C Basic & Clinical Pharmacology & Toxicology 2006, 98, 395–400.Printed in Denmark . All rights reserved

Copyright C

ISSN 1742-7835

Toxic Effects of Lipid-Mediated Gene Transfer in VentralMesencephalic Explant Cultures

Matthias Bauer1,5, Bjarne Winther Kristensen2, Morten Meyer2, Thomas Gasser3, Hans Ruedi Widmer4, Jens Zimmer2

and Marius Ueffing1

1GSF – National Research Center for Environment and Health, Institute for Human Genetics, Ingolstädter Landstraße1, D-85764 Neuherberg, Germany, 2Department of Anatomy and Neurobiology, University of Southern Denmark,

Odense, Winsløwparken 21, DK-5000 Odense C, Denmark, 3Department of Neurology, University of Tubingen,Hoppe-Seyler Str. 3, D-72076 Tübingen, Germany, 4Department of Neurosurgery, Inselspital, University of Bern,

Freiburgstraße, 3010 Bern, Switzerland and 5Department of Neurology, Technical University of Munich,Nohlstr. 28, 81675 Munchen, Germany

(Received August 12, 2005; Accepted October 26, 2005)

Abstract: Adverse effects of cDNA and oligonucleotide delivery methods have not yet been systematically analyzed. Weintroduce a protocol to monitor toxic effects of two non-viral lipid-based gene delivery protocols using CNS primarytissue. Cell membrane damage was monitored by quantifying cellular uptake of propidium iodide and release of cytosoliclactate dehydrogenase to the culture medium. Using a liposomal transfection reagent, cell membrane damage was alreadyseen 24 hr after transfection. Nestin-positive target cells, which were used as morphological correlate, were severely dimin-ished in some areas of the cultures after liposomal transfection. In contrast, the non-liposomal transfection reagentrevealed no signs of toxicity. This approach provides easily accessible information of transfection-associated toxicity andappears suitable for prescreening of transfection reagents.

Lipid-mediated gene transfer (‘‘transfection’’) to primaryCNS cell cultures is a procedure first introduced by Felg-ner & Ringold (1989). Since then, different transfection re-agents, including liposomes, non-liposomal lipids and acti-vated polyamidoamine dendrimers (Hudde et al. 1999;Mortimer et al. 1999) have been applied to CNS tissue.Liposomal transfection mixtures, including DOSPA/DOPEformulations have been successfully used to transfect vari-ous glioma cell lines (Bell et al. 1998), and shown to targetcells in CNS explant cultures (Bauer et al. 2001). Further-more, non-liposomal transfection reagents have served tointroduce plasmids coding for therapeutic genes into pri-mary CNS tissue, resulting in improved cell survival andfunction of the graft within experimental cell replacementtherapy (Bauer et al. 2000). Along this line, direct injectionof plasmid DNA-liposome complexes into the striato-nigralsystem has been shown to be sufficient to ameliorate symp-toms in parkinsonian rats (Imaoka et al. 1998). More re-cently, non-viral, liposomal and non-liposomal delivery ofoligonucleotides to silence genes by RNA interference(RNAi) have been successfully applied (Davidson & Paul-son 2004; Shoji & Nakashima 2004).

Adverse effects of transfection reagents administered sys-temically are known, e.g. lipid-DNA complexes applied in-travenously or by inhalation via the lungs result in inflam-matory responses, hence diminishing both transfectionyields and transgene expression (Scheule et al. 1997; Tou-

Author for correspondence: Marius Ueffing, GSF – National Re-search Center for Environment and Health, Institute for HumanGenetics, Ingolstädter Landstraße 1, D-85764 Neuherberg, Ger-many (fax π4989 3187 3297, e-mail marius.ueffing/gsf.de).

signant et al. 2000). In contrast, knowledge on toxic effectsand tissue damage following local administration of lipid-DNA complexes to various tissues including brain paren-chyma is thus far limited.

During the process of optimizing transfection efficiencieswe found it necessary to establish an in vitro test system formonitoring both toxic effects and morphological changesafter administration of lipid-DNA complexes. This was car-ried out in organotypic cultures derived from embryonicventral mesencephalon. Since the cellular environment oforganotypic explant cultures closely resembles that foundin vivo, exposure of such cultures to transfection mixturesshould mimic those conditions found after injection in cer-tain brain areas.

In a comparative approach, we analyzed the cytotoxiceffects of a widely used liposomal (lipofectamine) and anon-liposomal (effectene) transfection reagent, suitable todeliver cDNA (Bauer et al. 2000 & 2001) as well as oligonu-cleotides (Krichevsky & Kosik 2002) to primary tissue/cells.We applied these two conceptually different commerciallyavailable transfection reagents on primary mesencephalicorganotypic neuronal cultures as our test system. Thisallowed us to compare reagent-specific toxic effects on pri-mary neuronal target cells, as well as to establish criteriafor the prescreening of reagents for local toxicity and tissuedamage prior to experimental therapeutic in vivo appli-cations of transfection reagents on mesencephalic tissue.

Materials and Methods

Culture system. Ventral mesencephalon from embryonic day 15(E15) Sprague-Dawley rats were isolated and divided into 4 equal-

MATTHIAS BAUER ET AL.396

sized tissue blocks (1 mm¿1.5 mm¿0.5 mm). These were trans-ferred into conical tubes with serum-containing medium consistingof 88.5% RPMI (Gibco), 1.5% glucose (20% solution) and 10%foetal calf serum (Gibco), and then placed in a roller drum withinan incubator at 37 æ (Gähwiler 1981). Tissue samples were grown as‘‘free-floating roller tube’’ (FFRT) cultures as described in detail bySpenger et al. (1994).

Vector and transfection procedure. For transfection experiments, weused a plasmidal vector construct, pCMVeGFP, that codes for en-hanced green fluorescence protein and is driven by a cytomegalo-virus promoter. Plasmid DNA was purified with the EndoFreeTM

Kit (Qiagen) to minimize endotoxine contamination. Optimizationof liposome-mediated transfection was conducted by first com-plexing plasmid DNA (0.1 mg–6 mg) and lipofectamine (Gibco,Karlsruhe, Germany) (0.5 ml–40 ml) in 100 ml Optimem (Gibco),incubating the mixture for 45 min. and then applying it to the cul-tures for 12 hr (final volume 1 ml). Effectene (Qiagen, Hilden, Ger-many) was used as a non-liposomal transfection reagent as pre-viously described (Bauer et al. 2000). Briefly, 2 mg DNA were incu-bated with 16 ml Enhancer (Qiagen) for 5 min., and subsequentlycomplexed with 20 ml effectene in 150 ml Buffer CE (Qiagen) for anadditional 10 min. The mixture was then added into the roller tubecontaining one milliliter medium and incubated for 12 hr. Transfec-tion was done one day after dissection (‘‘day in vitro 1’’) (fig. 1).

Indicators of cell membrane damage: Propidium iodide uptake. Pro-pidium iodide (3,8-diamino-5-(3-(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide, Sigma) is a stable fluorescentdye, which absorbs blue-green light (493 nm) and emits red fluor-escence (630 nm). Being a polar compound, propidium iodide ac-cesses cells via damaged or leaky cell membranes, where upon itinteracts with DNA to yield a bright red fluorescence. Propidiumiodide is non-toxic to neurones and, due to its already mentionedproperties, is a widely used indicator of neuronal membrane integ-rity and cell damage (Pozzo et al. 1994).

In the present study propidium iodide was used to quantify celldegeneration in ventral mesencephalon tissue blocks after exposureto lipid transfection reagents. We utilized the protocol developed byKristensen et al. (1999) and Noraberg et al. (1999), whereby 20 mlof 0.1 mM propidium iodide were added to the culture medium toachieve a final concentration of 2 mM. The propidium iodide uptakein the individual cultures was recorded by fluorescence microscopy(Olympus IMT-2, 4X (Splan FL2)), using a standard rhodaminefilter and a digital camera (Sensys KAF 1400 G2, Photometrics,Tucson, AZ, USA) at ‘‘day in vitro 2, 4 and 6’’ (fig. 1). The digitalphotos were analyzed by densitometry using NIH Image 1.62 imageanalysis program (National Institute of Health, USA) after firstoutlining the culture area.

Lactate dehydrogenase measurement. Cytosolic lactate dehydrogen-ase (LDH), released into the culture medium from dead or degener-

Fig. 1. Flow diagram. DIVΩday in vitro. PIΩpropidium iodide.

ating cells with permeabilized membranes, was analyzed after ‘‘dayin vitro 2, 4 and 6’’ (fig 1.) by spectrophotometry (COBAS MIRA,Roche), according to the method of Vassault (1983). The mediumcollected for LDH determination was always immediately frozenand stored at ª20 æ until analysis. At the start of each session ofsample measurements, a standard absorbance curve was determinedby first measuring the alterations in absorbance of standard LDHsolutions (Boehringer Mannheim). Aliquots of media (20 ml each)were prepared with pyruvate (Sigma) and nicotinamide adenine di-nucleotide (NADH; Sigma) in TBS, and the absorbance (340 nm;37 æ) of the reaction mixture was recorded automatically for a periodof 120 sec., starting with a lag time of 30 sec. The LDH activitywas calculated from the slope of the linear range of the absorbancecurve.

Immunohistochemistry. For immunohistochemical investigations,cultures were fixed for 60 min. in 0.1 M phosphate buffer (PB) con-taining 4% paraformaldehyde. After equilibration in 10% sucrose-PB for 24 hr, cultures were cut in 20 mm slices on a freezing micro-tome. Visualization of eGFP, nestin, microtubule-associated pro-tein-2, NeuN, tyrosine hydroxylase (TH) and glia fibrillary acidicprotein (GFAP) expressing cells was done by immunofluorescence.Sections were washed in 0.1 M phosphate-buffered saline (PBS) for30 min., incubated in PBS containing 0.3% Triton X-100 and 10%horse serum for 60 min., and then incubated with rabbit anti-GFPantibody (1:800, Invitrogen), mouse anti-nestin (1:1000, Pharming-en), mouse anti-MAP2 (1:2000, Sigma), mouse anti-GFAP (1:800,Chemicon), anti-NeuN or mouse anti-TH (1:500, Chemicon) over-night at 4 æ. Sections were washed in PBS and incubated for 2 hr ina biotinylated anti-mouse antibody (1:200, Vector Lab.) and an avi-din-biotinylated horseradish peroxidase complex according to thesuppliers instructions (Vector Lab.). Finally, immunoreactive cellswere visualized by incubation with 0.1% DAB (Pierce) in 0.1 MPB. Sections were dehydrated in ascending alcohol concentrations,cleaned in xylene and cover-slipped. Specificity of immunostainingwas determined by omission of primary or secondary antibodies.

Estimation of transfection efficiency. Transfected GFP-positive cellswere counted by fluorescence microscopy (Zeiss) after blind codingof the transfection method. In order to be included in the GFP-positive cell count, both dense staining and a clearly defined cellbody were required. Estimations of GFP-immunoreactive cells inthe tissue blocks were done as previously described (Meyer et al.1999; Bauer et al. 2000). Cell counts were performed in three, 20mm thin sections spaced equidistant within 360 mm of the centralpart of the explants. The total volume of each culture was estimatedbased on the assumption that the explants were spherically shaped.One individual culture was assumed as one treatment group. Cellcounts were corrected according to Abercrombie’s formula (Ab-ercrombie 1946). Counts are presented as number of cells pervolume.

Statistics. Comparison of two treatment groups (GFP positive cells)was done by means of a t-test for independent means, and compari-son of multiple groups (propidium iodide uptake and LDH concen-tration) was performed by one-way analysis of variance (ANOVA)for independent groups, followed by the Tukey post-hoc test.

Results

Both liposomal (lipofectamine) and non-liposomal (effec-tene) transfection reagents were used for plasmid transfer(‘‘transfection’’) into E15 rat ventral mesencephalon tissue.Transfection protocols were optimized with the aim of max-imizing transfection efficiency for each reagent. In this re-spect, highest transfection rates were obtained when 20 mllipid solution were complexed with 2 mg plasmid DNA, for

397TRANSFECTION OF VENTRAL MESENCEPHALIC TISSUE

Fig. 2. Transfected cells within ventral mesencephalic explant cul-tures. Ventral mesencephalon tissue derived from E15 rat embryoswas transfected with pCMVeGFP at day ion vitro 1. Two mg vectorDNA was complexed with 20 ml lipid solution. Figures show trans-fected cells on the surface of free-floating rooler tube cultures usinglipofectamine as a liposomal (A) and effectene as a non-liposomal(B) transfection reagent (scale bar: 300 mm).

both the liposomal and non-liposomal transfection re-agents. Transfection yields differed markedly, whereby 3.9times higher counts of GFP-positive cells (P�0.05) in effec-tene-transfected (651∫199 cells/mm3, nΩ7) were observed,compared to lipofectamine-transfected ventral mesencepha-lon tissue (168∫94 cells/mm3, nΩ7) (fig. 2). Given a con-stant lipid-DNA ratio, doubling DNA amounts (4 mg) innon-liposome-DNA complexes did not alter transfectionyields (628∫138 cells/mm3, nΩ7), whereas a considerabledecrease in yields was noted for liposome-DNA complexes(33∫12 cells/mm3, nΩ6) (fig. 3). Transfection mixtures withDNA amounts below 1 mg did not result in any detectabletransgene expression in both transfection reagents.

Cellular uptake of propidium iodide was used as an indi-cator of possible cell membrane damage following exposureto the various transfection mixtures. A significant increase ofpropidium iodide uptake with exposure time in the culturemedium was observed in both transfected and non-transfect-ed control groups (P�0.05) (fig. 4). This time-dependent in-crease in PI fluorescence is believed to represent primarilynon-specific staining of viable cells, an effect that has beenalso shown in hippocampal slice cultures (Vornov et al. 1998).With liposomal transfection, when cultures were exposed to

Fig. 3. DNA concentration and transfection yields. DNA was com-plexed 1:10 with either effectene (EFF) or lipofectamine (LFA).Ventral mesencephalon cultures were transfected at day in vitro 1and fixed 5 days post-transfection (day in vitro 5) for evaluation ofGFP-positive cells. (.P�0.05 ANOVA, Tukey Test, 1 mg versus 2 mgand 4 mg; πP�0.05 ANOVA, Tukey Test).

2 mg plasmid DNA complexed with 20 ml lipofectamine, pro-pidium iodide uptake was 2.1 times (889∫72, nΩ12 versus420∫17, nΩ16), 1.9 times (1597∫84, nΩ12 versus 855∫74,nΩ16) and 2.0 times (1772∫68, nΩ12 versus 906∫77, nΩ16)higher than the propidium iodide uptake in non-transfectedcontrol groups measured one day, three and five days posttransfection, respectively (P�0.05) (fig. 4A).

Using non-liposomal transfection, no differences wereseen in cultures transfected with effectene-DNA complexescompared to controls (fig. 4A). Doubling the amount ofeffectene-plasmid DNA did not result in higher propidiumiodide uptake, whereas increasing lipofectamine to 40 mland DNA to 4 mg further increased propidium iodide up-take by up to 42% (data not shown). In separate experi-ments, ventral mesencephalon explants were also exposed toeither liposomes alone, non-liposomal lipids alone or DNAalone, in an attempt to analyze whether the lipids alone ora composite effect of compound mixtures would cause thetoxic effects on the target tissue (fig. 4B). Propidium iodideuptake with effectene or plasmid DNA alone (2 mg) de-veloped as with untreated control cultures (compare fig. 4Aand B). Effectene alone (20 ml) increased propidium iodideuptake slightly and comparable to the slight increase seenwith effectene-DNA mixtures. Lipofectamine alone (20 ml)caused a significant increase in propidium iodide uptakevery similar to that seen with lipofectamine-DNA mixtures(P�0.05) (fig. 4B). This strongly suggests that the liposomeitself is toxic to the primary cells used here.

Spectophotometric analysis of lactate dehydrogenase(LDH), a marker for cell lysis of culture medium from trans-fected and non-transfected cultures showed no significant dif-ferences between treatment groups and controls (fig. 5).

Immunohistochemical analysis of transfected and non-transfected cultures, for markers of mature neurones(MAP-2, NeuN, TH), glia cells (GFAP) and undifferenti-ated neuroepithelial precursor cells (nestin), revealed an in-solated loss of nestin immunoreactivity in 30% of all sec-tions studied when lipofectamine-DNA had been applied (9

MATTHIAS BAUER ET AL.398

Fig. 4. Propidium iodide (PI) uptake in rat ventral mesencephalontissue after lipid-mediated plasmid transfer. E15 rat ventral mesen-cephalon tissue was transfected at day in vitro (DIV) 1 with 2 mgplasmid DNA complexed with a liposomal (lipofectamine, LFA) ora non-liposomal (effectene, EFF) transfection reagent, non-trans-fected cultures served as controls (A). The PI assay was performedone day (DIV 1π1), three days (DIV 1π3) and five days (DIV1π5)post-transfection. Ventral mesencephalon tissue was exposed toLipofectamine (20 ml), effectene (20 ml) or DNA (2 mg) alone for 12hr (B). (*P�0.05 ANOVA, Tukey Test, DIV1π1 versus DIV 1π3and DIV 1π5; .P�0.05 ANOVA, Tukey Test).

out of 30, fig. 6). No decrease of any cellular marker couldbe observed in sections taken from effectene-DNA-exposedcultures or control cultures which suggests that this trans-fection mixture does not impair integrity of the organotypicprimary material.

Discussion

We studied toxicity of two widely used although chemicallydifferent types of transfection reagents in a concentrationrange applicable for achieving optimal transfection ef-ficiencies in the cell system used (Bauer et al. 2000). Themain physico-chemical parameters influencing transfectionefficiencies in various lipid-DNA systems are related to thesize, stability and charge density of the complexes (Ches-

Fig. 5. LDH measurements in the supernatant of rat ventral mesen-cephalon tissue. Medium derived from transfected (effectene (EFF)-DNA and lipofectamine (LFA)-DNA) and from non-transfectedcontrols was collected one day in vitro (DIV1π1), three days(DIV1π3) and five days (DIV1π5) post-transfection (no significantdifferences).

noy & Huang 2000). In addition, there is experimental evi-dence that the mitotic activity of the target cells, incubationtime, as well as the DNA-liposome ratio are crucial factorsthat affect transfection yields in cell lines and primary tissue(McQuillin et al. 1997; Mortimer et al. 1999). Further, ef-fectene-mediated transfection efficiencies are negatively cor-related with increasing lipid to DNA ratios, whereby maxi-mal yields are obtained with 10:1 lipid-DNA ratio and low-est yields with a 50:1 ratio (Bauer et al. 2000). In contrast,liposomal lipofectamine-mediated gene transfer in our cel-lular system were constant with 2 mg DNA and liposome-DNA ratios in a range of 3:1 to 10:1 (data not shown). Thisfinding may be explained by that transfection efficienciesare more uniform within a broader lipid-DNA ratio range

Fig. 6. Nestin-immunoreactive cells in transfected rat ventral mesen-cephalon tissue. Rat VM tissue was exposed to effectene (EFF)-DNA (A, B) and lipofectamine (LFA)-DNA (C,D) complexes oneday post dissection. Ventral mesencephalon tissue was cultured foradditional five days prior to immunohistochemical evaluation (leftpanel low magnification, right panel high magnification; scale bar:50 mm).

399TRANSFECTION OF VENTRAL MESENCEPHALIC TISSUE

with lipofectamine and other liposomal reagents (McQuillinet al. 1997; Bell et al. 1998).

Toxicity is an often overseen but very critical factorwithin non-viral gene delivery protocols. In this study toxic-ity of two transfection methods and their key componentswere analyzed, using propidium iodide-uptake, release ofLDH measurement to the medium and histological evalu-ation. Propidium iodide is a polar compound, which canonly enter cells when cell membranes have lost integrity.The dye has been used as an indicator of neuronal integrityand cell viability (Macklis & Madison 1990). When propidi-um iodide uptake was quantified and compared, using es-tablished standardized protocols in relation to excitotoxicand other toxic damage to brain slice cultures (Noraberg et

al. 1999), cell membrane damage above control was detect-able by increased propidium iodide uptake as early as 1day post-transfection after liposome mediated gene transfer,whereas no transfection related changes in propidium iod-ideuptake were seen in effectene-transfected cultures. Forliposomes, a positive correlation of transfection yields andthe degree of toxicity in glioma cell lines was observed byBell et al. (1998) leading to the proposal that some degree ofcell membrane damage is required for optimal transfection,bearing a fine margin between optimized transfection andcytotoxicity. However, effectene-DNA complexes success-fully transfect cells to a higher degree without causing meas-urable effects on the cell membrane what might be due toa different biophysical mechanism regarding the entry ofthe complexes into the cytosol. Interestingly, our data showthat the liposomal lipid compound alone is sufficient tocause cell membrane damage, pointing to a general adverseeffect of this reagent rather than specific toxicity due to up-take or processing of lipid-DNA complexes with a specificstructure, size or charge.

Immunohistochemical investigations revealed areas with aselective loss of nestin immunoreactive cells, considered asthe main targets of lipid mediated gene transfer (Bauer et al.2000 & 2001), in lipofectamine-transfected cultures. Im-munohistochemical analysis at 3 days post-transfection didshow some fading of nestin immunoreactivity in some cellsbut no total loss as observed 5 days post-transfection wasseen.

There was a tendency of LDH levels to increase in lipo-fectamine-transfected cultures, indicating delayed substan-tial damage of the target cells. Nevertheless, nestin-positivecells represent only a small subset of cells within the or-ganotypic ventral mesencephalon culture and even substan-tial loss of nestin positive cells will be accompanied withonly moderate increase of LDH in the supernatant. Thus,changes in propidium iodide uptake is the earliest and mostsensitive marker to assay transfection-mediated impairmentof cellular integrity in our test system.

Conclusion

We have introduced the use of foetal brain explants in com-bination with markers of cell death for testing the toxicity

of liposomal and non-liposomal transfection reagents.Using standardized protocols to quantify the cellular up-take of propidium iodide and LDH release in the culturemedium impairment of cell membrane integrity was the firstdetectable sign of transfection-associated toxicity, followedby morphological/immunohistochemical changes, whichwere indicators for more extensive and later damage in thissystem. Lactate dehydrogenase measurements seem to be aless sensitive marker for cell-membrane damage in this sys-tem, although including it might be useful to quantify moreextensive membrane-and cell-damage. We anticipate thatthis test system can be more generally applied to get a betterunderstanding of transfection-associated toxicity of differ-ent reagents and to prescreen transfection reagents prior toin vivo applications into the striato-nigral system in thebrain.

AcknowledgementsWe thank D. Lyholmer for technical assistance and Dr.

U. Olazabal for preparing the manuscript. Professor G.W.Bornkamm and Professor T. Meitinger for continuouslysupporting the project. This research was supported byDFG (Nr. UE59/21) and the Danish MRC.

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