Utrecht Nanobody Facility

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Mission

The Utrecht Nanobody Facility (UNF) aims to provide support to academic researchers interested in nanobody technology. We provide advice and expertise for development of new nanobodies or new applications with existing nanobodies. In a collaborative set-up we provide the technology for the selection, production, functionalization, and applications of nanobodies. We offer technology for the functionalization of nanobodies using different site-specific conjugation methods of fluorophores (Alexa, Atto, NIR dyes etc.), drugs, nanoparticles etc. Functionalized nanobodies are excellent tracers for imaging purposes and in collaboration with the Biology Imaging Center we provide for single molecule imaging, super-resolution light microscopy, and in vivo molecular imaging.

Technology

Nanobodies are small antibody fragments (15 kDa) derived from camelid heavy chain antibodies. These single domain antibodies are uniquely adaptable tools. Nanobodies can be selected from (custom built) immune libraries, or alternatively synthetic libraries, using phage display. Extensive equipment is available for the thorough characterization of the nanobodies. Important parameters are production yield, stability, specificity, binding affinity, and selectivity in vivo. Nanobodies can be produced at small scale and equipment is available for the large scale production both from E. coli and HEK cells.

Applications

Nanobodies can be used for different applications, such as stabilization of protein conformation for X-ray crystallography and cryo-electron microscopy, protein or vesicle purification, in vitro imaging (both light- and electron- microscopy), as biosensors, and for in vivo imaging. Furthermore, nanobodies can be employed for therapeutic applications, for instance: as antagonists, conjugated to drugs for cancer therapy or fibrosis, as antivirals, for targeted protein degradation, conjugated to nanoparticles carrying drugs, or in immune therapies such as nanobody-based T cell engagers or chimeric antigen receptor T cells.

Flow Cytometry Facility

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The selection and purification of molecules, cells, tissues, and organisms of interest are critical yet often time-consuming aspects of biomedical research. Flow cytometers have revolutionized the sorting and analysis of large numbers of individual cells, microorganisms, and other particles at high speed. The fluidics subsystem of a flow cytometer brings particles in suspension into the flow cell where they are excited by light sources of different wavelengths. With the use of multiple mirrors, band pass, and long pass filters, the emitted light is routed to the appropriate photomultipliers, where the incoming photons are converted into electrons and visualized for further analysis. Applications range from simple cellular readouts to complex phenotyping and high-throughput screening.

Our facility houses three different machines that support a broad range of applications:

BD FACSymphony A1 cell analyzer with small particle detector:

The BD FACSymphony A1 is used for analyzing particles, ranging from very small ones such as extracellular vesicles, to microorganisms such as yeasts, and a wide variety of blood and tissue cells. This analyzer supports high-resolution analysis of up to 16 parameters simultaneously. Examples of possible applications are measuring cell proliferation, apoptosis, transfection efficiency, cell cycle progression, cytometry bead array, and identification of rare subsets by multicolor staining (both intra- and extracellular).

BD FACSAria Fusion cell sorter:

The BD FACSAria Fusion is used for sorting particles, ranging from microorganisms such as yeasts to various blood or tissue cells. This is performed at high speed and in sterile conditions. Thereby facilitating the option to bring these cells back into culture, allowing creation of specific cell clones or performance of functional assays with the cells of interest. This sorter supports high-resolution analysis of up to 17 fluorescent markers simultaneously.

Union Biometrica Large Particle Biosorter:

Some objects are too large or too sensitive for conventional flow cytometry. Therefore, Union Biometrica has developed large particle flow cytometers for handling a wide range of object sizes (1 – 1500 µm). Examples of materials that can be analyzed and sorted with the Biosorter are plant protoplasts, seeds, organoids and whole organisms, including zebrafish embryos and C. elegans embryos and larvae.

Relative size and optical density are measured, as well as fluorescence intensities at three different wavelengths. Sorting and dispensing decisions are based on user-selected ranges using the FlowPilot™ Pro software. Of each object, an optical profile can be made, in which the location and intensity of all parameters can be graphically mapped. This feature makes it possible to distinguish between fluorescence in the head, middle, or tail of the organism.

Centre for Flow Cytometry and Cell Sorting (CFC)

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The Centre for Flow Cytometry and Cell Sorting at the Faculty of Veterinary Medicine provides expertise in single cell measurement and sorting since 1998. The facility supports a wide range of analyses and purifications, including immune and stem cells, sperm cells, chromosomes, bacteria, hybridomas for monoclonal antibody production, single-cell sorting for cloning or PCR analysis, fluorescent reporter–based cell selection and rare event purification. We have accumulated expertise in small particle flow cytometric analysis guiding novel developments in the field and acquiring high-sensitivity equipment capable of measuring particles as small as 40 nm in size. We have a strong track record of translating complex experimental demands into successful solutions through close collaboration with research groups, instrument manufacturers and continuous technical development.

Available Equipment:

Cell Sorting
BD Influx Cell Sorter – Equipped with 5 lasers (355 nm, 405 nm, 457 nm, 488 nm, 561 nm, 635 nm) enabling up to 21 fluorescent parameters. This jet-in-air system supports up to six-way sorting into a variety of tubes and plate formats.
BD FACSAria Fusion – Equipped with 4 lasers (405 nm, 488 nm, 561 nm, 635 nm) for up to 14 fluorescent parameters. This fixed-alignment cuvette flow cell sorter is integrated into a biosafety cabinet and supports four-way sorting into tubes and multiple plate formats.

Cell Analysis
CytoFLEX LX Analyzer – Equipped with 6 lasers (375 nm, 405 nm, 488 nm, 561 nm, 635 nm, 808 nm), allowing up to 21 fluorescent parameters. Supports both tube- and plate-based measurements.
Cytek Aurora ESP Spectral Analyzer – Equipped with 3 lasers (405 nm, 488 nm, 635 nm), supporting up to 25 fluorescent parameters. Capable of both tube- and plate-based measurements. Includes a feature to measure and subtract intrinsic cellular autofluorescence.

Small Particle Analysis (Extracellular Vesicles, Nanoparticles, etc.)
CytoFLEX Nano – Dedicated to particles smaller than 1 µm. Offers VSSC detection with reduced background levels. Equipped with 4 lasers (405 nm, 488 nm, 561 nm, 638 nm) providing 6 fluorescent parameters and 6 light scatter parameters. Supports tube-based measurements.
Cytek Aurora ESP – Equipped with a high-sensitivity secondary SSC detection channel for improved small particle analysis while retaining capability for larger particles.

Data Analysis
Three dedicated workstations with FlowJo licenses for routine downstream analysis.
High-end workstation supporting FlowJo, SpectroFlo, R Studio, FCMpass, and other software for advanced, high-dimensional data pipelines.

Intravacc

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Based in the Netherlands and nestled within the Utrecht Science Park in Bilthoven, Intravacc is a globally recognized leader in translational vaccinology. With decades of experience, we stand as an independent Contract Development and Manufacturing Organization (CDMO) within the vaccine industry. Our purpose is to serve as the vital link between your discoveries and early-stage clinical trials. Whether for human or veterinary applications, we offer expertise in diverse vaccine platforms, including Viral, Bacterial, Conjugate, and Protein expression.

Ombion Centre for Animal-free Biomedical Translation

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Ombion Centre for Animal-Free Biomedical Translation is a centre for valorisation and dissemination of animal-free innovations and expertise. We aim to improve and accelerate the translation of new biomedical innovations to patients and users, at lower costs, and without the use of animals. We offer a professional network, courses, and labs for automation, demonstration and education.

Facilities and Services

– Ombion offers unique expertise in model development, assay miniaturisation and standardisation
– Annual summer schools and trainings on automation and the latest advances in in vitro technology
– 600m2 of total lab space for animal-free research of which almost 200m2 dedicated to education
– Over 400m2 of office space, for Ombion personnel and guest researchers
– State-of-the-art cell culture labs and molecular biology labs
– An education and training lab featuring cell culture facilities and automated equipment
– Advanced confocal microscopy, tailored to 3D cell culture models and assays in miniaturised format
– Unique robotic automation to support assay miniaturisation and standardisation of advanced three-dimensional cell culture assays like organoids

Capabilities

– Assay development, miniaturisation and standardisation in ML-I or ML-II conditions
– Regulatory routing
– AI
– Automation and medium-to-high throughput screening in ML-I labs
– High-content screening and image analysis

Users

– Personnel of Ombion, including those of the Transition Projects based at several UMCs and research centres
– The Ombion network
– External professionals in the Netherlands and abroad

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Hubrecht Imaging Center

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Imaging is becoming increasingly important in the biomedical sciences and in research at the Hubrecht Institute. To meet the growing demand for advanced imaging equipment, the Hubrecht Imaging Center (HIC) was established in 2009. In recent years, the Hubrecht Institute has acquired a range of state of the art microscopes, which are now managed by the HIC.

The HIC supports a wide spectrum of imaging needs, from simple phase contrast imaging to high resolution imaging of living tissue. Advanced systems ar available at the HIC include confocal, two photon, TIRF, FRAP, STED and FRET microscopy, including FLIM. We have also spinning disk systems and light sheet microscopes.

The HIC ensures that the Hubrecht Institute’s advanced imaging equipment functions optimally and is used to its full potential. This is achieved through regular system testing and optimization, training new researchers, and providing support for imaging experiments.

USEQ: Utrecht Sequencing facility (Next generation sequencing and Spatial omics)

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We provide support for a broad range of services to academic partners, including DNA/RNA isolation, sample preparation, Next Generation Sequencing, SNP fingerprinting and Spatial Omics. A comprehensive state-of-the-art infrastructure is available, including NGS sequencers, liquid handling robotics, Bruker GeoMx/CosmX and ICT.You are welcome to take a look at our standard services or contact us for any customizations.

The Utrecht Sequencing Facility is sponsored by the University Medical Center Utrecht and the NWO infrasctructure grant X-omics.

Visit www.useq.nl for our full and up-to-date portfolio and pricing.

Pathology Research Core

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The Pathology Research Core (PRC) facility offers a broad range of tissue collection, processing and analysis techniques to facilitate biomedical and clinical researchers. We collect preclinical and clinical samples and process them for analyses and storage.

Samples are collected as fresh frozen specimen, processed for formalin-fixing and paraffin embedding (FFPE), or prepared for electron microscopy (EM).

We have a vast range of antibody-based protein expression detection techniques (chromogenic and fluorescent) and chemical procedures for histopathological assessment.

Our facility can also culture and process/analyze patient derived tumor organoid PDO models in specified extracellular matrix (ECM) gels upon request.

We provide high-quality histopathological services to support research into human and non-human (mouse, rat, fish) healthy and diseased tissues

Westerdijk Fungal Biodiversity Institute: Culture collections and databases

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The Collection and databases facility of WI-KNAW maintains two important collections of microbes: the world-renowned CBS Collection for filamentous fungi and yeasts, founded in 1904 and one of the oldest and largest culture collections for fungi in the world; and the NCCB collection, a historically important collection of wild-type and mutant bacterial strains and phages.

Its holdings cover a broad range of the culturable fungal diversity, with strains originating from various habitats on all continents and in the seas, and over 16 000 type strains that serve as important references for taxonomy. Yearly, 4 000 – 6 000 strains are supplied to requestors in around 50 countries for research and development, and 2 000 – 3 000 new strains are deposited from various origin and substrates. All strains are DNA sequenced for quality control and identification purposes, the fungi mostly by ITS and LSU rDNA, and bacteria by (partial) 16S. New data including on physiology, mass spectrometry (MALDI-TOF) and other data are continuously generated and linked to the strains. For many strains whole genome sequences are also available.

  • 100 000 strains of filamentous fungi and yeasts in CBS
  • 10 000 strains of bacteria and actinobacteria, and 560 phages in NCCB
  • Strains are preserved in a metabolically inactive state, by lyophilization (freeze-drying) and cryopreservation. A smaller part is also actively kept on agar media.
  • Online databases providing open (meta)data to search strains and to perform analyses
  • Supply of cultures in active state on agar media, as freeze-dried ampoules or genomic DNA extracts
  • Deposit of novel living strains in the public collections (free-of-charge, acceptance pending quality and compliance checks)
  • International Depository under the Budapest Treaty (patent strains, fee charged)
  • Safe deposit service (fee charged periodically)
  • Quality assurance according to ISO 9001

UMC Utrecht Biobank Research Ethics Committee (TCBio)

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TCBio reviews procedures that collect and/or use human biological materials for medical scientific research to ensure high quality biomedical research and responsible collection, storage, management and distribution of human tissue, cells and related data. The committee is composed of experts in clinical practice, biomedical research, genetics, methodology, ethics, law and participant experience ensuring compliance with Dutch and European regulations.

For a more information see: https://tcbio.umcutrecht.nl/en/

Ethics review, METC and non-WMO biobank research

Researchers looking for METC or MREC guidance for biobank research at UMC Utrecht may also need to consult the Biobank Research Ethics Committee (TCBio). TCBio reviews the collection, storage, management and research use of human biological material and related data when the research is not subject to the Dutch Medical Research Involving Human Subjects Act (WMO).

For WMO-regulated research, review by an accredited Medical Research Ethics Committee (METC/MREC) or the Central Committee on Research Involving Human Subjects (CCMO) may be required. When human biological material is used as part of a WMO research protocol already approved by a METC, additional TCBio approval is generally not required.