Culture apparatus for three dimensional cell growth
Cells that make up tissues in the body possess a complex three-dimensional (3D) architecture which is markedly different from the flat monolayer structure of their cultured counterparts commonly grown on the two-dimensional (2D) surface of tissue culture plastic. The 3D structure of cells and interactions with their neighbours significantly influences their ability to grow and function. Evidence for the functional superiority of cells cultured on 3D supports compared to 2D monolayer cultures has created much interest in the fabrication of materials that promote the growth and function of cells in a more realistic manner.
We have produced a cell culture device that provides an environment for routine 3D cell culture. We have developed thin layers of micro-cellular polymers with a well defined and uniform architecture (images below). Our matrices are made from inert polystyrene using emulsion templating (Barbetta et al. 2005) and their surface chemistry can be varied to influence cell adhesion, proliferation and function (Hayman et al. 2005). By changing the chemical composition of the emulsion and the processing conditions, the porosity of the material can be adjusted to suit alternative applications (Carnachan et al. 2006).
The architecture of the emulsion-templated polystyrene scaffold is revealed below using scanning electron microscopy. The structure is highly porous and consists of a voids linked to one another by pores.
The polystyrene scaffold is initially cast as a monolith which is then subsequently sliced into 200 micron thick membranes ready for mounting into culture apparatus. The scanning electron microscope image below shows the edge of such a membrane. Note the scale bar for reference.
Traditionally, cultured cells normally grow on treated-polystyrene 2D surfaces as in standard cell culture plastic-ware. Using our technology, we can present to cells the equivalent growth substrate but in a 3D format. These materials are readily adaptable to different types of existing tissue culture plastic-ware (e.g. multi-well plates, well inserts). The culture device is pre-fabricated, sterile, is ready to use off-the-shelf and can be handled in a similar manner as standard 2D plastic-ware. These are distinct advantages over existing 3D culture products which are technically more difficult to use, have a finite shelf life and are expensive.
Importantly, we have demonstrated that our technology can be used for routine 3D cell culture. As an inert plastic, our 3D culture product can be treated in the same manner as traditional 2D cell culture plastic. The scaffold can be plasma treated without any detrimental effect to its structure. It can be sterilised using gamma radiation. In other words, its manufacture is compatible with standard culture-ware production. Furthermore, the use of polystyrene as a cell growth substrate is well accepted and recognised. These are essential attributes that make our technology attractive as a solution for routine 3D cell culture in any research laboratory culturing cells.
We have exemplified the application of this technology by growing a broad selection of different cell types on these 3D supports and demonstrate enhanced functional activity compared to cells grown under identical conditions on 2D culture plastic (see examples below). We have also shown that the differentiation of cultured stem cells is significantly influenced by 3D growth (Hayman et al. 2004).
We propose that this technology enables the routine culture of cells in a 3D format in a simple and straightforward way. In preparation to making this patented technology available for use by biotechnologists worldwide, we have established alliances with third parties to further develop this device, exemplify its applications and plan for its manufacture and production.
See below for examples of current products, application this technology and associated publications. (View relevant publications)
Reinnervate products available for routine three dimensional cell culture:
Discs of 200 micron thick polystyrene scaffold have been fitted into the bottom of a 12-well culture plate ready to receive cell culture medium and seeded cells (below). The scaffolds are readily removed from the plate and can be handled gently with forceps. Scaffolds containing different cell types can be brought together within the same well so that interactions between alternative cell / tissue types can be studied in vitro.
Reinnervate has developed a bespoke well insert designed to carry the polystyrene scaffold. A version designed to fit into a 6-well plate will be available as seen below. There are multiply ways in which this can be used and an example is illustrated in the schematic below.
Reinnervate"s well insert is unique and provides flexibility for the user. The insert is made from polystyrene and is a two part assembly that comes together and clamps the substrate without crushing it. The substrate is cut to the diameter of the insert (achieved using a punch cutting tool) and inserted into the lower ring of the assembly. The larger "cup" component slides into the ring clamping the substrate. During assembly the user can change the position of where the ring and cup engage to cater for different thicknesses of substrate (from 0 to 500 microns). The substrate can be a rigid scaffold (such as a 200 micron thick polystyrene scaffold as seen below), or it can be a different type of membrane or scaffold as required by the user.
Well inserts are available as "kits" to enable user to construct different 3D cell culture models. Inserts that have been assembled and contain the scaffold are will be available from Reinnervate.
For the first time, investigators will be able to culture different cell types in 3D, then place the different 3D cultures together to simulate alternative tissue layers in vitro. Bringing together different 3D cultures can be achieved either in our well insert (see example below) or in a multi-welled cultured plate. This may be re-construction of specific tissue layers in a healthy system or modelling of the interaction between healthy and diseased tissues. For example, a 3D cell invasion assay of tumour tissue cells invading healthy stromal tissues. The user has freedom to choose and to develop and customise the system for their specific needs. There are multiple permutations which will be determined by the end user.
We can also supply the 6-well inserts alone that are not assembled as seen below. These can be used with the polystyrene scaffold developed by Reinnervate or membranes and scaffolds from other manufacturers. A handheld tool is also available to aid assembly of the insert if required. An insert for a 12-well culture plate is currently under development.
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Example application for routine use in cell culture:
The following examples depict the application of styrene-based scaffolds as thin layers adapted for use in existing cell culture vessels such as a multi-welled plate or well insert.
Notes:
Exemplification of apparatus that enables routine three dimensional cell growth in vitro:
Scanning electron micrograph showing MG63 osteoblasts cultured on polystyrene scaffolds for 14 days in vitro. The polystyrene scaffold represents the white areas whilst cells (in grey) grow in between (medium seeding density).
High magnification scanning electron micrograph showing HEPG2 hepatocytes attached to a strut of three-dimensional polystyrene scaffold for 14 days in vitro (low seeding density). Cells acquire complex 3D geometries and form intricate interactions with adjacent cells.
Low power scanning electron micrograph showing uniform and homogeneous coverage of cells over polystyrene scaffold.
Electron micrograph of transverse section through scaffold showing penetration of cells throughout the material from top to bottom. Cells completely fill the 3D space created by the scaffold and grow in 3D with their neighbours hence creating an in vitro derived tissue layer.
Three dimensional cell growth throughout polystyrene scaffold (below, Toluidine Blue staining). Cells adapt a 3D geometry and form complex interactions with adjacent cells. The technology enables the growth of 200 micron thick "slabs" of tissue in vitro. The thickness of the tissue layer is critical in this avascular system since cell survival is dependent on the diffusion of nutrients and gases from the culture medium.
Another example of a three dimensional cell growth throughout the polystyrene scaffold. The cultures can be fixed and processed just like tissues and prepared for histology using standard procedures. The below image shows some epidermal cells cultured for 7 days, fixed in PFA, embedded in wax and sectioned (7 micron) before staining with H&E and coverslipping on standard microscope slides.
Tissues grown using our in vitro 3D cell culture technology can also be prepared for resin embedding and thin sectioning, followed by staining with Toluidine Blue. The below example shows human keratinocytes cultured for 21 days prior to lifting to the air-liquid interface. The thickness of the overall "tissue slab" is approaching 250 microns. The polystyrene scaffold is seen in white.
Alternatively, the scaffold can first be impregnated with extracellular matrix proteins, such as collagen as seen below. Human keratinocytes were then seeded on top of the scaffold and brought to the air liquid interface to induce stratification. The scaffold is acting as a wick in this case, providing support for the developing culture creating an in vitro skin construct for use in biological assays.
The scaffold is compatible with a broad range of commonly used molecular and cellular techniques. Here for example, cells grown in 3D have produced a tissue layer that has been fixed and sectioned for immunoflourescence. The top panel shows the phase contrast image, the middle panel shows staining with the fluorescent nucleus marker, DAPI, the bottom panel shows staining for Ki67, a marker of proliferating cells. Note that the scaffold does not autofluoresce and is seen as black.
