RAFT™ 3D System Applications

Dermal fibroblasts cultured in RAFT™ System

New White Paper:

Modeling Tumor-Stroma Interactions with the RAFT™ 3D Cell Culture 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.


    Please select your area of interest from the navigation above to learn more.

    White Papers and Protocols

    New White Papers for RAFT™ 3D Cell Culture SystemModeling Tumor-Stroma Interactions with the RAFT™ 3D Cell Culture System 

    Tumor metastasis is influenced by the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs.

    Read this white paper to learn how to engineer a compartmentalized co-culture tumoroid model with HUVECs and the RAFT™ 3D Cell Culture System.


    NK Cell-Mediated Cytotoxicity Assay in 3D Cell Culture

    This white paper describes how colorectal cancer cells and Natural Killer (NK) cells were co-cultured in the RAFT™ 3D Culture System and compared to a conventional 2D format. Read about the superior results achieved with the 3D model.


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    More White Papers Available                      

    • A Three-Dimensional RAFT™ Co-Culture Model for Breast Cancer Drug Discovery
    • Co-Culturing Airway Cells in the RAFT™ 3D System
    • Analysis of cell viability and proliferation in RAFT™ 3D Cell Cultures
    • Using immunofluorescence microscopy with RAFT™ 3D Cultures
    • And more...




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

  • Cancer Co-Culture-Models

    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. 


    The Benefits of RAFT™ 3D Cancer Models

    • Cancer cell lines from breast, liver and lung cancer have shown tumoroid structure formation in RAFT™ 3D System
    • 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, and Protein phosphorylation assays  


    Cancer Models Discussed:


    Cancer Tumoroids and Immune Cells  

    Oncoimmunology is an emerging area and there is growing emphasis on utilizing better models to understand this application. To properly study the interaction between immune cells and target tumor cells, an appropriate in vitro model system must be established. However, much of the data published to date used cancer cells plated as a two-dimensional (2D) monolayer. A growing amount of data has shown that cells cultured in this manner lack the cell:cell and cell:matrix communication, metabolic gradients, and polarity demonstrated in vivo.

    Read the full white paper:

    Validation of an Image-Based 3D Natural Killer Cell Mediated Cytotoxicity Assay

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    The ability to perform matrix infiltration studies is also eliminated with the use of 2D cell culture. By embedding cancer cells into a three-dimensional (3D) matrix and allowing the formation of tumoroids, the shortcomings of using 2D cultured cells can be overcome as communication networks and cellular gradients observed within in vivo tumors are re-established.  


    HCT116 colorectal cancer cells in RAFT™ 3D Cell Culture 

    Video courtesy of Biotek Instruments, Inc.
    3D Natural Killer (NK) cell-mediated cytotoxicity (CMC) assay. Using the RAFT™ 3D Cell Culture System HCT116 colorectal cancer cells were embedded within a collagen hydrogel of defined concentration and thickness. Following cell propagation to create tumoroids within the matrix, NK cells were labeled with a cell tracking dye and added on top of the RAFT™ Cultures. Fluorescent apoptosis and necrosis probes were also added to track cytotoxic events within the tumoroids. Cellular imaging and analysis were performed at regular intervals to monitor NK cell induced apoptosis and necrosis of the HCT116 cells.


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    Angiogenesis and Cancer

    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. Tumor progression is mediated by micro-environmental conditions that include cell-cell and cell-extracellular matrix (ECM) interactions. Tumor metastasis is influenced by the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumor microenvironment.


    Read the full white paper

    Modeling Tumor-Stroma Interactions with the RAFT™ 3D Cell Culture System


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    Breast Cancer Model in RAFT™ System

    Our recent study demonstrated that the RAFT™ 3D Cell Culture System could be used to successfully co-culture normal human mammary fibroblasts (HMFs) along with normal human mammary epithelial cells (HMECs). This model was compared as a control to a three-dimensional co-culture model of breast cancer cells (MCF7 cell line) and mammary fibroblasts.

    White Paper:

    A Three-Dimensional RAFT™ Co-Culture as Advanced Model for Breast Cancer Drug Discovery


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  • 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. 


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    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.


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    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.


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    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.


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    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 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)


    Nervous System Injury Repair

    Artificial tissues constructed from cells offer a promising approach for improving the treatment of severe peripheral nerve injuries. In a new study, the effectiveness of using a conditionally immortalised human neural stem cell line, as a source of allogeneic cells for constructing living artificial nerve repair tissue was tested.

    Read the full publication


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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™ 3D Cell Culture System

     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.   

    Archived Webinar:

    Learn how Prof. Dr. Julie T. Daniels and her team have developed multi-layer corneal models using the RAFT™ 3D Cell Culture System.

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    RAFT Culture Transparency     HAM Cultures Transparency
    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.

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    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.


    Primary Human and Animal Hepatocytes Cultured in
    RAFT™ System 
      Read the full scientific poster:

    Comparison of Hepatocytes in Monolayer and RAFT™ 3D Cell Culture System


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    RAFT™ System allows for the creation of tissue-like structures. The 3D matrix of the type 1 collagen-based RAFT™ Culture provides a more natural cell culture environment and therefore a potentially superior model for in vitro screening. In a recent study, we compare cell viability and cell morphology of rat and human hepatocytes, and the maintenance of Cytochrome P450 (CYP) activity in human hepatocytes grown in the traditional Sandwich Model with that of cells cultured in the 3D RAFT™ System. Our results show that the RAFT™ 3D System represents a more robust model for the long-term maintenance of liver-specific functions.    

    iPSC-derived Hepatocytes Cultured in RAFT™ System


    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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  • Pancreatic Islets

    Diabetes mellitus (DM) affects millions of people worldwide. Development of novel tissue culture techniques for maintaining pancreatic islets is critical in order to improve access to donor material, better understand the disease group and to support the basis for improved transplantation therapies.1

    Pancreatic islets are clusters of non-proliferating cells with limited viability making them difficult to utilize for long-term studies. Constant access to donor material is also a challenge, in particular, for human-sourced pancreatic islets. Most of the donor pancreas are sought for islet transplantation procedures with limited availability for research use. As a result, there is an unmet need to improve the viability and maintenance of islets to alleviate the accessibility concerns for research. Currently, pancreatic islets are utilized in a variety of research areas with a primary focus on understanding and improving therapies for diabetes.

    In a recent study (Szebeni et al., Cytotechnology. 2017 Apr; 69(2): 359–369), pancreatic islets were cultured in the RAFT™ 3D System and compared to a conventional 2D system. The data from this study shows that islets embedded in the RAFT™ 3D System maintain their tissue integrity better than in monolayer and suspension cultures. "Overall the use of RAFT™ provided excellent results in preserving islet spheroid viability, structure integrity and insulin, glucagon production for at least 18 days ex vivo". (Szebeni et al., p. 369)

    For further details review the figure below or read the full publication.




    Viability staining and morphology of the islets in different culture conditions after the indicated time points detected by confocal laser scanning microscopy. (a, b) shows monolayer cultures, (c, d) RAFT™ Cultures, (e,f columns) suspension cultures. Between days 4 and 7, gradual morphological changes were noticed with the outspreading of fibroblast-like cells in the monolayer cultures (a, b columns),whereas islets inoculated directly within RAFT™ Gels preserved their globular shape (c,d columns). Between day 10 and 18 the continued outgrowth of fibroblast-like cells resulted in the loss of islet integrity in monolayer cultures (a, b)whereas RAFT™ Cultures maintained spheroid structures (c, d).

    Data courtesy of Szebeni et al Cytotechnology (2017) 69:359–369, CC BY 4.0.


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    1 Szebeni et al, Cytotechnology (2017) 69:359–369