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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.

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. 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.

Some of the advantages of 3D spheroids are:

Fig. HCT116 cells were seeded at 10,00 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 CellTiter-Glo reagent kit (Promega) was used to examine cell toxicity and data normalised to untreated spheroids.

We have grown numerous cells in a 3D format

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.

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

Some of the advantages of 3D electrospun scaffolds are:

Examples:

Fig. The incorporation of fluoresence dyes into fibres during manufacture means that 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 cell types or populations in the same well.

Fig. Recombinant HEK-293 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 iPSCs.

Fig. Proteins were isolated from cells on scaffolds, run in Western Blotting  and probed with antibodies to Pax-6, Ctip-2, and beta tubulin. Shown are Western blots of proteins isolated 14 day post-culture in differentiation media showing expression of mature cortical neurone proteins.

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

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