BioCision Consistent. Reproducible. Standardized. September 2011

Transcription

1 BioCision Consistent. Reproducible. Standardized. September 2011

2 CoolProducts CoolProducts enable biological sample consistency, reproducibility and standardization user to user, site to site, trial to trial. CoolRack Error-Free / Temperature Control CoolBox TM Ice-Free / Sample Handling CoolCell Alcohol-Free / Cell Freezing 2

3 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: NCI/NIH: NIH Office of Biorepositories and Biospecimen Research 2010 Symposium: BioCoR: Duke: Molecular Epidemiology Workshop, Shanghai,

4 CoolRack CoolRack Error-Free / Cooling, Freezing or Thawing 4

5 Ice Cooling 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) Current method of cooling samples in ice. The random layout can result in mis-identification, errors in aliquoting and temperature variability. CoolRack modules instantly introduce organization and consistent temperature to all samples (+/- 0.1 o C). 5

6 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 Biotech International (November 2010) 6

7 How does it work? CoolRack modules are made of a novel thermoconductive 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 7

8 CoolRack Versatile CoolRack rapidly adapts to any temperature from -150 o C through 100 o C+ cooling (ice), snap-freezing (dry ice, LN2), thawing/incubating (water bath, heat block) Uniform temperature +/-0.1 o C 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 8

9 Modular For a variety of biological specimens CoolRack tube holders CoolSink Plate holders CoolTray platforms 9

10 Versatile For a variety of applications -150 o C -78 o C 0 o to 4 o C 37 o C + Liquid Nitrogen 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 (25 o C) to LN2 temp (-150 o C) in minutes Dry Ice 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 -78 o C in 5-7 minutes Ice 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 <4 o C in 90 seconds Using ThermalTray, hours of <4 o C with one pan of ice Water Bath 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 BioCision s tools are unique and replace very rudimentary methods of sample preservation (freezing, snap-freezing) and handling (cooling, thawing) 10

11 CoolRack Performance Cooling rate on ice Cooling Rate - Ice Ambient to < 4 o C in 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 11

12 CoolSink Performance Ø Multi-well plates placed directly on ice for cooling do not achieve critical < 4 o C 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 <4 o C Curvature of the plate accounts for center well variability (Fig.1) Figure 1. Variance in height of undersurface of 96-well flatbottom 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 in. Data was collected using a ball dial indicator with a resolution of in. 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. Published in Biotechniques, November

13 Kits and Accessories CoolBox CoolSystem Kits Ice-free cooling container Application based Tube based Accessories Ice pans o Temperature strips (1-8 C) Rectangular (1L, 4L, 9L) and round (4L) LCD display adheres to all thermo-conductive modules CoolMat histology cutting mat Protects surface of ThermalTray and scalpel blade 13

14 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. 14

15 CoolBox CoolBox Ice-Free / Sample Cooling & Freezing 15

16 Alternative to Ice Cooling Current method of cooling samples in ice. Directly inserting tubes into ice can result in temperature variability, lost or illegible labels, misidentification, errors in aliquoting and increased risk of contamination. 16

17 CoolBox eliminates ice Consistent ice-free cooling and freezing cartridges provide up to 10 hours of ice-free o C cooling continuous +/- 0.1 o C 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 17

18 CoolBox 30 & CoolBox Microplate CoolBox Microplate System (for plates) Ice-free SBS-format plate & tube cooling & freezing Includes CoolBox, lid, blue +2 o C cartridge Accommodates all SBS format modules - CoolSinks, CoolRack PCRs, CoolRack 1mlx96 Sold separately OR with CoolRack/CoolSink CoolBox 30 System (for tubes) Ice-free tube cooling / freezing Includes CoolBox, lid, blue +2 o C cartridge, red stage, purple insulator pad Accommodates all 30-well CoolRacks Sold separately OR with CoolRack 18

19 CoolBox Performance mmmm -25 o C with thermal regulator pad placed between CoolRack and dry ice Snap-freezing at -78 o C with CoolRack directly on 200cc dry ice NOTE: Performance may vary depending on ambient conditions, starting temperature and other specific set-up criteria. See instructions for complete information. 19

20 CoolBox Advantage CoolBox Well-to-well Temperature Variance: +/-0.1 o C Gel-Filled Container Well-to-well Temperature Variance: Up to 4 o C Cooling Media: LN2, Dry ice, Wet ice, Cartridge Temperature range: -150 o C through +150 o C Tube & Plate Formats: 0.5ml conical, 1.5ml conical, ml cylindrical, 12.5mm dia cryos, mass spec tubes, PCR 96- and 384-well plates, 6-, 12-, 24-, 48-, 96-well plates Cooling Duration: 10h (2ºC), 6h (-12ºC), 5h (-25ºC), 6h (-78ºC) Down Time: None switch cartridge with another frozen one from the freezer or replenish dry ice, LN2 Cooling Media: Gel Temperature range: Fixed at -20 o C or 0 o C Tube & Plate Formats: 0.5ml conical, 1.5ml conical, PCR tubes Cooling Duration: 8h (0ºC), 3h (-21ºC) Down-Time: 4-6 hours to re-freeze gel-filled tube rack CoolBox provides temperature uniformity and versatility 20

21 Application-based kits Protein / DNA CoolSystem Ice Free PCR Prep CoolSystem Microbiology CoolSystem Tissue Dissection CoolSystem Protein / DNA CoolSystem 21

22 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 22

23 CoolCell CoolCell Alcohol-Free / Cell Freezing 23

24 -1 o C/min Controlled-Rate Cell Freezing Highest reproducibility vs. other methods Highest cell viability Alcohol-free rate control -1 o C/minute freeze rate in -80 o C 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 24

25 Controlled vs. Uncontrolled Freezing Post-thaw viability Freezing artifacts Gene/biomarker expression & function Cell therapy & diagnostic Controlled (-1 o C/min) Controlled freezing reproducibly minimizes cell disruption and improves viability Reduces freeze selection of sub-populations of cells and/ or biomarkers Ensures cells will be recovered in a predictable & consistent state (advantage when working with precious and small samples) Essential to cell therapy and diagnostics Uncontrolled/ Inconsistent Allows variation in cell viability and function with unpredictable result batch to batch Selects for sub-population of cells and/or function that can best survive the freezing conditions Cells will be recovered with an unpredictable and inconsistent state Roadblock to consistent diagnostic & clinical outcome 25

26 Multiple Formats Same Function Consistent -1 o C/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 26

27 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 27

28 CoolCell: Thermo-Dynamic Consistency Consistent freezing, reproducible results Five consecutive freeze cycles in -80 o C freezer using CoolCell (12-well, 2ml cryo vials). Cooling profiles and phase change are identical every time. 28

29 Advantage CoolCell No IPA/No maintenance No fluids No pre-cooling required No hazardous waste CoolCell No variability All vials have uniform freeze rate every time Radially symmetric design ensures consistency to vials and freeze runs No hidden cost Once you buy it, you re done paying for it!! Lid opens easily Cap comes off easily when frozen Not cold to the touch, shatter-proof Highest post-thaw cell viability & function Isopropanol Container 100% Isopropanol On-going purchase, maintenance, hazardous waste due to IPA degradation Pre-cool hazardous alcohol in refrigerator Inconsistent freeze rate Each run is slightly different due to IPA degradation Two circles of wells uneven heat removal from vials ~$350/year maintenance per unit Change IPA every five uses + hazardous waste disposal 10 units = $3,500 maintenance cost EVERY YEAR Screw cap very difficult to remove when frozen Protective glove required when handling frozen unit Unit is cold, slippery, breakable Ready to use again in 5 minutes Low impact on freezer 1/3 the heat impact on freezer compared to alcoholfilled units Takes >1 hour for device to return to room temp Large thermal mass impacts local freezer area Large heat capacity removed from alcohol impacts nearby samples 29

30 Cell Line Performance Comparison I CoolCell vs. Mr. Frosty 100% % GFP transfection efficiency 100% % Viability 80% 60% 40% 20% Mr.Frosty CoolCell (12 samples) CoolCell (30 samples) 80% 60% 40% 20% Mr.Frosty CoolCell (12 samples) CoolCell (30 samples) 0% HeLa CHO-K K562 NIH3T3 0% HeLa CHO-K K562 NIH3T3 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) 30

31 Cell Line Performance Comparison II CoolCell vs. Mr. Frosty Growth performance after 24 hours post-thaw 6 5 ATP RLU (ViaLight) Mr Frosty CoolCell (12 samples) CoolCell (30 samples) 1 0 HeLa CHO-K1 K562 NIH3T3 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) 31

32 Primary Cells Performance Comparison CoolCell vs. Mr. Frosty PBMC HUVEC CoolCell 92% CoolCell 94% Alcohol-based 89% Alcohol-based 82% !"#$%&'(")(''")*+,-" Figure 1. Post freeze viability, PBMC. 2x10 7 PBMC cells total were isolated and split into two vials, at 1x10 7 cells per vial, in Human Sera with 10% DMSO. One vial was frozen overnight using a BioCision CoolCell and one in a commercial IPA-based freezing system ( Mr. Frosty ). Following long term storage in LN2, cells were thawed and viability counts for each vial were obtained by the trypan blue stain method. Independent Performance Analysis!"#$%&'(")(''")*+,-" Figure 2. Post freeze viable cell count, HUVEC (n=5). Cells were resuspended in freezing medium at a concentration of 2x10 6 cells per ml. 1ml aliquots were proportioned into 1.8ml cryo vials and frozen at -1 o C/ 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. 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 32

33 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) 33

34 Dead Cells after Freezing CoolCell vs. other common methods 5-1:-;/$+2$<-3<$ :-,,9$3=-1$ &!$ &)$ %!$ %)$ %&"'$ %("'$ Figure 1. Determination of amount of dead RC10 cells (human embryonic stem cell) using Trypan Blue after thawing. Comparison of 3 difference freezing methods (n=3)!$!"#$ )$ *++,*-,,$./ $ 536-1$7+8-,9$ 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. 34

35 Stem Cell Performance Comparison CoolCell vs. other common methods morphology data CoolCell Styrofoam Box! Paper Towel!! Figure 2. Human embryonic stem cell line RC10 was observed at day 1 post resuscitation under x10magnification using three different freezing methods. 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. 35

36 Stem Cell Proliferation CoolCell vs. other common methods '"!$##%!"34'% % &" %" $" Figure 3. RC10 cells count using Trypan Blue at 3 days post-thaw using three different freezing methods (n=3) #"!"!""#!$##% &'()"*"+,%-".% /+0$)%1"2$#% 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 36

37 Stem Cell Proliferation over Time CoolCell vs. other common methods )*++"),-./" 01%! 2 3" (#$" (" '#$" '" &#$" &" %#$" %"!#$"!"!" '" 4567"8,7/9/:5;" ),,+)*++" Figure 4. RC10 cells count using Trypan Blue at days 0 and 3 post-thaw using three different freezing methods. Average of 3 samples. 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 37

38 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 38

39 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 39

40 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. 40

41 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 41

42 Summary BioCision Pre-Analytical Sample Standardization 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 42

43 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 Thank Otsuka America you 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. Copyright Patents pending. All rights reserved.