October 15, 2016 (Vol. 36, No. 18)

Vidya Murthy Ph.D. Senior Applications Scientist Harvard Bioscience

Electroporation-Based Immunotherapy: Potential Treatment Options for Metastatic Melanoma

A powerful approach to cancer therapy involves modifying the patient’s own immune cells in order to enhance the immune response against cancer cells. The most studied cell types for cancer immunotherapy are dendritic cells (DCs) and T cells, which are pivotal players in initiating immune responses.

DCs are increasingly used in vaccine research as immunotherapy for cancer and other diseases, as they are the most potent and proficient antigen-presenting cells and are critical to the onset of immunity. They have the highest surface density of major histocompatibility complex (MHC) and costimulatory molecules, together with a high motility, which allows them to traffic from the site of antigen uptake to the T-cell areas of lymph nodes, and they have the ability to produce immunostimulatory cytokines and chemokines. All this knowledge has led to the use of DCs as natural adjuvants to immunize cancer patients against tumor-specific self-antigens and to break T-cell tolerance against these tumor antigens.

For the generation of an effective therapeutic cancer vaccine, it is necessary to load tumor antigens onto DCs and to present antigens in the appropriate immune stimulatory environment to induce strong antigen-specific T-cell responses, especially CD4+ T-helper and CD8+ cytolytic T lymphocytes (CTLs) with a sufficiently broad repertoire and immunologic memory. DCs can be loaded by transfection with DNA or RNA to produce antitumor antigens that break tolerance to tumors and induce tumor-specific therapeutic immunity.

While there are several ways to transfect DCs, electroporation significantly enhances transfection efficiency. Several recent reports have indicated that electroporation of DCs with whole tumor-derived mRNA or defined mRNA represents an effective nonviral strategy to stimulate T-cell responses both in in vitro and in vivo models.

This paper describes the optimization of parameters for electroporation-mediated transfection (electrotransfection) of myeloid immature DCs with in vitro expanded RNA isolated from tumor tissue to produce clinical-grade DC vaccines. In this paper immature DCs were electroporated using the BTX Agilepulse Max system.

BTX AgilePulse Max System


The overall scheme of DC vaccine preparation was to separate immature dendritic cells from autologous CD14-positive cells and isolate the total RNA from autologous tumor tissue. RNA was reversely transcribed to obtain cDNA and amplified using cDNA as template incorporating a T7 RNA promoter. Amplified cDNA was in vitro transcribed and loaded into immature DCs by electroporation. The DCs were subsequently matured in the presence of inflammatory cytokines and cryopreserved as single aliquots prior to use.

For experiments aimed at optimizing electroporation conditions, a cDNA encoding the enhanced green fluorescent protein (eGFP) gene and containing a T7 promoter and polyadenylation signal suitable for DC transfection was prepared using standard methods. For transfection into DCs, mRNA was dissolved in water at 1.0 µg/mL. Electrotransfection parameters were optimized by monitoring transfection efficiency and DC viability as a function of electrode separation, pulse amplitude and length and amplified mRNA concentration in the medium (Figure 1).

In all experiments AgilePulse Max square-wave generator and the proprietary cGMP-grade low-conductivity (80 µS/cm) Cytoporation T medium (both BTX, Harvard Bioscience) were used.

DCs were transfected with mRNA encoding the eGFP gene, matured the cells for 48 hours and measured transfection efficiency (by eGFP fluorescence) and viability (by exclusion of 7-amino-actinomycin D, 7-AAD, Pharmingen) following transfection in BTX electroporation cuvettes with a 4-mm electrode separation (400 µL). The mRNA concentration was varied between 4.0 µg/mL and 25 µg/mL, pulse a mplitude between 0.5 kV/cm and 2.5 kV/cm and pulse width between 0.05 ms and 0.45 ms.

From the dependence of transfection efficiency and viability on mRNA concentration (Figure 1A), the effect of pulse amplitude for mRNA concentration was optimized in the 20–25 µg/mL range (Figure 1B). Because the pulse of 1.0 kV/cm resulted in acceptable transfection efficiency and reasonable viability, the effect of pulse width at 20–25 µg/mL RNA and 1.0 kV/cm was further studied (Figure 1C).

Immature DCs manufactured from the blood of melanoma patients were electrotransfected with mRNA isolated from autologous tumor tissue and in vitro amplified. The cells were prepared as above except that the DCs were washed, suspended in the Cytoporation T Medium (BTX, Harvard Bioscience) at a density of 1 X 107/mL in the presence of 20–50 µg/mL of autologous mRNA. The cell suspension was transferred to sterile, disposable electroporation cuvettes with a 4-mm electrode gap (BTX, Harvard Bioscience). The cells were subjected to two square 400-V pulses of 50 µs each from the AgilePulse Max square-wave generator.

Following electroporation the cells were rested in X-VIVO 15 medium (BioWhittaker) containing HABS, GM-CSF and IL-4 as above at 37°C in humidified 5% CO2 for one hour. Subsequently the cells were washed once and suspended in the maturation medium containing 1100 IU/mL TNF-a and 1.0 µg/mL prostaglandin E2 for two more days. MDCs were collected and assayed for compliance with release criteria.

Figure 1. Identifying conditions for electrotrans- fection of immature dendritic cells. Normal IDCs were electrotransfected with in vitro transcribed eGFP-mRNA. Following electro-transfection, the cells were matured for 48 hours when via-bility (open symbols) and transfection effic- iency (closed symbols) were quantified (by 7-AAD exclusion and eGFP fluorescence, respectively). Shown are the data from the final iteration in the analysis where mRNA concentration varied from 4.0 µg/mL to 25 µg/mL (A), pulse amplitude from 0.5 kV/cm to 2.5 kV/cm (B), and pulse width from 0.05 µs to 0.45 µs (C). Symbols denote mean values of measurements in cells from three or more individuals ± standard deviation (except in panel C that is an example of an entire experiment conducted with cells from one individual).


1. Operating parameters were optimized for electroporation-mediated transfection of eGFP RNA into normal immature dendritic cells (IDCs) (Figure 1). The optimal settings were 1.0-kV/cm pulses of 150-µs duration for 10 µg RNA/106 cells.*

2. Electrotransfection of patient DCs with mRNA isolated from tumor tissue shows strong expression of CD83 and CD86 surface markers (Figure 2). Feasibility, safety, and toxicity of an autologous DC vaccine, electrotransfected with in vitro amplified autologous tumor-derived genomic mRNA, injected into patients with metastatic melanoma was evaluated. Figure 2 shows the CD83 and CD86 expression histograms measured in RNA-transfected DCs used for vaccination as part of individual batch release data.


Immunotherapy holds the promise of contributing to the limited treatment options available to patients suffering from metastatic melanoma. Standardized preparation of viable clinical-grade DCs transfected with tumor derived and in vitro amplified mRNA is feasible and the administration of resulting cellular vaccine is safe. Electroporation-mediated transfection of DCs was optimized for maximal efficiency and cell viability.

Figure 2. Expression of CD83 (left) and CD86 (right) by patients’ RNA-transfected DCs (red) used for vaccination. Isotype controls are shown in green.
(Ref: Markovic, SN, Dietz, AB, Greiner, CW, Maas, ML, Butler, GW, Padley, DJ, Bulur, PA, Allred, JB, Creagan, ET, Ingle, JN, Gastineau, and Vuk-Pavlovic, S. Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma. J Transl. Medicine, 4:35-48).

*Note: the AgilePulse Max allows scaling up of sample volume up to 5 mL and 10 mL.

Vidya Murthy, Ph.D. ([email protected]), is senior applications scientist, Harvard Bioscience.

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