Microinjection Of Cells Research Articles

Endothelialized Bronchioalveolar Lung Organoids Model Endothelial Cell Responses to Injury.

Organoid 3D systems are powerful platforms to study development and disease. Recently, the complexity of lung organoid models derived from adult mouse and human stem cells has increased substantially in terms of cellular composition and structural complexity. However, a murine lung organoid system with a clear integrated endothelial compartment is still missing. Here, we describe a novel method that adds another level of intricacy to our published bronchioalveolar lung organoid (BALO) model by microinjection of FACS-sorted lung endothelial cells (ECs) into differentiated organoid cultures. Before microinjection, ECs obtained from the lung homogenate (LH) of young mice expressed typical ECs markers such as CD31 and vascular endothelial (VE)-Cadherin and showed tube formation capacity. Following microinjection, ECs surrounded BALO´s alveolar-like compartment aligning with both alveolar epithelial cells type I (AECI) and type II (AECII), as demonstrated by confocal and electron microscopy. Notably, expression of Car4 and Aplnr was as well detected, suggesting presence of EC microvascular phenotypes in the cultured ECs. Moreover, upon epithelial cell injury by lipopolysaccharides (LPS) and influenza A virus (IV), endothelialized BALO (eBALO) released proinflammatory cytokines leading to the upregulation of the intercellular adhesion molecule 1 (ICAM-1) in ECs. In summary, we characterized for the first time a organoid model that incorporates ECs into the alveolar structures of lung organoids, not only increasing our previous model ́s cellular and structural complexity but also providing a suitable niche to model lung endothelium responses to injury ex vivo.

118 Gene editing: A European perspective

Abstract Gene editing by CRISPR/Cas has revolutionized numerous facets of biotechnology within a remarkably short period of time. Its combined use with either somatic cell nuclear transfer or microinjection into fertilized oocytes is highly useful for mutating genes in a very precise and specific manner. This technology has profoundly transformed the world of animal research, particularly for biomedical and productive applications. Gene editing can now be applied to animal modeling and therapeutic research, bringing us closer to the possibility of using animals as organ donors for xenotransplantation. Furthermore, it has become an effective tool in animal production for purposes such as increasing disease tolerance or resistance and enhancing animal welfare. In this presentation, we will explore how this genome editing tool has influenced the research conducted in Europe, examining the current landscape and addressing the challenges, with a special focus on pigs. We will also discuss the regulation of gene-edited animals in the EU.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Precision Alignment in Cell Microinjection Based on Hybrid Triple-View Micro-Vision

Precision Alignment in Cell Microinjection Based on Hybrid Triple-View Micro-Vision

A Fractional Viscoelastic Mechanical Model for Speed Optimization of Robotic Cell Microinjection

A Fractional Viscoelastic Mechanical Model for Speed Optimization of Robotic Cell Microinjection

Deep Learning-Enhanced Dual-Module Large-Throughput Microinjection System for Adherent Cells

Despite the rapid development in providing precise delivery of extraneous samples to the vast majority of cells, robotic microinjection is still hindered by cumbersome operations and low throughput in practice. This study presents a new automated microinjection system equipped with two micromanipulators and a deep learning algorithm for cell identification. The introduction of two coordinated micromanipulators based on the same cell handling platform results in a large increase in injection throughput. A deep convolutional neural network, Mask R-CNN, is used to detect and segment stain-free adherent cells, leading to a considerable increase in operational efficiency and subsequent throughput. In the three independent experiments, over 10,000 MC3T3 mouse fibroblast cells are injected to evaluate the injection speed, success rate, and survival rate. Experimental results confirm that our system can inject around 4,000 cells in 1 h with an approximately 60.3% success rate and an 82.0% survival rate. This research’s success will make robotic microinjection a competitive tool in many biomedical applications, such as plasmid DNA transfection. <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Note to Practitioners</i> —The motivation of this study is to improve the throughput of cell microinjection, which has become a bottleneck problem hindering the clinical application of microinjection technology. Existing cell microinjection systems typically use only one micromanipulator and rely on manual identification of cells, resulting in a limited ability to process cells for one experimental cycle. This study proposes a novel method that identifies cells through deep learning automatically and uses two micromanipulators simultaneously to improve cell processing capabilities. The problem of manipulating two micromanipulators for nearby cells is formulated as a route optimization problem, which is also suitable for three or more micromanipulators. A deep learning algorithm is used to identify and select cells to increase the processing speed. Experiments are performed to demonstrate that the proposed method can greatly increase the throughput while maintaining satisfactory performance in microinjection.

Design and testing of a new microinjector with FBG force sensor for robot-assisted single cell microinjection

Microinjection with force sensing has demonstrated robust performance in improving the cell survival rate and adjusting robot motion parameters. In this article, a novel force-sensing microinjector is presented based on fiber Bragg grating (FBG) sensors to precept three-dimensional (3D) forces. Fibers are processed with acid to become thinner and more sensitive. Three optical fibers are affixed to the outer surface of the glass needle along the axial direction. Benefiting from the novel configuration of the FBG sensors, the hollow channel of the glass needle can be used to inject exogenous substances. Moreover, a prototype has been fabricated for the experiment. Experimental results demonstrate the proposed force sensor can achieve a resolution of 66.8 μN for puncture forces. Repeatability and temperature compensation experiments show the force sensor can provide a consistent and accurate measurement. The developed force sensor has been applied to the microinjection of the zebrafish embryos, which is a promising tool for perceiving cell manipulation microforces.

Dynamic Seeding versus Microinjection of Adipose-Derived Mesenchymal Stem Cells to Acellular Nerve Allograft Reconstructions.

Functional recovery after acellular nerve allograft (ANA) reconstruction remains inferior to that after autologous nerve grafting, but improved outcomes have been demonstrated with the addition of adipose-derived mesenchymal stem cells (MSCs). Controversy exists regarding the optimal cell-delivery method to enhance ANA reconstructions. The authors investigated the functional recovery of ANAs after dynamic seeding versus microinjection of MSCs. Forty Lewis rats underwent reconstruction of a 10-mm sciatic nerve defect. Animals were divided into 4 groups: reversed autograft, ANA alone, dynamically seeded ANA, or ANA injected with MSCs. During the survival period, ultrasound measurements of the tibialis anterior muscle cross-sectional area were performed. At 12 weeks, functional recovery was evaluated using measurements of ankle contracture, compound muscle action potential, maximum isometric tetanic force, muscle mass, histomorphometry, and immunofluorescence. The dynamic seeding and microinjection groups demonstrated higher cross-sectional tibialis anterior muscle area recovery than autografts and ANAs alone at week 8 and weeks 4 and 8, respectively. The ankle contracture and compound muscle action potential amplitude recovery were superior in autografts and both seeding methods compared with ANAs alone. The microinjection group demonstrated significantly higher isometric tetanic force, muscle mass, and number of axons compared with ANAs alone. Both seeding methods showed higher CD34 densities compared with ANAs alone. No significant differences between dynamic seeding and microinjection were observed in functional or histologic outcomes. The addition of MSCs to ANAs demonstrated earlier motor regeneration compared with autografts and ANAs alone. Both seeding methods improved functional outcomes in the rat sciatic nerve defect model.

A Rate-Dependent Cell Microinjection Model Based on Membrane Theory.

As an effective method to deliver external materials into biological cells, microinjection has been widely applied in the biomedical field. However, the knowledge of cell mechanical property is still inadequate, which greatly limits the efficiency and success rate of injection. Thus, a new rate-dependent mechanical model based on membrane theory is proposed for the first time. In this model, an analytical equilibrium equation between the injection force and cell deformation is established by considering the speed effect of microinjection. Different from the traditional membrane-theory-based model, the elastic coefficient of the constitutive material in the proposed model is modified as a function of the injection velocity and acceleration, effectively simulating the influence of speeds on the mechanical responses and providing a more generalized and practical model. Using this model, other mechanical responses at different speeds can be also accurately predicted, including the distribution of membrane tension and stress and the deformed shape. To verify the validity of the model, numerical simulations and experiments were carried out. The results show that the proposed model can match the real mechanical responses well at different injection speeds up to 2 mm/s. The model presented in this paper will be promising in the application of automatic batch cell microinjection with high efficiency.

51. Dynamic Seeding versus Microinjection of Adipose-derived Mesenchymal Stem Cells to Acellular Nerve Allograft Reconstructions

51. Dynamic Seeding versus Microinjection of Adipose-derived Mesenchymal Stem Cells to Acellular Nerve Allograft Reconstructions

Infrared Laser-Based Single Cell Permeabilization by Plasma Membrane Temperature Gradients.

Single cell microinjection provides precise tuning of the volume and timing of delivery into the treated cells; however, it also introduces workflow complexity that requires highly skilled operators and specialized equipment. Laser-based microinjection provides an alternative method for targeting a single cell using a common laser and a workflow that may be readily standardized. This paper presents experiments using a 1550 nm, 100 fs pulse duration laser with a repetition rate of 20 ns for laser-based microinjection and calculations of the hypothesized physical mechanism responsible for the experimentally observed permeabilization. Chinese Hamster Ovarian (CHO) cells exposed to this laser underwent propidium iodide uptake, demonstrating the potential for selective cell permeabilization. The agreement between the experimental conditions and the electropermeabilization threshold based on estimated changes in the transmembrane potential induced by a laser-induced plasma membrane temperature gradient, even without accounting for enhancement due to traditional electroporation, strengthens the hypothesis of this mechanism for the experimental observations. Compared to standard 800 nm lasers, 1550 nm fs lasers may ultimately provide a lower cost microinjection method that readily interfaces with a microscope and is agnostic to operator skill, while inducing fewer deleterious effects (e.g., temperature rise, shockwaves, and cavitation bubbles).

Anti-tumor and analgesic activity evaluation and mechanism of Compound Kushen Injection

This study aims to evaluate the anti-tumor and analgesic activities of Compound Kushen Injection(CKI) based on zebrafish model in vivo and investigate the anti-tumor mechanism. To be specific, zebrafish tumor xenotransplantation model was established by microinjection of murine LPC H12 cells into yolk sac. Then the high-dose CKI(H-CKI), medium-dose CKI(M-CKI), low-dose CKI(L-CKI) groups, and the model group were set. The anti-tumor activity of CKI was evaluated with the tumor area growth fold and integral absorbance(IA) growth fold 72 h after administration. The peripheral pain and central pain in zebrafish were respectively induced with acetic acid(AA) and phorbol myristate acetate(PMA). Zebralab ViewPoint system was employed to monitor behavioral trajectory of zebrafish, and movement times, movement time, movement distance, and movement velocity were used to evaluate the analgesic activity of CKI. Finally, real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression levels of apoptosis-related B lymphocyte tumor-2(Bcl-2) and phosphatidylinositol-3-kinase(PI3 K)/protein kinase B(Akt or PKB) pathway-related genes, for the verification of the anti-tumor mechanism. Compared with the model group, M-CKI and H-CKI significantly reduced the growth folds of tumor area and IA, relief the peripheral pain and central pain. The mechanism was that CKI can up-regulate the expression of cysteine aspartic acid specific protease-3(caspase-3, Casp3) and caspase-9(Casp9), down-regulate the expression of phosphoinositide 3-kinase(PI3 K) and Akt, and significantly reduce the expression of Bcl-2, hypoxia-inducible factor-1α(HIF-1α), and vascular endothelial growth factor(VEGF). In conclusion, CKI has significant inhibitory effect on tumor growth and pain, which is related to the PI3 K/Akt signaling pathway. The pathway mediates cell apoptosis, suppresses tumor growth, and alleviates tumor pain.

Application of transposon systems in the transgenesis of bovine somatic and germ cells

BackgroundSeveral DNA transposons including PiggyBac (PB), Sleeping Beauty (SB), and Tol2 have been applied as effective means for of transgenesis in many species. Cattle are not typically experimental animals, and relatively little verification has been presented on this species. Thus, the goal here was to determine the applicability of three transposon systems in somatic and embryo cells in cattle, while also investigating which of the three systems is appropriate for each cell type. Green fluorescent protein (GFP)-expressing transposon systems were used for electroporation and microinjection in the somatic cells and embryo stage, respectively. After transfection, the GFP-positive cells or blastocysts were observed through fluorescence, while the transfection efficiency was calculated by FACS.ResultsIn bovine somatic cells, the PB (63.97 ± 11.56) showed the highest efficiency of the three systems (SB: 50.74 ± 13.02 and Tol2: 16.55 ± 5.96). Conversely, Tol2 (75.00%) and SB (70.00%) presented a higher tendency in the embryonic cells compared to PB (42.86%).ConclusionsThese results demonstrate that these three transposon systems can be used in bovine somatic cells and embryos as gene engineering experimental methods. Moreover, they demonstrate which type of transposon system to apply depending on the cell type.

Dynamic seeding versus microinjection of mesenchymal stem cells for acellular nerve allograft: an in vitro comparison

Mesenchymal stem cell (MSC)-supplemented acellular nerve allografts (ANA) are a potential strategy to improve the treatment of segmental nerve defects. Prior to clinical translation, optimal cell delivery methods must be defined. While two techniques, dynamic seeding and microinjection, have been described, the seeding efficiency, cell viability, and distribution of MSCs in ANAs are yet to be compared. Sciatic nerve segments of Sprague-Dawley rats were decellularized, and MSCs were harvested from the adipose tissue of Lewis rats. Cell viability was evaluated after injection of MSCs through a 27-gauge needle at different flow rates (10, 5, and 1 µL/min). MSCs were dynamically seeded or longitudinally injected into ANAs. Cell viability, seeding efficiency, and distribution were evaluated using LIVE/DEAD and MTS assays, scanning electron microscopy, and Hoechst staining. No statistically significant difference in cell viability after injection at different flow rates was seen. After cell delivery, 84.1±3.7% and 87.8±2.8% of MSCs remained viable in the dynamic seeding and microinjection group, respectively (p=0.41). The seeding efficiency of microinjection (100.4%±5.6) was significantly higher than dynamic seeding (48.1%±8.6) on day 1 (p=0.001). Dynamic seeding demonstrated a significantly more uniform cell distribution over the course of the ANA compared to microinjection (p=0.02). MSCs remain viable after both dynamic seeding and microinjection in ANAs. Higher seeding efficiency was observed with microinjection, but dynamic seeding resulted in a more uniform distribution. In vivo studies are required to assess the effect on gene expression profiles and functional motor outcomes.

Helicase-like transcription factor-deletion from the tumor microenvironment in a cell line-derived xenograft model of colorectal cancer reprogrammed the human transcriptome-S-nitroso-proteome to promote inflammation and redirect metastasis.

Methylation of the HLTF gene in colorectal cancer (CRC) cells occurs more frequently in men than women. Progressive epigenetic silencing of HLTF in tumor cells is accompanied by negligible expression in the tumor microenvironment (TME). Cell line-derived xenografts (CDX) were established in control (Hltf+/+) and Hltf-deleted male Rag2-/-IL2rg-/- mice by direct orthotopic cell microinjection (OCMI) of HLTF+/+HCT116 Red-FLuc cells into the submucosa of the cecum. Combinatorial induction of IL6 and S100A8/A9 in the Hltf-deleted TME with ICAM-1 and IL8 in the primary tumor activated a positive feedback loop. The proinflammatory niche produced a major shift in CDX metastasis to peritoneal dissemination compared to controls. Inducible nitric oxide (iNOS) gene expression and transactivation of the iNOS-S100A8/A9 signaling complex in Hltf-deleted TME reprogrammed the human S-nitroso-proteome. POTEE, TRIM52 and UN45B were S-nitrosylated on the conserved I/L-X-C-X2-D/E motif indicative of iNOS-S100A8/A9-mediated S-nitrosylation. 2D-DIGE and protein identification by MALDI-TOF/TOF mass spectrometry authenticated S-nitrosylation of 53 individual cysteines in half-site motifs (I/L-X-C or C-X-X-D/E) in CDX tumors. POTEE in CDX tumors is both a general S-nitrosylation target and an iNOS-S100A8/A9 site-specific (Cys638) target in the Hltf-deleted TME. REL is an example of convergence of transcriptomic-S-nitroso-proteomic signaling. The gene is transcriptionally activated in CDX tumors with an Hltf-deleted TME, and REL-SNO (Cys143) was found in primary CDX tumors and all metastatic sites. Primary CDX tumors from Hltf-deleted TME shared 60% of their S-nitroso-proteome with all metastatic sites. Forty percent of SNO-proteins from primary CDX tumors were variably expressed at metastatic sites. Global S-nitrosylation of proteins in pathways related to cytoskeleton and motility was strongly implicated in the metastatic dissemination of CDX tumors. Hltf-deletion from the TME played a major role in the pathogenesis of inflammation and linked protein S-nitrosylation in primary CDX tumors with spatiotemporal continuity in metastatic progression when the tumor cells expressed HLTF.

Penetration-force estimation approach for a flexure-jointed micro-injection mechanism with Lorentz force actuation

This paper presents a novel linear-motion compliant mechanism with Lorentz force actuation and integrated force-sensing capability for automated cell micro-injection. A model-based force estimation approach is introduced such that no force sensor is required. Model identification is undertaken by applying a sinusoidal actuation signal while the mechanism contacts with objects of known stiffness. Displacement data is then used to calculate model coefficients via a least-squares optimization. By using an observer-based state estimation scheme with actuation and displacement signals as inputs, force sensing accuracy within 70 μN RMS error could be achieved within a sensing range of 0 - 5 mN. This sensing capability confirms the suitability of the system for penetration force measurement in certain cases of cell micro-injection.

Image-Based Force Estimation in Medical Applications: A Review

Minimally invasive robotic interventions have highlighted the need to develop efficient techniques to measure forces applied to the soft tissues. Since the last decade, many scholars have focused on micro-scale and macro-scale robotic manipulations. Early articles used the model of soft tissue mathematically and tracked the displacement of the contour of the object in the vision system to provide the corresponding force to the user. Lack of knowledge of different materials and the computational complexity led to a transition from model-based to learning-based approaches to interpret the relation between object deformations, extracted from the vision system, and the real forces applied to the object. The dramatic growth of machine learning techniques and its integration with computer vision has brought novel learning-based visual data processing methods to the area. The application of the image-based force estimation methods in a controlled medical intervention has also received significant attention in the last five years. A decent number of surveys have been published on micromanipulation in recent years, especially for cell microinjection. However, the state of the art in meso- and macro-scale medical robotic interventions has not been reviewed. The aim and contribution of this paper are to fill the stated gap by reviewing the recent advances in image-based force estimation in robotic interventions. The survey shows that learning-based force estimation methods are growing significantly by using deep learning-based methods. The survey will encourage researchers and surgeons to apply learning-based algorithms to real-time medical and health-related operations.

High-Order Fiber Mode Beam Parameter Optimization for Transport and Rotation of Single Cells.

Optical tweezers are becoming increasingly important in biomedical applications for the trapping, propelling, binding, and controlled rotation of biological particles. These capabilities enable applications such as cell surgery, microinjections, organelle extraction and modification, and preimplantation genetic diagnosis. In particular, optical fiber-based tweezers are compact, highly flexible, and can be readily integrated into lab-on-a-chip devices. Taking advantage of the beam structure inherent in high-order modes of propagation in optical fiber, LP11, LP21, and LP31 fiber modes can generate structured radial light fields with two or more concentrations in the cross-section of a beam, forming multiple traps for bioparticles with a single optical fiber. In this paper, we report the dynamic modeling and optimization of single cell manipulation with two to six optical traps formed by a single fiber, generated by either spatial light modulation (SLM) or slanted incidence in laser-fiber coupling. In particular, we focus on beam size optimization for arbitrary target cell sizes to enable trapped transport and controlled rotation of a single cell, using a point matching method (PMM) of the T-matrix to compute trapping forces and rotation torque. Finally, we validated these optimized beam sizes experimentally for the LP21 mode. This work provides a new understanding of optimal optical manipulation using high-order fiber modes at the single-cell level.

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection.

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.