Comparison of Various Transfection Methods in Human and Bovine Cultured Cells

Longxun Jin, Daehwan Kim, and Sangho Roh Cellular Reprogramming & Embryo Biotechnology Laboratory, Dental Research Institute and School of Dentistry, Seoul National University, Korea

(received September 15, 2014; revised November 20, 2014; accepted November 24, 2014)

Transfection is a gene delivery tool that is a popular means NeonTM electroporation can be used to deliver foreign genes of manipulating cellular properties, such as induced efficiently in human and bovine somatic cells. pluripotent stem cell (iPSC) generation by reprogramming factors (Yamanaka factors). However, the efficiency of Key words: transfection, magnetofection, non-liposomal transfection needs to be improved. In the present study, three transfection, electroporation transfection protocols - non-liposomal transfection (NLT), magnetofection and electroporation - were compared by analysis of their transfection efficiencies and cell viabilities Introduction using human dental pulp cells (hDPC) and bovine fetal fibroblasts (bFF) as cell sources. Enhanced green fluorescent Viral vectors are commonly used to generate induced protein gene was used as the delivery indicator. For pluripotent stem cells (iPSCs) because of their high efficiency magnetofection, Polymag reagent was administrated. NLT, to introduce foreign DNA into mammalian cells [1-3]. FuGENE-HD and X-treme GENE 9 DNA transfection reagents were used for NLT. For electroporation, the NeonTM However, virus-free and/or integration-free plasmids in and NEPA21TM electroporators were tested. NeonTM combination with non-viral transfection methods are often electroporation showed highest transfection efficiency when recommended due to the concerns of clinical application [4,5]. compared with NLT, magnetofection, and NEPA21TM So far, many scientific research groups have introduced electroporation, with transfection efficiency of about 33% in numerous devices or materials for non-viral transfection hDPC and 50% in bFF, based on viable cell population in methods including diethylaminoethyl-dextran (DEAE-dextran), each cell type. These results suggest that transfection by calcium phosphate, liposome (lipofection), non-liposomal based transfection (NLT) reagent, polymer, magnetic bead and *Correspondence to: Sangho Roh, Cellular Reprogramming and electroporator. Embryo Biotechnology Laboratory, Seoul National University DEAE-dextran and calcium phosphate are the chemicals School of Dentistry 101 Daehak-Ro Jongno-gu Seoul 110-744 that are traditionally used to increase transfection efficiency of Republic of Korea Tel: +82-2-740-8681; Fax: +82-2-745-1907 viral RNA and DNA into mammalian cells [6,7]. Due to their E-mail: [email protected] positively charged property, they can easily bind to DNA, form co-precipitates and ultimately deliver DNA either into This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creati- nucleus or cytoplasm of the cells [8,9]. Liposome is one of vecommons.org/licenses/by-nc/3.0) which permits unrestricted non- the widely used transfection methods, taking advantage of the commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ability to fuse with lipid bilayers of the cells, and this

177 178 Longxun Jin, Daehwan Kim, and Sangho Roh endocytosis-like action can easily uptake foreign DNA and the final vector product is more than twofold longer than the induce the expression of introduced gene [10]. Other than monocistronic vector, often larger than 10 kb, and this liposomal based transfection, recently developed non- reversely decreases transfection efficiency [33]. Therefore the liposomal based transfection reagent also became a potent optimal transfection protocol needs be investigated to elevate transfection tool [11]. Electroporation makes electric field transfection efficiency when delivering longer plasmid DNA which drives negatively charged DNA into targeting cells into cells. Despite a series of successful reports on [12,13]. Among polymers developed, polyethyleneimine (PEI) establishment of iPSCs, low efficiency is still observed in and poly-beta-amino ester are effective “proton sponge” that human or large animal iPSCs when compared with a murine can facilitate the transfection efficiently [14,15]. Using the model. Therefore, an optimal transfection protocol is required magnetic bead is in the limelight among recent transfection to the cells of these species. methods because of its relatively higher transfection efficiency In the present study, the episome-derived enhanced green and less toxic effects when compared with lipofection and fluorescent protein (eGFP) vector was used to transfect bovine NLT methods [16]. For magnetofection, PEI which binds to fetal fibroblasts (bFF) and human dental pulp cells (hDPC) to DNA is coated over magnetic bead [17]. Then magnetic force compare three different transfection protocols, NLT, exerted by magnetic plate attracts magnetic bead-PEI-DNA magnetofection and electroporation by examining their complex into cells [18]. transfection efficiency and cell viability. This may elucidate In general, primary cell cultures are more difficult to be transfection efficacy of larger plasmid when applying to transfected than immortalized cell lines. Recent reports human and bovine somatic cells. suggest that transfection of a specific factor could ameliorate some injured tissues when subsequently transplant the transfected primary cells [19]. Hence, improving transfection Materials and Methods efficiency for cultured primary cells is at the forefront of gene-, cell-based therapy, tissue engineering and regenerative Chemicals and plastic wares medicine [20]. Moreover, there is less information about All inorganic and organic compounds were obtained optimal transfection protocol for bovine primary cultured cells from Life Technologies (Grand Island, NY, USA) unless [21,22]. To increase the transfection efficiency other than indicated in the text. All plastic ware were purchased from viral infection in primary cell cultures, various transfection Corning (One Riverfront Plaza, NY, USA). methods has been developed [23-25]. Primary culture of human and bovine cells Recent advancement of transfection technologies can To isolate human dental pulp tissue, mesiodens (maxillary achieve iPSC generation using a couple of non-viral central supernumerary teeth) were extracted from children at transfection methods above mentioned [4,26]. Due to its high the Department of Pediatric Dentistry in Dental Hospital of efficiency for non-viral plasmid transfection, electroporation Seoul National University according to the guidelines has been applied to iPSC generation from the cells in various provided by ethics committee (S-D20100005). Dental pulp mammalian species [27-29]. Other than electroporation, tissues were dissociated using 1% (w/v) collagenase type I, polymer-based transfection and magnetofection were also 2.4 mg/ml dispase and 0.25% (v/v) trypsin, and then successfully applied to generate iPSCs in mouse model proceeded to whole-tissue culture. After culturing for several [30,31]. In spite of the iPSC's successful application, its days, hDPC can be observed propagating from center of the comparably lower efficiency of virus-free transfection than tissue to the peripheral area. The hDPC were isolated from viral transfection is remained as the biggest hurdle to be two individual children were named to hDPC-1 and hDPC-2, overcome [32]. When generating iPSCs, poor efficiency is respectively. mainly caused by individual co-transfection of several vectors. The bFF were isolated from femoral skins of 60 – 90 days To overcome this problem, a polycistronic vector is cloned old fetuses. Bovine femoral skins were dissociated with and applied by many groups [29]. In this case, however, as 0.25% trypsin for 30 min and dissected using blades. Cells several factors need to be cloned in one polycistronic vector, are well propagated after culturing in dishes. To eliminate Comparison of Various Transfection Methods in Human and Bovine Cultured Cells 179 heterogeneous cell populations, the cells were cultured at least OptiMEM and subsequently mixed with triple volume of for five passages before transfection. Polymag (Chemicell GmbH, Berlin, Germany) for 20 min in room temperature. After replacing culture medium, Plasmid DNA preparation Polymag-DNA complex dissolved in OptiMEM was added to pCXLE episomal DNA (plasmid 27082; Addgene, the cells in culture, then incubated in a humidified 37℃ Cambridge, MA, USA) was obtained from Addgene. Midi incubator. prep was carried out either by using HiSpeed Midi Kit Electroporation was conducted using either NEPATM or (Qiagen, Valencia, CA, USA) or Purelink Maxi Kit. Final NeonTM transfection system. In NEPATM electroporation plasmid DNA concentration was used between 1.0 μg/μl to system, for poring pulse, voltage, length, interval, number, 1.5 μg/μl. decay rate, polarity were set respectively as 150 V, 5 ms, 50 ms, 2, 10%, ‘+’ for hDPC, and 275 V, 1.5 ms, 50 ms, 2, 10% Cell culture and ‘+’ for bFF. For transfer pulse, voltage, length, interval, The bFF were cultured in Dulbecco’s modified Eagle’s number, decay rate, polarity were set respectively as 20 V, 50 medium (DMEM; Welgene, Daegu, Korea), supplemented ms, 50 ms, 5, 40% and ‘+/-’ for both hDPC and bFF. In with 10% fetal bovine serum (FBS; Atlas, Fort Collins, NeonTM electroporation system, voltage and length tested here CO, USA), 100 U/ml penicillin – 100 μg/ml streptomycin were 1,400 V and 20 ms for hDPC, and 1,150 V and 30 ms (Sigma-Aldrich, Yong-in, Korea), 250 ng/ml fungizone. The for bFF, and the pulse was repeated twice in both cell groups. hDPC were cultured in minimum essential medium – alpha (MEM-α), supplemented with 10% FBS, 100 U/ml Fluorescence detection penicillin–100 μg/ml streptomycin. Both bFF and hDPC Fluorescence was observed using fluorescence microscope were thawed at passage 4 and at least re-plated twice (Nikon Eclipse TE2000-U; Nikon, Tokyo, Japan) either on before transfection. Day 1 or Day 2 to verify its GFP expression and cell condition. Cell transfection For NLT and magnetofection, cells were seeded in 6-well Measurement of cell viability after transfection 5 plates at the density of 1.5×10 per well 24 h prior to Control and transfected cells were dissociated and harvested 5 transfection to match the cell density to 2.0×10 approximately on Day 2 using trypsin. Cells were centrifuged and resuspended as in electroporation. Electroporation was performed at the in 1 ml PBS. Then 10 μl samples are mixed with 10 μl trypan 5 density of 2×10 cells per well. Before bFF and hDPC blue. Dead cells which stained by trypan blue were included transfection, medium was suctioned out, washed with PBS for to count potential survived cells. three times and freshly supplied with new medium for 1.8 ml in 6-well plates at least 30 min ago. Three transfection protocols Fluorescence activated cell sorting are as follows. Cells were sorted using fluorescence activated cell sorting For NLT, FuGENE-HD (Promega, Madison, WI, USA) and Calibur (FACS Calibur; BD biosciences, San Jose, CA, USA) X-treme GENE 9 DNA transfection reagent (Roche, to measure transfection efficiency. Cells were all dissociated Mannheim, Germany) were used. The ratio of DNA and using 0.05% trypsin (v/v) and transferred into 15 ml tube. Then reagent were 2 to 8 (FuGENE-HD) and 2 to 6 (X-treme cells were centrifuged for 5 min to form pellets at the bottom GENE 9) in hDPC, and 2 to 7 (FuGENE-HD) and 2 to 6 of the tube. Supernatants are suctioned out and pellets are (X-treme GENE 9) in bFF. First, OptiMEM was aliquoted for re-suspended with PBS. After another 5 min centrifugation and 200 μl in 1.5 ml tube. Then transfection reagent and DNA suction, 2% paraformaldehyde was added into tube to fix cells. mixture were added and mixed vigorously to make a Then centrifugation was performed twice and re-suspension homogeneous solution. After 15 min of incubation at 20-25℃ , using PBS was carried out. For each sample, 10,000 cells were mixture solution was evenly scattered onto cell culture sorted, and gated region was matched to control group. Within medium. gated region, the transfection efficiency was measured For magnetofection, plasmid DNA was dissolved in 200 μl according to FL-1 detected eGFP cell number. 180 Longxun Jin, Daehwan Kim, and Sangho Roh

showed 35.6, 57.0, 7.9 and 25.6% of cell viabilities (Figure Results 4a), and 14.7, 6.7, 8.6 and 33.2% of transfection efficiencies based on viable cell population (Figure 4b), respectively. For the transfection to the hDPC, cells were seeded 24 h On contrary to hDPC, magnetofection was applicable to bFF prior to NLT and magnetofection. Electroporation was as the cells showed a well condition after 2 days of transfection performed immediately after trypsinization. Fluorescence of eGFP was detected 48 h after transfection (Figure 1), and most hDPC were in normal condition and exhibited GFP except magnetofection group which showed large damages with no fluorescence activity (Figure 2). Fluorescence activated cell sorting by SSC and forward scatter (FSC) indicated that the cell condition is mostly defined by cell size and granularity in magnetofection group was also largely deviated from non-transfected control (Figure 3a) whereas most cells were in normal condition when using other transfection methods (Figure 3b, c, d and e). This made exclude magnetofection from the further experiments in hDPC. FuGENE-HD, X-tremeGENE 9, NEPATM and NeonTM

Fig 1. Time course of three transfection methods. NLT and magnetofection are applied 1 day after cell seeding. Electroporation is adopted directly after cell detachment by trypsin treatment. Fluorescence is detected 2 days after transfection.

Fig 3. Cell damage was observed after magnetofection in Fig 2. Morphology and GFP fluorescence of hDPC after hDPC by FACS showing deviated SSC and FSC parameters, magnetofection, NLT or electroporation. NeonTM electroporation different from other transfection methods. (a) Magnetofection. group shows good condition in morphology and the highest (b) NLT (Fugene-HD). (c) NLT (X-treme GENE 9). (d) TM TM GFP-fluorescence expressing cells. Bar, 100 μm. NEPA electroporation. (e) Neon electroporation. Comparison of Various Transfection Methods in Human and Bovine Cultured Cells 181

Fig 4. (a) Viability of hDPC after NLT and electroporation. (b) Transfection efficiency of hDPC after NLT or electroporation. Three replicates. *P<.001.

Fig 6. FACS analysis demonstrates all transfection methods Fig 5. Morphology and GFP fluorescence of bFF after TM were applicable to transfecting bFF. (a) Magnetofection. (b) magnetofection, NLT or electroporation. Neon electroporation NLT (Fugene-HD). (c) NLT (X-treme GENE 9). (d) NEPATM group shows good condition in morphology and the highest electroporation. (e) NeonTM electroporation. GFP-fluorescence expressing cells. Bar, 100 μm. as well as other transfection methods demonstrating c, d and e). theirapplicability either by its cell morphology or GFP After applying these conditions, magnetofection, FuGENE- TM TM fluorescence under microscope (Figure 5). Cell morphology HD, X-tremeGENE 9, NEPA and Neon protocols exhibited analyzed by cell sorter also revealed that the cells in all 82.3, 67.3, 68.9, 24.2 and 29.4% for viabilities (Figure 7a) and experimental groups presented little damages as there was 6.9, 4.2, 8.5, 19.0 and 49.5% for transfection efficiencies limited deviation from non-transfected control (Figure 6a, b, (Figure 7b) respectively. 182 Longxun Jin, Daehwan Kim, and Sangho Roh

The yield of final transfected cells from total cells were calculated and presented in Table 1 and 2. The cells which seeded on Day -1 for magnetofection and NLT are presumed to be proliferated to the density of 2.0×105 on Day 0, and followed by applying doubling time as 20 h for hDPC and 15 h for bFF in the following two days. Hence the number of cells on Day 2 for hDPC is 10.56×105 and 18.36×105 for bFF. The formula for calculating final transfected cells is multiplication of the number of cells on Day 2. The results demonstrates NeonTM transfection system is most suitable transfection method among magnetofection, NLT and electroporation, which could obtain 0.89×105 and 2.67×105 transfected cells after 2 days in culture from initial 2.0×105 cells in hDPC and bFF, respectively.

Discussion

Recently, iPSCs became one of the most important cell sources in studying differentiation and dedifferentiation Fig 7. (a) Viability of bFF after magnetofection, NLT and electroporation. (b) Transfection efficiency of bFF after processes, drug discovery and regenerative medicine by magnetofection, NLT or electroporation. Three replicates. transplanting the cells [34-36]. However, the efficiency of *P<.001. iPSC generation is still low, mainly because of low simultaneous transfection efficacy of several key factors such Table 1. Comparison of transfected cells in hDPC. as Oct4, Sox2, Klf4, C-myc. Viral transfection into Transfection methods Transfected cells [×105] mammalian cells is commonly used to generate iPSCs [1]. Magnetofection Despite its high transfection efficiency, it has several concerns Polymag ND NLT in human medicine such as the possibility of unknown genetic FuGENE-HD 0.55 mutation and tumor formation [37]. To avoid these concerns, X-tremeGENE 9 0.40 virus-free transfection methods are required to apply to a Electroporation human being [38,39]. In this study, NLT, magnetofection and NEPATM 0.07 electroporation methods were compared using hDPC and bFF NeonTM 0.89* as the cell sources. The results show that NeonTM *P<.001. ND: not determined electroporation is the best transfection method when compared with NLT, magnetofection and NEPATM electroporation. Table 2. Comparison of transfected cells in bFF. Magnetofection is a recently introduced gene delivery Transfection methods Transfected cells [×105] technique and is known to be effective in many cell lines Magnetofection [40]. Furthermore, it shortens the duration of transfection with Polymag 1.05 high cell viability [41]. These advantages have been reported NLT FuGENE-HD 0.48 in many previous studies [42]. However, aggravated condition X-tremeGENE 9 1.08 of transfected hDPC with deviated morphology indicates that Electroporation magnetofection is an improper transfection method because it NEPATM 0.84 confers cell damage to hDPC. Although it exhibited NeonTM 2.67* comparably higher cell viability than other methods in bFF, *P<.001. magnetofection is so far behind electroporation in transfection Comparison of Various Transfection Methods in Human and Bovine Cultured Cells 183 efficiency. (MAFRA; 111160-04), Republic of Korea. Lipofection mediated transfection is one of the earliest transfection methods and its partially charged feature enables foreign genes to be delivered into cells [43]. Recently NLT Conflict of interest also became more highlighted than conventional liposome-based transfection reagent [11,44]. In this study, The authors declare that there is no conflict of interest transfection efficiency of NLT turns out to be low in that would prejudice the impartiality of this work. transfecting DNA into hDPC and bFF than NeonTM electroporation method and little difference than NEPATM electroporation. This contrasts the previous report References demonstrating its higher transfection yield than electroporation [45]. There are also some reports suggesting that dental 1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures follicle cells or dental pulp stem cells are able to be by defined factors. Cell. 2006;126:663-676. transfected with other liposome-based transfection reagents, 2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, FuGENE-HD and Fugene 6. However, the efficiencies of Tomoda K, Yamanaka S. 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