High-throughput and dosage-controlled intracellular delivery of large cargos by an acoustic-electric micro-vortices platform

ABSTRACTIntracellular delivery of cargos for cell engineering plays a pivotal role in transforming medicine and biomedical discoveries. Recent advances in microfluidics and nanotechnology have opened up new avenues for efficient, safe, and controllable intracellular delivery, as they improve precision down to the single-cell level. Based on this capability, several promising micro- and nanotechnology approaches outperform viral and conventional non-viral techniques in offering dosage-controlled delivery and/or intracellular delivery of large cargos. However, to achieve this level of precision and effectiveness, they are either low in throughput, limited to specific cell types (e.g., adherent vs. suspension cells), or complicated to operate with. To address these challenges, here we introduce a versatile and simple-to-use intracellular delivery microfluidic platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP). Hundreds of acoustic microstreaming vortices form the production line of the AESOP platform, wherein hundreds of thousands of cells are trapped, permeabilized, and mixed with exogenous cargos. Using AESOP, we show intracellular delivery of a wide range of molecules (from <1 kDa to 2 MDa) with high efficiency, cell viability, and dosage-controlled capability into both suspension and adherent cells and demonstrate throughput at 1 million cells/min per single chip. In addition, we demonstrate AESOP for two gene editing applications that require delivery of large plasmids: i) eGFP plasmid (6.1 kbp) transfection, and ii) CRISPR-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and sgRNA. Compared to alternative platforms, AESOP not only offers dosage-controlled intracellular delivery of large plasmids (>6kbp) with viabilities over 80% and comparable delivery efficiencies, but also is an order of magnitude higher in throughput, compatible with both adherent and suspension cell lines, and simple to operate.

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Investigation of Rhabdomyosarcoma Cell Electrofusion

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.254.207 ◽

2011 ◽

Vol 254 ◽

pp. 207-210

Author(s):

Chong Xian Felix Yeo ◽

Kian Hwa Tan ◽

Eng Lee Tan ◽

Chu Sing Daniel Lim

Keyword(s):

Cell Fusion ◽

High Efficiency ◽

Promising Result ◽

Human Cancer ◽

Cell Types ◽

Myeloma Cell ◽

Chromosomal Mapping ◽

Wide Range ◽

Rd Cells ◽

Fusion Yield

Cell-cell fusion is an important natural and engineered process for in-depth studies into hybridomas, developmental biology, immunology and various cellular therapies. It is also a powerful tool for analysis of gene expression, chromosomal mapping, antibody production, cloning mammals, and cancer immunotherapy. However, research so far has primarily focused on cell models such asC.elegans, drosophila, myoblasts, spleen-myeloma cell hybrids and various plant protoplasts. Rhabdomyosarcoma cells are a rare form of musculoskeletal cancer cells found in the head, neck, and other less skeletal areas of the human cancer patient’s body. As these cells do not normally undergo fusion naturally, they are an interesting model for studying cell fusion. Among all the techniques for fusion, electrofusion (or electroporation) can be applied to a wide range of cell types with high efficiency and high post-fusion viability. By coupling these cells with this technique, the effectson cell proliferation, growth pattern, and hybridoma count wereinvestigated. Overall, the experimental results showed that an adequate electrical stimulation helped to facilitate the fusion and proliferation of the RD cells. Furthermore,a DC current produced the highest number of hybridomas, while maintaining the highest proliferation rate.After subtracting for the control samples, an average fusion yield of 24% was obtained under this DC setting, which is comparable to the fusion yield of 20% obtained using the same technique by other researchers. This is a promising result for its application in the production of monoclonal antibodies for cancer research and treatment.

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The gene regulatory basis of genetic compensation during neural crest induction

10.1101/314534 ◽

2018 ◽

Author(s):

Christopher M. Dooley ◽

Neha Wali ◽

Ian M. Sealy ◽

Richard J. White ◽

Derek L. Stemple ◽

...

Keyword(s):

Neural Crest ◽

Target Genes ◽

Gene Knockout ◽

Pigment Cell ◽

Specific Cell ◽

Multiple Time ◽

Genetic Ablation ◽

Wide Range ◽

Gene Regulatory ◽

Genetic Compensation

AbstractBackgroundThe neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors but detailed interactions within the network remain to be resolved.ResultsWe have used gene knockout and RNA sequencing strategies to dissect NC differentiation in zebrafish. We establish that initiation of the NC GRN takes place just after genome activation. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that although a single allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, expression of many target genes remains abnormal and sensitive to tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation.ConclusionsStepwise genetic ablation of the NC identifies the core gene module required for neural crest induction. This work extends and refines the NC GRN while also uncovering the complex transcriptional basis of genetic compensation via paralogues.

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A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.880 ◽

1994 ◽

Vol 14 (2) ◽

pp. 880-887 ◽

Cited By ~ 56

Author(s):

J R Rayner ◽

T J Gonda

Keyword(s):

Cell Types ◽

Retroviral Vector ◽

Expression Cloning ◽

Target Cells ◽

Specific Cell ◽

Functional Screening ◽

Efficient Procedure ◽

Plasmid Library ◽

Wide Range ◽

Expression Libraries

cDNA expression cloning is a powerful method for the rescue and identification of genes that are able to confer a readily identifiable phenotype on specific cell types. Retroviral vectors provide several advantages over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express genes in a wide range of cell types, including those that form important experimental systems such as the hemopoietic system. We describe here a straightforward and efficient method for generating expression libraries by using a murine retroviral vector. Essentially, the method involves the directional cloning of cDNA into the retroviral vector and the generation of pools of stable ecotropic virus producing cells from this DNA. The cells so derived constitute the library, and the virus they yield is used to infect appropriate target cells for subsequent functional screening. We have demonstrated the feasibility of this procedure by constructing several large retroviral libraries (10(5) to 10(6) individual clones) and then using one of these libraries to isolate cDNAs for interleukin-3 and granulocyte-macrophage colony-stimulating factor on the basis of the ability of these factors to confer autonomous growth on the factor-dependent hemopoietic cell line FDC-P1. Moreover, the frequency at which these factor-independent clones were isolated approximated the frequency at which they were represented in the original plasmid library. These results suggest that expression cloning with retroviruses is a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes.

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30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Mineralocorticoid receptor null mice: informing cell-type-specific roles

Journal of Endocrinology ◽

10.1530/joe-17-0155 ◽

2017 ◽

Vol 234 (1) ◽

pp. T83-T92 ◽

Cited By ~ 19

Author(s):

Timothy J Cole ◽

Morag J Young

Keyword(s):

Mineralocorticoid Receptor ◽

Gene Knockout ◽

Cell Signalling ◽

Plasma Renin ◽

Cell Types ◽

Specific Cell ◽

Salt Wasting ◽

Corticosteroid Hormones ◽

Stress Behaviour

The mineralocorticoid receptor (MR) mediates the actions of two important adrenal corticosteroid hormones, aldosterone and cortisol. The cell signalling roles of the MR in vivo have expanded enormously since the cloning of human MR gene 30 years ago and the first MR gene knockout in mice nearly 20 years ago. Complete ablation of the MR revealed important roles postnatally for regulation of kidney epithelial functions, with MR-null mice dying 1–2 weeks postnatally from renal salt wasting and hyperkalaemia, with elevated plasma renin and aldosterone. Generation of tissue-selective MR-deficient mice using Cre recombinase-LoxP gene targeting has made it possible to analyse mice lacking MR only in specific cell types. Targeting renal-specific MR has differentiated roles in specific compartments of the kidney. Ablating MR in neurons of the forebrain reinforced important roles of the MR in response to stress, behaviour and anxiety, but suggested a minimal role in maintaining basal HPA axis tone. Deletion of the MR in macrophages and other cell types of the cardiovascular system clearly defined important roles for the regulation of cardiovascular physiology and pathophysiology. Knockdown of MR mRNA in vivo using antisense/siRNA approaches, and similarly MR overexpression, has provided useful rodent models to study physiological roles of MR signalling in vivo. More recently, targeted mutation of specific domains of the MR such as the DBD has defined genomic vs non-genomic roles in vivo. New tissue-selective MR-null models are required to define roles of MR signalling in other regions of the brain, the eye, gastrointestinal tract, lung, skin, breast and gonadal organs.

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The Endocannabinoid System in Glial Cells and Their Profitable Interactions to Treat Epilepsy: Evidence from Animal Models

International Journal of Molecular Sciences ◽

10.3390/ijms222413231 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13231

Author(s):

Jon Egaña-Huguet ◽

Edgar Soria-Gómez ◽

Pedro Grandes

Keyword(s):

Glial Cells ◽

Endocannabinoid System ◽

Cell Types ◽

Specific Cell ◽

Neuronal Viability ◽

Wide Range ◽

Novel Target ◽

Different Cell Types ◽

The Impact

Epilepsy is one of the most common neurological conditions. Yearly, five million people are diagnosed with epileptic-related disorders. The neuroprotective and therapeutic effect of (endo)cannabinoid compounds has been extensively investigated in several models of epilepsy. Therefore, the study of specific cell-type-dependent mechanisms underlying cannabinoid effects is crucial to understanding epileptic disorders. It is estimated that about 100 billion neurons and a roughly equal number of glial cells co-exist in the human brain. The glial population is in charge of neuronal viability, and therefore, their participation in brain pathophysiology is crucial. Furthermore, glial malfunctioning occurs in a wide range of neurological disorders. However, little is known about the impact of the endocannabinoid system (ECS) regulation over glial cells, even less in pathological conditions such as epilepsy. In this review, we aim to compile the existing knowledge on the role of the ECS in different cell types, with a particular emphasis on glial cells and their impact on epilepsy. Thus, we propose that glial cells could be a novel target for cannabinoid agents for treating the etiology of epilepsy and managing seizure-like disorders.

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CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses

10.1101/2020.03.13.991224 ◽

2020 ◽

Author(s):

Kristina Sakers ◽

Yating Liu ◽

Lorida Llaci ◽

Michael J. Vasek ◽

Michael A. Rieger ◽

...

Keyword(s):

Rna Binding ◽

Gene Knockout ◽

Binding Protein ◽

Rna Binding Protein ◽

Cell Types ◽

Specific Gene ◽

Specific Cell ◽

Normal Brain ◽

Oligodendrocyte Development

AbstractQuaking RNA binding protein(QKI) is essential for oligodendrocyte development as myelination requires MBP mRNA regulation and localization by the cytoplasmic isoforms(e.g. QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we show via CLIPseq that QKI-6 binds 3’ UTRs of a subset of astrocytic mRNAs, including many enriched in peripheral processes. Binding is enriched near stop codons, which is mediated partially by QKI binding motifs(QBMs) yet spreads to adjacent sequences. We developed CRISPR TRAPseq: a viral approach for mosaic, cell-type specific gene mutation with simultaneous translational profiling. This enabled study of QKI-deleted astrocytes in an otherwise normal brain. Astrocyte-targeted QKI deletion altered translation and maturation, while also increasing synaptic density within the astrocyte’s territory. Overall, our data indicate QKI is required for astrocyte maturation and demonstrate an approach for a highly targeted translational assessment of gene knockout in specific cell-types in vivo.

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The derivation and potential use of human embryonic stem cells

Reproduction Fertility and Development ◽

10.1071/rd01101 ◽

2001 ◽

Vol 13 (8) ◽

pp. 523 ◽

Cited By ~ 28

Author(s):

Alan O. Trounson

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Regenerative Medicine ◽

Human Embryonic Stem Cells ◽

High Efficiency ◽

Es Cells ◽

Cell Mass ◽

Embryonic Stem ◽

Cell Types ◽

Specific Cell

Human embryonic stem cells lines can be derived from human blastocysts at high efficiency (>50%) by immunosurgical isolation of the inner cell mass and culture on embryonic fibroblast cell lines. These cells will spontaneously differentiate into all the primary embryonic lineages in vitro and in vivo, but they are unable to form an integrated embryo or body plan by themselves or when combined with trophectoderm cells. They may be directed into a number of specific cell types and this enrichment process requires specific growth factors, cell-surface molecules, matrix molecules and secreted products of other cell types. Embryonic stem (ES) cells are immortal and represent a major potential for cell therapies for regenerative medicine. Their use in transplantation may depend on the formation of a large bank of suitable human leucocyte antigen (HLA) types or the genetic erasure of their HLA expression. Successful transplantation may also require induction of tolerance in recipients and ongoing immune suppression. Although it is possible to customize ES cells by therapeutic cloning or cytoplasmic transfer, it would appear unlikely that these strategies will be used extensively for producing ES cells compatible for transplantation. Embryonic stem cell research may deliver a new pathway for regenerative medicine.

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Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair

Nucleic Acids Research ◽

10.1093/nar/gkaa085 ◽

2020 ◽

Vol 48 (7) ◽

pp. e38-e38 ◽

Cited By ~ 4

Author(s):

Sara E DiNapoli ◽

Raul Martinez-McFaline ◽

Caitlin K Gribbin ◽

Paul J Wrighton ◽

Courtney A Balgobin ◽

...

Keyword(s):

Genome Editing ◽

High Efficiency ◽

Cell Types ◽

Specific Cell ◽

Loss Of Function ◽

Genomic Deletions ◽

Homology Directed Repair ◽

Linear Dsdna ◽

Rapid Generation

Abstract CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

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A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.880-887.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 880-887

Author(s):

J R Rayner ◽

T J Gonda

Keyword(s):

Cell Types ◽

Retroviral Vector ◽

Expression Cloning ◽

Target Cells ◽

Specific Cell ◽

Functional Screening ◽

Efficient Procedure ◽

Plasmid Library ◽

Wide Range ◽

Expression Libraries

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Cell therapy in orthopaedics

The Bone & Joint Journal ◽

10.1302/0301-620x.101b4.bjj-2019-0013.r1 ◽

2019 ◽

Vol 101-B (4) ◽

pp. 361-364 ◽

Cited By ~ 20

Author(s):

S. A. Rodeo

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Adipose Tissue ◽

Cell Therapy ◽

Bone Cells ◽

Cell Types ◽

Specific Cell ◽

Cell Therapies ◽

Native Bone ◽

Wide Range

Stem cells are defined by their potential for self-renewal and the ability to differentiate into numerous cell types, including cartilage and bone cells. Although basic laboratory studies demonstrate that cell therapies have strong potential for improvement in tissue healing and regeneration, there is little evidence in the scientific literature for many of the available cell formulations that are currently offered to patients. Numerous commercial entities and ‘regenerative medicine centres’ have aggressively marketed unproven cell therapies for a wide range of medical conditions, leading to sometimes indiscriminate use of these treatments, which has added to the confusion and unpredictable outcomes. The significant variability and heterogeneity in cell formulations between different individuals makes it difficult to draw conclusions about efficacy. The ‘minimally manipulated’ preparations derived from bone marrow and adipose tissue that are currently used differ substantially from cells that are processed and prepared under defined laboratory protocols. The term ‘stem cells’ should be reserved for laboratory-purified, culture-expanded cells. The number of cells in uncultured preparations that meet these defined criteria is estimated to be approximately one in 10 000 to 20 000 (0.005% to 0.01%) in native bone marrow and 1 in 2000 in adipose tissue. It is clear that more refined definitions of stem cells are required, as the lumping together of widely diverse progenitor cell types under the umbrella term ‘mesenchymal stem cells’ has created confusion among scientists, clinicians, regulators, and our patients. Validated methods need to be developed to measure and characterize the ‘critical quality attributes’ and biological activity of a specific cell formulation. It is certain that ‘one size does not fit all’ – different cell formulations, dosing schedules, and culturing parameters will likely be required based on the tissue being treated and the desired biological target. As an alternative to the use of exogenous cells, in the future we may be able to stimulate the intrinsic vascular stem cell niche that is known to exist in many tissues. The tremendous potential of cell therapy will only be realized with further basic, translational, and clinical research. Cite this article: Bone Joint J 2019;101-B:361–364.

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