Short Guide to 3D Cell Culture by Ewa_sem

SHORT GUIDE to 3D CELL CULTURE Comprehensive overview of available 3D technologies to help you choose the right solution for your research

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ABOUT AMSBIO Accelerating Discovery through Innovative Life Science Founded in 1987, AMSBIO (AMS Biotechnology) is recognized today as a leading company contributing to the acceleration of discovery through the provision of cutting-edge life science technology products and services for research and development in the medical, nutrition, cosmetics and energy industries. The AMSBIO range includes specialist antibodies, peptides and recombinant proteins. In addition, the company is able to draw upon in-depth expertise in extracellular matrices to provide elegant solutions for studying cell motility, migration, invasion and proliferation. Widely acknowledged as experts in cell culture, AMSBIO partners with clients in tailoring cell systems to enhance screening outcomes and eventual prognosis. With a range of molecular detection reagents, and a significant Biorepository the company can also provide tissue DNA, RNA, protein and microarray products. Key research areas for these products include: Oncology, Regenerative Medicine, Environmental Analysis, Cytotoxicity Screening, Glycomics and Stem Cell Biology.

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WHAT is 3D culture & WHY use it? Traditional cell culture on plastic dishes or in flasks, rarely recapitulates the conditions cells experience in vivo. Over the past 20 years, many researchers looked for ways to culture cells in a more physiologically relevant manner. It is important to note that there is no silver bullet, each cell type needs a different environment and many biological questions may need specific methods to study them. AMSBIO offers many matrices and other solutions for 3D culture. Below is a comprehensive guide to 3D culture matrices and applications. These are also colour coded to highlight what are the typical types of 3D objects generated in culture: spheroid (red), organotypic (blue) or directional cultures (green). We also offer customised advise and in-house 3D culture services. You can also explore our bio-repository to find cells or tissues to culture. This guide will assist those interested in 3D cell culture to be able to perform it in the most optimal way. If you have any further questions, please contact us: 3Dcellculture@amsbio.com or for an interactive version of this guide with more detailed information, visit www.amsbio.com/Guide-to-3D-Cell-Culture.aspx

APPLICATIONS

WHICH 3D CULTURE ?

Viability & cytotoxicity

Invasion & motility

Cell signalling & communication

Tissue regeneration

Natural Hydrogels

Scaffold-free

Artificial scaffolds

Synthetic matrices

Typical types of 3D objects generated in culture :

- spheroid - organotypic - directional FURTHER READING: Asthana A, Kisaalita WS. Biophysical microenvironment and 3D culture physiological relevance. Drug Discovery Today. doi:10.1016/j.drudis.2012.12.005. [Epub ahead of print] Elliott NT, Yuan F. A review of three-dimensional in vitro tissue models for drug discovery and transport studies. J Pharm Sci. 2011;100(1):59-74. Williams DF. On the nature of biomaterials. Biomaterials. 2009;30(30):5897-909.

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Spheroid culture The most common use of 3D cultures is in spheroid format. This format is particularly useful in cancer research as it enables quick discovery of morphological changes in transformed cells. Typically, cells are embedded in extracellular matrix (ECM) and left to proliferate and polarise according to the organ of origin. This results in the formation of a perfect sphere if the cells are normal, or a distorted structure if malignant. The most common types of ECM

The most common use of 3D cultures is in spheroid format

used are basement membrane extract or collagen, although other native ECM proteins are also in use. A variation on this principle is scaffold-free spheroid culture. In this type, cells are growing suspended in media. This could be achieved either by continues spinning or by using low-adherence plates. While this is 3D culture, it is important to bear in mind that no adherence cue is provided to the cells and the culture is largely dependent on cell-cell contacts. The 96 well spheroid invasion utilizes specialized matrices for spheroid formation & invasion.

Analysis of cell invasion from spheroid culture, using 3D Culture 96 Well BME Cell Invasion Assay . Further reading: Benton G, Kleinman HK, George J, Arnaoutova I. Multiple uses of basement membrane-like matrix (BME/Matrigel) in vitro and in vivo with cancer cells. I. Int J Cancer. 2011 Apr 15;128(8):1751-7.

Organotypic culture The origins of organotypic cultures of epithelial tissues lie in the need to model skin layers. Many of these cultures still involve both an epithelial layer (usually on top) and a mixture of extracellular matrix protein, such as collagen, and fibroblasts (usually as a supportive lower layer). Nowadays, organotypic cultures also use synthetic matrices or artificial scaffolds. These permit further manipulation of cells beyond the culture. For example, alvetex 速 is a plastic scaffold which will not interfere with extraction of proteins from cells cultured in 3D. alvetex 速 can be used for normal tissue functional studies (such as liver cultures) and oncology.

Scanning electron micrograph showing full thickness skin construct grown on a layer of collagen on top of alvetex速

HepG2 liver cells forming bile canaliculus (characteristic of liver tissues in the body) on

Further reading: Egeblad M, Nakasone ES, Werb Z. Tumors as organs: complex tissues that interface with the entire organism. Dev Cell. 2010 Jun 15;18(6):884-901. www.amsbio.com

Directional culture Directional culture is a highly specialised type of 3D cell culture. This type of culture is highly suitable for tissue regeneration applications such as muscular or neuronal repair. Typically, directional culture uses native extracellular proteins, especially laminin or collagen, with other matrices such bio-mimetics now also being considered. Further reading: Phillips JB, Brown R. Micro-structured materials and mechanical cues in 3D collagen gels. Methods Mol Biol. 2011;695:183-96.

Natural Hydrogels Various components of the natural extracellular matrix have been used for many years to study organogenesis and diseases. Particularly popular materials are collagen I and basement membrane extracts (BME). Over the years, BMEs have been modified into multiple formats including growthfactor reduced BME. The use of BME spread to supporting tumour growth in vivo with several specialised types. One difficulty in working with natural hydrogels are the changes in volume during culture. This has been addressed by using non-ECM materials such as the natural product alginate. More recently, dedicated BME-based kits also emerged in which the volume size is controlled enabling investigation into processes such as cell invasion. Further reading: Seliktar D. Designing cell-compatible hydrogels for biomedical applications. Science. 2012;336(6085):1124-8.

Scaffold-free cell culture The use of native components to generate 3D culture could be challenging for several applications, in particular those involving protein extraction and/or purification. Several longstanding methods, such as hanging-drop, have been revised recently to generate multicellular spheroids in multiple-well formats. However, ultra-low adhesion plates provide the most straight forward method to generate cell spheroids. These are particularly effective in studying processes such as tumour hypoxia.

Cells assembling into spheroids when grown in absence of scaffold on low adhesion plate

Further reading: Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol. 2013;31(2):108-15.

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Artificial scaffolds In recent years, one of the areas in which 3D culture has seen most advanced development is the usage of artificial scaffolds to generate a stable 3D environment. These scaffold, similarly to scaffold-free techniques, also benefit from the absence of protein in the preparation. Plastic scaffolds are those most widely used and are based on several rendering techniques. alvetex ® is a product using tissue culture plastic to generate ordered 3D space with well defined and connected cavities. This approach is very useful for applications such as liver modelling for toxicity and is now reaching high-throughput formats. Another option is biodegradable plastics, an even newer addition to 3D culture, which has the advantage of disappearing as the culture progresses.

Further reading: Marga F, Jakab K, Khatiwala C, Shepherd B, Dorfman S, Hubbard B, Colbert S, Gabor F. Toward engineering functional organ modules by additive manufacturing. Biofabrication. 2012;4(2):022001.

Synthetic matrices A need for balance between natural microenvironment materials and the requirement for robust 3D culture has created the idea of synthetic matrices. These are often made by end user by mixing a commercial matrix such as alginate with bio-mimetic peptides to enhance binding through integrins. Other options include the mussel adhesion protein based MAPTrix™ HyGels, which also contain various bio-mimetics. MAPTrix™ HyGel provides an in vivo like extracellular microenvironment by presenting combinatorial peptide motifs to induce a nd/or regulate processes such as tube formation as demonstrated in the above figure Further reading: Woolfson DN, Mahmoud ZN. More than just bare scaffolds: towards multi-component and decorated fibrous biomaterials. Chem Soc Rev. 2010;39(9):3464-79.

Viability & cytotoxicity Mapping the effects of drugs or genes on the viability of cancer cells is the mainstay of tumour biology. Scaffold-free cell culture provides a straightforward way to translate existing protocols to the 3D context. However, measurements of viability (or cytotoxicity) are also possible when using the artificial scaffold alvetex ® or natural hydrogels such as BME. 3D Culture of MCF-10A mammary endothelial cells on 3D Culture Matrix™ BME in Assay Medium with 2% BME and stained with Calcein-AM. Further reading: Laurent J, Frongia C, Cazales M, Mondesert O, Ducommun B, Lobjois V. Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D. BMC Cancer. 2013 Feb 8;13(1):73. www.amsbio.com

OrisPro™ Collagen I

Invasion & motility

Pre-migration

Control

100µm UO126

50µm Blebbistain

The study of invasive properties of cells is well established in 3D systems using natural hydrogels. However, quantification of the invasion process remained challenging for many years. Boyden chambers measure how many cells invade through a known width of matrix in a trans-well format and are useful for investigating chemotaxis, but do not allow invasion to be analysed in real time. Newer higher throughput solutions now exist: one is the OrisPro™ designed to measure mobility on ECM proteins. A second, is a 96 well plate invasion assay in which one spheroid per well invades into a specially formulated BME. These latest developments should enhance the scientific understanding of a critical process in tumour biology.

Cell signalling & communication Perhaps the greatest challenge in transition to 3D cell culture is the biochemical study of cell behaviour. While assays relaying on soluble factors such as cytokines are easy to adapt to most 3D formats, other assays require special protocols. Protocols are readily available for immuno-staining based microscopy techniques. Other solutions involve cell lysis for applications such as Western blotting, DNA or RNA purification.

Tissue regeneration Biomaterials of all kinds are critical to the regeneration of tissue in vitro before it can be introduced into the patient. This is typically achieved from culture of stem cells. Popular scaffolds for regenerative medicine applications include natural hydrogels and synthetic scaffolds.