Microfluidic Parallel Patterning and Cellular Delivery of Molecules with a Nanofountain Probe

Kang, Wonmo; McNaughton, Rebecca L.; Yavari, Fazel; Minary‐Jolandan, Majid; Safi, A.; Espinosa, Horacio D.

doi:10.1177/2211068213495395

Cited by 17 publications

(25 citation statements)

References 36 publications

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“…If only partial sealing is achieved, significant electrical leakage may occur, and therefore a much higher input voltage would be required to trigger formation of nanopores in the cell membrane 36, 37 . Applying high voltage is undesirable because it may damage cells by Joule heating or bubble formation.…”

Section: Resultsmentioning

confidence: 99%

“…Two 1-sec input square wave signals (input duration) with a 1-sec time interval (resting duration) between pulses were applied (see Figure S4a). These parameters were chosen based on previous studies of transfection using NFP-E, which is also a localized electroporation technique 36, 37 . After electroporation, we flushed the device to remove the PBS/PI solution by circulating cell media through the microchannels, stained the cells with Calcein AM, and stored the device in an incubator for 20 min before imaging.…”

Section: Resultsmentioning

confidence: 99%

“…However, bulk electroporation methods, including nucleofection 35 (modified electroporation) and microporation, suffer from significant disadvantages: i) the entire cell population is exposed to very high voltages, which routinely causes cell death rates of up to 50%, and/or ii) cells need to be suspended during the process. To address these disadvantages while still utilizing electroporation, the Espinosa group developed nanofountain probe electroporation (NFP-E) for single-cell transfection of adherent cells with cell selectivity, dosage control, and high transfection efficiency and viability 36, 37 . This method uses a microfluidic cantilever to apply a localized electric field to an adherent cell for transfection.…”

Section: Introductionmentioning

confidence: 99%

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Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons

et al. 2014

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New techniques to deliver of nucleic acids and other molecules for gene editing and gene expression profiling, which can be performed with minimal perturbation to cell growth or differentiation, are essential for advancing biological research. Studying cells in their natural state, with temporal control, is particularly important for primary cells that are derived by differentiation from stem cells and are adherent, e.g., neurons. Existing high-throughput transfection methods either require cells to be in suspension or are highly toxic and limited to a single transfection per experiment. Here we present a microfluidic device that couples on-chip culture of adherent cells and transfection by localized electroporation. Integrated microchannels allow long-term cell culture on the device and repeated temporal transfection. The microfluidic device was validated by first performing electroporation of HeLa and HT1080 cells, with transfection efficiencies of ~95% for propidium iodide and up to 50% for plasmids. Application to primary cells was demonstrated by on-chip differentiation of neural stem cells and transfection of postmitotic neurons with a green fluorescent protein plasmid.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons

et al. 2014

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“…Electroporation causes transient nanopores to form in the cell membrane when the cell is subjected to a sufficiently large electric field through which molecules can be delivered inside cells [3, 17, 18, 61–63]. As the electric field is typically created by applying an input voltage between two electrodes, the appropriate placement of the electrodes is crucial for reproducibility and consistent yield.…”

Section: Electroporationmentioning

confidence: 99%

Micro- and Nanoscale Technologies for Delivery into Adherent Cells

Kang

McNaughton

Espinosa

2016

Trends in Biotechnology

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Several recent micro- and nano-technologies have provided novel methods for biological studies of adherent cells because the small features of these new biotools provide unique capabilities for accessing cells without the need for suspension or lysis. These novel approaches have enabled gentle, yet effective delivery of molecules into specific adhered target cells, with unprecedented spatial resolution. Here we review recent progress in the development of these technologies with an emphasis on in vitro delivery into adherent cells utilizing mechanical penetration or electroporation. We discuss major advantages and limitations of these approaches and propose possible strategies for improvements. Finally, we discuss the impact of these technologies on biological research concerning cell-specific temporal studies, e.g., non-destructive sampling and analysis of intracellular molecules. Need For Techniques To Study Adherent Cells A mechanistic understanding of cell biology is often limited by both the complexity of the processes and limitations of commonly available research tools that lack temporal or spatial resolution. The lack of tools capable of providing cell-specific, non-destructive biomolecular delivery and analysis is a particular barrier for advancing fundamental discoveries of cell heterogeneity, single-cell behavior within a complex environment, and the mechanisms that govern disease states, responses to drugs or other stimuli, and differentiation of stem cells. To gain new mechanistic understanding, advances in methods for precise intracellular delivery and non-destructive biochemical analyses of non-secretory molecules (e.g., mRNA and proteins) are greatly needed so that individual cells can be experimentally controlled and repeatedly analyzed over time and/or within a particular location of the cell. For example, developing neurons must undergo a series of sequential changes in gene expression to achieve a mature phenotype; hence, understanding the process will require the ability to accurately monitor the sequence of intracellular events, within individual cells, in a non-destructive manner. In addition, neuronal maturation is influenced by interactions with surrounding cells and with extracellular matrix, so it is necessary to be able to simultaneously monitor events occurring in multiple cells that are interacting with each other and with the matrix. While the requirements are challenging, these experimental capabilities would provide unprecedented insight into the determinants of both the timing of cellular processes and their phenotype, the principles of cell heterogeneity, and the role of cell-cell communication in homogeneous cell populations and co-cultures. Because most cells adhere to a substrate or to other cells during their growth or differentiation [1], it is advantageous for new technologies to be capable of accessing adhered cells to avoid the need to disrupt cell processes by suspension and replating. Several technologies for studying adhered cells are currently being developed, and ...

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“…Despite the variety of transfection techniques available to deliver MBs, the only minimally invasive technique that would enable transfection of single cells within a population and immediate optical/fluorescent imaging after transfection is nanofountain probe electroporation (NFP‐E) . The NFP‐E system consists of a microfluidic device with a nanoscale cantilever probe tip together with an integrated electronic component and software to deliver a localized voltage to a cell membrane for electroporation.…”

Section: Introductionmentioning

confidence: 98%

Single‐Cell Detection of mRNA Expression Using Nanofountain‐Probe Electroporated Molecular Beacons

Giraldo-Vela

Kang

McNaughton

et al. 2015

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New techniques for single-cell analysis enable new discoveries in gene expression and systems biology. Time-dependent measurements on individual cells are necessary, yet the common single-cell analysis techniques used today require lysing the cell, suspending the cell, or long incubation times for transfection, thereby interfering with the ability to track an individual cell over time. Here a method for detecting mRNA expression in live single cells using molecular beacons that are transfected into single cells by means of nanofountain probe electroporation (NFP-E) is presented. Molecular beacons are oligonucleotides that emit fluorescence upon binding to an mRNA target, rendering them useful for spatial and temporal studies of live cells. The NFP-E is used to transfect a DNA-based beacon that detects glyceraldehyde 3-phosphate dehydrogenase and an RNA-based beacon that detects a sequence cloned in the green fluorescence protein mRNA. It is shown that imaging analysis of transfection and mRNA detection can be performed within seconds after electroporation and without disturbing adhered cells. In addition, it is shown that time-dependent detection of mRNA expression is feasible by transfecting the same single cell at different time points. This technique will be particularly useful for studies of cell differentiation, where several measurements of mRNA expression are required over time.

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Microfluidic Parallel Patterning and Cellular Delivery of Molecules with a Nanofountain Probe

Cited by 17 publications

References 36 publications

Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons

Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons

Micro- and Nanoscale Technologies for Delivery into Adherent Cells

Single‐Cell Detection of mRNA Expression Using Nanofountain‐Probe Electroporated Molecular Beacons

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