A Variant of A Slam Freezing Device for Electron Microscopy

A home-made slam freezing device is presented that allows reproducible results in freezing various unfixed tissues. The heart of the device is an aluminium socket, which harbors a plunger that is set in motion by a spring. At the end of the plunger there is an electromagnet which holds the sample on a sheet metal planchette. During stop freezing the electrical contacts are interrupted and the plunger can be withdrawn leaving the specimen on the cooled copper block. This guarantees freezing of not only solid tissues, but also cell suspensions, such as blood or bone marrow.     

对吸附式电极记录装置的技术改进

本文介绍一种用吸附式电极记录合体细胞组织生物电位的改进装置。它的特点是在建造负压的注射器和吸附式电极之间设置一分离的小室。这一小室既保障了放大器与实验标本之间的电路连系,又可直接放置在实验标本附近,负压由改进的注射器经过充有空气的塑料管抽吸,注射器可以放在任何方便的位置上。该方法在记录小动物,如蜗牛、青蛙、等的心脏、消化道等组织器官的生物电位时都能获得比较理想的效果,对研究小动物合体细胞组织的正常机能及药物作用等都具有较好的适用价值,并具有定位准确、操作方便的优点。     

Application of atomic force microscopy to the study of micromechanical properties of biological materials

Atomic force microscopy (AFM) has been used to study the micromechanical properties of biological systems. Its unique ability to function both as an imaging device and force sensor with nanometer resolution in both gaseous and liquid environments has meant that AFM has provided unique insights into the mechanical behaviour of tissues, cells and single molecules. As a surface scanning device, AFM can map properties such as adhesion and the Young's modulus of surfaces. As a force sensor and nanoindentor AFM can directly measure properties such as the Young's modulus of surfaces or the binding forces of cells. As a stress-strain gauge AFM can study the stretching of single molecules or fibres and as a nanomanipulator it can dissect biological particles such as viruses or DNA strands. The present paper reviews key research that has demonstrated the versatility of AFM and how it can be exploited to study the micromechanical behaviour of biological materials.     

Three-dimensional bioprinting human cardiac tissue chips of using a painting needle method

Three-dimensional (3D) printers are attracting attention as a method for arranging and building cells in three dimensions. Bioprinting technology has potential in tissue engineering for the fabrication of scaffolds, cells, and tissues. However, these various printing technologies have limitations with respect to print resolution and due to the characteristics of bioink such as viscosity. We report a method for constructing of 3D tissues with a “microscopic painting device using a painting needle method” that, when used with the layer-by-layer (LbL) cell coating technique, replaces conventional methods. This method is a technique of attaching the high viscosity bioink to the painting needle tip and arranging it on a substrate, and can construct 3D tissues without damage to cells. Cell viability is the same before and after painting. We used this biofabrication device to construct 3D cardiac tissue (LbL-3D Heart) using human-induced pluripotent stem cell–derived cardiomyocytes. The constructed LbL-3D Heart chips had multiple layers with a thickness of 60 µm, a diameter of 1.1 mm, and showed synchronous beating (50–60 beats per min). The aforementioned device and method of 3D tissue construction can be applied to various kinds of tissue models and would be a useful tool for pharmaceutical applications.     

Novel Cell and Tissue Acquisition System (CTAS): Microdissection of Live and Frozen Brain Tissues

We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS - Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.     

Concentration independent modulation of local micromechanics in a fibrin gel

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues.     

Remote electrical stimulation by means of implanted rectifiers

Miniaturization of active implantable medical devices is currently compromised by the available means for electrically powering them. Most common energy supply techniques for implants--batteries and inductive couplers--comprise bulky parts which, in most cases, are significantly larger than the circuitry they feed. Here, for overcoming such miniaturization bottleneck in the case of implants for electrical stimulation, it is proposed to make those implants act as rectifiers of high frequency bursts supplied by remote electrodes. In this way, low frequency currents will be generated locally around the implant and these low frequency currents will perform stimulation of excitable tissues whereas the high frequency currents will cause only innocuous heating. The present study numerically demonstrates that low frequency currents capable of stimulation can be produced by a miniature device behaving as a diode when high frequency currents, neither capable of thermal damage nor of stimulation, flow through the tissue where the device is implanted. Moreover, experimental evidence is provided by an in vivo proof of concept model consisting of an anesthetized earthworm in which a commercial diode was implanted. With currently available microelectronic techniques, very thin stimulation capsules (diameter <500 μm) deliverable by injection are easily conceivable.     

Liquid Scintillation Vial for Cumulative and Continuous Radiometric Measurement of In Vitro Metabolism

A two-compartment vial is described in which suspensions of bacteria, cells, or tissues may be cultured and their growth and metabolism measured radiometrically by using a liquid scintillation counter. The device consists of a scintillation vial lined with a cylinder of scintillating paper into which is placed a sterilized inner culture vial containing a carbon-14 substrate. The assembled device can be carried by the sample transport systems of conventional liquid scintillation counters. Evolved (14)CO(2) is collected and measured cumulatively and continuously. The device can be constructed simply and economically from readily available reagents and glassware. Data are given on relative sensitivity and on the effect of the color and transparency of the inner vial. A pilot experiment with bacteria (Escherichia coli) is described.     

Tissue engineering of a bioartificial kidney

Tissue engineering is a rapidly growing field in biotechnology. The use and packaging of synthetic materials, biologic compounds, and cellular components of specific tissues can be envisioned to replace physiologic function of diseased organs. Long-term ex vivo therapy for kidney failure has been achieved, so that the kidney may be the first solid organ in which tissue engineering concepts can produce an implantable device for long-term in vivo replacement therapy. To replace the kidney's excretory function, an implantable bioartificial kidney requires both a device to replace blood ultrafiltration performed by renal glomeruli and a device to replace transport regulatory function of the renal tubule. The initial concepts for these devices are just beginning to be considered and developed. (c) 1994 John Wiley & Sons, Inc.     

Methodology to determine failure characteristics of planar soft tissues using a dynamic tensile test

Predicting the injury risk in automotive collisions requires accurate knowledge of human tissues, more particularly their mechanical properties under dynamic loadings. The present methodology aims to determine the failure characteristics of planar soft tissues such as skin, hollow organs and large vessel walls. This consists of a dynamic tensile test, which implies high-testing velocities close to those in automotive collisions. To proceed, I-shaped tissue samples are subjected to dynamic tensile tests using a customized tensile device based on the drop test principle. Data acquisition has especially been adapted to heterogeneous and soft biological tissues given that standard measurement systems (considered to be global) have been completed with a non-contact and full-field strain measurement (considered to be local). This local measurement technique, called the Image Correlation Method (ICM) provides an accurate strain analysis by revealing strain concentrations and avoids damaging the tissue. The methodology has first been applied to human forehead skin and can be further expanded to other planar soft tissues. The failure characteristics for the skin in terms of ultimate stress are 3 MPa +/- 1.5 MPa. The ultimate global longitudinal strains are equal to 9.5%+/-1.9% (Green-Lagrange strain), which contrasts with the ultimate local longitudinal strain values of 24.0%+/-5.3% (Green-Lagrange strain). This difference is a consequence of the tissue heterogeneity, clearly illustrated by the heterogeneous distribution of the local strain field. All data will assist in developing the tissue constitutive law that will be implemented in finite element models.     

Standardization of a method for characterizing low-concentration biogels: elastic properties of low-concentration agarose gels

Low-concentration biogels, which provide an extracellular matrix for cells in vitro, are involved in a number of important cell biological phenomena, such as cell motility and cell differentiation. In order to characterize soft tissues, which collapse under their own weight, we developed and standardized a new experimental device that enabled us to analyze the mechanical properties of floating biogels with low concentrations, i.e., with values ranging from 2 g/L to 5 g/L. In order to validate this approach, the mechanical responses of free floating agarose gel samples submitted to compression as well as stretching tests were quantified. The values of the Young's moduli, measured in the range of 1000 to 10,000 Pa, are compared to the values obtained from other experimental techniques. Our results showed indeed that the values we obtained with our device closely match those obtained independently by performing compression tests on an Instron device. Thus, the floating gel technique is a useful tool first to characterize and then to model soft tissues that are used in biological science to study the interaction between cell and extracellular matrix.     

Microfluidic immobilization of physiologically active Caenorhabditis elegans

We present a protocol for building and operating a microfluidic device for mechanical immobilization of Caenorhabditis elegans in its physiologically active state. The system can be used for in vivo imaging of dynamic cellular processes such as cell division and migration, degeneration, aging and regeneration, as well as for laser microsurgery, Ca2+ imaging and three-dimensional microscopy. The device linearly orients C. elegans, and then completely restrains its motion by pressing a flexible membrane against the animal. This technique does not involve any potentially harmful anesthetics, gases or cooling procedures. The system can be installed on any microscope and operated using only one syringe and one external valve, making it accessible to most laboratories. The device fabrication begins by patterning photoresist structures on silicon wafers, which are then used to mold features in elastomeric layers that are thermally bonded to form the device. The system can be assembled within 3 d.     

The stem cell niche: theme and variations

Stem cells in animal tissues are often located and controlled by special tissue microenvironments known as niches. Studies of stem cell niches in model systems such as Drosophila have revealed adhesive interactions, cell cycle modifications and intercellular signals that operate to control stem cell behavior. Candidate niches and regulatory molecules have also been identified in many mammalian tissues, including bone marrow, skin, gut and brain. While niches are an ancient evolutionary device with conserved features across diverse organisms, we suggest that certain niches display important differences in their organization and function.     

Do-It-Yourself device for recovery of cryopreserved samples accidentally dropped into cryogenic storage tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 °C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues (1,2). The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn' (2), which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces (2). In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks (1). These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials (3). However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use. In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.     

Towards microbial tissue engineering?

Tissue engineering involves the creation of multicellular tissues from individual cells. It was previously perceived that tissues were only formed by higher organisms such as plants and animals. However, it is now known that multicellular systems of microorganisms, such as microbial colonies, biofilms, flocs and aggregates, can also show extensive spatial organization. Here, we discuss methods that can be used to spatially organize microorganisms--bacteria, in particular--into tissue-like materials with defined internal architectures. Some potential uses of such "microbial tissues" are covered.     

Development of a cell culture system loading cyclic mechanical strain to chondrogenic cells

Mechanical stimulation is considered to be one of the major epigenetic factors regulating the metabolism, proliferation, survival and differentiation of cells in the skeletal tissues. It is generally accepted that the cytoskeleton can undergo remodeling in response to mechanical stimuli such as tensile strain or fluid flow. Mechanically induced cell deformation is one of the possible mechanotransduction pathways by which chondrocytes sense and respond to changes in their mechanical environment. Mechanical strain has a variety of effects on the structure and function of their cells in the skeletal tissues, such as chondrocytes, osteoblasts and fibroblasts. However, little is known about the effect of the quality and quantity of mechanical strain and the timing of mechanical loading on the differentiation of these cells. The present study was designed to investigate the effect of the deformation of chondrogenic cells, and cyclic compression using a newly developed culture device, by analyzing mechanobiological response to the differentiating chondrocytes. Cyclic compression between 0 and 22% strains, at 23 microHz was loaded on chondrogenic cell line ATDC5 by seeding in a mass mode on PDMS membrane, assuming direct transfer of cyclic deformation from the membrane to the cells at the same frequency. The compressive strain, induced within the membrane, was characterized based on the analysis of the finite element modeling (FEM). The results showed that the tensile strain inhibits the chondrogenic differentiation of ATDC5 cells, whereas the compressive strain enhances the chondrogenic differentiation, suggesting that the differentiation of the chondrogenic cells could be controlled by the amount and the mode of strain. In conclusion, we have developed a unique strain loading culture system to analyze the effect of various types of mechanical stimulation on various cellular activities.     

A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.     

High-throughput droplet PCR

The polymerase chain reaction has facilitated the ready analysis of nucleic acids. A next challenge requires the development of means to unravel the complexity of heterogeneous tissues. This has presented the task of producing massively parallelized quantitative nucleic acid data from the cellular constituents of tissues. The production of aqueous droplets in a two phase flow is shown to be readily and routinely facilitated by miniaturized fluidic devices. Droplets serve as ideal means to package a future generation of PCR, offering an enhanced handling potential by virtue of reactant containment, to concurrently eliminate both contamination and sample loss. This containment also enables the measurement of nucleic acids from populations of cells, or molecules by means of high throughput, single cell analysis. Details are provided for the production of a prototype micro-fluidic device which shows the production and stable flow of droplets which we suggest will be suitable for droplet-based continuous flow micro-fluidic PCR. Suggestions are also made as to the optimal fabrication techniques and the importance of device calibration.     

Adaptation of a planar microbiaxial optomechanical device for the tubular biaxial microstructural and macroscopic characterization of small vascular tissues

Murine models of disease are a powerful tool for researchers to gain insight into disease formation, progression, and therapies. The biomechanical indicators of diseased tissue provide a unique insight into some of these murine models, since the biomechanical properties in scenarios such as aneurysm and Marfan syndrome can dictate tissue failure and mortality. Understanding the properties of the tissue on the macroscopic scale has been shown to be important, as one can then understand the tissue's ability to withstand the high stresses seen in the cardiac pulsatile cycle. Alterations in the biomechanical response can foreshadow prospective mechanical failure of the tissue. These alterations are often seen on the microstructural level, and obtaining detailed information on such changes can offer a better understanding of the phenomena seen on the macroscopic level. Unfortunately, mouse models present problems due to the size and delicate features in the mechanical testing of such tissues. In addition, some smaller arteries in large-animal studies (e.g., coronary and cerebral arteries) can present the same issues, and are sometimes unsuitable for planar biaxial testing. The purpose of this paper is to present a robust method for the investigation of the mechanical properties of small arteries and the classification of the microstructural orientation and degree of fiber alignment. This occurs through the cost-efficient modification of a planar biaxial tester that works in conjunction with a two-photon nonlinear microscope. This system provides a means to further investigate how microstructure and mechanical properties are modified in diseased transgenic animals where the tissue is in small tube form. Several other hard-to-test tubular specimens such as cerebral aneurysm arteries and atherosclerotic coronary arteries can also be tested using the described modular device.     

Regulation of cytokine production by virus-specific CD8 T cells in the lungs

Inflammation and the elimination of infected host cells during an immune response often cause local tissue injury and immunopathology, which can disrupt the normal functions of tissues such as the lung. Here, we show that both virus-induced inflammation and the host tissue environment combine to influence the capacity of virus-specific CD4 and CD8 T cells to produce cytokines in various tissues. Decreased production of cytokines, such as IFN-γ and TNF-α, by antigen-specific T cells is more pronounced in peripheral tissues, such as the lung and kidney, than in secondary lymphoid organs, such as the spleen or lymph nodes. We also demonstrate that tissues regulate cytokine production by memory T cells independently of virus infection, as memory T cells that traffic into the lungs of naïve animals exhibit a reduced ability to produce cytokines following direct ex vivo peptide stimulation. Furthermore, we show that cytokine production by antigen-specific memory CD4 and CD8 T cells isolated from the lung parenchyma can be rescued by stimulation with exogenous peptide-pulsed antigen-presenting cells. Our results suggest that the regulation of T-cell cytokine production by peripheral tissues may serve as an important mechanism to prevent immunopathology and preserve normal tissue function.