Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Mater Today Bio ; 19: 100601, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37063248

RESUMO

Membrane disruption using Bulk Electroporation (BEP) is a widely used non-viral method for delivering biomolecules into cells. Recently, its microfluidic counterpart, Localized Electroporation (LEP), has been successfully used for several applications ranging from reprogramming and engineering cells for therapeutic purposes to non-destructive sampling from live cells for temporal analysis. However, the side effects of these processes on gene expression, that can affect the physiology of sensitive stem cells are not well understood. Here, we use single cell RNA sequencing (scRNA-seq) to investigate the effects of BEP and LEP on murine neural stem cell (NSC) gene expression. Our results indicate that unlike BEP, LEP does not lead to extensive cell death or activation of cell stress response pathways that may affect their long-term physiology. Additionally, our demonstrations show that LEP is suitable for multi-day delivery protocols as it enables better preservation of cell viability and integrity as compared to BEP.

2.
Nano Lett ; 23(8): 3653-3660, 2023 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-36848135

RESUMO

Delivery of proteins and protein-nucleic acid constructs into live cells enables a wide range of applications from gene editing to cell-based therapies and intracellular sensing. However, electroporation-based protein delivery remains challenging due to the large sizes of proteins, their low surface charge, and susceptibility to conformational changes that result in loss of function. Here, we use a nanochannel-based localized electroporation platform with multiplexing capabilities to optimize the intracellular delivery of large proteins (ß-galactosidase, 472 kDa, 75.38% efficiency), protein-nucleic acid conjugates (protein spherical nucleic acids (ProSNA), 668 kDa, 80.25% efficiency), and Cas9-ribonucleoprotein complex (160 kDa, ∼60% knock-out and ∼24% knock-in) while retaining functionality post-delivery. Importantly, we delivered the largest protein to date using a localized electroporation platform and showed a nearly 2-fold improvement in gene editing efficiencies compared to previous reports. Furthermore, using confocal microscopy, we observed enhanced cytosolic delivery of ProSNAs, which may expand opportunities for detection and therapy.


Assuntos
Sistemas CRISPR-Cas , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Edição de Genes , Eletroporação , Proteínas/genética
3.
ACS Nano ; 16(10): 15653-15680, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36154011

RESUMO

The emerging field of cell therapy offers the potential to treat and even cure a diverse array of diseases for which existing interventions are inadequate. Recent advances in micro and nanotechnology have added a multitude of single cell analysis methods to our research repertoire. At the same time, techniques have been developed for the precise engineering and manipulation of cells. Together, these methods have aided the understanding of disease pathophysiology, helped formulate corrective interventions at the cellular level, and expanded the spectrum of available cell therapeutic options. This review discusses how micro and nanotechnology have catalyzed the development of cell sorting, cellular engineering, and single cell analysis technologies, which have become essential workflow components in developing cell-based therapeutics. The review focuses on the technologies adopted in research studies and explores the opportunities and challenges in combining the various elements of cell engineering and single cell analysis into the next generation of integrated and automated platforms that can accelerate preclinical studies and translational research.


Assuntos
Engenharia Celular , Nanotecnologia , Fluxo de Trabalho , Nanotecnologia/métodos , Terapia Baseada em Transplante de Células e Tecidos , Pesquisa Translacional Biomédica
4.
Sci Adv ; 8(29): eabn7637, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35867793

RESUMO

Manipulation of cells for applications such as biomanufacturing and cell-based therapeutics involves introducing biomolecular cargoes into cells. However, successful delivery is a function of multiple experimental factors requiring several rounds of optimization. Here, we present a high-throughput multiwell-format localized electroporation device (LEPD) assisted by deep learning image analysis that enables quick optimization of experimental factors for efficient delivery. We showcase the versatility of the LEPD platform by successfully delivering biomolecules into different types of adherent and suspension cells. We also demonstrate multicargo delivery with tight dosage distribution and precise ratiometric control. Furthermore, we used the platform to achieve functional gene knockdown in human induced pluripotent stem cells and used the deep learning framework to analyze protein expression along with changes in cell morphology. Overall, we present a workflow that enables combinatorial experiments and rapid analysis for the optimization of intracellular delivery protocols required for genetic manipulation.


Assuntos
Aprendizado Profundo , Células-Tronco Pluripotentes Induzidas , Engenharia Celular , Eletroporação/métodos , Humanos , Fluxo de Trabalho
5.
Small ; 18(20): e2107795, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35315229

RESUMO

Genome engineering of cells using CRISPR/Cas systems has opened new avenues for pharmacological screening and investigating the molecular mechanisms of disease. A critical step in many such studies is the intracellular delivery of the gene editing machinery and the subsequent manipulation of cells. However, these workflows often involve processes such as bulk electroporation for intracellular delivery and fluorescence activated cell sorting for cell isolation that can be harsh to sensitive cell types such as human-induced pluripotent stem cells (hiPSCs). This often leads to poor viability and low overall efficacy, requiring the use of large starting samples. In this work, a fully automated version of the nanofountain probe electroporation (NFP-E) system, a nanopipette-based single-cell electroporation method is presented that provides superior cell viability and efficiency compared to traditional methods. The automated system utilizes a deep convolutional network to identify cell locations and a cell-nanopipette contact algorithm to position the nanopipette over each cell for the application of electroporation pulses. The automated NFP-E is combined with microconfinement arrays for cell isolation to demonstrate a workflow that can be used for CRISPR/Cas9 gene editing and cell tracking with potential applications in screening studies and isogenic cell line generation.


Assuntos
Aprendizado Profundo , Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Eletroporação/métodos , Edição de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo
6.
SLAS Technol ; 26(1): 26-36, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33449846

RESUMO

Single-cell delivery platforms like microinjection and nanoprobe electroporation enable unparalleled control over cell manipulation tasks but are generally limited in throughput. Here, we present an automated single-cell electroporation system capable of automatically detecting cells with artificial intelligence (AI) software and delivering exogenous cargoes of different sizes with uniform dosage. We implemented a fully convolutional network (FCN) architecture to precisely locate the nuclei and cytosol of six cell types with various shapes and sizes, using phase contrast microscopy. Nuclear staining or reporter fluorescence was used along with phase contrast images of cells within the same field of view to facilitate the manual annotation process. Furthermore, we leveraged the near-human inference capabilities of the FCN network in detecting stained nuclei to automatically generate ground-truth labels of thousands of cells within seconds, and observed no statistically significant difference in performance compared to training with manual annotations. The average detection sensitivity and precision of the FCN network were 95±1.7% and 90±1.8%, respectively, outperforming a traditional image-processing algorithm (72±7.2% and 72±5.5%) used for comparison. To test the platform, we delivered fluorescent-labeled proteins into adhered cells and measured a delivery efficiency of 90%. As a demonstration, we used the automated single-cell electroporation platform to deliver Cas9-guide RNA (gRNA) complexes into an induced pluripotent stem cell (iPSC) line to knock out a green fluorescent protein-encoding gene in a population of ~200 cells. The results demonstrate that automated single-cell delivery is a useful cell manipulation tool for applications that demand throughput, control, and precision.


Assuntos
Aprendizado Profundo , Edição de Genes , Inteligência Artificial , Computadores , Eletroporação , Humanos
7.
Small ; 16(43): e2002616, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33006271

RESUMO

Introducing exogenous molecules into cells with high efficiency and dosage control is a crucial step in basic research as well as clinical applications. Here, the capability of the nanofountain probe electroporation (NFP-E) system to deliver proteins and plasmids in a variety of continuous and primary cell types with appropriate dosage control is reported. It is shown that the NFP-E can achieve fine control over the relative expression of two cotransfected plasmids. Finally, the dynamics of electropore closure after the pulsing ends with the NFP-E is investigated. Localized electroporation has recently been utilized to demonstrate the converse process of delivery (sampling), in which a small volume of the cytosol is retrieved during electroporation without causing cell lysis. Single-cell temporal sampling confers the benefit of monitoring the same cell over time and can provide valuable insights into the mechanisms underlying processes such as stem cell differentiation and disease progression. NFP-E parameters that maximize the membrane resealing time, which is essential for increasing the sampled volume and in meeting the challenge of monitoring low copy number biomarkers, are identified. Its application in CRISPR/Cas9 gene editing, stem cell reprogramming, and single-cell sampling studies is envisioned.


Assuntos
Eletroporação , Edição de Genes , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Plasmídeos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA