Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

March 2012

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Gel Base Bioreactor Technology

At the cutting edge of cell based assays

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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The problem

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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All chemical and biological processes are influenced by environmental elements such as temperature, pH and chemical composition.

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

CellBasedAssays

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Environmental Concerns

Mechanical disturbances (Vibrations etc)

Alterations in water media hydration

Variations in dissolved CO2

Fluctuations in thermal conditions

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Uncontrolled changes in any of these factors can exert unwanted physical, chemical and/or biological effects on the specimen/sample in question leading to poor reproducibility or disruption of a given scientific and/or manufacturing process

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

Disruption &

Poor Reproducibility

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Negative outcomes

Increased Heterogeneity in cell populations

Cellular stress

Aberrant gene expression

Retardation of growth

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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With the emergence of high throughput and multiplexed biological, chemical and materials screening, the use of multi-welled assay plates has become standard for almost all automated experimental and storage applications.

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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However many of these experimental procedures are compromised by positional inequalities between wells on outer and inner locations on the micro-plate.

These inequalities or “edge effects” become increasingly apparent as micro-plate densities increase.

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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To improve reproducibility when conducting chemical and biological experiments, optimal environmental conditions must be maintained at all times.

To achieve this, it is often necessary to deploy costly and cumbersome environmental control systems. These systems dramatically reduce the experimental design flexibility of automated screening platforms and often do not solve the problem.

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Our solution

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

Phase-lockTechnology

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ThermalBuffering

Prevents evaporation

ImprovedCellGrowth

Betterwell to well consistency

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Plate

Gel

Side view and plan of gel plate showing construction of wells of plate surrounded by gel.

Wells

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Heat, CO2 passes into Gel down

thermal and concentration gradient

Loading in Incubator

Un-loading out of incubator

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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A 384 well gel buffered bioreactor

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Top view of the 384 bioreactorplate with gel buffering

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Thermal buffering

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Cooling rate reduced

Thermal retention ~ 3 X Improved

SBS format plate

Comparison of cooling between normal and Biocroí plates

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Protection against evaporation

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Wells of 96 welled plates were filled with 100ul serum free culture medium and then maintained in a standard drying oven set at 50ºC for 48 hours. (Data expressed as percentage volume remaining after incubation time.

(See plate diagram to identify wells used in all experiments).

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Improved cell growth & reproducibility

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Wells of buffered and normal 96 well plates were seeded at (equally) low density with cells of immortalised cell line. Prior to maintenance in a standard tissue culture incubator (set at 37C, 95% air/ 5% CO2) for 48 hours. Both buffered and normal plates were placed in middle of incubator side by side.

Normal Plates Buffered Plates

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Non-insulated 96 well Insulated 96 well

DRASTIC REDUCTION OFEDGE EFFECTS

14710 (1871)

CV=12.7% 6.12%16805 (1029)

CV=

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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The excellent environmental buffering properties of Biocroí plate technologies allows miniaturisation of cell based assays down to nanoliter volumes.

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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High Content Image of cells reverse transfected with SiGlo in a gel buffered 192 nanoslide. (Volume 100nl)Miniaturised 1000XAs compared to 96-well plate assays normally performed at 100ul

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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High Content Image of cells incubated in 100nl in a standard 384-well buffered plate

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Reproducibility of spotting 50 nanoliter siRNA in 384 buffered gel plate

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Dried 50 nanoliter siRNA spot overlain with 100 nanoliter lung carcinoma cells

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Advantages

Biocroí Ltd. Unit 3, 80 The Trinity CentreSt James’s Hospital, Dublin 8, Ireland

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Improved data quality

Substantial decrease in reagent & cell usage

Proven reduction in

edge effects

Increase in throughput productivity