OPEN An efficient low cost means of biophysical gene transfection in primary cells

OPEN An efficient low cost means of biophysical gene transfection in primary cells

Efficient, facile gene modification of cells has become an indispensable part of modern molecular biology. For the majority of cell lines and several primary populations, such modifications can be readily performed through a variety of methods. However, many primary cell lines such as stem cells frequently suffer from poor transfection efficiency. Though several physical approaches have been introduced to circumvent these issues, they often require expensive/specialized equipment and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral methods are capable of transducing difficult cellular populations, however such methods can be time consuming for large arrays of gene targets, present biohazard concerns, and result in expression of viral proteins; issues of concern for certain experimental approaches. We report here a widely applicable, low-cost (less than one hundred Canadian dollars) method of electroporation, applicable to small (one to ten microliter) cell volumes and composed of equipment readily available to the average investigator. Using this system we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process improves the survival of sensitive stem cell populations while reducing reagent requirements for protocols such as Cas9/gRNAs transfections.

Facile genetic transfection of target cell populations is an indispensable aspect of modern molecular biology, involving the introduction of DNA/RNA substituents or other cell impermeable reagents (biologic drugs, peptides, etc.) to alter signaling responses of target cells. Often, this results in a transient or stable modification of cellular responsivity depending on whether the modification is integrated into the genome, or remains extragenomic. Particularly with the advent of CRISPR (clustered regularly interspace short palindromic repeats) and genome base-editing technologies, efficient means of performing a wide array of gene modifications have become readily available. However the success of these and many other forms of cell modification depend upon efficient intracellular introduction of the modifying agent; the difficulty of which varies with cell type. Thus while a number of straightforward chemical and biophysical methods exist for the transfection of (particularly transformed) cell lines, such methods are limited for difficult to transfect primary cell lines such as embryonic stem cells. In addition, cell types such as patient-derived primary cells from resections, biopsies, etc. are often difficult to obtain or isolate in sufficient quantity, further complicating transfection studies. Of methods which do exist for difficult to transfect cell lines, many are often inefficient, biohazardous, and/or require use of costly specialized equipment and consumables. Such considerations become significant at scale, as modifications become more complex (multiple RNA/protein targets), and in clinical settings. Though viral transduction methods (lentivirus, adenovirus, adeno-associated virus, etc.) exist to transduce difficult to transfect cell types, these too possess certain drawbacks. Despite known advantages including transduction efficiency toward non-dividing cells, and their ability to be utilized in vivo, ex vivo and in vitro; viral transducers require additional processing and purification in order to produce infective particles, adding time and cost particularly for operations involving a large number of target genes. With respect to biosafety concerns, these include the induction of antiviral and immunologic cellular responses, potential for generation of replication-competent viruses, vector mobilization in some instances and the possibility of integration-associated oncogenesis.

Additionally adeno-associated virus vectors exhibit a typical insert size less than five kilo-base, limiting modifications in projects requiring multiple RNA or large DNA segments.

By contrast, chemical (modified lipids, polyethylenimine, calcium phosphate) and physical (microinjection, biolistics, ultrasound, electroporation, nucleofection, cell squeezing and laser-poration) techniques are technically straightforward, more adaptable, less time-consuming and do not pose biohazard risks to laboratory personnel. For example lipofection and polyethylenimine mediated transfection are quick, easy procedures based on the condensation of nucleic acids with cationic lipids or organic polyamine polymers respectively to facilitate fusion-based phospholipid entry into target cells. These techniques however demonstrate variable efficiencies depending on cell type. For example typically only one to five percent of primary neurons are transfected using lipofection, despite up to eighty-five percent efficiency in many transformed cell lines. Additionally these agents demonstrate significant toxicity toward sensitive cell types such as embryonic stem cells and neurons. Alternatively with respect to physical methods, some difficulties include the relatively high input and per unit costs for modifications due to equipment requirements (microinjection, biolistics, ultrasound, electroporation, nucleoporation, laser-poration), consumables (nucleofection, cell squeezing, electroporation, microinjection), temporal efficiency (microinjection, laser-poration), and/or sustained throughput (microfluidic channel /cell squeezing).

In theory, electroporation (EP) is a fast simple method involving exposure of cells to brief electric pulses inducing pore formation through the plasma membrane in order to allow plasmids and other ectopic molecules to enter the cell cytoplasm. However the electrode gap distance (four to ten millimeters) and associated voltages (two hundred to eight hundred volts) typically utilized for mammalian cells require significant electrical capacity with substantial electrolytic effects (acidification, alkalinization) at anode and cathode interfaces respectively with such effects potentially causing significant damage to sensitive cell types. Additionally larger chamber sizes (and thus electrode gap distance) can reduce homogeneity of the applied electric field, ultimately decreasing cell viability and transfection efficiency. Minimum electroporation volumes required in these systems (two hundred to one thousand microliter) can impart additional significant reagent costs (outside of equipment/consumable costs) for gene-editing experiments utilizing ribonucleoprotein complexes containing defined RNA guides plus Cas9, single-stranded DNA deaminase (base-editing) or equivalents (CRISPR), which must be maintained at critical concentrations for maximum efficiency. Given the current application range and relative efficiency of the above gene modification approaches, considerable interest has arisen in more cost effective, efficient means of performing such biophysical gene transfection.

We describe herein a novel, efficient, low-cost means of performing biophysical gene transfection with no additional equipment or EP consumables: microcell electroporation (ME). Using material and expertise widely available to the average investigator at a total cost of less than one hundred Canadian dollars, a range of programmable EP experiments could be performed over varied field strengths, pulse widths and sequences for mammalian EP cell transfections, demonstrating improved efficiency compared to standard commercial EP systems. With this approach, for CRISPR-mediated gene modifications of embryonic stem cells, sufficient modified lines could routinely be derived from a single five microliter electroporation event utilizing twenty-five thousand cells. This implementation thus addresses cost issues associated with bulk EP in addition to enhancing transfection efficiency of sensitive, difficult to transfect cell types while maintaining the speed and ease of use issues inherent to EP.

Results

In order to optimize conditions for ME of difficult to transfect cells, we designed a durable, dynamic, low-cost electroporation chamber. As shown in Figure one A and B, polished one by one centimeter sections of zero point zero two four inches thick three one six stainless steel were separated at fixed gap distances ranging from two hundred to one thousand micrometers, serving as electrodes. Electrodes were then affixed and protected with epoxy on a standard glass slide (Figure one B, arrows). While experiments were routinely performed in a class two A two biosafety cabinet with cell solutions confined within the electroporation channel, samples could be cover-slipped for real-time analysis on a standard upright or inverted microscope (Figure one C and D). Due to the reduced electrical requirements resulting from the diminished gap size (seven hundred micrometers shown), electronics such as those commonly employed for electrophysiology are capable of supplying sufficient power for electroporation (Figure one E). In fact, further experimentation demonstrated that the electronics required could be wholly replaced by a battery powered Arduino based system with equal efficiency at a cost of less than one hundred dollars Canadian (Figure five). Using this system, relative cell permeability was assessed by monitoring the relative rate of fluorescence enhancement of cells to the cell-impermeant marker propidium iodide. Under conditions appropriate for electroporation, a rapid increase in propidium iodide fluorescence could be observed in a substantial subpopulation of ES cells within two minutes of electroporation (Figure one F).