RAFT™ 3D System Applications

Dermal fibroblasts cultured in RAFT™ System

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  • Selected Applications

    The RAFT™ 3D Cell Culture System is compatible with a variety of cell types and has already been used to successfully generate 3D cultures in a number of research areas, including oncology, toxicology and barrier modeling.

     

    Learn more by choosing the application of interest from the navigation above.


       

    Application Highlights:


    Co-Culturing Airway Cells in 3D

    With 2D methods, it is challenging to layer multiple cell types to achieve the complexity of an airway model. In order to better understand cellular interactions, we developed a preliminary co-culture system with Lonza's bronchial epithelial and smooth muscle cells from normal and asthmatic donors using the RAFT™ 3D Cell Culture System.

     

    Choose Airway Research from the navigation above to learn more and register to download our new white paper.

     

    BSMC stained with anti-β-tubulin and cultures in 3D.  

    Human Bronchial Smooth Muscle Cells in 3D Cell Culture

         
    Apply Standard Cell Analysis Tools to Your 3D Culture

    It can be very challenging to apply standard cell analysis techniques on cells cultured in 3D. The tight structure of some spheroids, which form a necrotic core, the lack of transparency of many plastic materials used in 3D methods or the dense fiber network of some 3D hydrogels can interfere with basic techniques such as imaging, transfection, cytotoxicity assays or biotherapeutic applications. The RAFT™ 3D System addresses these challenges. The system is well-supported with optimized protocols and white papers to analyze cells cultured in 3D.

     

    Choose 3D Culture Analysis from the navigation above to learn  more.



         

    Contact us to find out if the RAFT™ System is applicable to your project or if you have any questions.


  • 3D Culture Analysis

    3D culture is transforming cell biology research and tissue engineering applications. These advanced tools are allowing researchers to develop higher-order structures with cells in vitro. This, in turn, allows cells to grow and interact in an environment more closely mimicking in vivo. However, such revolution in research is also coupled to new challenges. It can be very difficult to apply standard cell analysis techniques on 3D cultures which have long been established in 2D environment. The tight structure of some spheroids, which form a necrotic core, the lack of transparency of many plastic materials used in 3D methods or the dense fiber network of some 3D hydrogels can interfere with basic techniques such as imaging, transfection, cytotoxicity assays or biotherapeutic applications.

    RAFT™ 3D Culture System is attempting to address such challenges with 3D methods enabling researchers to work with a 3D system that is well supported with protocols to conduct downstream assays. Lonza continues to develop and support the RAFT™ System with additional optimized protocols that allow for applying standard histological, biochemical and imaging techniques to 3D cultures. 

     

    Visualization of RAFT™ 3D Cultures with Standard Microscopy Techniques

    Applying standard microscopy techniques can be challenging on 3D cultures. Due to the translucent properties of RAFT™ Scaffolds, immunofluorescently stained 3D cultures can be visualized with subcellular resolution under a standard fluorescence microscope. The breast cancer cell line MCF7 and human dermal fibroblasts were cultured in RAFT™ 3D System for several days prior to fixation and immunocytochemical staining. The protocol demonstrated an efficient permeability of the antibodies through the RAFT™ Matrix to visualize these 3D cultures. 

      

    Register to read the full white paper





    Optimization of Cell-Based Assays Made Simple

    The assessment of cell viability in 3D cultures can be equally challenging because most cell based assays have been optimized for traditional 2D culture. It is generally recommended that the assays are optimized by researchers to make them work for 3D method in use. The higher density of the cells and the abundant presence of extracellular matrix molecules in certain 3D methods add to the complexity of using these assays. Lonza attempts to make this optimization step easier. With slight modifications to standard 2D protocol, Lonza’s Vialight™ Assays could seamlessly assess viability and proliferation of HCT116 colon cancer cell line and human dermal fibroblasts cultured in RAFT™ 3D Cultures.

      

    Register to read the full white paper

     


    Transfecting 3D Cultures Efficiently

    Transfection of cells in 2D culture is already challenging if it comes to hard-to-transfect cell types. Given the complexity of 3D cultures, standard 2D transfection techniques pose bigger challenges. Lonza’s Nucleofector™ Technology and RAFT™ 3D Culture System attempt to bridge these gaps. The Nucleofector™ System has been an established method to accomplish efficient transfection in hard-to-transfect cell types. When coupled with the RAFT™System, achieving high transfection efficiency becomes seamless in 3D environment. In a recent technical note, we show a more successful approach where cells can be transfected in 2D prior to transfer into 3D method. The prerequisite for this is achieving high transfection efficiencies while keeping cells viable so that a large number of cells transferred into 3D express the gene of interest and stay viable over a longer period. In combination with Lonza’s Nucleofector™ Transfection Technology, the RAFT™ System used this efficient approach to create 3D cultures which maintained transfected substrate for over 4 days.

     

    Register to read the full white paper

       

     

    Contact us to find out if the RAFT™ System is applicable to your project or if you have any questions.


  • Airway Research

    The development of more complex in vitro airway models is needed for the assessment of novel drugs and chemicals because of the limited biological relevance of animal models to humans as well as ethical considerations1. Many cell-based assays are usually developed in 2D with limited cellular and functional representation of the native tissue. An optimal co-culture model is needed to truly understand the cellular interactions and mimic the features of airway remodeling in the diseased states. For instance, tissue injury is associated with airway remodeling in several airway diseases including asthma, chronic obstructive pulmonary disease, and fibrosis alveolitis2. In the case of epithelial injury, certain airway epithelial-derived mediators can stimulate the proliferation of smooth muscle cells.

     

    With current 2D and 3D methods, it is sometimes challenging to layer multiple cell types to achieve the complexity of an airway model. In order to better understand cellular interactions, we developed a preliminary co-culture system with bronchial epithelial and smooth muscle cells from normal and asthmatic donors using the RAFT™ 3D Cell Culture System. As a next step, we seek to develop a stratified air-liquid interface model using bronchial epithelial cells and smooth muscle cells.

     

    bronchial smooth muscle cells in 2D culture

     

    bronchial smooth muscle cells in 3D culture

     

     

     

      

    Human Bronchial Smooth Muscle Cells (BSMC)
    in 2D Culture
      Human Bronchial Smooth Muscle Cells (BSMC)
    in RAFT™ 3D Culture

    The morphology and the growth pattern of bronchial smooth muscle cells appeared to be influenced by the RAFT™ 3D Cell Culture System.

     

    Register to download the full white paper:

    Comparison of Normal and Asthmatic Bronchial Epithelial Cells and SmoothMuscle Cells in Monolayer and RAFT™ 3D Cell Culture System

     


    Related Application: Daniels et. al (2015) demonstrates that the RAFT™ System can be used to support the air liquid interface model with human corneal epithelial cells in a co-culture with limbal melanocytes. After one week in submerged culture followed by another week of air-lifting, multi-layering and stratification of the epithelial sheet was observed. Limbal melanocytes served as a feeder layer and supported the formation of thicker epithelial cell sheets.

         


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    1 Mol Pharm. 2014 Jul 7; 11(7): 2082–2091. doi:  10.1021/mp5000295

    2 Am J Respir Cell Mol Biol. 2009 Sep;41(3):297-304. doi: 10.1165/rcmb.2008-0358OC

  • Blood Brain Barrier Models

    Many tissues are composed of multiple cell types that are often organized within well-defined layers. Examples of such tissues are the human skin, the cornea or the blood-brain-barrier. Classic 2D cell culture systems are often not suitable for mimicking the complex structure of these tissues. With the RAFT™ 3D Cell Culture System, spatially defined organotypic blood-brain-barrier models mimicking epithelial and endothelial tissues can be made simply. 

     

    Human Brain Endothelium

    The blood-brain barrier is formed by microvascular endothelial cells, pericytes and astrocytes. It prevents the entry of most large hydrophilic molecules and many potentially harmful toxins from the blood into the brain. On the other hand it also prevents the entry of many therapeutic agents into the brain. Considerable efforts are made to develop therapeutics that can cross the blood-brain-barrier and these efforts can be supported by high-value 3D in vitro blood-brain-barrier models. See below an example of a 3D blood-brain barrier model developed by researchers based on the RAFT™ System.

     blood-brain-barrier-model-in-raft

    Blood-Brain Barrier Model in RAFTCultures. Figure A shows primary human astrocytes in RAFT™ Cultures.  Figure B shows co-culture of astrocytes with brain endothelial cells hCMEC/D3 and transport of glucose-coated gold nanoparticles in primary human astrocytes and/or brain endothelial cells (hCMEC/D3) . Data courtesy of Gromnicova et al (2013) PlosOne 8(12)

       

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  • Corneal Models

    Classic 2D cell culture systems are often not suitable for mimicking the complex structures of human cornea, skin or blood brain barrier. With the RAFT™ 3D Cell Culture System, spatially defined organotypic barrier models mimicking epithelial and endothelial tissues can be made simply. Generation of an artificial human cornea in vitro could have applications as a tool for pre-clinical development of novel therapies. Cornea, in vivo, is high in collagen type 1 concentration and the RAFT™ System provides an ideal solution for researchers to develop an in vitro corneal model within rat and bovine type 1 collagen.
    Corneal_model_in_RAFT
     

     A 3D in vitro human cornea model containing biomimetic corneal limbal crypts established in RAFT™ System. H&E stained paraffin embedded section shows that the HLE (human limbal epithelial) cells formed a healthy, 3-4 cell multi-layered epithelium on the flat surface of the HLF (human limbal fibroblasts) embedded in collagen.
    Data Courtesy of Levis et al (2013) Biomaterials.


     

    Limbal Stem Cell Deficiency Research (LSCD)
    Limbal epithelial stem cells (LESCs) are a population of cells responsible for maintenance and repair of the corneal surface. Injury or loss of these cells can lead to limbal stem cell deficiency (LSCD) in which the cornea becomes opaque, vascularized, and inflamed1. Transplantation of cultured human limbal epithelial cells (hLE) on a carrier known as human amniotic membrane (HAM) can restore vision. However, this treatment has its challenges since clinical graft manufacture using HAM can be costly, unreliable due to supply issues, and inconsistent from donor variability. Research has aimed to develop alternative carrier methods to HAM to increase success rate of the LSCD treatment. In order to serve as a carrier for hLE cells to the cornea, it is important that the alternative method has the right optical and mechanical properties (i.e. material should be as transparent as possible) as well as the capability to expand and carry cells to the cornea.

     

    RAFT™ 3D Culture System – Translating LSCD Research into Clinical Applications

    RAFT™ 3D Culture System uses high density collagen scaffolds which are very robust and transparent. Our customers have leveraged this capability of RAFT™ constructs to understand if they can potentially be utilized as a reliable and robust tissue equivalent (TE) to HAM. In a study by Julie T. Daniels and her team at University College London, RAFT™ 3D Constructs were able to support optimal hLE expansion and stratification conditions as well as provide a tunable option to develop a consistent production process for an alternative method to HAM.

     

     Floating acellular RAFT™ 3D Cultures

    Sample acellular
    RAFT™ 3D Cultures in a vial



     

    Data Courtesy of Julie Daniels and the team. Figure shows subjective assessment of RAFT™ TE and HAM transparency. Macroscopic images of text through either RAFT™ TEs (A) or HAM (B) were captured for qualitative comparison. As the image demonstrates, RAFT™ TEs showed comparable transparency at certain collagen concentrations which is an important criteria to serve as an alternative to HAM treatment.

      

    Read the full paper

      

    Please also watch the recent webinar where Prof. Dr. Julie T. Daniels explains how her lab at the University College London has developed multilayer corneal models and is using the RAFT™ System to address the current gaps in limbal stem cell deficiency (LSCD) treatment. Her lab aspires to take their research to the next level into clinical applications.

     

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    References

    1. Daniels et al. STEM CELLS 2011;29:1923–1932
    2. Daniels et al. Acta Biomaterialia Volume 24, 15 September 2015, Pages 241–250
  • Liver Models

    It is widely accepted that hepatocytes lose their function rapidly when cultured on a planar 2D surface. There is a desire within drug discovery and academic research to create a liver model that maintains higher levels of functionality for a longer period of time, ideally for many weeks. This would enable chronic exposure experiments to be carried out with some confidence that the results would bear similarities to results from primary cells and humans. The liver model is also an area where co-culturing of different cell types has proven to be of the most use. In addition to the parenchymal hepatocytes, researchers have been adding the non-parenchymal stellate and kupffer cells which clearly aid in supporting culture longevity and hepatocyte function.

     

    iPSC-derived hepatocytes in RAFT™ form 3D canalicular structures and exhibit cell polarisation. Data courtesy of Gieseck et al., 2014, PLOSOne

    Recent data established in the RAFTTM 3D Cell Culture System has demonstrated that human iPS-derived hepatocytes show enhanced levels of maturation markers and cytochrome P450 3A4 activity levels when compared to cells maintained in a 2D culture . 

      

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  • Tumor Formation

    It has been shown that an estimated 70% of cancer cells can cluster together and form higher-order structures that are referred to as tumoroids or spheroids, which then proliferate to make larger, more complex and multi-layered structures. The cells in the center of the tumoroid are exposed to a hypoxic environment and the core can become necrotic, which more closely resembles the inner tumor mass in vivo. This special behavior of cancer cells can’t easily be mimicked in a classical 2D cell culture environment. This provides a compelling argument for the adoption of tumoroid-enabling 3D cell culture techniques for oncology research. 

    There are, however, additional factors to consider when choosing the right cell culture model. The properties and behavior of cancer cells and tumoroids are strongly influenced by the surrounding extracellular matrix. Therefore each meaningful oncology model should contain a representative extracellular matrix. The right choice of the extra-cellular matrix is also important for the estimated 30% of cancer cell lines that will not form a ‘spheroid’ or form a structure with any substantial cohesion. Despite this lack of strong cell-cell or cell-matrix interactions, the cells are still influenced by their surroundings.

    colon-cancer-cell-lines-in-raft

        

    Colon cancer cell lines form "tumoroids" in RAFT™ 3D Cell Culture System. Data Courtesy of Magdeldin et. al, 2014, J Tissue Eng

     

    The Benefits of RAFT™ 3D Cancer Models

    The RAFT™ 3D Cell Culture System is the ideal system to model and study these behaviors: choose a cancer cell line, find the right cell seeding density, monitor cell proliferation with appropriate assays like the MTT assay, study cell signaling and protein phosphorylation levels and determine IC50 values with dose response curves.

    • Cancer cell lines from breast, liver and lung cancer have shown tumoroid structure formation in RAFT™ 3D System
    • Cancer cells that will not form these structures can still be studied in RAFT™ 3D Cultures
    • The cancer cells are being studied in a biologically relevant collagen environment. Therefore the cells can interact with the matrix through matrix-metallo-protease activity and integrin/DDR cell surface receptors and signaling cascades
    • The cultures are amenable to various assays allowing the generation of dose-response curves. Assays that have been shown to be compatible with RAFT™ are:
      • Cell proliferation assays
      • Mitochondrial function assays
      • Cell signaling assays
      • Protein phosphorylation assays

     

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