Cellular Manipulation Research Articles

Antifouling coatings for optoelectronic tweezers

Optoelectronic tweezers enables parallel manipulation of individual single cells using optical addressing and optically induced dielectrophoretic force. This provides a useful platform for performing a variety of biological functions, such as cell manipulation, cell sorting, and cell electroporation. However, in order to obtain more reliable cellular manipulation, especially of adherent mammalian cells, antifouling coatings need to be used to avoid non-specific cell adherence. Two antifouling coatings are discussed here, which can reduce the amount of non-specific adherence by as much as a factor of 30.

Simultaneous intracellular delivery of targeting antibodies and functional nanoparticles with engineered protein G system

Cellular internalization of functional nanoparticles that have optical and magnetic properties is very important in the cellular imaging and manipulation of specifically targeted biomolecules. In this study, a robust method to deliver functional nanoparticles and targeting antibodies into cells was suggested. The engineered protein G system, which contains an affinity tag and a cell penetration peptide in the N- and C-terminals, respectively, can capture surface-modified nanoparticles and antibodies without chemical reaction, and then non-invasively deliver them into the cells. Finally, gold-coated iron oxide nanoparticle/engineered protein G hybrid systems were successfully employed as multifunctional cargo systems for the targeting, imaging, and manipulation of mitochondria.

Induction of Cell Death by Magnetic Actuation of Nickel Nanowires Internalized by Fibroblasts

Magnetic nanomaterials with multimodal functionalities have emerged as a versatile platform for biomedical applications that range from basic cellular interrogation to clinical nanomedicine. In this work, we have prepared electrodeposited ferromagnetic nickel nanowires for efficient internalization into 3T3 fibroblasts. Agitation of the nanowires by a low external field induced cell death, as assessed by MTT viability assays. The response of the interleukin-6 (IL-6) gene expression of the fibroblasts to nanowire-mediated cellular manipulation was examined by quantitative real-time polymerase chain reaction (qRT-PCR). These nanowires exhibited significant potential as therapeutic and interrogative platforms for biomedicine.

Allogeneic endometrial regenerative cells: an "Off the shelf solution" for critical limb ischemia?

Critical limb ischemia (CLI) is an advanced form of peripheral artery disease which is responsible for approximately 100,000 amputations per year in the US. Trials to date have reported clinical improvement and reduced need for amputation in CLI patients receiving autologous bone marrow or mobilized peripheral blood stem cells for stimulation of angiogenesis. While such treatments are currently entering Phase III trials, practical and scientific pitfalls will limit widespread implementation if efficacy is proven. Hurdles to be overcome include: a) reduced angiogenic potential of autologous cells in aged patients with cardiovascular risk factors; b) invasiveness/adverse effects of bone marrow extraction and G-CSF mobilization, respectively; and c) need for on-site cellular manipulation. The Endometrial Regenerative Cell (ERC) is a mesenchymal-like stem cell derived from the menstrual blood that is believed to be associated with endometrial angiogenesis. We discuss the possibility of using allogeneic ERCs as an "off the shelf" treatment for CLI based on the following properties: a) High levels of growth factors and matrix metalloprotease production; b) Ability to inhibits inflammatory responses and lack of immunogenicity; and c) Expandability to great quantities without loss of differentiation ability or karyotypic abnormalities.

Generation of transducible versions of transcription factors Oct4 and Sox2

The transcription factors Oct4 and Sox2 are two of the main regulators of pluripotency in embryonic stem cells. Since the importance of non-genetic modification is continually increasing, particularly for therapeutic application of manipulated cells, the aim of the present study was to generate cell-permeant Oct4 and Sox2 proteins for the direct cellular delivery of active proteins. Protein transduction allowing cellular manipulation to circumvent genetic modification of target cells has recently been developed. We present a new expression vector system, pSESAME, that facilitates the generation of transducible proteins. Using pSESAME, both Oct4 and Sox2 were genetically fused with a TAT protein transduction domain that promotes cellular penetration. The recombinant purified Oct4 and Sox2 fusion proteins display DNA-binding properties comparable to their endogenous counterparts, and exhibit cellular entry and the ability to modulate the transcriptional machinery maintaining pluripotency of mouse embryonic stem cells. In a rescue assay we demonstrate that transducible Oct4 and Sox2 fusion proteins can compensate knockdown of Pou5f1 and Sox2, respectively. This study provides powerful tools for the modulation of stem cell properties without genetic interference.

A self-renewing, tissue-engineered vascular graft for arterial reconstruction

Various tissue-engineered vascular grafts have been studied to overcome the clinical disadvantages of conventional prostheses. Previous tissue-engineered vascular grafts have generally required preoperative cellular manipulation or use of bioreactors to improve performance, and their mechanical properties have been insufficient. We focused on the concept of in situ cellularization and developed a tissue-engineered vascular graft for arterial reconstruction that would facilitate renewal of autologous tissue without any pretreatment. The graft comprised an interior of knitted polyglycolic acid compounded with collagen to supply a scaffold for tissue growth and an exterior of woven poly-L-lactic acid for reinforcement. All components were biocompatible and biodegradable, with excellent cellular affinity. The grafts, measuring 10 mm in internal diameter and 30 mm in length, were implanted into porcine aortas, and their utility was evaluated to 12 months after grafting. All explants were patent throughout the observation period, with no sign of thrombus formation or aneurysmal change. Presence in the neomedia of endothelialization with proper integrity and parallel accumulation of functioning smooth muscle cells, which responded to vasoreactive agents, was confirmed in an early phase after implantation. Sufficient collagen synthesis and lack of elastin were quantitatively demonstrated. Dynamic assessment and long-term results of the in vivo study indicated adequate durability of the implants. The graft showed morphologic evidence of good in situ cellularization, satisfactory durability to withstand arterial pressure for 12 postoperative months, and the potential to acquire physiologic vasomotor responsiveness. These results suggest that our tissue-engineered vascular graft shows promise as an arterial conduit prosthesis.

Chemical Analysis of Single Cells

Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques. Microseparation techniques, coupled to varying detection schemes including absorbance and fluorescence detection, electrochemical detection, and mass spectrometry, have allowed researchers to examine a number of processes inside single cells. This review also touches on a promising direction in single cell cytometry: the development of microfluidics for integrated cellular manipulation, chemical processing, and separation of cellular contents.

Involvement of the vascular wall in regenerative processes after CA1 ischemic neuronal death

The involvement of vascular wall in response to neuronal death was challenged here using a transient forebrain ischemia model in gerbil, which causes CA1 neuronal death and trigger neurogenesis in hippocampus. We found an important vascular reaction in CA1 5 days after ischemia evaluated by Von Willebrand factor and Vimentin immunoreactivity, as well as increased expression of angiogenic and neurogenic regulators: Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). Analysing the morphology and cell phenotype by confocal microscopy, we confirmed the colocalization of the neurogenic markers (bromodeoxyuridine–neuronal nuclei–TOPRO-3) in newborn cells associated to vascular walls in CA1 and dentate gyrus of hippocampus 32 days after ischemia. The results indicate that vascular tissues may participate in neurogenesis after brain ischemia, reinforce the notion that blood vessels represent a source of neuronal progenitor cells in damaged brain areas and suggest that molecular and cellular manipulation of the vascular wall may expand the possibilities of novel regenerative therapies.

원격조작을 위한 2차원 영상정보에 기반한 저속 변형체의 실시간 햅틱 렌더링

Haptic rendering is a process providing force feedback during interactions between a user and a virtual object. This paper presents a real-time haptic rendering technique for deformable objects based on visual information of intervention between a tool and a real object in a remote place. A user can feel the artificial reaction force through a haptic device in real-time when a slave system exerts manipulation tasks on a deformable object. The models of the deformable object and the manipulator are created from the captured image obtained with a CCD camera and the recognition of objects is achieved using image processing techniques. The force at a rate of 1 kHz for stable haptic interaction is deduced using extrapolation of forces at a low update rate. The rendering algorithm developed was tested and validated on a test platform consisting of a one-dimensional indentation device and an off-the shelf force feedback device. This software system can be used in a cellular manipulation system providing artificial force feedback to enhance a success rate of operations.

Optically integrated microfluidic systems for cellular characterization and manipulation

Expanding interest in microfluidic techniques for biomedical applications has driven the recent need for micro-integrated optics capable of both traditional characterization and emerging optical manipulation techniques. We discuss here how ultrafast laser micromachining can be used to create optical waveguides directly within microfluidic systems. We then utilize this fabrication approach to create a unique microfluidic platform for optical characterization and sorting of cells and particles. This new platform employs optically fabricated waveguides to scatter and refract light from individual particles, allowing accurate in situ size detection and sorting within a microfluidic channel.

Transcriptional Profiling of Hematologic Malignancies with a Low-Density DNA Microarray

High-density microarrays are powerful tools for expression analysis of thousands of genes simultaneously; however, experience with low-density microarrays in gene expression studies has been limited. We developed an optimized procedure for gene expression analysis based on a microarray containing 538 oligonucleotides and used this procedure to analyze neoplastic cell lines and whole-blood samples from healthy individuals and patients with different hematologic neoplasias. Hierarchical clustering and the Welch t-test with adjusted P values were used for data analysis. This procedure detects 0.2 fmol of mRNA and generates a linear response of 2 orders of magnitude, with CV values of <20% for hybridization and label replicates. We found statistically significant differences between Jurkat and U937 cell lines, between blood samples from 15 healthy donors and 59 chronic lymphocytic leukemia (CLL) samples, and between 6 acute myeloid leukemia patients and 4 myelodysplastic syndrome patients. A classification system constructed from the expression data predicted healthy or CLL status from a whole-blood sample with a 97% success rate. Transcriptional profiling of whole-blood samples was carried out without any cellular or sample manipulation before RNA extraction. This gene expression analysis procedure uncovered statistically significant differences associated with different hematologic neoplasias and made possible the construction of a classification system that predicts the healthy or CLL status from a whole-blood sample.

A Low Density DNA Microarray for Comparing Gene Expression Profiles in Hematological Malignancies.

High density microarrays (HDM) are powerful tools for simultaneously profiling the expression levels of thousands of genes. The application of this technology to study of neoplastic hematological disorders.has identified new sub groups of disease not related previously and new prognosis markers. However there is a limited experience in the gene expression studies using low density microarrays (LDM) in neoplastic hematological disorders.A gene expression analysis system based on a LDM containing 538 oligonucleotides has been developed. Whole technical process was optimized to improve the analysis of differential expression. We have analyzed mRNA from cell line cultures (Jurkat, U937), whole blood samples from healthy subjects and different hematological malignancies (HM) using this chip. A hierarchical clustering procedure applying Welch t-statistics with Bonferroni correction was used to analysis gene expression data The LDM generated a linear response of 2 magnitude orders and a CV values less than 20% for hybridization and label replicates. This procedure detects 0,2 fmols of mRNA. We have found genes with statistically significant differences between Jurkatt and U937 cells cultures, and blood samples from 15 healthy donors, 59 lymphocyte leukemia and 13 myeloid leukemia and myelodisplasia syndrome patients. A classification system based on gene expression data was constructed with an accuracy of 97%.to predict healthy or lymphocyte leukemia status. To identify different subsets of patients in the B-CLL group, whole blood samples from 12 B-CLL patients were collected and defined as stables, according to clinical and analytical criteria at the time of diagnosis, “stable” (n=6) if disease stability was maintained for more than five years after the diagnosis and “progressive” (n=6) if the disease progressed less than one year after the diagnosis. Applying Welch statistical test without correction and a p<0.05 yielded two lists of 29 and 19 probes differentially hybridized from VSN and quantile-robust normalized data, respectively. The supervised hierarchical clustering of B-CLL samples with 29 statistically significant probes shown that samples grouped together based on their stable or progressive behavior. Eighteen probes were statistically significant in both normalized data. In order to confirm the data expression of POU2F2, PSMB4, FCER2, LCP1, and ABCC5 genes represented by 5 of the 18 statistically significant probes, real-time RT-PCR was performed. Three out of 5 genes -POU2F2, PSMB2, and FCER2- were over-expressed in B-CLL stable patients. Differences were statistically significant (P<0.05) and, therefore, results obtained from the chip for POU2F2, PSMB2, and FCER2 genes were confirmed.In conclusion, a viable LDM for gene expression analysis and a simple procedure has been developed useful for analysis of whole blood samples, without any cellular or sample manipulation prior to RNA extraction with variability and reproducibility similar to others commercial HDM. The application to different samples is capable to establish significant differences in gene clusters and could be useful for clinical application in HM

Reprogramming somatic cells into stem cells

Recent scientific achievements in cell and developmental biology have provided unprecedented opportunities for advances in biomedical research. The demonstration that fully differentiated cells can reverse their gene expression profile to that of a pluripotent cell, and the successful derivation and culture of human embryonic stem cells (ESCs) have fuelled hopes for applications in regenerative medicine. These advances have been put to public scrutiny raising legal, moral and ethical issues which have resulted in different levels of acceptance. Ethical issues concerning the use of cloned human embryos for the derivation of stem cells have stimulated the search for alternative methods for reversing differentiated cells into multi/pluripotent cells. In this article, we will review the present state of these reprogramming technologies and discuss their relative success. We also overview reprogramming events after somatic cell nuclear transfer (SCNT), as they may further instruct ex ovo strategies for cellular manipulation.

Heart valve tissue engineering: concepts, approaches, progress, and challenges.

Potential applications of tissue engineering in regenerative medicine range from structural tissues to organs with complex function. This review focuses on the engineering of heart valve tissue, a goal which involves a unique combination of biological, engineering, and technological hurdles. We emphasize basic concepts, approaches and methods, progress made, and remaining challenges. To provide a framework for understanding the enabling scientific principles, we first examine the elements and features of normal heart valve functional structure, biomechanics, development, maturation, remodeling, and response to injury. Following a discussion of the fundamental principles of tissue engineering applicable to heart valves, we examine three approaches to achieving the goal of an engineered tissue heart valve: (1) cell seeding of biodegradable synthetic scaffolds, (2) cell seeding of processed tissue scaffolds, and (3) in-vivo repopulation by circulating endogenous cells of implanted substrates without prior in-vitro cell seeding. Lastly, we analyze challenges to the field and suggest future directions for both preclinical and translational (clinical) studies that will be needed to address key regulatory issues for safety and efficacy of the application of tissue engineering and regenerative approaches to heart valves. Although modest progress has been made toward the goal of a clinically useful tissue engineered heart valve, further success and ultimate human benefit will be dependent upon advances in biodegradable polymers and other scaffolds, cellular manipulation, strategies for rebuilding the extracellular matrix, and techniques to characterize and potentially non-invasively assess the speed and quality of tissue healing and remodeling.

Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells

Recent work in neuroscience has shown that the adult central nervous system (CNS) contains neural progenitors, precursors and stem cells that are capable of generating new neurons, astrocytes and oligodendrocytes. While challenging the previous dogma that no new neurons are born in the adult mammalian CNS, these findings bring with them the future possibilities for development of novel neural repair strategies. The purpose of this review is to present the current knowledge about constitutively occurring adult mammalian neurogenesis, highlight the critical differences between 'neurogenic' and 'non-neurogenic' regions in the adult brain, and describe the cardinal features of two well-described neurogenic regions-the subventricular zone/olfactory bulb system and the dentate gyrus of the hippocampus. We also provide an overview of presently used models for studying neural precursors in vitro, mention some precursor transplantation models and emphasize that, in this rapidly growing field of neuroscience, one must be cautious with respect to a variety of methodological considerations for studying neural precursor cells both in vitro and in vivo. The possibility of repairing neural circuitry by manipulating neurogenesis is an intriguing one, and, therefore, we also review recent efforts to understand the conditions under which neurogenesis can be induced in non-neurogenic regions of the adult CNS. This work aims towards molecular and cellular manipulation of endogenous neural precursors in situ, without transplantation. We conclude this review with a discussion of what might be the function of newly generated neurons in the adult brain, and provide a summary of present thinking about the consequences of disturbed adult neurogenesis and the reaction of neurogenic regions to disease.

Neural stem cell properties of Müller glia in the mammalian retina: Regulation by Notch and Wnt signaling

The retina in adult mammals, unlike those in lower vertebrates such as fish and amphibians, is not known to support neurogenesis. However, when injured, the adult mammalian retina displays neurogenic changes, raising the possibility that neurogenic potential may be evolutionarily conserved and could be exploited for regenerative therapy. Here, we show that Müller cells, when retrospectively enriched from the normal retina, like their radial glial counterparts in the central nervous system (CNS), display cardinal features of neural stem cells (NSCs), i.e., they self-renew and generate all three basic cell types of the CNS. In addition, they possess the potential to generate retinal neurons, both in vitro and in vivo. We also provide direct evidence, by transplanting prospectively enriched injury-activated Müller cells into normal eye, that Müller cells have neurogenic potential and can generate retinal neurons, confirming a hypothesis, first proposed in lower vertebrates. This potential is likely due to the NSC nature of Müller cells that remains dormant under the constraint of non-neurogenic environment of the adult normal retina. Additionally, we demonstrate that the mechanism of activating the dormant stem cell properties in Müller cells involves Wnt and Notch pathways. Together, these results identify Müller cells as latent NSCs in the mammalian retina and hence, may serve as a potential target for cellular manipulation for treating retinal degeneration.

Selective loss of dopaminergic neurons and formation of Lewy body‐like aggregations in α‐synuclein transgenic fly neuronal cultures

A key pathological feature of Parkinson's disease (PD) is the selective loss of dopaminergic neurons accompanied by the formation of Lewy bodies (LB). Given the complex nature of the disease, it is imperative to develop a model system suitable for molecular and cellular manipulation in order to study the mechanisms underlying the pathogenesis of PD. Here, we report that a new in vitro model of PD has been developed by using Drosophila melanogaster primary neuronal cultures expressing a human mutant alpha-synuclein (alpha-Syn; A30P). The selective loss of dopaminergic (DA) neurons was observed when alpha-Syn was pan-neuronally expressed while non-dopaminergic neurons (e.g. GABAergic) were not influenced. This degeneration was also observed even when alpha-Syn was specifically expressed in DA neurons, demonstrating alpha-Syn toxicity is DA cell-autonomous. In all experiments, cultures 5 days or older showed clear degeneration of DA neurons whereas this degeneration was not significant in 3-day-old cultures. In addition, there were intracellular aggregations in 5-day or older alpha-Syn neurons that were recognized by anti-alpha-Syn or ubiquitin antibodies, demonstrating the formation of LB-like inclusions. By contrast, no such aggregations were found in 3-day-old neurons. The results demonstrate that mutated human alpha-Syn expressed in Drosophila primary neuronal cultures causes the selective loss of DA neurons and the formation of cellular aggregations. Therefore, this is one of the first in vitro models recapitulating two important cellular features of PD and will be useful in examining mechanisms underlying selective neurodegeneration mediated by alpha-Syn.

Magnetic cell manipulation utilizing magnetic particles and diamagnetic collagen fibers

This paper reports a method of controlling the orientation of adherent fibroblastic cells (osteoblasts) by utilizing a ferromagnetic particle chain and a diamagnetic collagen under exposure to magnetic fields. A cell culture medium with cells and magnetic particles (γ-Fe2O3) was mixed with a collagen solution and incubated for 24h. During the first 3h of incubation, the medium in the cell culture dish was exposed to magnetic fields of up to 8T. The exposure caused the aggregation of magnetic particles (MPs), which formed chains in parallel with the magnetic field and at the same time oriented the polymerized collagen fibers perpendicular to the magnetic field. A lattice pattern of MP chains and collagen fibers was observed on the bottom of the culture flask. After 24h of incubation, the spindlelike cells had become oriented in parallel with the MP chains or the collagen fibers. The results indicate the possibility of a technique of cellular manipulation with MP-collagen hybrid that could be used to control cellular orientation.

Cellular Manipulation of Human Embryonic Stem Cells by TAT-PDX1 Protein Transduction

Human embryonic stem cells (hESCs) are an in vitro model system for the study of human early development and a potential source for cell-based therapies. An efficient strategy for cellular manipulation of hESCs may be highly valuable for the analysis of gene function involved in human embryogenesis and the development of cell-based therapies via induced differentiation into particular cell types. However, plasmid transfection of hESCs has low efficiency and viral transduction may not be the method of choice for cell-based therapies due to genome integration. To overcome these limitations, we applied protein transduction technology that can transfer proteins into cells via direct penetration across the lipid bilayer. Here, we show that the FITC dye fused to the TAT protein transduction domain (PTD) was efficiently transferred into hESCs. In addition, the PDX1 transcription factor, which plays a central role in pancreatic development, was transferred into hESCs as a fusion form of TAT PTD. The transduced TAT-PDX1 activated its downstream target genes and induced insulin protein production in hESCs. These results demonstrate that protein transduction could be used in the cellular manipulation of hESCs and would provide a significant breakthrough for basic and therapeutic research in hESCs.

Transport, retention and fluorescent measurement of single biological cells studied in microfluidic chips.

Cellular manipulation and fluorescent measurement were performed on two types of biological cells. First, transport and retention of yeast cells were demonstrated on a glass microfluidic chip, which consists of special U-shaped microstructures. These microstructures have the openings parallel to the liquid flow and weirs perpendicular to the flow. These allow the retention of yeast cells in the U-shaped pocket and drainage of liquid over the weirs. Thereafter, the same chip was used to carry out real-time fluorescent measurement for the cellular changes in single Jurkat T cells. In this case, the Jurkat cells were localized inside the straight portion of a microchannel. Fluorescent imaging on the same, single suspension cell was carried out to study two cellular processes occurring in viable cells, (1) the intracellular conversion of fluorescein diacetate (FDA) to fluorescein; (2) the degradation of an inhibitory protein, IkappaB, as involved in the NF-kappaB signalling pathway. In the former, the increase in fluorescent intensity of single Jurkat T cells (due to fluorescein formation) was measured; whereas in the latter, the decrease in the fluorescent intensity of a single transfected Jurkat cell (due to the degradation of the IkappaB-EGFP fusion protein) was monitored. In addition, we employed a Jurkat cell expressed with IkappaB-EGFP to probe any possible action of an herbal compound, isoliquiritigenin (IQ), on the degradation of IkappaB-EGFP. These examples have demonstrated that Jurkat cells remain viable within microfluidic channels for cellular studies and that the microfluidic chip can facilitate monitoring of cellular changes of biological cells at the single cell level and in the same cell.