Technological platforms at CIM
The Center for Infectious Medicine (CIM) has invested a significant amount of economic resources to amass cutting edge technology and scientific expertise. CIM offers advanced equipment for visualization and quantification of immune cell functions, as well as advanced flow cytometry. Researchers at CIM also have developed methods for isolating large numbers of specific primary cells and experimental In vivo models.
Imaging at the cellular and tissue level
To perform complete studies of immunological and pathogenic mechanisms, it is essential to have advanced equipment for visualization and quantification of immune cell functions.
At CIM, researchers have established an advanced visualization facility that provides researchers with front-line imaging technology, technical support and imaging-related expert advice to researchers. Specific acquired computerized image analyses programs for quantification of biological and immunological mediators have been developed in the research environment.
CIM also holds advanced equipment for fluorescence microscopy analyses, including a Nikon A1R confocal microscope, which is an excellent tool for studies of immune cell functions, viral and bacterial entry into host cells, as well as intracellular vesicular transport and diffusion mechanisms. It allows studies of single molecule dynamics, surface structures, exocytic and endocytic trafficking, receptor/ligand and cell-cell interactions in living and fixed specimens. The Nikon A1R is equipped with the total internal reflection fluorescence (TIRF) application, which is an ideal tool for live imaging investigating both the mechanisms and dynamics of protein interactions at the cell membrane surface.
As a joint effort with S. Strömblad and R. Toftgård at the Novum Research Center in Huddinge, CIM has established a state-of-the art imaging facility. The facility offers a wide range of microscopes for advanced fluorescence microscopy and includes a multiphoton confocal microscope Zeiss 710, which allows extended time lapse experiments on live cells.
This includes four-dimensional (4D, i.e. 3D over time) imaging of dynamic cellular events and quantitative modeling of molecular mechanisms regulating immune cell behavior in more complex settings such as, e.g., organotypic cultures, tissue explants, and whole body organs. This microscope allows simultaneous analyses of a range of fluorophores. The microscope also provides a cutting edge technology for imaging of live human immune cells and host pathogen interactions under more physiological conditions.
Tissue model systems
At CIM, researchers have access to unique biopsies from patient cohorts.
Analyses of sections or isolated cells from biopsies at the cellular or molecular level provide information of high clinical relevance that are further strengthened by detailed analyses in appropriate human cell and tissue model systems.
From simple co-culture models of human tissue cells and, ultimately, to the generation of whole organs, or representations of whole organs in the laboratory, scientists in this research environment develop human tissue model systems in vitro. These can complement essential work currently achievable only in vivo.
Our commitment to this development is essential. It includes specialized cultures of tissue cells as well as ex vivo explant models. Several approaches have been adapted to study immune cell interactions with tissue specific cells in monolayer-based cultures. Although these culture systems have provided important information with respect to tissue-specific influences on immune cell function, moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues.
Scientists in the research environment engineer three-dimensional (3D) tissue models, so-called organotypic cultures that also contain immune cells. Our approach of developing and using 3D tissue models that mimic real tissues, provides unique tools to study human immune cell functions. These human immune cell functions are present in live tissue and often missed in monolayer-based cell cultures.
In 3D cultures, tissue specific cells acquire a polarized phenotype and a large number of cell-cell contacts occur, which are likely to affect immune cell function and responses to external stimuli, such as pathogens. In addition, the 3D tissue model allows performance of live imaging of immune cells within tissue using 4D, i.e. 3D over time fluorescence imaging techniques. For this purpose we generate tissue models with fluorescent cells, e.g. epithelial cells, fibroblasts, dendritic cells and monocytes.
Thus, creating tissue models with immune cells provide us with a technological platform to increase our understanding of human immune cell responses, migration and positioning in tissue, in addition to interactions with pathogens in more physiological models.
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Advanced flow cytometry
Researchers at CIM are constantly striving to be at the fore front of flow cytometry analysis.
The advanced flow cytometry lab at CIM is equipped with three 5-laser instruments with 29, 28 and 18 fluorescence detectors respectively;
- one BD Symphony A5
- one BD Symphony A3
- one BD LSR Fortessa
The advanced flow cytometry lab at CIM is also equipped with one 2-laser instrument (BD Accuri with 4 fluorescence detectors). This technology allows characterization of immune cell composition at single cell level and at high resolution, maximizing in depth analysis of patient samples with low cell numbers.
In addition to the analysis instruments, there are two Sony MA900 cell sorters (2 lasers, 12 fluorescence detectors) available for users at ANA Futura, enabling sorting of live cells in both BSL2 and BSL3 environment for downstream applications.
The instruments are currently used by many groups in the environment in research related to human immune cell function and infectious diseases.
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Isolation of human immune cells from tissues
To study functional aspects of human immune cells it is crucial to have efficient methods of obtaining primary human cells. Researchers at CIM have developed several sophisticated methods for isolating specific subsets of primary cells. As an example, our researchers now isolate rare differentiated immune cell subsets and hematopoietic progenitor cells (HPC) from various tissues including human cord blood, gut, lung and liver.
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In vivo models
Experimental models constitute an important complement to in vitro experiments and studies on patient material.
Researchers at CIM use experimental models with an aim to gain increased understanding for complex host-microbe interactions, disease mechanisms and for testing new vaccine- and drug candidates. CIM is also actively involved in the generation of needs-driven, novel in vivo models for studies within these and related research areas.
10X Genomics single cell partitioning for DNA, RNA and protein analyses
To study molecular aspects of human immune cells at the single-cell resolution, CIM offers advanced instruments such as 10X Genomics single cell partitioning for DNA, RNA and protein analyses.