LNCaP microaggregates are metabolically active and proliferative over 14 days; whereas RWPE-2 microaggregates are metabolically dormant and non-proliferative.

<p>The viability of LNCaP (A and B) and RWPE-2 (C and D) microaggreagtes versus the same number of cells cultured in 2D was assessed via Alamar blue (A and C) and PicoGreen assay (B and D). LNCaP microaggregates demonstrate an increase in metabolism over 14 days (A) and an increase in DNA content (B). However, RWPE-2 microaggregates show a decrease in metabolism (C) and a decrease in DNA content (D). Mean +/- SD; n = 4. Data represents three experiments. *p<0.05, a paired students t-test was used to determine significance between 2D and 3D.</p

LNCaP microaggregates are more resistant to docetaxel compared to cells grown as monolayers.

<p>LNCaP cells (50,000 cells/well) were treated with docetaxel over 48 hrs (A) or 72 hrs (B) at day 2 of growth either in 2D and 3D. Alamar blue was used to assess cell viability. Mean +/- SE, n = 4 biological replicates *P<0.05; the data shown is representative of three independent experiments. (C) Phase contrast images show the effects of docetaxel after 72 hrs on the morphology of the microaggregates (3D) compared to cells grown in monolayer (2D).</p

The PDMS microwell system produces prostate cancer cell aggregates that are viable and of a controlled size.

<p>(A) The diameters of LNCaP and RWPE-2 cell aggregates were measured over 14 days; mean +/- SD, n = 50 from *p<0.05, a paired students t-test was used to show significant differences in diameter at day 7 and 14. (B) FDA/PI stain was used to stain viable cells green and dead cells red on the days shown and the percentage area of dead cells per microaggregate has been quantified to show that the non-cancer prostate cells (RWPE-1) have greater cell death (percentage red pixels) in the microwell inserts compared to the prostate cancer cells (LNCaP and RWPE-2); mean +/- SD, n = 10. A paired students t-test was used to calculate significance*p<0.05. (C) The confocal images and phase contrast images (Day 14 Phase) show that prostate cancer cell lines (RWPE-2 and LNCaP) grow as compact smooth microaggregates until day 14. Conversely, the non-cancer cells (RWPE-1) form dispersed loose clusters which are non-viable and with no definable diameter at day 14. Scale bar is 100 µm.</p

The microaggregates lack polarity as would be observed in a tumor.

<p>Microaggregates were fixed at day 7 and immunostained for apical and basal polarity markers (green). E-Cadherin (E-Cad) and β-catenin were used as apical markers and the basal markers were α6-integrin (α6-INT) and Laminin 5 (LM5). Nuclei were stained with DAPI (magenta) and the scale bar is 100 µm.</p

Apoptotic core can be controlled for using differently sized microwells.

<p>(A) Cleaved caspase-3 (CASP3; green), an apoptosis marker, was used to stain sections of RWPE-1, RWPE-2 and LNCaP microaggregates. (B) Due to the absence of cleaved caspase-3 in the small LNCaP aggregates, a large microwell (800 µm ×800 µm ×800 µm) was used to create large LNCaP microaggregates with an apoptotic core (CASP3 Lge). (C) The diameter of the LNCaP microaggregates (Sml) was compared with LNCaP microaggregates grown in the large microwells (Lge). A minimum of 50 aggregates were measured per condition. (D) Ki67 (green) was also used to stain proliferating cells within the large LNCaP aggregate. Nuclei were stained with DAPI (magenta); scale bar is 100 µm.</p

Fabrication of the PDMS micro-patterned surface system and surface coating with pluronic or multi-layering.

<p>(A) A polystyrene mold <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111029#pone.0111029-Babur1" target="_blank">[13]</a>, was used to cast PDMS sheets with a micro-patterned surface. Discs were punched out of the PDMS sheet to form inserts for multiwell plates, and these surfaces were coated with either 5% pluronic or multilayered with CHI and HA. LNCaP cells (50,000) were seeded on to either (B) pluronic coated (3D pluronic) or (C) multi-layered (3D multi) microwells and the Alamar blue assay was used to measure metabolism in 3D versus 2D cells. Both coatings produced viable aggregates that increased in metabolism over 7 days at a comparable rate to cells grown in 2D culture. Three technical replicates were carried out per time-point.</p

Description of packed bed bioreactor and results from the steady state oxygen mass balance.

<p>(A) Process flow diagrams of the single pass small-scale 160cm² bioreactor and (C) recirculating perfusion for the large-scale 2800cm² bioreactor. (B) Picture of 160 cm² left and 2800 cm² right bioreactor scaffolds made from 3 mm polystyrene pellets (scale bar is 10 mm). (D) 2800 cm² bioreactor inside the incubator. (E) Mass transport model result for radial oxygen diffusion with cell density of 100,000 cells/cm² in small-scale (160 cm²) and (F) large-scale (2800 cm²) packed bed bioreactors.</p

Characterisation of plasma modified surface and GFP-mMSCs expansion in our packed bed bioreactor in static and perfusion conditions.

<p>(A) Surface composition of the air plasma treated polystyrene scaffold determined by XPS. (B) GFP-mMSC attachment after one and half hours, initially seeded at 3000 cells/cm² (n = 4). (C) The growth after 3 days of culture of GFP-mMSC seeded at 1000 cells/cm² (n = 6). (D) Growth of GFP-mMSC in the bioreactor (BR) under static conditions (n = 4) and (E) under 5 mL/day perfusion (n = 4). (F) Cell harvest recovery and viablity from the bioreactor. (G, H, I and J) Fluorescent microscopy showed that the GFP-mMSC attached to the scaffold (scale bar is 500 μm). (K) IVIS imaging of the fluorescent intensity of PI stained GFP-mMSC in the bioreactor under static and (L) 5 mL/day perfusion conditions. IVIS images are a red (low) / yellow (high) heat map of fluorescent intensity.</p

Pre-isolated passage four pMSC expanded in our bioreactor in static and perfusion conditions.

<p>(A) pMSC expansion in the small-scale 160 cm² bioreactor (BR) in static (n = 4) and (B) 5 mL/day perfusion (n = 4), with the 2D controls (2D). (C) IVIS imaging of PI stained pMSC under perfusion conditions. IVIS images are a red (low) / yellow (high) heat map of fluorescent intensity. (D) Two week tri-lineage mesodermal differentiation induction of bioreactor expanded pMSC and 2D controls down the adipogenic (Oil Red O, 10x, scale bar is 100 μm), osteogenic (Alizarin Red, 5x, scale bar 500 μm) and chondrogenic (Alcian Blue, 10x, scale bar 500 μm) lineages. Quantification of (E) triglycerides (n = 4), (F) ALP activity (n = 4) and (G) GAG production.</p

GFP-mMSC expansion in a scaled-up packed bed bioreactor (A) The fold expansion of GFP-mMSC in a scaled-up bioreactor under 0.5 ml/min perfusion with T175 flask control (n = 4). (B) Glucose and lactate levels in the bioreactor (n = 3). (C, D, E & F) IVIS imaging of the fluorescent intensity of PI stained GFP-mMSC in the bioreactor.

<p>IVIS images are a red (low) / yellow (high) heat map of fluorescent intensity.</p