3D101 Part 2: Keep Calm and Culture On: InSphero s easy to use GravityPLUS Hanging Drop System and GravityTRAP TM ULA Plate

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1 3D101 Part 2: Keep Calm and Culture On: InSphero s easy to use GravityPLUS Hanging Drop System and GravityTRAP ULA Plate GravityTRAP TM ULA Plate 1

2 A complete portfolio of 3D cell culture products and services MT production Cultivation and Dosing Endpoint Reporting GravityPLUS Hanging Drop System GravityTRAP ULA Plate 3D InSight Microtissues 3D InSight Services 3D InSight Cell Culture Medium 3D InSight Assay Kits Cell 3 imager 3D optimized protocols 2

3 InSphero 3D cell culture platforms GravityPLUS Hanging Drop System GravityTRAP ULA Plate Now available exclusively through 3

4 Components of the two systems GravityPLUS Hanging Drop System GravityTRAP ULA Plate 4

5 Microtissue production systems THE GRAVITYPLUS HANGING DROP SYSTEM GravityPLUS GravityTRAP Hanging TM ULA Drop Plate System and GravityTRAP Plate Technology 5

6 The hanging drop culture method Benefits of system: Scaffold-free and highly functional Long lifetime Assay-ready Highly reproducible 6

7 Scalable production of microtissues Process steps Single Channel pipette Multichannel pipette 96-channel semiautomatic Fully automated MT Production in GravityPLUS Transfer to GravityTRAP Culture Maintenance GravityTRAP Compound Treatments, Assays ( ) ( ) 7

GravityPLUS (<30 µl/sec) Medium aspiration from GravityPLUS (<10 µl/sec) Microtissue

8 Manual pipetting - considerations Use of multi-channel pipette recommended Electric pipette for speed control preferred Apply low speed pipetting for: Seeding in GravityPLUS (<30 µl/sec) Medium aspiration from GravityPLUS (<10 µl/sec) Microtissue transfer into GravityTRAP (<10 µl/sec) Medium aspiration from GravityTRAP (10-30 µl/sec) 8

9 The GravityPLUS TM Plate: Distinct features Reg. 96-well Contact-free GravityPLUS Contact Spring-loaded wells ensure tight contact with tips while using multi-channel pipette heads 9

10 Manual pipetting - guidelines GravityPLUS GravityTRAP Good Best Good 1. Make tight contact with well inlet at upright or slightly angled position 2. Rest pipette tip on SureXchange ledge for safe pipetting (remaining volume after complete aspiration: 5-7 µl) 10

11 Manual pipetting - guidelines GravityPLUS GravityTRAP Individual well with spring element Pitch 0% 1 mm 50% 100 % 1. Make tight contact with well inlet at upright or slightly angled position 2. Rest pipette tip on SureXchange ledge for safe pipetting (remaining volume after complete aspiration: 5-7 µl) 11

12 Hanging Drop System step-by-step: 1. Loading step Single Microtissue per well for manual or robotic handling and optical analysis Performed with Starlet Pipetting Station 12

13 Hanging Drop System step-by-step: 2. 3D Microtissue formation Seeding After 1 hour After 2-4 days Tumor tissue Medium/air interface Cells Medium 13

Specific pins and pinholes allow for easy connection

14 Hanging Drop System step-by-step: 3. Alignment to the GravityTRAP TM Plate GravityTRAP plate GravityPLUS Pin holes Pins GravityTRAP Merge the GravityTRAP plate with the GravityPLUS plate Specific pins and pinholes allow for easy connection of the 2 plates Additional pins provide standard distance between plates 14

15 Hanging Drop System step-by-step: 4. Microtissue transfer to GravityTRAP 15

16 Hanging Drop System step-by-step: 5. Medium exchange in GravityTRAP Aspiration 16

compared to manual pipetting with respect to time

17 Semi-automated pipetting with Viaflo96 (Integra) Comparative study: Microtissue production process compared to manual pipetting with respect to time requirements and accuracy 96-channel bench-top pipetting device 17

18 Semi-automated pipetting with Viaflo96 Increased productivity at comparable accuracy 18

19 Microtissue production technologies THE GRAVITYTRAP ULA PLATE webina summer GravityTRAP school TM ULA GravityPLUS Plate and TRAP technology 19

20 Limitations of common U-shaped ULA plates Common problems : Fluffy mass of cells at the bottom of U-bottom well Pseudo-spheroids that are not representative of solid tissues Loss of spheroids from U-bottom wells during medium exchange Difficulties imaging through curved plastic 20

GravityTRAP ULA Plate An enhanced ultra-low attachment solution for longterm 3D culture and assays GravityTRAP ULA: a costeffective, transfer-free ULA Plate that can be used for the generation,

21 GravityTRAP ULA Plate An enhanced ultra-low attachment solution for longterm 3D culture and assays GravityTRAP ULA: a costeffective, transfer-free ULA Plate that can be used for the generation, longterm cultivation, observation and testing of scaffold-free spheroids. Features: Ultra-low attachment (ULA) coated wells promote scaffold-free selfassembly Simple medium exchange (SureXchange ledge) Improved imaging (flat bottom) Components of the GravityTRAP TM System 21

22 The GravityTRAP Plate - Advantages: (a) Avoids loss of spheroids Novel well design stops inadvertent spheroid loss 22

23 The GravityTRAP Plate - Advantages: (b) Makes imaging easier Base of well is flat and made of clear thin plastic Clearly visible spheroid ideal for publication quality imaging 23

24 Design & Use of GravityTRAP ULA 24

25 GravityTRAP TM ULA produces consistently sized spheroids HCT116 HEY 100 cells/mt 500 cells/mt 2500 cells/mt cells/mt 25

26 Which plate to use? GravityPLUS Hanging Drop System For primary cells and co-cultures GravityTRAP ULA For tumor cells and cell lines 26

27 Examples of scaffold-free 3D Tumor microtissues >150 mono- or co-culture assay-ready tumor microtissue models available Optional GFP/RFP labeling of tumor/stromal cell populations 27

28 Comparison of GravityPLUS and GravityTRAP plates with competitor ULA Primary cells and complex cocultures Tumor cell lines and simple cocultures GravityPLUS hanging-drop system (tissue transfer required) GravityTRAP ULA Other ULA plates Throughput Medium exchange Risk of tissue loss Size measurement Imaging (Brightfield and Fluorescence) Biochemical assays

29 Summary 1. GravityPLUS Hanging Drop system Complete system contains GravityPLUS and GravityTRAP plates Gold standard solution for primary cells and complex co-cultures More handling steps involved, less throughput 2. GravityTRAP ULA plate For tumor cells and cell lines, which easily form microtissues Cost effective, high throughput 29

30 How to order GravityPLUS or GravityTRAP plates? The GravityPLUS TM Hanging Drop System and GravityTRAP TM ULA Plates are now EXCLUSIVELY available through PerkinElmer PerkinElmer Part Number Product Description ISP GravityPLUS Hanging Drop System, pack of 10 ISP GravityPLUS Hanging Drop System, pack of 3 ISP GravityTRAP ULA Plate, pack of 10 ISP GravityPLUS plate only, pack of 10 How do I order? Online: through the website By phone: please call your local customer care center of PerkinElmer By please your local customer care center of PerkinElmer 30

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