Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

Abstract

Gene electrotransfer is an efficient and reproducible nonviral gene transfer technique useful for the nonpermanent expression of therapeutic transgenes. The present study established optimal conditions for the electrotransfer of reporter genes into mesenchymal stem cells (MSCs) isolated from rat bone marrow by their selective adherence to tissue-culture plasticware. The electrotransfer of the lacZ reporter gene was optimized by adjusting the pulse electric field intensity, electric pulse type, electropulsation buffer conductivity and electroporation temperature. LacZ electrotransfection into MSCs was optimal at 1500 V cm À1 with pre-incubation in Spinner's minimum essential medium buffer at 22 1C. Under these conditions b-galactosidase expression was achieved in 29 ± 3% of adherent cells 48 h post transfection. The kinetics of b-galactosidase activity revealed maintenance of b-galactosidase production for at least 10 days. Moreover, electroporation did not affect the MSC potential for multidifferentiation; electroporated MSCs differentiated into osteoblastic, adipogenic and chondrogenic lineages to the same extent as cells that were not exposed to electric pulses. Thus, this study demonstrates the feasibility of efficient transgene electrotransfer into MSCs while preserving cell viability and multipotency.

Optimization of gene delivery to HEK293T cells by microporation using a central composite design methodology

“…It was recently reported that electroporation of cells in a capillary system gave rise to an enhancement of transfection rate and cell viability when compared to the cuvette system (Kim et al 2008). In the conventional electroporation, using a cuvette, the reported cellular viabilities are generally below 70 % and most importantly the cell recoveries are rarely mentioned, with the exception of few studies with primary cells (Aluigi et al 2006;Ferreira et al 2008). In this work we achieved cell viabilities up to 90 % and cell recoveries near 50 %.…”

“…The number of pulses (1-3) did not greatly affect the number of GFP + cells contrarily to some reported studies, although using different cell lines and electroporation equipments (Lin et al 2009). It is well known that when cells are submitted to high electric pulses a major amount of DNA enter into the cells though concurrently an increase of cell death is observed (Ferreira et al 2008). Several studies, using different cells, have shown that a way to circumvent this fact is by decreasing electric pulse and increasing the duration of pulse (pulse width) or within low voltages use more than one pulse (Cemazar et al 2009).…”

“…In fact, previous reports have shown that the percentage of surviving cells increases with the decreasing medium conductivity (Ferreira et al 2008) suggesting that electroporation buffer is other parameter that might be considered on electrotransfer optimization protocols. Even though each electroporation equipment has a specific buffer suggested by the supplier, some authors have recently reported promising results of gene delivery efficiencies using accessible buffers of known composition (Ferreira et al 2008;Kang et al 2009). Actually, in one experiment transfection efficiency obtained with RB was slightly higher than those obtained with SB-PBS because 78 % of GFP + cells were obtained using 1 g of DNA.…”

“…In fact, high cell viabilities may be obtained even when few cells are recovered after being submitted to electric voltages. To our best knowledge few authors have published results regarding cell recovery and yield of transfection after electroporation of mammalian cells (Aluigi et al 2006;Ferreira et al 2008).…”

Nonviral gene delivery of erythropoietin by mesenchymal stromal cells

“…Nucleofection has been used successfully for transient transfection of human, bovine, rat and porcine MSCs, but has never been tested in BM-derived mMSCs. [34][35][36][37] Magnetically enhanced nucleic acid delivery (magnetofection) has not explored for any type of MSCs to date. We found that nucleofection is a very efficient transfection technique for mMSCs with transfection rates of B60%.…”