RESUMO
Cellular therapies have the potential to advance treatment for a broad array of diseases but rely on viruses for genetic reprogramming. The time and cost required to produce viruses has created a bottleneck that constricts development of and access to cellular therapies. Electroporation is a non-viral alternative for genetic reprogramming that bypasses these bottlenecks, but current electroporation technology suffers from low throughput, tedious optimization, and difficulty scaling to large-scale cell manufacturing. Here, we present an adaptable microfluidic electroporation platform with the capability for rapid, multiplexed optimization with 96-well plates. Once parameters are optimized using small volumes of cells, transfection can be seamlessly scaled to high-volume cell manufacturing without re-optimization. We demonstrate optimizing transfection of plasmid DNA to Jurkat cells, screening hundreds of different electrical waveforms of varying shapes at a speed of ~3 s per waveform using ~20 µL of cells per waveform. We selected an optimal set of transfection parameters using a low-volume flow cell. These parameters were then used in a separate high-volume flow cell where we obtained similar transfection performance by design. This demonstrates an alternative non-viral and economical transfection method for scaling to the volume required for producing a cell therapy without sacrificing performance. Importantly, this transfection method is disease-agnostic with broad applications beyond cell therapy.
Assuntos
Eletroporação , Microfluídica , Humanos , Transfecção , Terapia Baseada em Transplante de Células e Tecidos , EletricidadeRESUMO
Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing.
Assuntos
Metilação de DNA , DNA/genética , Técnicas Analíticas Microfluídicas/métodos , Nanotecnologia/métodos , DNA/análise , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fluorescência , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia Confocal , Nanotecnologia/instrumentação , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Viral vectors represent a bottleneck in the manufacturing of cellular therapies. Electroporation has emerged as an approach for non-viral transfection of primary cells, but standard cuvette-based approaches suffer from low throughput, difficult optimization, and incompatibility with large-scale cell manufacturing. Here, we present a novel electroporation platform capable of rapid and reproducible electroporation that can efficiently transfect small volumes of cells for research and process optimization and scale to volumes required for applications in cellular therapy. We demonstrate delivery of plasmid DNA and mRNA to primary human T cells with high efficiency and viability, such as > 95% transfection efficiency for mRNA delivery with < 2% loss of cell viability compared to control cells. We present methods for scaling delivery that achieve an experimental throughput of 256 million cells/min. Finally, we demonstrate a therapeutically relevant modification of primary T cells using CRISPR/Cas9 to knockdown T cell receptor (TCR) expression. This study displays the capabilities of our system to address unmet needs for efficient, non-viral engineering of T cells for cell manufacturing.
Assuntos
Eletroporação , Linfócitos T , Humanos , Transfecção , Eletroporação/métodos , Vetores Genéticos , RNA MensageiroRESUMO
Cellular therapies have the potential to advance treatment for a broad array of diseases but rely on viruses for genetic reprogramming. The time and cost required to produce viruses has created a bottleneck that constricts development of and access to cellular therapies. Electroporation is a non-viral approach for genetic reprogramming that bypasses these bottlenecks, but current electroporation technology suffers from low throughput, tedious optimization, and difficulty scaling to large-scale cell manufacturing. Here, we present an adaptable microfluidic electroporation platform with the capability for rapid, multiplexed optimization with 96-well plates. Once parameters are optimized using small volumes of cells, transfection can be seamlessly scaled to high-volume cell manufacturing without re-optimization. We demonstrate optimizing transfection of plasmid DNA to Jurkat cells, screening hundreds of different electrical waveforms of varying shapes at a speed of ~3 s per waveform using ~ 20 µL of cells per waveform. We selected an optimal set of transfection parameters using a low-volume flow cell. These parameters were then used in a separate high-volume flow cell where we obtained similar transfection performance by design. This demonstrates an economical method for scaling to the volume required for producing a cell therapy without sacrificing performance.
RESUMO
Fluidic systems with nanometre length scales enable sensitive analysis of DNA molecules. Nanofluidic systems have been used to probe conformational, dynamic, and entropic properties of DNA molecules, to rapidly sort DNA molecules based on length dependent interactions with their confining environment, and for determining the spatial location of genetic information along long DNA molecules. In this critical review, recent experiments utilizing fluidic systems comprised of nanochannels, nanoslits, nanopores, and zero-mode waveguides for DNA analysis are reviewed (161 references).
Assuntos
DNA/química , Microfluídica/instrumentação , Microfluídica/métodos , Nanotecnologia , Modelos Moleculares , Polímeros/química , Análise de Sequência de DNARESUMO
Epigenetic states are governed by DNA methylation and a host of modifications to histones bound with DNA. These states are essential for proper developmentally regulated gene expression and are perturbed in many diseases. There is great interest in identifying epigenetic mark placement genome wide and understanding how these marks vary among cell types, with changes in environment or according to health and disease status. Current epigenomic analyses employ bisulfite sequencing and chromatin immunoprecipitation, but query only one type of epigenetic mark at a time, DNA methylation, or histone modifications and often require substantial input material. To overcome these limitations, we established a method using nanofluidics and multicolor fluorescence microscopy to detect DNA and histones in individual chromatin fragments at about 10 Mbp/min. We demonstrated its utility for epigenetic analysis by identifying DNA methylation on individual molecules. This technique will provide the unprecedented opportunity for genome wide, simultaneous analysis of multiple epigenetic states on single molecules.
Assuntos
Cromatina/química , Metilação de DNA , DNA/análise , Histonas/química , Microfluídica/instrumentação , Microscopia de Fluorescência/métodos , Epigênese Genética , Desenho de Equipamento , Células HeLa , HumanosRESUMO
We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for off-chip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level.
Assuntos
Fracionamento Químico/instrumentação , Cromossomos Humanos/genética , DNA/análise , DNA/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única/instrumentação , Linhagem Celular Tumoral , DNA/química , Humanos , Conformação de Ácido Nucleico , Hibridização de Ácido NucleicoRESUMO
Single DNA molecules confined to nanoscale fluidic channels extend along the channel axis in order to minimize their conformational free energy. When such molecules are forced into a nanoscale fluidic channel under the application of an external electric field, monomers near the middle of the DNA molecule may enter first, resulting in a folded configuration with less entropy than an unfolded molecule. The increased free energy of a folded molecule results in two effects: an increase in extension factor per unit length for each segment of the molecule, and a spatially localized force that causes the molecule to spontaneously unfold. The ratio of this unfolding force to hydrodynamic friction per DNA contour length is measured in nanochannels with two different diameters.