TrypLE™ Select and TrypLE™ Express

1 A highly purified, animal origin-free cell dissociation enzyme; replaces porcine trypsin Room temperature-stable easier to store and use; saves valuable freezer storage space Gentle on cells trypsin inhibitors not required Ideal for both serum and serum-free conditions Direct substitute for trypsin with no protocol changes Economical more cost-efficient than standard trypsin in research applicationsGIBCO TrypLE Select and TrypLE Express for cell dissociation are newadditions to Invitrogen's ever-growingline of serum-free media and non-ani-mal-derived reagents. TrypLE Select isdesigned specifically for the needs of thebioproduction industry. TrypLE Expressuses the same formulation as TrypLE Select but is manufactured under condi-tions allowing pricing comparable totrypsin.

2 Both are stable at 4 microbially produced alterna-tive to trypsinPorcine- or bovine-derived trypsin iswidely used in cell culture researchand bioproduction to remove adherentcells from and manufacturers whowant to ensure that their mammaliancell culture and bioproduction process-es are free of animal-derived materialscan now replace trypsin with micro-bially-produced trypsin-like enzymesTrypLE Select or TrypLE TrypLE has similar kineticsand cleavage specificity to trypsin, ithas significantly better is stable under conditions inwhich trypsin activity is lost. Workingstocks can be kept at room tempera-ture for six months or 37 C for oneweek without loss of stability(Figures 1 and 2). Likewise, it can befreeze/thawed without loss of activity(Figure 3).

3 Refrigerated long-termstorage is TrypLE is a microbially pro-duced alternative to animal trypsin. Itis manufactured in a controlled fer-mentation process that is completelyfree of animal- and human-derivedcomponents. The process produces anTrypLE Select and TrypLE ExpressTrypsin Replacement EnzymesPRODUCT NEWS exceptionally pure reagent (Figure 4),which is gentle on cells (Figure 5).The source of GIBCO TrypLE isreadily available for supply at anyscale, unlike other potential sources ofnon-animal trypsin alternatives suchas plant-derived enzymes, which aredependent on farming cycles andaffected by environmental regulationsof genetically modified employed in a serum-free orserum-supplemented system, GIBCO TrypLE demonstrates equivalent per-formance and superior stability totrypsin and, in many instances, can beeasier to use.

4 Trypsin inhibitors are notneeded (Figure 6). Additionally,TrypLE Express is available in bothphenol red-free and phenol red-contain-ing versions. Phenol red is a pH indica-tor that can exhibit estrogen-like proper-ties and may be toxic to certain celltypes. TrypLE Select is phenol has been proven effectivewith many different cell lines underboth serum-supplemented and serum-free conditions (Figure 7).Request a sampleTo try TrypLE Select or TrypLE Express , ask for a free 100 ml samplefrom Technical Service at 800 955 cultureProductQuantityCat. ExpressStable trypsin-like enzyme with phenol red100 ml12605-010500 ml12605-02820 100 ml12605-036 TrypLE ExpressStable trypsin-like enzyme without phenol red100 ml12604-013500 ml12604-02120 100 ml12604-039 TrypLE SelectAnimal origin-free trypsin-like enzyme100 ml12563-011500 ml12563-029 Ordering informationTrypLE Express and animal trypsin were incubated at 37 C for twodays.

5 Test samples were left on cell monolayers until all MDCK cellsappeared to dissociate. Although all cells eventually dissociated from theplastic, the time required for cells treated with TrypLE Expressremained constant while the time required for cells treated with porcinetrypsin more than doubled after 2 Express was stored at 4 C, room temperature, or 37 C. At the times indi-cated, samples were tested for the ability to cleave acetyl arginine p-nitroaniline(Ac-Arg-pNA). The results were calculated in moles/min/ml*.Figure 1 TrypLE Express stabilityrPU/ml*Time0 Day2 Day4 Day8 Week2 Week4 Week8*Assay measures Ac-Arg-pNA cleavage at ambient temp, pH CRoom Temperature37 2 Minutes required for cells to dissociate01020304050607002 DaysStorage Time at 37 CMinutesTrypLE Porcine TrypsinLeftHPLC Chromatograph illustrates a typical result with standard trypsin.

6 Note the multiple peaks indicating impurities. RightHPLC Chromatograph demonstratesthe purity of TrypLE activity was determined using the Ac-Arg-pNA assay. Values obtained are expressed as per-cent of starting activity (Freeze/Thaw #1). Errorbars are 1 standard deviation. No loss in activi-ty was seen on 4 different lots of TrypLE Expressfollowing 6 freeze/thaw 3 TrypLE Express stability following 6 freeze/thaw cyclesFigure 4 Demonstrated increased purity of TrypLE Select vs. trypsin0%20%40%60%80%100%120%140%12 3456 Freeze/Thaw Cycle NumberPercent of Starting ActivityLot #1 Lot #2 Lot #3 Lot #4 AUTrypLE Select1 2 3 4 5 6 7 8 9 10111213141516171819 AUMinutes12345678910 11 12 13 14 1516 MinutesPorcine were treated with animal trypsin or TrypLE Express and replated in new T-flasks in OptiPro SFM.

7 Cells were not washed following dissociationand no protease inhibitors were used. Morphologywas observed 24 hours later. Unlike trypsin, noprotease inhibitors are needed with TrypLE Express or TrypLE cultures of each cell line were serially passed 6 times. After enzyme dissociation, the cells were washed by centrifugation, counted, and subcultured atappropriate seeding densities. These values of time and viability represent the averages of the replicates over the 6 were detached with TrypLE Select or trypsin and plated in 6-well plates at100-200 cells per well. Colonies were counted 7-14 days post seed (cell line depend-ent). The animal trypsin gave plating efficiency values of and 55%, respec-tively, with the two cell lines.

8 A549 cells were grown in D-MEM + 5% FBS. MDCK cells were grown in OptiPRO Line MediaMean Time Requiredfor Cell ReleaseMean ViabilityVEROVP-SFM5 min100%VEROE-MEM + 5% FBS8 min100%VEROOptiPro 7 min98%PK-15E-MEM + 10% FBS27 min98%PK-15 OptiPro 11 m i n99%MDCKOptiPro 28 min98%MDBKD-MEM + 5% FBS15 min100%A549D-MEM + 10% FBS9 min98%293FD-MEM + 5% FBS2 min97%CHO-K1 CHO III A7 min95%020406080100120140A549 MDCK% of Animal Trypsin ControlTrypLE Animal TrypsinTrypLE ExpressAnimal TrypsinFigure 5 Colony plating efficiency assayFigure 6 MDCK cell dissociation without washing or trypsin inhibitor with TrypLE ExpressFigure 7 TrypLE Select and TrypLE Express activity on various cell linesTo try TrypLE Select or TrypLE Express .

9 Ask for a free 100 ml sample from Technical Service at 800 955 6288 These products are for research use, and where appropriate, as raw material components in further cell culture manufacturing are not intended for human or animal diagnostic, therapeutic, or other clinical uses, unless otherwise in the 2004 Invitrogen Corporation. All rights reserved. Reproduction without permission Headquarters:Invitrogen Corporation 1600 Faraday Avenue Carlsbad, California 92008 Tel: 1 760 603 7200 Tel (Toll Free): 1 800 955 6288 Toll Free Fax: 1 800 331 2286 E-mail: Headquarters:Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 9RF, UK Tel: +44(0) 141 814 6100 Fax: +44(0) 141 814 6260 E-mail: 031704 5 MProductQuantityCat.

10 ExpressStable trypsin-like enzyme with phenol red100 ml12605-010500 ml12605-02820 100 ml12605-036 TrypLE ExpressStable trypsin-like enzyme without phenol red100 ml12604-013500 ml12604-02120 100 ml12604-039 TrypLE SelectAnimal origin-free trypsin-like enzyme100 ml12563-011500 ml12563-029 Ordering informationCross Reference Product Selection GuideGIBCO animal-origin-free products do not contain material directly derived from animal tissues, cells, orbody fluids of higher eukaryotic organisms, such as mammals (including humans), fish, birds, insects, term "animal-origin" does not pertain to lower eukaryotic organisms such as the higher plants, fungi,protozoa, and algae, nor does it include prokaryotic organisms such as bacteria or blue-green Express ,w/ Phenol RedTrypLE Express ,w/o Phenol RedTrypLE SelectrProtease (1X), liquid12563-011100 ml12563-01112563-029500 ml12563-029 Trypsin-EDTA ( Trypsin, EDTA 4Na) (1X), liquid25300-054100 ml12605-01012604-01312563-01125300-06250 0 ml12605-02812604-02112563-02920 100 ml12605-03612604-039 Trypsin-EDTA ( Trypsin, EDTA 4Na) (10X), liquid15400-054100 ml*500 ml*20 100 mlTrypsin-EDTA ( Trypsin, EDTA 4Na) (1X)