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3D Cell Culture Technology

Spheroids

 

How it works:

  • Aurelia Bioscience uses Ultra Low Attachment (ULA) plates to generate and culture spheroids. These plates have an ultra-low attachment surface coating and a well geometry that favours the formation of a single spheroid within each well.
  • Spheroids of different sizes develop gradients of oxygen, nutrients and metabolites, creating a hypoxic core and replicating cells on the outer edges.
  • Drug potency and efficacy is monitored in either an imaging-based assay (on a wide-field imaging plate reader) or by using commercially available assays that track parameters such as cell viability.

 

Fig. U87 cells form a spheroid – stained with Hoechst (blue), Calcein AM (green) and propidium iodide (red), imaged with CellInsight™ CX5 High Content Screening Platform. The spheroid was formed from seeding 1000 cells per well and culturing in 384 well Costar ULA spheroid plates for 7 days.

Fig. Spheroids formed from U87 cells then treated with Paclitaxel. The spheroid decreases in size upon treatment and incubation with compound over 48 hours in culture.

Advantages:

  • Retain 3D architecture of cellular environment in culture along with associated physiological functions
  • Uniformity in size and shape make them desirable for high throughput screening formats in drug discovery projects
  • Generate highly reproducible results
  • Increased viability of cells that are otherwise difficult to culture, including primary cells
  • Ability of spheroids to replicate the hypoxic core during tumour growth makes it a reliable model for cancer drug discovery projects
  • Allow for the investigation of cellular changes such as cell toxicity or cell viability in real-time

Fig. HCT116 cells were seeded at 1000 cells per well and grown for 7 days. Spheroids were then treated with increasing concentrations of compounds over a 48 hour period to examine cell toxicity. A 3-D Cell titre GLO reagent kit (Promega) was used to examine cell toxicity and data normalised to untreated spheroids.

We have grown numerous cell lines in a 3D format:

  • MCF7 – breast cancer cell – loose spheroid
  • HEK – embryonic kidney – loose spheroid
  • U2OS – osteosarcoma – loose spheroid
  • HCT 116 – colon – Tight spheroid
  • U87 – glioblastoma – tight spheroid
  • HT-29 – colon – tight spheroid

We have treated the spheroids with various drug treatments and have monitored their response using Draq 7 (which only penetrates dead cells), Calcein AM (which only penetrates live cells) and Hoeshct for an overall cell count. We have also used to CellTire Glo to determine viability by looking at ATP levels.

Micro-tissues on Electrospun Scaffolds

 

How it works:

  • Aurelia Bioscience has developed a 3-D micro-tissue system from electrospun material that can be used in conjunction with well plates for higher throughput screening
  • Electrospun material mimics the natural extracellular matrix and provides an ideal substrate for cells to adhere to
  • We have re-engineered electrospun material to form micro-scaffold islands on to which we seed, grow and differentiate cells prior to performing more conventional assays in well plates. Cells grow on, around and into the material, forming a micro-island of adherent cells that are effectively “micro-tissues in solution”
  • The incorporation of iron particles into fibres during manufacture results in scaffolds that can be physically manipulated using magnetism

Advantages:

  • Robust and reproducible 3-D culture environment
  • Applicable to any cell type (recombinant, human primary cells and iPSC’s plus differentiation)
  • Movable: from vessels-to-well and well-to-well with magnetism
  • Scalable to any assay throughput
  • Integrates seamlessly with all current screening and assay workflows

Fig. The incorporation of fluorescence dyes into fibres during manufacture means that fibres can be seen when imaging and can be used as a ‘barcode’ – two colours can be used to distinguish different cells types or populations in the same well.

Fig. Recombinant HEK293 cells stably expressing CRE-luciferase (Promega) – monitors cyclic AMP (cAMP) levels in cells. Treatment with forskolin increases cAMP levels, which acts as a transcription factor and increases synthesis of luciferase.

Fig. Scaffolds containing cortical neurons differentiated from iPSC’s

Fig. Protein expression examined from cells grown on scaffolds using WES high throughput Western blotting. Expression of PAX-6, C-tip-2 and beta-tubulin were all detected in protein isolated for scaffolds after differentiation from iPSC cells.

Fig. Optical Coherent Tomography was used to examine the growth of cells in scaffold material. HCT116, A549, U87 and U937 cells were seeded onto scaffold material and allowed to grow for 24, 48 and 72 hours post seeding. Show here for HCT116 cells, after 24 hrs the cells have infiltrated the material and by 72 hrs the material is completely engulfed by the cells.

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