Cellartis Definitive Endoderm Differentiation Kit with DEF-CS Culture System User Manual

Transcription

1 Takara Bio Europe AB Cellartis Definitive Endoderm Differentiation Kit with DEF-CS Culture System User Cat. No. Y30035 (121515) Takara Bio Europe AB A Takara Bio Company Arvid Wallgrens backe 20, SE Göteborg, Sweden Europe Technical Support: tech-cellartis@takara-clontech.eu United States/Canada Asia Pacific Europe +46.(0) Japan +81.(0) Page 1 of 21

2 Table of Contents I. Introduction... 4 II. List of Components... 4 III. Additional Materials Required... 4 IV. General Considerations... 5 A. Storage and Handling... 5 V. Differentiation of Human ips Cells to Definitive Endoderm Cells... 6 VI. The Cellartis DEF-CS Culture System... 7 A. Transferring Human ips Cells to the Cellartis DEF-CS Culture System... 7 B. Coating Cell Culture Vessels... 8 C. Preparing Cellartis DEF-CS Medium... 8 D. Thawing Human ips Cell Lines... 8 E. Passaging Human ips Cell Lines... 9 F. Changing Medium for Human ips Cell Lines G. Start of Definitive Endoderm Differentiation VII. Cellartis Definitive Endoderm Differentiation Kit A. Starting Material B. Coating and Media Volumes C. Day 0: Start Definitive Endoderm Differentiation D. Day 1: Change Medium E. Day 2: Change Medium F. Day 3: Change Medium G. Day 4: Change Medium H. Day 6: Change Medium I. Day 7: Differentiation to Downstream Lineages, or Assays for Definitive Endoderm Formation VIII. Images of Cellartis Human ips Cell Lines Maintained in the Cellartis DEF-CS Culture System IX. Images of hips Cells Differentiated Using the Cellartis Definitive Endoderm Differentiation Kit X. RNA Profile of hips Cells Differentiated Using the Cellartis Definitive Endoderm Differentiation Kit Appendix A. Troubleshooting Guide Cellartis DEF-CS Culture System Appendix B. Troubleshooting Guide for the Cellartis Definitive Endoderm Differentiation Kit Page 2 of 21

3 Table of Figures Figure 1. Schematic presentation of the Cellartis human ips cell line work flow Figure 2. ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF-CS Culture System Figure 3. ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF-CS Culture System Figure 4. ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF-CS Culture System Figure 5. Day 1 3 morphology of two human ips cell lines differentiated using the Cellartis Definitive Endoderm Differentiation Kit Figure 6. Day 4 7 morphology of two human ips cell lines differentiated using the Cellartis Definitive Endoderm Differentiation Kit Figure 7. RNA profile of hips cell line 1 differentiated using the Cellartis Definitive Endoderm Differentiation Kit.. 19 Figure 8. RNA profile of hips cell line 2 differentiated using the Cellartis Definitive Endoderm Differentiation Kit.. 20 Table of Tables Table I. Recommended Culture Schedule for Definitive Endoderm Differentiation... 6 Table II. Weekly schedule for medium changes and passaging Table III. Media Volumes for Cellartis Definitive Endoderm Differentiation Kit Table IV. Troubleshooting Guide for the Cellartis DEF-CS Culture System Table V. Troubleshooting Guide Cellartis Definitive Endoderm Differentiation Kit Contact Us Customer Service/Ordering Technical Support tel: +33.(0) tel: +46.(0) fax: +33.(0) fax: +46.(0) web: web: orders@takara-clontech.eu tech-cellartis@takara-clontech.eu Notice to Purchaser This product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc. This product may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from Takara Bio Europe AB. If you require licenses for other use, please contact us by phone at Your use of this product is also subject to compliance with any applicable licensing requirements as detailed in our catalogues, on our website at on the label or other documentation accompanying the goods. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. STEM-CELLBANKER is a registered trade mark of Nippon Zenyaku Kogyo Co., Ltd.Takara and the Takara logo are trademarks of TAKARA HOLDINGS INC., Kyoto, Japan. Cellartis and DEF-CS are trademarks of Takara Bio Europe AB. All other trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. Takara Bio Europe AB is a Takara Bio company Takara Bio Europe AB This document has been reviewed and approved by the Takara Bio Europe AB Quality Assurance Department. Page 3 of 21

4 I. Introduction The Cellartis Definitive Endoderm Differentiation Kit with DEF-CS Culture System is used for differentiation of human induced pluripotent stem (ips) cells to definitive endoderm (DE) and includes two components; the Cellartis DEF-CS 100 Culture System and Cellartis Definitive Endoderm Differentiation Kit. The Cellartis Definitive Endoderm Differentiation Kit is optimized for the use with the Cellartis DEF-CS Culture System, for culture of undifferentiated human ips cells prior to the start of DE differentiation. The DE cells obtained with this kit can be used for generating further-specified cells of endodermal origin, such as hepatocytes and pancreatic endoderm. This product should only be handled by persons who have been trained in laboratory techniques and should only be used in accordance with the principles of good cell culture practice. Takara Bio Europe AB recommends the use of media and reagents according to this manual. Takara Bio Europe AB cannot guarantee correct technical feedback on customer cultures unless the below culture instructions have been followed. II. III. List of Components Cellartis Definitive Endoderm Differentiation Kit with DEF-CS Culture System (Cat. No. Y30035) o Cellartis Definitive Endoderm Differentiation Kit (Cat. No. Y30030; not sold separately) 10 ml Definitive Endoderm Differentiation Coating 18 ml Definitive Endoderm Differentiation Day 0 18 ml Definitive Endoderm Differentiation Day 1 18 ml Definitive Endoderm Differentiation Day 2 25 ml Definitive Endoderm Differentiation Day 3 47 ml Definitive Endoderm Differentiation Day 4 47 ml Definitive Endoderm Differentiation Day 6 o Cellartis DEF-CS 100 Culture System (Cat. No. Y30020; not sold separately. A larger 500 ml version of this product is sold as Cat. No. Y30010.) 100 ml DEF-CS Basal Medium 800 µl DEF-CS COAT-1 (for 100 ml) 300 µl DEF-CS GF-1 (for 100 ml) 100 µl DEF-CS GF-2 (for 100 ml) 40 µl DEF-CS GF-3 (for 100 ml) Additional Materials Required The following materials are required but not supplied: Human ips cells: Takara Clontech offers several different human ips cell lines Cell culture vessels, tissue culture treated polystyrene surface General cell culture equipment used in cell culture laboratory Appropriate medium for DE cell dissociation* PBS Dulbecco's with Ca 2+ & Mg 2+ (D-PBS +/+) PBS Dulbecco s w/o Ca 2+ & Mg 2+ (D-PBS / ) TrypLE Select Enzyme (1X), without phenol red * Needed for DE cell dissociation Page 4 of 21

5 IV. General Considerations A. Storage and Handling 1. Cellartis DEF-CS 100 Culture System Store Cellartis DEF-CS Basal Medium and Cellartis DEF-CS COAT-1 at 2 8 C; shelf life specified on product label. The Cellartis DEF-CS Basal Medium formulation contains penicillin and streptomycin. Store Cellartis DEF-CS Additives (GF-1, GF-2 and GF-3) at 20 C; shelf life specified on product label. At first use, thaw provided vials, mix gently and aliquot into appropriate volumes. Store at 20 C according to expiry date on original vial. Thawed vials may be stored at 2 8 C for up to one week. Do not re-freeze aliquots after thawing. NOTE: All three Cellartis DEF-CS Additives (GF-1, GF-2 and GF-3) are used when thawing and passaging human ips cells. Only Additives DEF-CS GF-1 and DEF-CS GF-2 are needed when changing medium on human ips cells. 2. Cellartis Definitive Endoderm Differentiation Kit Store all components of the Cellartis Definitive Endoderm Differentiation Kit at 20 C; shelf life specified on product label. Use thawed Cellartis Definitive Endoderm Differentiation Coating and Cellartis Definitive Endoderm Differentiation Day 0, 1, 2, 3, 4, and 6 the same day that they are thawed. Leftover Cellartis Definitive Endoderm Differentiation Coating and Cellartis Definitive Endoderm Differentiation Day 0, 1, 2, 3, 4 and 6 can be refrozen on the same day as they are thawed, if stored at 2 8 C prior to refreezing. Always discard warmed, leftover Cellartis Definitive Endoderm Differentiation Coating and Cellartis Definitive Endoderm Differentiation Day 0, 1, 2, 3, 4 and 6. Page 5 of 21

6 V. Differentiation of Human ips Cells to Definitive Endoderm Cells The human ips cell line of choice is transferred to the Cellartis DEF-CS Culture System, adapted, and expanded before start of differentiation to definitive endoderm (referred to as day zero). After seven days of differentiation definitive endoderm cells are obtained. The Cellartis DEF-CS Culture System is a complete system for efficient expansion and scale-up manufacturing of human ips cells in a feeder-free and defined environment. It consists of basal medium, additives, and a coating compound. 3 x 10 6 undifferentiated human ips cells cultured in the Cellartis DEF-CS Culture System are needed for the complete Cellartis Definitive Endoderm Differentiation Kit. While cells cultured in Essential 8 Medium or in mtesr1 can be used with the Cellartis Definitive Endoderm Differentiation Kit with some protocol optimization, we recommend transferring the cells to the Cellartis DEF-CS Culture System for optimal performance. The Cellartis Definitive Endoderm Differentiation Kit consists of six complete media and one ready-to-use coating compound for the differentiation of human ips cells to DE cells in 2D culture. One kit is enough to differentiate human ips cells to DE cells in 75 cm 2. The smallest recommended format is a 6-well plate. The recommended culture schedule is presented in Table I. This protocol for DE Differentiation has generated DE cells from 25 human pluripotent stem cell lines, resulting in 80% SOX17 positive cells. The RNA profile of two hips cell lines during differentiation using Cellartis Definitive Endoderm Differentiation Kit is shown in section X. Examples of cell morphology during differentiation are shown in section IX. Morphology may vary depending on cell lines and differences in cell densities. Table I. Recommended Culture Schedule for Definitive Endoderm Differentiation Day 0 Coating: Definitive Endoderm Differentiation Coating (0.1 ml/cm 2 ) Cell Seeding: Definitive Endoderm Differentiation Day 0 (0.2 ml/cm 2 ) Day 1 Medium change: Definitive Endoderm Differentiation Day 1 (0.2 ml/cm 2 ) Day 2 Medium change: Definitive Endoderm Differentiation Day 2 (0.2 ml/cm 2 ) Day 3 Medium change: Definitive Endoderm Differentiation Day 3 (~0.27 ml/cm 2 ) Day 4 Medium change: Definitive Endoderm Differentiation Day 4 (~0.52 ml/cm 2 ) Day 5 - Day 6 Medium change: Definitive Endoderm Differentiation Day 6 (~0.52 ml/cm 2 ) Day 7 DE cells ready to use NOTE: Always work under aseptic conditions. Page 6 of 21

7 VI. The Cellartis DEF-CS Culture System A schematic picture of thawing or transfer, medium changes, and passage of human ips cell lines in Cellartis DEF-CS Culture System is shown in Figure 1. Thawing (VI.D) or Transfer (VI.E) Coating (VI.B) Medium Preparation (VI.C.1) Medium Changes (VI.F) Medium Preparation (VI.C.2) Differentiation to Definitive Endoderm (VII) Passage (VI.E) Coating (VI.B) Medium Preparation (VI.C.1) Figure 1. Schematic presentation of the Cellartis human ips cell line work flow. Corresponding sections of this user manual are referenced in brackets. Human ips cell lines that are maintained in Cellartis DEF-CS Culture System should be passaged every three to four days, with daily medium changes. When the cell density is sparse, you can change the medium every other day; however, it is important to change medium the day after passage or thawing, and the day before passage or freezing. It is recommended that the cells are grown to a maximum confluence of x 10 5 cells/cm 2. A suggested weekly schedule is depicted in Table II. Table II. Weekly schedule for medium changes and passaging. Monday Tuesday Wednesday Thursday Friday Saturday Sunday Passage Change Change Passage Change - Change medium medium medium medium All procedures described in the manual are optimised for Cellartis human ips cell lines. If you wish to use Cellartis DEF-CS Culture System for other human induced pluripotent stem cells, please be aware that procedures and protocols may have to be adjusted. A. Transferring Human ips Cells to the Cellartis DEF-CS Culture System Undifferentiated human ips cells maintained in other culture systems can be readily transferred to the DEF-CS Culture System. Fresh cultures can be transferred at passage and cryopreserved cultures can be thawed directly using the Cellartis DEF-CS Culture System. It takes between two and five passages to adapt a cell line to the Cellartis DEF-CS Culture System. When initially transferring ips cells to this system, some cell characteristics might be different from what was observed in previously used culture systems: First, the Cellartis DEF-CS system utilizes single-cell passaging, and therefore the morphology of cells cultured in the Cellartis DEF-CS Culture System differs from that of cells cultured in systems using aggregate passaging methods. Second, newly passaged cells tend to spread out. However, when proliferating, the cells get denser, and the typical undifferentiated stem cell morphology (i.e., high nucleus to cytoplasm ratio, defined borders, and prominent nucleoli) reappears. Page 7 of 21

8 B. Coating Cell Culture Vessels 1. Dilute the required volume of Cellartis DEF-CS COAT-1 1:20 in D-PBS +/+ before use. 2. Mix the diluted Cellartis DEF-CS COAT-1 solution gently and thoroughly by pipetting up and down. 3. Add the appropriate volume of diluted Cellartis DEF-CS COAT-1 solution to the cell culture vessels (use 0.1 ml/cm 2 ), and make sure that the entire surface is covered. 4. Place the cell culture vessels in the incubator at 37 C ± 1 C for at least 20 min, or at RT for 30 min to 3 hr. 5. Aspirate the Cellartis DEF-CS COAT-1 solution from the cell culture vessels just before use. C. Preparing Cellartis DEF-CS Medium 1. Medium for Thawing or Passaging Human ips Cells 1. Decontaminate the external surface of all additives and the medium bottle with an appropriate disinfectant and place in the biological safety cabinet. 2. Prepare the appropriate volume of supplemented Cellartis DEF-CS medium by adding DEF-CS GF-1 (dilute 1:333), GF-2 (dilute 1:1000) and GF-3 (dilute 1:1000) to the Cellartis DEF-CS Basal Medium. 3. Prepare fresh medium on the day of intended use. Discard any leftover warm medium. 2. Medium for Maintenance of Human ips Cells 1. Decontaminate the external surface of all additives and the medium bottle with an appropriate disinfectant and place into the biological safety cabinet. 2. Prepare the appropriate volume of supplemented Cellartis DEF-CS medium by adding DEF-CS GF-1 (dilute 1:333) and GF-2 (dilute 1:1000) to the Cellartis DEF-CS Basal Medium. Do not add DEF-CS GF-3 to maintenance medium. 3. Prepare fresh medium on the day of intended use. Discard any left-over warm medium. D. Thawing Human ips Cell Lines When thawing human ips cells in Cellartis DEF-CS Culture System, seed approximately x 10 5 viable cells/cm 2 in ml medium/cm Preparation Coat cell culture vessels as described above (Section VI.B). Prepare supplemented Cellartis DEF-CS medium as described above (Section VI.C.1) and warm it to the appropriate temperature. See below for recommended volumes. 2. Thawing Cells NOTE FOR YOUR PROTECTION: Wear a protective face mask and protective gloves. Use forceps when handling a frozen vial. Never hold the vial in your hand as the cryovial may explode due to rapid temperature changes. 1. Transfer 4 ml of supplemented Cellartis DEF-CS medium to a sterile centrifuge tube and warm to RT. 2. Using forceps, transfer the vial directly from liquid nitrogen into a container of 37 C ± 1 C water. Thaw the vial by gently pushing it under the surface of the water. Do not submerge the cap of the vial in the water bath, as this could contaminate the cells. 3. Allow the vial to thaw until the cell suspension can be poured out of the vial. (It is okay if the suspension has a slushy consistency, as long as it can be poured out.) Page 8 of 21

9 4. Decontaminate the vial in an appropriate disinfectant. 5. Pour the entire contents of the vial into the sterile tube containing 4 ml supplemented Cellartis DEF-CS medium (RT). 6. Rinse the vial with 1 ml supplemented Cellartis DEF-CS medium, warmed to RT. Add to the cell suspension. 7. Centrifuge at 300 x g for 1 minute. 8. After centrifugation, aspirate the supernatant and gently resuspend the pellet in supplemented Cellartis DEF-CS medium (37 C ±1 C) to a density of approximately 1 x 10 6 cells/ml. 9. Count the cells in a hemocytometer or in a cell counter (optimized for the cell type). 10. Dilute the cell suspension to achieve a seeding density of x 10 5 cells/cm 2 in ml medium/cm Pipet the cell suspension into the cell culture unit. 12. Ensure that the cells and medium are evenly distributed across the surface of the cell culture unit, and place the cell culture unit in the incubator at 37 C ± 1 C, 5% CO 2, and 90% humidity. 3. Thawing Cells from Other Culture Systems Cryopreserved cells can be thawed directly into the DEF-CS Culture System. The standard thawing protocol should be followed, although some modifications may increase the success of transfer: The cells may benefit from a higher concentration of Cellartis DEF-CS COAT-1. Use a dilution of 1:10 or 1:5 at thawing and the first few passages to provide extra support during the adaptation period. The cells might initially grow at a slightly slower rate. A suitable passage interval might therefore be between three and seven days for the first few passages. The cells should adapt morphology as displayed in Figure 3 and Figure 4 prior to passage. If the cells are sparse after seven days in culture, a passage is still recommended. E. Passaging Human ips Cell Lines As a general rule, cells should be seeded at a density of x 10 4 cells/cm 2 (use 4.0 x 10 4 cells/cm 2 if leaving the cells four days between passages and 5.0 x 10 4 cells/cm 2 if leaving three days between passages). Adjust the density to suit your particular cell line as appropriate. When passaging the cells, we strongly recommend growing them to a confluence of x 10 5 cells/cm 2 (see Figure 2 Figure 4 for images of a variety of Cellartis human ips cell lines in culture). 1. Preparation Coat cell culture flasks as described above (Section VI.B). Prepare the appropriate volume of supplemented Cellartis DEF-CS maintenance medium as described above (Section VI.C.1) and warm it to 37 C ± 1 C before use. Warm all other reagents to RT before use. 2. Passaging 1. Check cells under microscope; photo document as necessary. 2. Aspirate medium from cell culture flasks and wash the cell layer once with D-PBS /. 3. Add 20 µl/cm 2 of TrypLE Select to the cell culture flasks and incubate for 5 minutes or until the cell layer has detached. Detachment can be aided by swirling the cell culture flask or by tapping the side of the cell culture flask firmly but gently. Page 9 of 21

10 4. Resuspend the cells in the supplemented Cellartis DEF-CS medium and pipet up and down several times to ensure a single cell suspension. (The cells will aggregate if left too long in TrypLE Select). 5. OPTIONAL: (To remove TrypLE Select if present at greater than 1:10 ratio.) Centrifuge the cells at 200 x g for 2 5 minutes. There is no need to centrifuge the cell suspension after dissociation if the TrypLE Select was diluted at least 1: Count the cells in a hemocytometer or in a cell counter (optimized for the cell type). 7. Add the appropriate volume of cell suspension and medium to the newly coated cell culture flasks to obtain the selected density. The seeding volume of supplemented Cellartis DEF-CS medium should be ml/cm Tilt the flask backwards and forwards gently to ensure that the cell suspension is dispersed evenly over the surface, then place in the incubator at 37 C ± 1 C, 5% CO 2, and 90% humidity. 3. Transfer from Other Culture Systems at Passage Fresh cultures can be transferred to the Cellartis DEF-CS Culture System at passage. The cells should be dissociated according to the protocol of the previous system, and seeded as single cells or aggregates according to the Cellartis DEF-CS Culture System protocol using a 1:1 split ratio based on culture area. Some modifications may increase the success of transfer: The cells may benefit from a higher concentration of Cellartis DEF-CS COAT-1. Use a dilution of 1:10 or 1:5 during the first few passages to provide extra support during the adaptation process. Newly transferred cells might initially grow at a slightly slower rate. A suitable passage interval might therefore be between three and seven days for the first passages. The cells are ready for passage when they have acquired the morphology displayed in Figure 3 and Figure 4. If the cells are sparse after seven days in culture, a passage is still recommended. F. Changing Medium for Human ips Cell Lines Medium change is recommended daily (except the day of passage). Use ml/cm 2 of medium. If the medium turns yellow due to high metabolic activity, increase the medium volume. 1. Preparation Prepare the appropriate volume of supplemented Cellartis DEF-CS medium as described above (Section VI.C.2) and warm it to 37 C ± 1 C before use. Do not add Cellartis DEF- CS GF-3 at medium change. Discard any leftover warm medium. 2. Medium Change 1. Check cells under microscope; photo document as necessary. 2. Carefully aspirate the medium and pipet newly warmed medium into the cell culture flask. Avoid pipetting medium directly onto the cell layer. 3. Place the cell culture flask in the incubator at 37 C ± 1 C, 5% CO 2, and 90% humidity. G. Start of Definitive Endoderm Differentiation The human ips cell line are ready to be used for differentiation once they are adapted to the Cellartis DEF-CS Culture System, i.e. the cells should display the morphology depicted in Figure 3 and Figure 4, and x 10 5 cells/cm 2 should be obtained at passage using the recommended passage intervals. Page 10 of 21

11 VII. Cellartis Definitive Endoderm Differentiation Kit All methods described in this user manual are for differentiation of human ips cells to DE cells in 2D monolayer cultures on a total area of 75 cm 2. Examples of cell morphology during differentiation of human ips cells to DE cells are shown in section IX. Morphology may vary depending on cell lines and differences in cell densities. A. Starting Material The Cellartis Definitive Endoderm Kit is optimised for the use with the Cellartis DEF-CS Culture System. All procedures described in the manual are optimised for human ips cells cultured in the Cellartis DEF-CS Culture System. If cells maintained in culture systems other than the Cellartis DEF-CS Culture System are used with this kit, the seeding density may have to be adjusted to get the cell densities shown in Figure 5 and Figure 6. If seeded too sparsely, the cells will die and if seeded too densely, differentiation will not be efficient enough. B. Coating and Media Volumes Media volumes for different cell culture vessels are listed in Table III. The smallest recommended format is a 6-well plate. Table III. Media Volumes for Cellartis Definitive Endoderm Differentiation Kit Format Coating Days 0, 1 and 2 Day 3 Day 4 and 6 6-well plate 1 ml 2 ml 2.7 ml 5 ml T25 flask 2.5 ml 5 ml 7 ml 13 ml T75 flask 7.5 ml 15 ml 20 ml 40 ml C. Day 0: Start Definitive Endoderm Differentiation 1. Preparation Thaw Definitive Endoderm Differentiation Coating and warm the appropriate volume (from Table III above) to RT. Thaw Cellartis Definitive Endoderm Differentiation Day 0 and warm the appropriate volume (from Table III above) to 37 C ± 1 C. 2. Coating Cell Culture Vessels 1. Add Definitive Endoderm Differentiation Coating to the cell culture vessels (0.1 ml/cm 2 ). Make sure the entire surface of each vessel is covered. 2. Incubate at RT for min. 3. Aspirate Definitive Endoderm Differentiation Coating solution from the cell culture vessels just before seeding. 3. Seeding Cells 1. Detach and dissociate the human ips cells into a single cell suspension. 2. Count the cells. 3. Transfer the appropriate number of cells to obtain x 10 4 cells/cm 2 to a 15-ml tube. 4. Centrifuge the human ips cell suspension for 5 minutes at 200 x g at RT. 5. Remove the supernatant and resuspend the cells in Cellartis Definitive Endoderm Differentiation Day 0 ( x 10 5 cells/ml) and seed in the coated cell culture vessels at a density of x 10 4 cells/cm 2 in 0.2 ml medium/cm Place the cell culture vessels in the incubator at 37 C ± 1 C, 5% CO 2, and 90% humidity. Page 11 of 21

12 D. Day 1: Change Medium 1. Preparation Thaw Cellartis Definitive Endoderm Differentiation Day 1 and warm the appropriate volume (from Table III above) to 37 C ± 1 C. 2. Medium Change 1. Remove the medium from the culture vessels with a pipette or vacuum pump. 2. Add warm Definitive Endoderm Differentiation Day 1 according to the volumes stated in Table III. 3. Return the cell culture vessels to the incubator (37 C ± 1 C, 5% CO 2, and 90% humidity). E. Day 2: Change Medium NOTE: Use nitrile gloves when preparing and changing medium, and discard any remaining medium in a closed container as hazardous waste. 1. Preparation Thaw Cellartis Definitive Endoderm Differentiation Day 2 and warm the appropriate volume (from Table III above) to 37 C ± 1 C. 2. Medium Change 1. Remove the medium from the culture vessels with a pipette or vacuum pump. 2. Add warm Definitive Endoderm Differentiation Day 2 according to the volumes stated in Table III. 3. Return the cell culture vessels to the incubator (37 C ± 1 C, 5% CO 2, and 90% humidity). F. Day 3: Change Medium NOTE: Use nitrile gloves when changing medium, and discard the old medium in a closed container as hazardous waste. 1. Preparation Thaw Cellartis Definitive Endoderm Differentiation Day 3 and warm the appropriate volume (from Table III above) to 37 C ± 1 C. 2. Medium Change 1. Remove the medium from the culture vessels with a pipette. Discard the old medium in a closed container as hazardous waste. 2. Add warm Definitive Endoderm Differentiation Day 3 according to the volumes stated in Table III. 3. Return the cell culture vessels to the incubator (37 C ± 1 C, 5% CO 2, and 90% humidity). Page 12 of 21

13 G. Day 4: Change Medium 1. Preparation Thaw Cellartis Definitive Endoderm Differentiation Day 4 and warm the appropriate volume (from Table III above) to 37 C ± 1 C. 2. Medium Change 1. Remove the medium from the culture vessels with a pipette or vacuum pump. 2. Add warm Definitive Endoderm Differentiation Day 4 according to the volumes stated in Table III. 3. Return the cell culture vessels to the incubator (37 C ± 1 C, 5% CO 2, and 90% humidity). H. Day 6: Change Medium 1. Preparation Thaw Cellartis Definitive Endoderm Differentiation Day 6 and warm the appropriate volume (from Table III above) to 37 C ± 1 C. 2. Medium Change 1. Remove the medium from the culture vessels with a pipette or vacuum pump. 2. Add warm Definitive Endoderm Differentiation Day 6 according to the volumes stated in Table III. 3. Return the cell culture vessels to the incubator (37 C ±1 C, 5% CO 2, and 90% humidity). I. Day 7: Differentiation to Downstream Lineages, or Assays for Definitive Endoderm Formation On day seven, the cells are ready to be differentiated to downstream linages, or to be assayed for the formation of definitive endoderm. If you wish to use the DE cells for differentiation into hepatocytes, we strongly recommend using the Cellartis Hepatocyte Differentiation Kit (Cat. No. Y30050) according to its user manual, which contains methods for dissociating the DE cells and for further differentiation. If you wish to differentiate the DE cells into other lineages or if the DE cells are to be assayed, suggestions for DE cell dissociation and freezing of DE cells are listed below. 1. Suggested Protocol for DE Cell Dissociation 1. Warm the appropriate volumes of D-PBS / and TrypLE Select Enzyme to 37 C ± 1 C. 2. Remove the media from the culture units with a pipette or vacuum pump. 3. Wash the cell culture vessels with warm D-PBS /. 4. Remove the D-PBS from the culture units with a pipette or vacuum pump. 5. Add the warm TrypLE Select Enzyme to the cell culture vessels (0.1 ml/cm 2 ). 6. Incubate the cell culture vessels in the incubator at 37 ± 1 C and 5% CO 2 for 3 5 minutes, until the cells have detached. 7. Transfer the cell suspension to the required number of 50-ml tube(s). 8. Rinse the cell culture vessels with appropriate medium (0.1 ml/cm 2 ) and transfer to the 50-ml tube(s) to achieve a 1:1 dilution of the cell suspension. 9. Centrifuge the cell suspension for 5 minutes at 300 x g at RT and resuspend the cell pellet in medium appropriate for further applications. Page 13 of 21

14 2. Freezing of DE Cells The DE cells can be frozen according to standard slow-freeze protocols, using STEM- CELLBANKER (Takara Clontech, Cat. No. CB041). VIII. Images of Cellartis Human ips Cell Lines Maintained in the Cellartis DEF- CS Culture System Cell line: ChiPSC4 Flattened homogenous layer Cell line: ChiPSC18 Flattened homogenous layer 4X magnification 10X magnification 20X magnification Figure 2. ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF-CS Culture System. Cell density 5 x 10 4 cells/cm 2. Page 14 of 21

15 Cell line: ChiPSC4 Flattened homogenous layer Cell line: ChiPSC18 Flattened homogenous layer 4X magnification 10X magnification 20X magnification Figure 3. ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF-CS Culture System. Cell density 1.5 x 10 5 cells/cm 2. Page 15 of 21

16 Cell line: ChiPSC4 Flattened homogenous layer Cell line: ChiPSC18 Flattened homogenous layer 4X magnification 10X magnification 20X magnification Figure 4. ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF-CS Culture System. Cell density >2 x 10 5 cells/cm 2. Page 16 of 21

17 IX. Images of hips Cells Differentiated Using the Cellartis Definitive Endoderm Differentiation Kit Figure 5 and Figure 6 show examples of morphology on days one to seven, for two human ips cell lines differentiated using the Cellartis Definitive Endoderm Differentiation Kit. Morphology may vary depending on the cell line used and differences in cell density. Day hipsc line 1 hipsc line Figure 5. Day 1 3 morphology of two human ips cell lines differentiated using the Cellartis Definitive Endoderm Differentiation Kit. For all images, the scale bar is 100 µm. Page 17 of 21

18 Day hipsc line 1 hipsc line Figure 6. Day 4 7 morphology of two human ips cell lines differentiated using the Cellartis Definitive Endoderm Differentiation Kit. For all images, the scale bar is 100 µm. Page 18 of 21

19 X. RNA Profile of hips Cells Differentiated Using the Cellartis Definitive Endoderm Differentiation Kit Figure 7 and Figure 8 show examples of RNA profiles from two different hips cell lines, both differentiated using the Cellartis Definitive Endoderm Differentiation Kit. The mrna levels were analyzed using RTqPCR. Similar patterns have been seen in other hips cell lines, but the actual levels may vary from cell line to cell line. Figure 7. RNA profile of hips cell line 1 differentiated using the Cellartis Definitive Endoderm Differentiation Kit. Page 19 of 21

20 Figure 8. RNA profile of hips cell line 2 differentiated using the Cellartis Definitive Endoderm Differentiation Kit. Page 20 of 21

21 Appendix A. Troubleshooting Guide Cellartis DEF-CS Culture System Table IV. Troubleshooting Guide for the Cellartis DEF-CS Culture System Problem Possible Explanation Solution Cells do not detach at passage TrypLE Select Enzyme is too cold. Make sure TrypLE Select Enzyme is at room temperature before use. Cell density too low at passage. Cells are normally easier to detach The cell density at passage varies considerably The cells seem to differentiate Transferred cells do not adapt to Cellartis DEF-CS Culture System Cells do not adhere at passage or thawing Cells are sparse even after seven days in culture Recommended seeding density and passage interval are not optimal for the cell line being used. Cells have been seeded too sparsely. Cell growth inhibition due to too-high density in the previous passage. Too-small media volumes used between passages. (Some cell lines have a higher metabolic activity, though they do not necessarily divide faster.) The cells are not used to the new environment. DEF-CS COAT-1 has been diluted in D-PBS /. Too short incubation with DEF-CS COAT-1. Too few cells are attached at passage. The cell line being used has a slow growth rate. at higher densities. Optimize the seeding density and passage interval. Make sure that the seeding density is at least 4.0 x 10 4 cells/cm 2 ; some cell lines may require higher seeding densities. Increase the seeding density slightly if the cells are extremely dense at passage. Increase the media volumes used, especially if the medium has turned yellow before the medium change. The cells could benefit from a higher seeding density for the first few passages, e.g. 8x10 4 cells/cm 2. The cells may benefit from a higher concentration of Cellartis DEF-CS COAT-1. Use a dilution of 1:10 or 1:5 during the first few passages to provide extra support during the adaptation process. Newly transferred cells might initially grow at a slightly slower rate. Extend the passage interval up to 7 days for the first passages. Make sure to use D-PBS with Mg 2+ and Ca 2+. Prolong the incubation time with DEF-CS COAT-1. Increase the seeding density. Increase the volume of GF-1; use maximum a 1:111 dilution. Appendix B. Troubleshooting Guide for the Cellartis Definitive Endoderm Differentiation Kit Table V. Troubleshooting Guide Cellartis Definitive Endoderm Differentiation Kit Problem Possible Explanation Solution Cells die during DE Cells are seeded too sparsely. Increase seeding density at start of differentiation. DE differentiation (referred to as day Differentiation to DE cells is not efficient; low yield of DE cells. Cells are seeded too densely. 0). Decrease seeding density at start of DE differentiation (day 0). Page 21 of 21