Living Animal Cells Research Articles

A practical guide to quantify cell adhesion using single-cell force spectroscopy

Quantitative analysis of cellular interactions with the extracellular environment is necessary to gain an understanding of how cells regulate adhesion in the development and maintenance of multicellular organisms, and how changes in cell adhesion contribute to diseases. We provide a practical guide to quantify the adhesive strength of living animal cells to various substrates using atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS). We describe how to control cell state and attachment to the AFM cantilever, how to functionalize supports for SCFS measurements, how to conduct cell adhesion measurements, and how to analyze and interpret the recorded SCFS data. This guide is intended to assist newcomers in the field to perform AFM-based SCFS measurements.

Dynamic tracing of immune cells in an orthotopic gastric carcinoma mouse model using near-infrared fluorescence live imaging

Adoptive cellular immunotherapy (ACI) has been demonstrated to be a promising cancer therapeutic, however, the distribution of immune cells injected into a tumor-bearing body is unclear. In this study, we investigated the tumor-targeting capacity of cytokine-induced killer (CIK) cells and cytotoxic T lymphocytes (CTLs) in a human gastric carcinoma orthotopic mouse model using a near-infrared fluorescence imaging system. CIK cells and tumor-specific CTLs were prepared with the near-infrared fluorescent dye DiR. As expected, no significant change in the proliferation rate or antitumor activity of CIK cells and CTLs was noted after labeling with DiR. Furthermore, a gastric carcinoma orthotopic model was established using a fibrinogen-thrombin method in nude mice followed by intraperitoneal infusion of the labeled immune cells into nude mice with established gastric carcinoma. Dynamic tracing of the immune cells was performed using a fluorescence-based live imaging system. Concentrated fluorescence signals were observed for a minimum of two weeks at the tumor site in mice infused with either CIK cells or CTLs with a peak signal at 48 h. Notably, CTLs were more persistent at the tumor site and exhibited a more intense antitumor activity than CIK cells following infusion. These results provided visual evidence of the tumor-targeting capacity of immune cells in live animals.

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility. Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA(+) neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor(1). A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

Phenylboronic acid-containing block copolymers: synthesis, self-assembly, and application for intracellular delivery of proteins

The boronic acid group has been known to bind to sugars and living animal cells. Herein, a novel amphiphilic block copolymer poly(2-lactobionamidoethyl methacrylate)-block-poly(3-acrylamidophenylboronic acid) (p(LAMA-b-AAPBA)) was prepared by reversible addition fragmentation chain transfer (RAFT) polymerization. Due to the interaction between lactose moieties and phenylboronic acid moieties in p(LAMA-b-AAPBA), the copolymer could easily form nanoparticles in a spherical shape. The p(LAMA-b-AAPBA) nanoparticles had mean sizes from 238 to 403 nm with a zeta potential of about −20 mV. To study the feasibility of p(LAMA-b-AAPBA) nanoparticles acting as the potential nanocarrier for protein delivery, insulin, as a drug model, was encapsulated into the nanoparticles, the loading capacity was about 11%. Moreover, the nanoparticles demonstrated a sustained release of insulin and had no cytotoxicity on Chinese hamster ovary cells (CHO) and human colorectal carcinoma (Caco-2) cells. Confocal laser scanning microscopy showed that the nanoparticles could be taken up by Caco-2 cells, indicating that the stimuli-response of phenylboronic acid to carbohydrates on the cell surface facilitated the nanoparticles to bind to Caco-2 cells. Thus, the p(LAMA-b-AAPBA) nanoparticles can be considered as a promising carrier for protein transport.

Preparing photochromic nanofibers and animal cells using a photochromic compound of 1′,3′,3′-trimethyl-6-nitrospiro (2H-1-benzopyran-2,2′-indoline)

In this work, the photochromic compound 1′,3′,3′-trimethyl-6-nitrospiro (2H-1-benzopyran-2,2′-indoline) (NOSP) was synthesized by a two step process. The photochromic properties of NOSP were investigated by ultraviolet–visible (UV–Vis) spectrophotometry. The results showed that NOSP was very sensitive to UV irradiation with absorption peaks at about 336nm and 567nm. Our hypothesis was that both photochromic nanofibers and photochromic living animal cells could be obtained by combining them with NOSP. To test the hypothesis, photochromic nanofibers were fabricated by electrospinning from various mixed solutions of NOSP and polymers (including a synthetic polymer of poly(methyl methacrylate) and a natural polymer of gelatin); NOSP/ethanol solution was dissolved in culture medium to stain pig iliac endothelial cells (PIEC) and endow them with photochromic capability. Polymer nanofibers from electrospinning were characterized by water contact angle measurements, ultraviolet–visible (UV–Vis) spectrophotometry and fluorescence microscopy. Morphology of photochromic PIEC was observed by fluorescence microscopy after being irradiated. It was shown that nanofibers from electrospun polymers and NOSP-treated PIEC had photochromic properties. The bio-toxicity of the photochromic compound was also evaluated and it was shown that ∼50% of PIEC remained viable for at least 20min. The photochromic compound NOSP could be a potentially powerful tool for development of multi-functional nanofibers and biological applications.

Rigidity Sensing Explained by Active Matter Theory

The magnitude of traction forces exerted by living animal cells on their environment is a monotonically increasing and approximately sigmoidal function of the stiffness of the external medium. We rationalize this observation using active matter theory, and propose that adaptation to substrate rigidity results from an interplay between passive elasticity and active contractility.

Live imaging of apoptotic cells in zebrafish

Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.

Development of a Ruthenium(II) Complex Based Luminescent Probe for Imaging Nitric Oxide Production in Living Cells

A unique ruthenium(II) complex, bis(2,2'-bipyridine)(4-(3,4-diaminophenoxy)-2,2'-bipyridine)ruthenium(II) hexafluorophosphate ([(Ru(bpy)(2)(dabpy)][PF(6)](2)), has been designed and synthesized as a highly sensitive and selective luminescence probe for the imaging of nitric oxide (NO) production in living cells. The complex can specifically react with NO in aqueous buffers under aerobic conditions to yield its triazole derivative with a high reaction rate constant at the 10(10) M(-1) s(-1) level; this reaction is accompanied by a remarkable increase of the luminescence quantum yield from 0.13 to 2.2 %. Compared with organic probes, the new Ru(II) complex probe shows the advantages of a large Stokes shift (>150 nm), water solubility, and a wide pH-availability range (pH independent at pH>5). In addition, it was found that the new probe could be easily transferred into both living animal cells and plant cells by the coincubation method, whereas the triazole derivative was cell-membrane impermeable. The probe was successfully used for luminescence-imaging detection of the exogenous NO in mouse macrophage cells and endogenous NO in gardenia cells. The results demonstrated the efficacy and advantages of the new probe for NO detection in living cells.

Optimization of enzyme–substrate pairing for bioluminescence imaging of gene transfer using Renilla and Gaussia luciferases

Bioluminescence imaging (BLI) permits the non-invasive quantification and localization of transduction and expression by gene transfer vectors. The tendency of tissue to attenuate light in the optical region, however, limits the sensitivity of BLI. Improvements in light output from bioluminescent reporter systems would allow the detection of lower levels of expression, smaller numbers of cells and expression from deeper and more attenuating tissues within an animal. With the goal of identifying substrates that allow improved sensitivity with Renilla luciferase (RLuc) and Gaussia luciferase (GLuc) reporter genes, we evaluated native coelenterazine and three of its most promising derivatives in BLI of cultured cells transduced with retroviral vectors encoding these reporters. Of the eight enzyme-substrate pairs tested, the two that performed best were further evaluated in mice to compare their effectiveness for imaging vector-modified cells in live animals. In cell culture, we observed striking differences in luminescence levels from the various enzyme-substrate combinations and found that the two luciferases exhibited markedly distinct abilities to generate light with the substrates. The most effective pairs were RLuc with the synthetic coelenterazine derivative ViviRen, and GLuc with native coelenterazine. In animals, these two pairs allowed similar detection sensitivities, which were eight- to 15-fold higher than that of the prototypical RLuc-native coelenterazine combination. Substrate selection can dramatically influence the detection sensitivity of RLuc and GLuc and appropriate choice of substrate can greatly improve the performance of reporter genes encoding these enzymes for monitoring gene transfer by BLI.

LacZ as a genetic reporter for real-time MRI

Molecular imaging based on MRI is currently hampered by the lack of genetic reporters for in vivo imaging. We determined that the commercially available substrate S-Gal can be used to detect genetically engineered beta-galactosidase expressing cells by MRI. The effect and specificity of the reaction between beta-galactosidase and S-Gal on MRI contrast were determined both in vitro and in vivo. beta-galactosidase activity in the presence of S-Gal resulted in enhanced T(2) and T*(2) MR-contrast, which was amplified with increasing magnetic field strengths (4.7-17.6 T) in phantom studies. Using both lacZ(+) transgenic animals and lacZ(+) tissue transplants, we were able to detect labeled cells in live animals in real time. Similar to phantom studies, detection of the labeled cells/tissues in vivo was enhanced at high magnetic fields. These results demonstrate that the genetic reporter, lacZ, can be used as an in vivo marker gene using high-field-strength MRI.

Migrating Brain Cells Stick Together

Migrating Brain Cells Stick Together

In Situ Nanoliter-Scale Polymer Fabrication for Flexible Cell Patterning

Drug-testing technologies, biosensor fabrication, tissue engineering, and basic biological research depend strongly on the patterning of live animal cells. Current techniques for controlling cellular adhesion are restricted with two primary limitations. Firstly, the complexity of the available patterns is very limited and, secondly, the pallet of materials that induce cellular patterning is exhaustible. Here, we demonstrate a method for computer-aided control of cell patterning using a scientific inkjet printer that yields a highly complex cellular pattern suitable for applications in regenerative medicine and rapid prototyping, and a strategy for using in situ polymerization for fabrication polymeric patterns directly on-chip.

High-efficiency labeling of sialylated glycoproteins on living cells

We describe a simple method for efficiently labeling cell-surface sialic acid-containing glycans on living animal cells. The method uses mild periodate oxidation to generate an aldehyde on sialic acids, followed by aniline-catalyzed oxime ligation with a suitable tag. Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell-surface sialylated glycoproteins while maintaining high cell viability.

Active transport on disordered microtubule networks: The generalized random velocity model

The motion of small cargo particles on microtubules by means of motor proteins in disordered microtubule networks is investigated theoretically using both analytical tools and computer simulations. Different network topologies in two and three dimensions are considered, one of which has been recently studied experimentally by Salman [Biophys. J. 89, 2134 (2005)]. A generalization of the random velocity model is used to derive the mean-square displacement of the cargo particle. We find that all cases belong to the class of anomalous superdiffusion, which is sensitive mainly to the dimensionality of the network and only marginally to its topology. Yet in three dimensions the motion is very close to simple diffusion, with sublogarithmic corrections that depend on the network topology. When details of the thermal diffusion in the bulk solution are included, no significant change to the asymptotic time behavior is found. However, a small asymmetry in the mean microtubule polarity affects the corresponding long-time behavior. We also study a three-dimensional model of the microtubule network in living animal cells. Three first-passage-time problems of intracellular transport are simulated and analyzed for different motor processivities: (i) cargo that originates near the nucleus and has to reach the membrane, (ii) cargo that originates from the membrane and has to reach the nucleus, and (iii) cargo that leaves the nucleus and has to reach a specific target in the cytoplasm. We conclude that while a higher motor processivity increases the transport efficiency in cases (i) and (ii), in case (iii) it has the opposite effect. We conjecture that the balance between the different network tasks, as manifested in cases (i) and (ii) versus case (iii), may be the reason for the evolutionary choice of a finite motor processivity.

Porcine endogenous retroviruses and xenotransplantation.

Xenotransplantation is defined by the PHS as any procedure that involves the transplantation, implantation or infusion into a human recipient of either (a) live cells, tissues or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs (Public Health Service Guideline on Infectious Disease Issues in Xenotransplantation). Use of pigs for human xenotransplantation raises concerns about the risks of transfer of infectious agents from the pig cells to xenotransplantation recipients. The observation that the porcine germline harbors genetic loci encoding porcine endogenous retroviruses (PERVs) that are in some cases infectious for human cells has resulted in renewed scientific interest in PERVs. However, in spite of the past 10 years of investigation, the actual risk for PERV infection, replication, and pathogenic outcome in human recipients of xenotransplantation products is still undefined. (Part of a multi-author review).

Basics in clinical nutrition: Metabolic response to injury and sepsis

All processes, in living animal cells, are dependent on a constant supply of substrate to generate high-energy phosphate bonds e.g. ATP. Energy is then harnessed to cellular activity by the hydrolysis of ATP. The turnover of ATP is extremely high reaching 1.3 mmol/ kg/sec. As the total body pool of ATP in an individual of average size is between 60 and 100 mmol, this would cover energy requirements for less than a minute evenwhen all ATP is fully hydrolyzed. It can be estimated that the mass of ATP hydrolyzed daily is equivalent to the bodyweight of the individual and that insufficient generation of ATP leads to irreversible destruction of the whole organism within less than one 1 minute. Continuous regeneration of ATP is therefore essential for survival under all conditions. Carbohydrates (CHO), fat and proteins are the substrates, which are oxidized to generate ATP. Under normal circumstances these substrates are supplied in food, which is, after absorption, processed by different metabolic pathways. As food intake is not a continuous process the organism in between meals has to utilize

Nondestructive micropatterning of living animal cells using focused femtosecond laser-induced impulsive force

Micropatterning of mouse NIH3T3 fibroblast cells was performed using focused femtosecond laser-induced impulsive force in a culture medium. The cells were detached from an upper substrate by the force and transferred to an underlying substrate with less than spatial resolution of 80μm full width at half maximum. About 80% of the cells were confirmed to be alive at 3h after the patterning. The force exerted to the cell was investigated by high-speed imaging and estimated to be an order of micronewtons. The force origin was not only due to cavitation bubble but also due to shockwave and jet flow.

Identification of a crystal cell‐specific enhancer of the black cells prophenoloxidase gene in drosophila

In Drosophila, Black cells (Bc) encodes a Prophenoloxidase and is expressed late in the maturation of crystal cells, which are blood cells involved in wound healing and immune encapsulation. Enhancer analysis of Bc revealed a 1,025-bp upstream sequence that regulates gene expression in a crystal cell exclusive pattern. Expression of this fragment is altered by mutations in the GATA family serpent (srp) and RUNX family lozenge (lz) genes; Srp and Lz are required for crystal cell specification. Deletional analysis uncovered a 330-bp crystal cell-specific sequence, which contains two GATA and three Lz binding sites. Mutational analysis revealed that both GATA sites are necessary, but not sufficient for crystal cell expression. However, one of the Lz sites is essential for crystal cell expression. Thus, Srp and Lz do not just specify the crystal cell lineage, but also regulate the later differentiation of these cells. Additionally, we now have a sensitive tool for marking crystal cells in live animals.

Clinical Trials of Xenotransplantation: Waiver of the Right to Withdraw from a Clinical Trial Should Be Required

Xenotransplantation pits clinical research ethics against public health needs because recipients must undergo long-term, perhaps life-long, surveillance for infectious diseases. This surveillance requirement is effectively an abrogation of the right to withdraw from a clinical trial. Ulysses contracts, which are advance directives for future care, may be an ethical mechanism by which to balance public health needs against limitation of individual rights.

Raman acoustic levitation spectroscopy of red blood cells and Plasmodium falciparum trophozoites

Methods to probe the molecular structure of living cells are of paramount importance in understanding drug interactions and environmental influences in these complex dynamical systems. The coupling of an acoustic levitation device with a micro-Raman spectrometer provides a direct molecular probe of cellular chemistry in a containerless environment minimizing signal attenuation and eliminating the affects of adhesion to walls and interfaces. We show that the Raman acoustic levitation spectroscopic (RALS) approach can be used to monitor the heme dynamics of a levitated 5 microL suspension of red blood cells and to detect hemozoin in malaria infected cells. The spectra obtained have an excellent signal-to-noise ratio and demonstrate for the first time the utility of the technique as a diagnostic and monitoring tool for minute sample volumes of living animal cells.