BioCision Consistent. Reproducible. Standardized.

September 2011

CoolBoxTM Ice-Free /

Sample Handling

CoolCell® Alcohol-Free / Cell Freezing

CoolRack®

Error-Free / Temperature Control

CoolProducts enable biological sample consistency, reproducibility and standardization – user to user, site to

site, trial to trial.

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CoolProducts

Problem: Lack of Standardization “The lack of standardized, high-quality biospecimens has

been widely recognized as one of the most significant roadblocks to the progress of cancer research.”

- NIH / NCI / OBBR

•  Worldwide effort to develop new standards for collecting, handling, and processing pre-analytical samples (blood, tissue, tumor, other biological specimens or sample materials)

•  EU: http://www.devicelink.com/ivdt/archive/09/04/003.html •  NCI/NIH: http://biospecimens.cancer.gov/practices/default.asp •  NIH Office of Biorepositories and Biospecimen Research 2010 Symposium:

http://www.brnsymposium.com/meeting/brnsymposium/2010/ •  BioCoR: http://biocor.umn.edu/ •  Duke: http://web.genome.duke.edu/cores/biorepository/protocols/ •  Molecular Epidemiology Workshop, Shanghai, 2008

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CoolRack

Error-Free / Cooling, Freezing or

Thawing

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CoolRack

CoolRack modules instantly introduce organization and consistent temperature to all samples (+/- 0.1oC).

Ice Cooling

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Current method of cooling samples in ice. The random layout can result in mis-identification, errors in aliquoting and temperature variability.

“BioCision’s proprietary and patented portable bench top tools reduce variability and ensure consistency in clinical sample handling. How samples are handled prior to analysis is critical to the outcome of experiments in basic and clinical research. "Biotech International” (November 2010)

Solution: CoolRack Reproducible and standardized cooling, (snap)freezing, thawing

CoolRack in water bath

CoolRack in dry ice

Five consecutive freeze/thaw cycles using a CoolRack module show highly reproducible freezing and thawing profiles

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Biotech International (November 2010)

How does it work? •  CoolRack modules are made of a novel thermo-

conductive alloy material •  Replaces “direct immersion” into temperature

sources such as ice, dry ice, LN2, water bath •  Thermo-conductivity is the transfer of heat from

higher temperature region to lower temperature region

•  CoolRack on Ice: Heat from the relatively warmer CoolRack module is transferred to the ice (dry ice, LN2) until equilibrium is reached

•  CoolRack in Water Bath: Heat is transferred from water bath toward relatively cooler CoolRack until equilibrium is reached

•  CoolRack novel alloy evenly distributes the temperature across all wells providing very uniform and consistent temperature to all samples

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CoolRack Versatile •  CoolRack rapidly adapts to any temperature

•  from -150oC through 100oC+ •  cooling (ice), snap-freezing (dry ice, LN2), •  thawing/incubating (water bath, heat block)

Uniform temperature •  +/-0.1oC temp uniformity to all wells •  standardize assay to assay, user to use, site to site

Patent-pending novel alloy construction •  very rapid thermo-conductivity/adaptivity •  temperature assurance – no mess, no uncertainty

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CoolRack tube holders

Modular For a variety of biological specimens

CoolSink Plate holders

CoolTray platforms

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Dry Ice Liquid Nitrogen Water Bath

-150oC -78oC 0o to 4oC 37oC +

•  Eliminate direct immersion into LN2 •  All tubes are upright •  Same freezing profile for all samples •  ThermalTray provides elevation of samples & increased distance between hands and LN2 •  Rapidly adapts from ambient (25oC) to LN2 temp (-150oC) in 12-14 minutes

•  Replaces direct contact with dry ice •  Eliminates ethanol bath on the bench – cost savings, reduced hazard & waste •  CoolRack PF provides equal or faster snap-freezing than ethanol slurry •  Standardized freezing - minimized standard deviation in freeze profiles •  Improved work flow •  Adapts to -78oC in 5-7 minutes

•  Gain organization & stability in ice •  Even temperature distribution to all samples even as ice melt •  Labels stay dry and legible for better sample identification •  Reduced risk of sample contamination •  Rapidly goes from ambient to <4oC in 90 seconds • Using ThermalTray, 10-15 hours of <4oC with one pan of ice

•  Keeps tubes out of water – reduced contamination, labels stay dry •  Consistent thawing profile for all samples •  All wells at identical temperature - eliminates “edge effect” on multi-well plates •  Stable support of tubes & plates

Ice

Versatile For a variety of applications

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BioCision’s tools are unique and replace very rudimentary methods of sample preservation (freezing, snap-freezing) and handling (cooling, thawing)

Cooling Rate - Ice §  Ambient to < 4oC in 60-90 seconds

§  4-6 hours of use when placing CoolRack directly on ice

§  Up to 14 hours cooling with a single pan of ice with ThermalTray

Snap-Freezing Rate – Dry Ice §  Equal or better freeze rate than dry ice/

ethanol bath

§  No alcohol required – cost savings, reduced hazard

§  Consistent freeze profile for all samples

Freeze rate on dry ice Standard deviation

Cooling rate on ice

CoolRack Performance

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CoolSink Performance

Figure 2. Final equilibrium well temperature for a 96-well flat bottom plate in direct contact with (A) crushed ice, (B) CoolSink 96Fwith a dry interface, and (C) CoolSink 96F with aqueous conductive medium in the interface. Colors represent 0.5°C temperature intervals of the corresponding plate wells. The white cell represents the well that was fitted with the thermocouple probe.

Ø  Multi-well plates placed directly on ice for cooling do not achieve critical < 4oC temperature in any of the wells (Fig. 2, A)

Ø  CoolSink modules evenly distribute the ice temperature to all wells (Fig. 2, B and C)

•  All wells are <4oC •  Curvature of the plate accounts for center

well variability (Fig.1)

Figure 1. Variance in height of undersurface of 96-well flat-bottom plate. The x and y axes represent the well number and letter, and the z axis represents the relative deviation of the surface in units of 0.001 in. Data was collected using a ball dial indicator with a resolution of 0.0001 in.

Published in Biotechniques, November 2010

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Kits and Accessories •  CoolBox

•  Ice-free cooling container

•  CoolSystem Kits •  Application based •  Tube based

•  Accessories •  Ice pans

•  Rectangular (1L, 4L, 9L) and round (4L) •  Temperature strips (1-8oC)

•  LCD display adheres to all thermo-conductive modules •  CoolMat histology cutting mat

•  Protects surface of ThermalTray and scalpel blade

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Customer Feedback - CoolRack Steffan Vartanian - Genentech Don’t you hate it when you spend all morning preparing RNA or harvesting lysates, only to come back from lunch to find your precious samples drowning in a pool of melted ice? ……But after using the Thermal Tray and CoolRack a few times, my soggy-sample days are officially over! Not only do these tools keep your samples chilled for hours, but they also keep everything organized, safe, and secure. BioCision's products are very well made, well designed, and just plain look cool. Highly recommended! Hans B - Perkin Elmer We have been using the CoolRack for snap freezing our samples for the past weeks with great success. Since we were on a tight schedule we performed some validation in house and were very pleased with the results.

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CoolBox Ice-Free /

Sample Cooling & Freezing

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CoolBox

Alternative to Ice Cooling

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Current method of cooling samples in ice. Directly inserting tubes into ice can result in temperature variability, lost or illegible labels, mis-identification, errors in aliquoting and increased risk of contamination.

CoolBox eliminates ice Consistent ice-free cooling and freezing •  cartridges provide up to 10 hours of ice-free 0.5 – 4oC cooling •  continuous +/- 0.1oC consistency to all wells Cool, freeze or snap-freeze •  five temperature options using cartridges, dry ice or LN2 •  portable •  accommodates variety of tubes & plates

Highly recommended for •  PCR and RNA work, cell culture, cooling in biosafety cabinet, GMP/GLP

suites and vivarium, bio-hazardous containment, short transport

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CoolBox 30 & CoolBox Microplate

CoolBox 30 System (for tubes) • Ice-free tube cooling / freezing • Includes CoolBox, lid, blue +2oC cartridge, red stage, purple insulator pad • Accommodates all 30-well CoolRacks • Sold separately OR with CoolRack

CoolBox Microplate System (for plates) • Ice-free SBS-format plate & tube cooling & freezing • Includes CoolBox, lid, blue +2oC cartridge • Accommodates all SBS format modules - CoolSinks, CoolRack PCRs, CoolRack 1mlx96 • Sold separately OR with CoolRack/CoolSink

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-25oC with thermal regulator pad placed between CoolRack and dry ice

Snap-freezing at -78oC with CoolRack directly on 200cc dry ice

CoolBox Performance

NOTE: Performance may vary depending on ambient conditions, starting temperature and other specific set-up criteria. See instructions for complete information.

mmmm

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CoolBox Advantage CoolBox Gel-Filled Container

Well-to-well Temperature Variance: +/-0.1oC

Well-to-well Temperature Variance: Up to 4oC

Cooling Media: LN2, Dry ice, Wet ice, Cartridge

Cooling Media: Gel

Temperature range: -150oC through +150oC

Temperature range: Fixed at -20oC or 0oC

Tube & Plate Formats: 0.5ml conical, 1.5ml conical, 1.5-2.0ml cylindrical, 12.5mm dia cryos, mass spec tubes, PCR 96- and 384-well plates, 6-, 12-, 24-, 48-, 96-well plates

Tube & Plate Formats: 0.5ml conical, 1.5ml conical, PCR tubes

Cooling Duration: 10h (2ºC), 6h (-12ºC), 5h (-25ºC), 6h (-78ºC)

Cooling Duration: 8h (0ºC), 3h (-21ºC)

Down Time: None – switch cartridge with another frozen one from the freezer or replenish dry ice, LN2

Down-Time: 4-6 hours to re-freeze gel-filled tube rack

CoolBox provides temperature uniformity and versatility

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Application-based kits

Protein / DNA CoolSystem Tissue Dissection CoolSystem Microbiology CoolSystem

Protein / DNA CoolSystem – Ice Free PCR Prep CoolSystem

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Customer Feedback - CoolBox Fraser Moss Ph.D. - Case Western Reserve University, Dept. of Physiology and Biophysics, President Scientist Solution BioCision has now eliminated the ice factor and have the CoolBox in which you can keep the 96 well CoolRack…..I'm using it to keep my RNA cold for oocyte injections right now. I'll be using it to assemble my PCR reactions in the not to distant future. This has been a great new little toy to have on the bench recently because our departmental ice machine broke in April and still is not fixed! Laura Matthews - Sarah Cannon Research Institute (SCRI) I am writing regarding the demo CoolRack and CoolBox that we have been testing. They are both great products. The CoolRacks have eliminated the mess of mushy cardboard freezer boxes. The samples also freeze much faster in the CoolRacks. The CoolBox was especially helpful in the transport of fresh biopsies on dry ice from one location to another

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CoolCell Alcohol-Free / Cell Freezing

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CoolCell

-1oC/min Controlled-Rate Cell Freezing Highest reproducibility vs. other methods

Highest cell viability

Alcohol-free rate control

•  -1oC/minute freeze rate in -80oC freezer •  Identical freezing profiles every time •  Format: 1ml – 5ml cryo vials or injectable vials •  Cell lines, PBMC, primary cells, stem cells,

yeast, algae •  Requires no isopropanol, no fluids •  No on-going expense •  No toxic waste •  No alcohol-induced variability

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Controlled vs. Uncontrolled Freezing Controlled (-1oC/min)

Uncontrolled/ Inconsistent

Post-thaw viability Controlled freezing reproducibly minimizes cell disruption and improves viability

Allows variation in cell viability and function with unpredictable result batch to batch

Freezing artifacts Reduces freeze selection of sub-populations of cells and/or biomarkers

Selects for sub-population of cells and/or function that can best survive the freezing conditions

Gene/biomarker expression & function

Ensures cells will be recovered in a predictable & consistent state (advantage when working with precious and small samples)

Cells will be recovered with an unpredictable and inconsistent state

Cell therapy & diagnostic

Essential to cell therapy and diagnostics

Roadblock to consistent diagnostic & clinical outcome

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Multiple Formats – Same Function Consistent -1oC/min freezing

1ml and 2ml cryo vials 12 wells

1ml and 2ml cryo vials 30 wells

- Vial module can be easily transferred to cryo storage

box

4ml and 5ml cryo vials 12 wells

2ml serum vials 12 wells

10ml serum vials 6 wells

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What is CoolCell?

Passive Controlled Cell Freezing* •  Radially-symmetric thermal exchange to

ensure uniform cooling profiles to all vial positions

•  HD Materials used in aerospace industry for lightweight & insulative properties

•  Highly Thermo-conductive core •  Conduction and/or micro-convection air

control (FTS30)

* Patents Pending

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CoolCell: Thermo-Dynamic Consistency Consistent freezing, reproducible results

Five consecutive freeze cycles in -80oC freezer using CoolCell (12-well, 2ml cryo vials). Cooling profiles and phase change are identical every time.

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Advantage CoolCell CoolCell Isopropanol Container

No IPA/No maintenance • No fluids • No pre-cooling required • No hazardous waste

100% Isopropanol • On-going purchase, maintenance, hazardous waste due to IPA degradation • Pre-cool hazardous alcohol in refrigerator

No variability • All vials have uniform freeze rate every time • Radially symmetric design ensures consistency to vials and freeze runs

Inconsistent freeze rate • Each run is slightly different due to IPA degradation • Two circles of wells – uneven heat removal from vials

No hidden cost • Once you buy it, you’re done paying for it!!

~$350/year maintenance per unit • Change IPA every five uses + hazardous waste disposal • 10 units = $3,500 maintenance cost EVERY YEAR

Lid opens easily • Cap comes off easily when frozen • Not cold to the touch, shatter-proof

Screw cap very difficult to remove when frozen • Protective glove required when handling frozen unit • Unit is cold, slippery, breakable

Highest post-thaw cell viability & function

Ready to use again in 5 minutes Takes >1 hour for device to return to room temp

Low impact on freezer • 1/3 the heat impact on freezer compared to alcohol-filled units

Large thermal mass impacts local freezer area • Large heat capacity removed from alcohol impacts nearby samples

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0%

20%

40%

60%

80%

100%

HeLa CHO-K K562 NIH3T3

% GFP transfection efficiency

Mr.Frosty

CoolCell (12 samples)

CoolCell (30 samples)

Cell Line Performance Comparison I CoolCell vs. “Mr. Frosty”

0%

20%

40%

60%

80%

100%

HeLa CHO-K K562 NIH3T3

% Viability

Mr.Frosty

CoolCell (12 samples)

CoolCell (30 samples)

Independent Performance Analysis by LONZA/Germany •  Identical transfection efficiencies and viabilities after freezing & thawing of cells •  CoolCell is more durable than Mr. Frosty (i.e. CoolCell is shatter-proof)

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Cell Line Performance Comparison II CoolCell vs. “Mr. Frosty”

0

1

2

3

4

5

6

HeLa CHO-K1 K562 NIH3T3

ATP RLU (ViaLight)

Growth performance after 24 hours post-thaw

Mr Frosty

CoolCell (12 samples)

CoolCell (30 samples)

Independent Performance Analysis by LONZA/Germany •  Identical growth of cells observed 24 hours after thawing •  Freezing and thawing is easier and more convenient with CoolCell (no isopropanol)

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0 20 40 60 80 100

Alcohol-based

CoolCell

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82%

HUVEC

0 20 40 60 80 100

Alcohol-based

CoolCell

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89%

PBMC

Primary Cells Performance Comparison CoolCell vs. “Mr. Frosty”

Figure 1. Post freeze viability, PBMC. 2x107 PBMC cells total were isolated and split into two vials, at 1x107 cells per vial, in Human Sera with 10% DMSO. One vial was frozen overnight using a BioCision CoolCell and one in a commercial IPA-based freezing system (“Mr. Frosty”). Following long term storage in LN2, cells were thawed and viability counts for each vial were obtained by the trypan blue stain method.

Independent Performance Analysis •  In primary human PBMC and HUVEC, freezing with CoolCell produces >90% viable cell

count post-thaw without the use of isopropanol (a flammable, hazardous solvent) which is used in the alcohol-based method

Figure 2. Post freeze viable cell count, HUVEC (n=5). Cells were resuspended in freezing medium at a concentration of 2x106 cells per ml. 1ml aliquots were proportioned into 1.8ml cryo vials and frozen at -1oC/minute in either a BioCision CoolCell or in an alcohol-filled freezing unit. Five vials frozen by either method were rapidly thawed and resuspended in growth media. Live cell counts were obtained by the trypan blue exclusion method.

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Stem Cells CoolCell vs. other common methods

Common un-standardized methods of stem cell cryopreservation •  Styrofoam box •  Paper towel insulation

Performance comparison with CoolCell •  Independent evaluation of CoolCell cyropreservation system initiated

by Roslin Cellab, a leading stem cell research institute •  Determine effect of different freeze/thaw methods on critical stem cell

growth parameters (viability)

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Independent Performance Analysis by Roslin Cellab •  Cryopreservation with CoolCell results in less cell death upon thaw (P<0.05) •  CoolCell ensures less variability when freezing multiple vials.

Dead Cells after Freezing CoolCell vs. other common methods

Figure 1. Determination of amount of dead RC10 cells (human embryonic stem cell) us ing Trypan B lue a f te r thawing. Comparison of 3 difference freezing methods (n=3)

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Stem Cell Performance Comparison CoolCell vs. other common methods morphology data

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Independent Performance Review – Roslin Cellab •  Stem cell morphology was visually improved when using the CoolCell compared to other methods.

This complements the data showing the higher viability and proliferative capacity results, as seen with CoolCell.

Figure 2. Human embryonic stem cell line RC10 was observed at day 1 post resuscitation under x10magnification using three different freezing methods.

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Independent Performance Analysis by Roslin Cellab •  Significant increase in viable cell count using a CoolCell (P<0.001)

1 Million cells (33%) more than other methods •  Direct impact in various experimental results (i.e. detection of proteins secreted in low levels) •  Decrease in patient sample size required for diagnostics, treatment or research

Stem Cell Proliferation CoolCell vs. other common methods

Figure 3. RC10 cells count using Trypan Blue at 3 days post-thaw using three different freezing methods (n=3)

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Independent Performance Analysis by Roslin Cellab •  Stem cells cryopreserved with a standardized and temperature controlled CoolCell recover better

and their proliferation rate is much higher

Stem Cell Proliferation over Time CoolCell vs. other common methods

Figure 4. RC10 cells count using Trypan Blue at days 0 and 3 post-thaw using three different freezing methods. Average of 3 samples.

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CoolSystem® CRYO Kit for ice-free vial preparation and alcohol-free cell freezing

CoolSystem CRYO (Item #BCS-142) Includes:

CoolBox CFT30 for ice-free vial cooling during preparation in the biosafety cabinet CoolCell 12-place 2ml vial controlled-rate freezing container

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TruCool™ Cryogenic Tubes Leak-proof caps, individually barcoded

•  Thermally-fused gasket prevents leakage and O-ring contamination •  Sterile, medical-grade polypropylene – will not discolor upon autoclaving •  Individually barcoded – no external labeling required (Barcodes are Code 128

and 10 characters) •  Internal- and external-threading options

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Customer Testimonials John Gardner – Roslin Cellab “This is by far and away the best benchtop technology to enter the field of cell cryopreservation for decades.” Rohit G. - Stanford University “We run a registry, in which large amounts of PBMCs are processed for long-term cyropreservation.………Overall, the CoolCell has proven to us to set a new bar in cryopreservation.” Sam Knight - Ceramisphere (Australia) “I spent nearly a year getting terrible viability-recovery on cell freezing with dry ice (using an alcohol device) followed by storage in LN2, and this on HeLa cells which aren't exactly hard to store. I then bought a CoolCell and now I can just use standard cryo-preservation techniques (DMSO, 20% NBS) and get nearly 100% viability on thawing.”

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CoolCell Summary §  Controlled cryopreservation results in

§  greater cell recovery (less dead cells) §  better “functional” recovery (higher rate of proliferation)

§  Highest reproducibility between samples §  Less selection of cell subsets §  Smaller sample size (patient sample/biopsy, hard to

transfect cells, flow cytometry sorted cells, generation of primary cells)

§  Easy to use, no toxic waste, easy transport to freezer farms

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Sample handling has become increasingly important •  Growing number of multi-national companies, labs, studies •  Increasing use of biomarkers and highly sensitive analytical methods

BioCision = Consistency. Reproducibility. Standardization. •  Test to test •  Researcher to researcher •  Lab to lab •  Site to site

Watch the VIDEO

Summary BioCision Pre-Analytical Sample Standardization

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Genentech Amgen Pfizer Roche Harvard MIT CalTech UCSF Glaxo Bristol Myers Squibb Novartis Translational Genomics Gilead Sciences Merck MedImmune ATCC Genpharm Elan Pharmaceuticals Centocor Boehringer Ingelheim Oncomed NIH/NCI Takeda Pharmaceuticals CDC Covance XOMA Quest Diagnostics Otsuka America Pharmaceuticals Salk Institute Burnham Institute Scripps Institute Stanford University Yale Genzyme EMD Serono Shire HGT Alnylam Wyeth Cell-Systems Jackson Labs Beth Israel Deaconess Hospital HHMI Whitehead Institute Harvard Systems Biology Exelixis Charles River Labs Sanofi Pasteur Penn State Lawrence Berkeley Lab Batelle UC Berkeley Cedars Sinai Hospital Cleveland Clinic IDEXX Biogen Perkin Elmer Genetics Blood Systems Research Inst.

Thank you

Copyright 2012. Patents pending. All rights reserved.