|Year : 2014 | Volume
| Issue : 2 | Page : 164-168
Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture
M Majumdar1, R Ratho1, Y Chawla2, MP Singh1
1 Department of Virology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Hepatology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
|Date of Submission||01-Aug-2013|
|Date of Acceptance||25-Nov-2013|
|Date of Web Publication||2-Apr-2014|
Department of Virology, Post Graduate Institute of Medical Education and Research, Chandigarh
Source of Support: None, Conflict of Interest: None
The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.
Keywords: Centrifugation enhanced transfection, lipofectamine, MTT, PBMCs, RNA interference, siRNA
|How to cite this article:|
Majumdar M, Ratho R, Chawla Y, Singh M P. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture. Indian J Med Microbiol 2014;32:164-8
|How to cite this URL:|
Majumdar M, Ratho R, Chawla Y, Singh M P. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture. Indian J Med Microbiol [serial online] 2014 [cited 2019 Jun 26];32:164-8. Available from: http://www.ijmm.org/text.asp?2014/32/2/164/129806
| ~ Introduction|| |
Transfection is the process of introducing nucleic acids into eukaryotic cells by non-viral methods. It satisfactorily introduces negatively charged molecules (e.g. phosphate backbones of nucleic acids) into a negatively charged cell membrane. By coating over the nucleic acid, the transfecting reagents either neutralise or impart an overall positive charge to the molecule. Transfection through DEAE-dextran , or calcium phosphate coprecipitation is amenable to a small change in pH (±0.1) compromising the efficacy of transfection.  On the other hand Cationic lipids "liposome" refers to lipid bilayers that form colloidal particles in an aqueous medium.  Liposome-mediated delivery offers an added advantage of relatively high efficiency of gene transfer, ability to transfect cell types resistant to calcium phosphate or DEAE-dextran, in vitro and in vivo applications, successful delivery of DNA of various sizes, , delivery of RNA  and delivery of protein.  Direct microinjection  into cultured cells, electroporation using an electrical pulse to form transient pores allowing passage of nucleic acids  and biolistic particle  (particle bombardment) through high-velocity delivery of nucleic acids on micro-projectile needs fine-tuning and optimisation. Requirement of expensive instruments, significant loss of cell viability and involvement of labourious procedures are other added disadvantages.
PBMCs are an important component of immunological compartment of human body, which plays a major role in immunological reactions. Following an infection PBMCs are able to recognise the pathogen-associated molecular patterns and mount an immunological response. PBMCs can be cultured in vitro and has become an essential component towards understanding in vitro immunological responses like cytokine production following stimulation with specific antigens. Based on these implications standardisation of PBMCs transfection experiments to simulate near in vivo situation are of great importance.
Gene transfer to human peripheral blood mononuclear cells (PBMCs) can be very challenging and various approaches have been tried to overcome this problem. Neill et al., coupled polyethylenimine to anti-CD3 antibody and demonstrated it as an efficient transfection agent for gene delivery to human PBMCs. Zhao et al.,  have observed a rapid and durable gene expression post-transfection of green fluorescence protein (GFP) or CD26L in T-cell lymphocytes by electroporation of transcribed mRNA. Similarly Okano et al., have successfully transfected enhanced GFP in human dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs) using mRNA lipofection. In search of a simple and cost-effective technique, in the present study the authors have evaluated the effect of low-speed centrifugation in comparison to conventional chemical transfection for studying transfection efficiency in suspension culture of PBMCs.
| ~ Materials and Methods|| |
PBMC separation and culture
Approximately 5 ml of anti-coagulated (acid citrate dextrose) venous blood was collected from 10 apparently healthy controls after obtaining written informed consent. The blood was double diluted and over-layered on the ficoll-Hypaque (Hi-sep, specific gravity: 1.0770) (Himedia, Mumbai, India) in the ratio of 3:1, followed by centrifugation at 1,600 rpm for 20-30 mins. The PBMCs were carefully taken out from the plasma-ficoll interface, washed three times with RPMI-1640 at 1,600 rpm for 7-10 minutes each. Cell viability was checked by trypan blue (Gibco, Auckland, New Zealand) staining and cells were counted using haemocytometer. Finally cells at a density of 2 × 10 5 were plated in flat-bottomed 96-well tissue culture plates (BD Falcon, NJ, USA) in Opti-MEM ® (Life technologies, NY, USA) reduced-serum medium which is an improved minimal essential medium (MEM) with reduced foetal bovine serum supplementation and without antibiotics was incubated overnight at 37°C in the presence of 5% CO 2 .
Lipofectamine (Lipofectamine ® 2000) (Life technologies) was used as a promising transfecting reagent with high transfection efficiency. , BLOCK-iT™ Fluorescent Oligos (siRNA) were taken as positive controls to detect the transfection efficiencies. BLOCK-iT™ is a fluorescein-labelled, double-stranded RNA duplex containing chemical modifications that enhance the stability and allows assessment of fluorescent signal for a significantly longer time period than is obtained with other unmodified, fluorescently labelled RNA. The sequence of BLOCK-iT™ Fluorescent Oligo (Life technologies) is not homologous to any known gene, ensuring against induction of non-specific cellular events caused by introduction of the Oligo into cells.
Transfection by conventional method
PBMCs were plated overnight before transfection. Different concentrations of BLOCK-iT™ Fluorescent Oligos (20 picomole μl−1 ), i.e.; 20, 40 and 60 picomole were diluted in 50 μl of opti-MEM-reduced serum medium. Also two different concentration of lipofectamine 2000 (1 μg μl−1 ) i.e.; 1 μg and 1.5 μg were added to 50-μl opti-MEM reduced serum medium and incubated at 37°C for 5 min. Diluted siRNA and Lipofectamine-2000 reagent was mixed together and incubated at 37°C for 20 minutes. The siRNA-lipid complexes were added to each well and the plates were incubated at 37°C in a CO 2 incubator. The transfected cells were observed under fluorescent microscope (Nikon, E600, USA) for apple-green fluoresence and studied by flow cytometry for quantitation of transfection 18 and 36 hours post-transfection. (Filter setting for flow cytometric detection: λex = 494 nm, λem = 519 nm). The total cell population under study was put in gate P1, the untransfected cells were gated in P2 and the siRNA-transfected cells were put in gate P3 (Canto, Becton Dickinson, NJ, USA).
Here the transfection method was modified by incorporating a centrifugation step after adding the siRNA-liposomal complex to cells. The plates were centrifuged at 1000 rpm for 30 mins at 37°C followed by incubation at 37°C in 5% CO 2 atmosphere. The transfected cells were observed under fluorescent microscope for apple-green fluorescence and studied by flow cytometry 18-hour post-transfection.
Cytotoxicity of the transfection reagents in conventional and CET method were compared using commercially available MTT Cell Growth Assay Kit (Millipore, MA, USA) according to the manufacturer's instructions. MTT is a colorimetric assay that measures the reduction of MTT by mitochondrial succinate dehydrogenase present in the live cells into an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent and the released formazan reagent is measured spectrophotometrically. Briefly 10 μl of (5 mg ml -1 ) MTT solution was added to each well post-transfection, incubated at 37ºC for 4 hour. This was followed by addition of 100 μl of isopropanol with 0.04-N HCl and mixing thoroughly by repeated pipetting for colour development. The percentage of cell viability was calculated by comparing the appropriate luminescent signal obtained at 570 nm from the transfected cells to the signal obtained with non-transfected control cells multiplied by 100.  Each value represents the mean ± standard deviation from quadruplicates.
Post-transfection cells were qualitatively assessed for red and apple-green fluorescence showing the presence of intact nuclei and siRNA, respectively. The efficiency of siRNA transfection by conventional and centrifugation-enhanced transfection was determined quantitatively by flow cytometry. Untransfected PBMCs served as control in the siRNA transfection experiments.
| ~ Results and Discussions|| |
Qualitative demonstration of siRNA transfection
Using BLOCK-iT™ Fluorescent Oligo and Lipofectamine complex it was qualitatively demonstrated that BLOCK-iT™ Fluorescent Oligo (siRNA) was transfected into the cytoplasm of PBMCs (apple-green fluorescence), counter staining with PI demonstrates the intact nucleus (red fluorescence) [Figure 1].
|Figure 1: Qualitative demonstration of siRNA transfection using BLOCK-iT™ following centrifugation enhanced transfection method. The images show the presence of siRNA (apple green) in the cytoplasm of the transfected cells. The nucleus of the cells is stained with propidium iodide and appears red. (a) luorescent microscopic picture of BLOCK-iT™ Oligos (scrambled siRNA) tranfected PBMCs counterstained with PI under ×10, excited at 536 nm showing nuclear component of the cells (b) The same region under ×10 magnifi cation, excited at 494 nm showing the presence of scrambled siRNA. (c) ×40 magnifi cation of transfected PBMC excited at 536 nm clearly showing the counter stain taken up by the nucleic acid and giving a red fl uorescence. (d) The same region excited at 494 nm showing the presence of transfected siRNA in the cytoplasm. (e, f) Cytoplasmic apple green fl uorescence of tranfected BLOCK-iT™ Oligos at ×20 and ×40, excited at 494 nm without propidium iodide counter staining|
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Quantification of siRNA transfection efficiency of conventional and CET method
Flow cytometry performed after 18 hrs of incubation demonstrated that at 20, 40 and 60-picomole concentration siRNA was incorporated into only 3.7%, 28.3% and 23.7% of cells, respectively. As the percentage efficiency of transfection was not satisfactory in the next set of experiments, we increased the incubation period to 36 hrs. The transfection efficiency with 20, 40 and 60 picomoles of siRNA even after 36 hours of incubation remains comparable showing 17%, 21.2% and 22.2% of transfected cells, respectively [Figure 2]a, 2b]. Following introduction of low-speed centrifugation, flowcytometry suggest that there was significant increase in the percentage of transfected cells using CET method in comparison to conventional transfection method. At a siRNA concentration of 20, 40, 60 picomoles the siRNA was incorporated into 63.1%, 99.8% and 92.6% of cells. On further increasing the siRNA concentration to 100 picomoles the transfection efficiency was reduced to 73.2%. This may have occurred due to improper liposomal: siRNA interaction resulting in reduced liposomal complex formation [Figure 2]c].
|Figure 2: Total cell population after each run was put in gate P1, the control tubes (untransfected cells) were gated in P2. Test samples were run and transfected population shows a shift along the X-axis and gated in P3. (a) 1st and 2nd row represents the transfection using 1 µg and 1.5 µg of Lipofectamine-2000 and 20, 40 and 60 picomoles of siRNA, respectively, 18 hrs post-transfection. (b) 36 hrs post-transfection. (c) Represents centrifugation enhanced transfection using 1 µg of Lipofectamine 2000 and 20, 40, 60 and 100 picomoles of siRNA|
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On comparison of the transfection efficiency of the two methods, a significant increase in the percentage of cells transfected using CET was observed even at low siRNA concentration of 40 picomoles. The concentration of siRNA: liposomal formulation that is able to transfect >85% of cell was found to be 40 picomoles: 1 μg and 60 picomoles: 1 μg using CET modification. Further 40 picomoles of siRNA gave significantly higher transfection than 1:20 (63.1% vs 99.8%; P < 0.0001); therefore, the optimum concentration of siRNA: lipofectamine mixture was narrowed down to 40 picomoles of BLOCK-iT™ (siRNA) and 1 μg of lipofectamine. The comparison of the transfection efficiency of conventional and CET method is given in the [Figure 3]. CET yielded significantly higher number of transfected cells as compared to conventional transfection at each concentration.
|Figure 3: Increased transfection effi ciency in the PBMC culture by introducing CET at a concentration of 1 µg of Lipofectamine-2000 and 20, 40 and 60 picomoles (P < 0.0001) of BLOCK-iT™ Fluorescent Oligos|
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Effects of transfection on cell viability
On comparing the percentage viability of cells in the conventional vs CET group using 1 μg of lipofectamine and 40 picomoles of BLOCK-iT™ no significant difference in the viability of the cells was observed (88.56 ± 10.23 vs 85.89 ± 8.68; P = 0.587). This suggests that low-speed centrifugation does not hinder the cell viability and can be performed for enhanced transfection without compromising normal functionality of the cell [Figure 4].
|Figure 4: Bar diagram demonstrating the comparison of cell viability of conventional transfection and centrifugation enhanced transfection method at a concentration of 1 µg of lipofectamine and 40 picomole of BLOCK-iT™ Fluorescent Oligos|
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| ~ Conclusions|| |
Among the cell lines, suspension cells have traditionally proven to be very difficult to transfect. The common reason for this is a reduced attachment of the transfection complex to the surface of the cells that results in a reduced uptake of the target DNA. PBMCs represent the immunological compartment of the human body and therefore, these cells are being widely used by biomedical researchers to understand the immunopathogenesis of various diseases. To list a few, successful transfection experiments of PBMCs have been used to generate immunotherapy vaccines in clinical trials against cancer,  in vitro monitoring of natural or vaccine-induced immune response  and for studying pathogenesis model of micro-organisms. , The present study analyzed the efficacy of low-speed centrifugation on transfection of human primary PBMCs in suspension culture as compared to conventional method. We demonstrated a statistically significant rise in the number of transfected cells in each group of treated cells with 20, 40 and 60 picomoles of siRNA. A significantly high number of cells got transfected (99.8%) even at a low concentration of 40 picomoles of siRNA, which may prove to be helpful in counteracting the off-target effects of siRNA. We also report that CET can efficiently enhance the number of transfected cells without compromising the cell viability. Gene transfection is a widely used technique for molecular studies and therapeutics. Modifications using egg white for coating cells in suspension,  ultrasonic waves,  and gene delivery by gold particle  have been introduced to increase the transfection efficiency; however, low-speed centrifugation appeared to be a simple and cost-effective approach of enhancing RNA interference.
| ~ Acknowledgments|| |
Manasi Majumdar is a recipient of senior research fellowship from the Council of Scientific and Industrial Research, Govt. of India.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]