus-pension have a mean radius equal to 6.5 Wm. Cellsgrown in suspension can be replated readily on Petridishes and kept at 37³C in a 5% CO2 incubator(Jouan, France). The ability of cells in suspensionto replate is evidence of their viability.

Plasmid pUT531 used to transfect cells is apBR322 shuttle vector carrying the L-galactosidase

gene under the control of the SV40 early promoter.It is extracted from Escherichia coli and puri¢ed bystandard procedures (Qiagen plasmid kit).

2.2. Cell electropulsation

The cell electropulsation protocol has been de-scribed elsewhere [13]. Brie£y, cells were centrifugedfor 5 min at 350Ug (1000 rpm, Jouan C500 centri-fuge, France) and resuspended in the `pulsing bu¡er'(10 mM phosphate, 250 mM sucrose, 1 mM MgCl2,pH 7.2, conductivity of 1400 WS) at a concentrationof 107 cells per ml. The low ionic content saline bu¡-er allows the delivery of pulses of long duration (ms)at high voltages (up to 1000 V). One-hundred Wl ofthe cell suspension was put at 21³C between two £atand parallel electrodes seated on the bottom of aculture dish that formed the pulsation chamber.The electrodes were connected to a voltage generatorwhich gave square-wave electric pulses (CNRS cellelectropulser, Jouan, France). The voltage pulseswere monitored with an oscilloscope. The experi-ments were performed under a laminar £ow hood.Ten pulses lasting 5 ms were applied with di¡erent¢eld strengths at a 1 Hz frequency. These long pulseduration, mild electric ¢eld strength conditions leadto the transfer of macromolecules into mammaliancells while preserving their viability [13].

In the case of experiments performed with serum,new-born calf serum was used (Gibco BRL, France).It was added to the cell suspension either beforepulsation or immediately after it at concentrationsranging from 5 to 50% (v/v). To keep constant the¢nal volumes of cell suspension, precise amounts ofpulsing bu¡er were added. Under these conditions,the osmolarity did not change (0.28 Osm/kg withoutserum, 0.30 Osm/kg with 50% serum).

2.3. Electropermeabilization of cells

Electropermeabilization was quanti¢ed by penetra-tion of dyes to which the membrane was normallyimpermeable. Propidium iodide, PI (100 WM in puls-ing bu¡er, P4170 Sigma, USA), was used to monitorpermeabilization. Cells were pulsed, incubated 10 minat room temperature and then tested for permeabili-zation. Pulsed cells were analyzed by £ow cytometry(Becton Dickinson, FACScan, USA). Flow cytomet-

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ry can be used to measure both the percentage of£uorescent cells and the amount of associated £uo-rescence. Laser excitation was 488 nm, £uorescencewas detected above 600 nm.

2.4. Determination of electropulsed cell viability

Cells were pulsed, kept 5 min at room temperatureand then grown (37³C, 5% CO2) in Petri dishes afteradding 2 ml of culture medium. Viability was meas-ured by observing the growth of cells over 24 h bythe crystal violet coloration method [13].

2.5. Electrotransfection procedure

0.8U106 cells were incubated on ice in 100 Wl puls-ing bu¡er containing the plasmid (25 Wg/ml) 10 minbefore electropulsation. They were loaded betweenelectrodes and pulsed as described above (10 times,5 ms, 1 Hz, 0.6 kV/cm) at room temperature. Theywere immediately incubated for 10 min at 37³C. Twoml of culture medium was added and the cells werecultured on dishes for 24 h. As only viable cells areprone to plating, plating e¤ciency gives an easy as-say of cell viability. Plated cells were then tested forthe expression of the L-galactosidase activity as de-scribed in [13]. Cells expressing L-galactosidase ap-peared deep blue. Transfection frequency is de¢nedas the ratio of blue cells over the total number ofsurviving cells. 300^500 cells were observed under aninverted digitized video microscope (Leica, Ger-many). Expression of L-galactosidase was used toevaluate the occurrence of DNA delivery and expres-sion at the single cell level. Detection of the activityis the evidence that at least one copy of plasmidsintroduced into the cell has been expressed.

2.6. Determination of intracellular ATP content

Cells were centrifuged and resuspended in pureDMSO (D2650 Sigma), at a concentration of4U107 cells/ml. After a 4 min incubation at roomtemperature, they were kept at 320³C until ATPwas measured. For this, cells were mixed (10 s, Genie2 vortex, Scienti¢c Industries, USA) and centrifuged.Fifty Wl of supernatant was added to a 250 Wl lucif-erin^luciferase solution (4 mg/ml, L0633 Sigma) (pH7.4). ATP was measured in a luminometer (LKB

1250 Luminometer, Sweden). Calibration of resultswas performed by using ATP standard solutions.

All experiments were repeated at least three timesat 2 or 3 day intervals in order to avoid possible£uctuations due to di¡erent physiological states ofthe cells, mainly due to the age of the culture.

3. Results

E¡ect of DNase activity of serum on DNA. Serumis known to exhibit an intrinsic DNase activity [14].Plasmid DNA was incubated with cells for 10 min onice (i.e. under conditions used in the transfection ex-periments) in the presence or not of serum and ana-lyzed by agarose gel electrophoresis. Plasmid in puls-ing bu¡er was present in supercoiled and relaxed

Fig. 1. E¡ect of serum on the electropermeabilization e¤ciencyof CHO cells, observed by PI uptake. Ten pulses lasting 5 msat a 1 Hz frequency were applied. Serum (5 or 20%) was addedto the cell suspension 10 min before pulsation. PI penetrationwas quanti¢ed by £ow cytometry measurements. (a) representsthe control cells, (b) represents cells in the presence of 5% se-rum, and (R) represents cells in the presence of 20% serum.The percentage of permeabilized cells (A) and the amount of£uorescence associated with this permeabilization (B) are re-ported as a function of electric ¢eld intensity.

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form (55% and 45%, respectively). The proportion ofthe supercoiled form was 55%, 22% and 14% with5%, 20% and 50% serum, respectively; in a corre-sponding way, the relaxed form of the plasmid was45%, 70% and 75% with 5%, 20% and 50% serum,respectively. Linear plasmid was detected; it repre-sented 8% for 5% and 20% serum and 10% for 50%serum. No fragments were visible, a result in agree-ment with previous observations [14]. Under our ex-perimental conditions, plasmid DNA is not dramat-ically degraded by the presence of serum.

E¡ect of serum on cell electropermeabilization.The e¡ect of the electric ¢eld intensity on cell per-meabilization is reported in Fig. 1. As soon as theelectric ¢eld was higher than a threshold of 0.3 kV/cm, membrane permeabilization was detected bothby an increase in the percentage of permeabilizedcells and in the intracellular PI £uorescence. The£uorescence intensity is related to the number ofmolecules incorporated into electropermeabilizedcells. The threshold of 0.3 kV/cm was not a¡ectedby the presence of serum. No signi¢cant di¡erencecould be detected between cells pulsed in the pres-

ence of serum or in pulsing bu¡er alone. A slight butsigni¢cant decrease in e¤ciency was observed for¢eld values between 0.7 and 1 kV/cm only at 20%serum. The e¡ect of the addition sequence of serumand PI to cells was therefore studied at 0.8 kV/cmand 20% serum. The results reported on Fig. 2 showthat serum present during pulsation only slightly de-creased permeabilization by decreasing both the per-centage of permeabilized cells and the number ofelectroloaded molecules. This e¡ect was more pro-nounced when cells were incubated with serum be-fore the mixing with PI. When serum was added justafter pulsation, PI being present during pulsation,the same results were obtained, i.e. a slight decreasein permeabilization e¤ciency. This result is in agree-

Fig. 2. E¡ect of the addition sequence of serum on permeabili-zation of CHO cells. PI £uorescence was the assay of permeabi-lization as in Fig. 1. Serum at 20% was added to the cell sus-pension either: (1) and (2) before pulsation, or (4) afterpulsation. (3) represents the control with no serum. Ten pulseslasting 5 ms at a 1 Hz frequency and 0.8 kV/cm were appliedafter 10 min incubation of the cells. Percentage of permeabi-lized cells (white symbol) and the associated £uorescence inten-sity (dashed symbol) of electroloaded PI are reported. In condi-tion (1), PI was added to the cell suspension 10 min beforeaddition of serum, pulses being immediately applied after serumaddition. In condition (2), PI was added to the cell suspension10 min after the addition of serum, pulses being applied imme-diately after PI addition.

Fig. 3. E¡ect of the presence of serum during electric ¢eld ap-plication on transfection e¤ciency and viability of CHO cells.Increasing percentages of serum were added to the cell suspen-sion already in contact with DNA 10 min before pulsation. Tenpulses lasting 5 ms at a 1 Hz frequency and 0.8 kV/cm wereapplied. Cell viability (A) and transfection e¤ciency (B) weremeasured 24 h later.

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ment with the previous conclusion that penetrationof PI into electropermeabilized cells occurred alsoafter pulsation [13].

E¡ect of serum on ATP leakage. CHO cells havean intracellular concentration of ATP of 1 mM. Elec-tropulsation performed under conditions of genetransfer (10 pulses, 5 ms duration, 0.8 kV/cm inten-sity) induced a decrease of 40% in ATP content ofcells which is in agreement with previous studies [15].In the presence of 20% serum (added before thepulse), the associated ATP leakage was limited to10%.

E¡ect of serum on cell viability, gene transfer andexpression. As shown in Fig. 3, addition of serumbefore the pulses led to an increase in cell viabilityin a dose dependent manner. Whatever the concen-tration used, serum had no signi¢cant e¡ect on celltransfection e¤ciency, when measured by the per-centage of surviving cells expressing the reportergene activity. But, taking into account the positivee¡ect of serum on cell viability, transfection e¤-ciency, i.e. the percentage of total pulsed cells ex-

Fig. 4. E¡ect of the post-pulse addition of serum on transfec-tion e¤ciency and viability of CHO cells. Increasing percen-tages of serum were added to the cell suspension just after pul-sation. Ten pulses lasting 5 ms at a 1 Hz frequency and 0.8kV/cm were applied. Cell viability (A) and transfection e¤-ciency (B) were measured 24 h later.

Fig. 5. E¡ect of the addition sequence of serum on transfectione¤ciency and viability of CHO cells. 20% serum was added tothe cell suspension either: (1) and (2) before pulsation, (3) afterpulsation; (4) represents the control with no serum. Ten pulseslasting 5 ms at a 1 Hz frequency and 0.8 kV/cm were appliedafter a 10 min incubation of the cells on ice. Cell viability (A)and transfection e¤ciency (B) were measured 24 h later. In con-dition (1), DNA was added to the cell suspension 5 min beforeaddition of serum, pulses being applied 5 min later. In condi-tion (2), DNA was added to the cell suspension 5 min after theaddition of serum, pulses being applied 5 min later. In condi-tions 3 and 4, DNA was added 10 min before pulsing.

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pressing the reporter gene activity, was in fact in-creased up to 4-fold. When serum was added justafter electric pulse application, a viability enhance-ment was dependent on serum concentration. Inthose conditions, cell transfection was increased upto3-fold (Fig. 4), and the total number of cells ex-pressing L-galactosidase increased 10 times. Theseresults are in agreement with previous results onmurine myelomonocytic leukemia cells showing that20% of horse serum added after electric ¢eld appli-cation while modestly improving cell survival signi¢-cantly improved cell transfection [16].

The e¡ect of the sequence of addition of plasmidDNA and serum to the cell suspension with respectto pulsation was then tested. DNA must be presentduring pulsation [17]. Results are reported in Fig. 5.Serum induced an increase in cell viability, the serumbeing present during the electric pulses or added aftertheir application. Interestingly, the e¡ect was morepronounced when serum, present during electricpulses, was added before plasmid DNA. When serumpresent during ¢eld application was added after plas-mid DNA addition, viability was increased in a waysimilar to that obtained when serum was added afterpulses application. Transfection e¤ciency was notincreased when serum was present during electric¢eld applications, plasmid DNA being added beforeor after serum addition.

4. Discussion

The present results show that serum has no nega-tive e¡ect on electrically mediated gene transfer andexpression in mammalian cells, while the oppositeobservation was reported for cationic lipids [1,2]. In-deed, serum present during electric pulses has a pos-itive e¡ect on cell viability and does not decrease celltransfection e¤ciency. Moreover, serum added afterelectric pulses induces an increase both in cell viabil-ity and in cell transfection. Such an increase in cellviability could be due to a protection of cells byserum against the free exchange across the membranedue to the associated long-lived permeabilization ofcells. The ATP leakage and the PI penetration wereless pronounced when serum was present. Serum ad-dition after the pulses has a positive e¡ect on celltransfection. In the case of serum added before

pulses, serum as a macromolecule could limit the£ow of exchange of molecules across the permeabi-lized membrane. This could have a negative e¡ect oncell transfection, but this is not the case due to theconcommitant protective e¡ect of the cell viability.In contrast to small size molecules that can di¡useacross the permeabilized membrane both during thepulses and in the minutes following it, the transfer ofplasmid DNA across the permeabilized membrane isonly detected when the plasmid is present during thepulses [17]. This process depended on the physiologyof the cells, the more their viability was preserved themore e¤cient the transfection obtained [15].

Electropermeabilization leads to e¤cient transfec-tion in vitro even in the presence of serum. In vivo,the method has already been used to transfect cells[7^12]. A decrease in transfection e¤ciency by oneorder of magnitude is generally observed eventhough in the liver [8] and melanoma in mice [9]e¤ciencies up to 20% and 5% have been obtained.This decrease in transfection e¤ciency is not due toserum. The sequence of events is : serum bathingcells, DNA addition and pulse application. This isshown in vitro to increase the number of cells ex-pressing L-galactosidase activity. Other factors canbe responsible for the decrease in transfection ob-served in vivo. One can suggest the heterogeneityof the electric properties of tissues leading to a het-erogeneity in the ¢eld strength, the di¡usion of mol-ecules which is slowed down due to steric hindrancesinherent in the cell contacts and the local decrease inDNA concentration due to washing e¡ect when thedelay between plasmid injection and pulse applica-tion is too long [18,19].

Acknowledgements

Thanks are due to Claire Millot for her help in cellculture, Dr. J.L. Rols for his help in rereading themanuscript, the AFM (Association Franc°aise contreles Myopathies) and the Region Midi-Pyrenees fortheir ¢nancial support.

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