**4. Application of the mechanical agitation method to** *ex vivo* **gene therapy**

One of the main obstacles for the application of adult stem cells in *ex vivo* gene therapy is the low efficiency of gene transfer to these cells. For example, electroporation or transfection in mesenchymal stem cells yields 5-10% gene delivery efficiency (Cai et al., 2002; Ding et al., 1999; Eiges et al., 2001; Lakshmipathy et al., 2004; Peister et al., 2004). Therefore, improved gene delivery methods would potentially be very beneficial for the practical application of *ex vivo* gene therapy in patient care. In current gene transfer protocols, virus particles or lipoplexes passively diffuse through the liquid culture medium to reach their target cells, which are layered on the bottom of a culture dish (Chuck & Palsson, 1996). Because the virus particles or lipoplexes contact the target cells by passive diffusion, increasing the chance of contact between virus particles or lipoplexes and their target cells would increase the chance of gene transfer and to promote higher transfer efficiencies. One simple way to increase contact between viruses or lipoplexes and target cells is through mechanical agitation. Based on this hypothesis, we developed a mechanical agitation method for retroviral transduction of primary cells or transfection by lipoplexes (Park et al., 2009). In this method, we simply implemented a step in which virus-containing or lipoplexescontaining cell suspensions are agitated to increase the movement of viruses or lipoplexes and target cells with the purpose of generating more frequent contact between them. Suspended target cells have a better chance of making physical contact with virus particles or lipoplexes than adherent target cells because of the possibility for three-dimensional contact between the cells and viruses or lipoplexes. The simple addition of the mechanical agitation step to the conventional transduction or transfection protocol increased gene transfer efficiency two-fold above the current rates these protocols (Fig. 4). In the following

The Mechanical Agitation Method of Gene Transfer for *Ex-Vivo* Gene Therapy 101

**4.2 One example protocol of retroviral transduction into mesenchymal stem cells**  1. Trypsinize pure rat mesenchymal stem cells (0.1 ml/cm2) for 3 min at 37°C.

virus stock in the presence of 6 μg/ml polybrene (Calbiochem, USA).

7. Replace the supernatant containing virus particles with fresh growth medium.

fluorescence microscope (TE2000-S, Nikon, Japan) after an 86 h incubation.

3. Mix a 1 ml aliquot of the trypsinized rat mesenchymal stem cells directly with 1 ml

5. Mechanically agitate the plate containing the mixture of rat mesenchymal stem cells and virus on a rocker (SLS4, Seoulin, Korea) at 20 rpm for 50 min while incubating at

8. Observe EGFP fluorescence in the transduced rat mesenchymal stem cells with a

Since the first clinical trial of gene therapy in 1990, 1703 gene therapy clinical trials have been completed as of March of 2011 (http://www.wiley.com/legacy/wileychi/genmed /clinical/). However, little progress has been made since the first gene therapy clinical trial, and therefore, the Food and Drug Administration of the United States has not yet approved any human gene therapy for actual patient treatments. Although current gene therapy is still in the experimental stage, *ex vivo* gene therapeutic approaches show a great potential to treat monogenic genetic diseases as shown in the clinical trial results of adenosine deaminase deficiency and familial hypercholesterolemia (Cappelli et al., 2010 & Kassim et al., 2010). The clinical trial results of these diseases were encouraging to continually pursue *ex vivo*

Primary cells, including adult stem cells, have limited self-renewal ability and are vulnerable to epigenetic modification (Dube & Denis, 1995; Muller-Sieburg & Sieburg, 2006; Tseng et al., 2006; Nehlin & Barington, 2009). The long-term culture of primary cells is not possible. Therefore, it is absolutely necessary to deliver therapeutic DNA molecules into isolated cells promptly with high efficiency. However, the transfer efficiency of exogenous DNA into primary cells is very low (Beyer & Da sliva, 2006; Tonti & Mannello, 2008). The gene transfer efficiency into primary cells is several fold less than those of cell lines in current gene transfer methods. This means that improvement of the transfer efficiency of exogenous DNA into primary cells is the first obstacle for the practical use of for *ex vivo* gene

8. TrypLE Express (Invitrogen).

11. Rocker (SLS4, Seoulin, Korea). 12. Incubator at 37°C under 5% CO2. 13. 24-well plates and 96-well plates.

37°C under 5% CO2.

**5. Conclusion and prospect** 

gene therapy.

therapy.

9. Carl Zeiss LSM510 Meta microscope. 10. 0.45 μm cellulose acetate filter (Millipore).

14. E-Max micro-well reader (Molecular Devices, USA). 15. Fluorescence microscope (TE2000-S, Nikon, Japan). 16. FACSCalibur instrument (Becton Dickinson, USA).

2. Adjust the cell suspension to contain 5 × 105 cells/ml.

6. Incubate the plate at 37°C under 5% CO2 for 24 h.

4. Seed the mixing solution in a six-well plate (Falcon, USA).

section, we describe one example of retroviral transduction using our mechanical agitation protocol.

Fig. 4. A Typical Example of Application of Machanical Agitation Method to Transduction of EGFP-Carrying Retrovirus (Park et al., 2009). (A) The representative FACS plots of rat mesenchymal stem cells after transduction with Retro-EGFP using the static method (left panel), mechanical agitation of viruses with adhered cells (middle panel) and simultaneous mechanical agitation of retroviruses with suspended cells (right panel). (B) Numerical representation of the transduction efficiencies of EGFP retrovirus under the static protocol versus the new agitation protocol. The transduction efficiency is defined as the percentage of cells expressing EGFP as measured using FACSCalibur. The mean percentage of GFPpositive cells is presented as the average of three independent transduction experiments (+/- SEM, n=3).
