Cellartis Hepatocyte Diff Kit User Manual

Takara Bio Europe AB Cellartis Hepatocyte Diff Kit User Manual Cat. No. Y30050 (061715) Takara Bio Europe AB A Takara Bio Company Arvid Wallgrens backe 20, SE-413 46 Göteborg, Sweden Europe Technical Support: tech-cellartis@takara-clontech.eu United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +46.(0)31.758.0900 Japan +81.(0)77.543.6116 Page 1 of 12

Table of Contents I. Introduction... 4 II. List of Components... 4 III. Additional Materials Required... 4 IV. General Considerations... 4 A. Storage and Handling... 4 V. Differentiation of Definitive Endoderm Cells to Hepatocytes... 5 A. Starting Material... 5 B. Media Volumes... 5 C. Medium Changes... 5 VI. Coating of Cell Culture Vessels... 6 VII. Day 0: Thawing and Seeding of Cellartis Definitive Endoderm Cells... 7 A. Preparation... 7 B. Thawing Cells... 7 VIII. Day 0: Passage and Seeding of Definitive Endoderm Cells... 8 A. Preparation... 8 B. Dissociation and Seeding Cells... 8 IX. Day 2 and 4: Medium Changes... 8 A. Preparations... 8 B. Medium Change Day 2 and 4... 8 X. Day 7 and 9: Medium Changes... 9 A. Preparations... 9 B. Medium Change Day 7... 9 C. Medium Change Day 9... 9 XI. Day 11 and Onwards: Medium Changes... 9 A. Preparations... 9 B. Medium Change... 9 XII. Images of Cells Differentiated using Cellartis Hepatocyte Diff Kit... 10 Appendix A. CYP Activity Assay... 11 A. Additional Material Required... 11 B. Preparation... 11 C. Activity Assay... 11 Table of Figures Figure 1 Day 2 after start of hepatocyte differentiation... 10 Figure 2 Day 7 after start of hepatocyte differentiation... 10 Figure 3 Day 21 after start of hepatocyte differentiation... 10 Page 2 of 12

Table of Tables Table I. Suggested schedule for Cellartis Hepatocyte Diff Kit... 6 Table II. CYP Substrate Cocktail... 11 Page 3 of 12

I. Introduction Cellartis Hepatocyte Diff Kit contains complete media and ready-to-use coating for the differentiation of definitive endoderm (DE) cells to hepatocytes in 2D culture. We recommend that this product is handled only by persons who have been trained in laboratory techniques and that it is 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 Hepatocyte Diff Kit (Cat. No. Y30050) o 1 bottle Hepatocyte Thawing and Seeding Medium (1) (25 ml) o 1 bottle Hepatocyte Progenitor Medium (2) (50 ml) o 2 bottles Hepatocyte Maturation Medium Base (3A) (24 ml) o 1 tube Hepatocyte Maturation Medium Supplement (3B) (2.6 ml) o 3 bottles Hepatocyte Maintenance Medium (4) (50 ml) o 1 tube Hepatocyte Coating (7.5 ml) Additional Materials Required The following materials are required but not supplied: Human definitive endoderm cells. Takara Clontech offers DE cells and kit for DE differentiation: o Cellartis Definitive Endoderm ChiPSC18 (Cat. No. Y10040) o Cellartis Definitive Endoderm Diff Kit (Cat. No. Y30030) Fetal bovine serum (FBS) or KnockOut Serum Replacement (KOSR)* PBS Dulbecco s w/o Ca 2+ & Mg 2+ (D-PBS / )* TrypLE Select Enzyme* Cell culture vessels, tissue culture treated polystyrene surface General cell culture equipment used in cell culture laboratory * Needed if Cellartis Definitive Endoderm Diff Kit is used for DE differentiation IV. General Considerations A. Storage and Handling All components of Cellartis Hepatocyte Diff kit should be stored at 20 C and expires as indicated on the label. Thaw Hepatocyte Thawing and Seeding Medium (1) at 37 C ± 1 C and use the same day as thawing. Thaw Hepatocyte Progenitor Medium (2) at room temperature (RT, 15 25 C), or overnight at 4 C, and use within 2 weeks. Thaw Hepatocyte Maturation Medium Base (3A) at RT and Hepatocyte Maturation Medium Supplement (3B) at 4 C. Use Hepatocyte Maturation Medium Base (3A) to prepare complete medium directly when thawed. One vial of Hepatocyte Maturation Medium Supplement (3B) is enough for two medium changes and should be kept cold and used within 2 days of thawing. Complete Hepatocyte Maturation Medium should be used fresh. Thaw Hepatocyte Maintenance Medium (4) at RT. Thawed Hepatocyte Maintenance Medium (4) should be used within 3 days or aliquoted and frozen the same day as thawing. Page 4 of 12

Both Hepatocyte Maturation Medium Base (3A) and Hepatocyte Maintenance Medium (4) contain DMSO. Therefore, use nitrile gloves when preparing and changing medium and discard old medium in a closed container as hazardous waste. Both Hepatocyte Maturation Medium Base (3A) and Hepatocyte Maintenance Medium (4) are light sensitive; therefore avoid unnecessary exposure to light. Thaw Hepatocyte Coating at RT and use directly when thawed. V. Differentiation of Definitive Endoderm Cells to Hepatocytes Cellartis Hepatocyte Diff Kit consists of four different media and one coating compound. One kit is enough to differentiate DE cells to hepatocytes in 50 cm 2. The kit is optimized for differentiating cells in 24- or 96-well plates. Other formats can be used, but optimization may be required. All methods described in this user manual are for differentiation of DE cells to hepatocytes in 2D monolayer cultures on a total area of 50 cm 2. Table I shows a suggested workflow for hepatocyte differentiation. Coating can be performed on any of the days suggested, using a minimum coating time of 30 minutes in 37 C ± 1 C or overnight, prior seeding the cells. For examples of cell morphology during differentiation, please refer to Figure 1, Figure 2, and Figure 3. The images are just examples, and the morphology is not necessarily identical between experiments at the exact same time. The differentiated hepatocyte cells are ready to be used from day 14 after start of differentiation and can be maintained at least until day 25 if handled according to instructions,. The usage window for applications that requires high CYP activity is between day 16 and day 25. For optimal results, change the medium the day before starting an assay. NOTE: Always work under aseptic conditions. A. Starting Material Human Cellartis Definitive Endoderm cells or DE cells derived using Cellartis Definitive Endoderm Diff Kit can be used as starting material. If using Cellartis Definitive Endoderm cells, please see section 0 for thawing and seeding instructions. If Cellartis Definitive Endoderm Diff Kit is used to derive the staring material, please see section 0 for dissociation and seeding of DE cells. B. Media Volumes 0.5 ml medium/cm 2 is used throughout the differentiation procedure. C. Medium Changes The following are general considerations for medium changes: The medium changes should be performed with care. It is recommended to use a pipette and aspirate roughly 75% of the medium in each well. Do not use a vacuum pump but pipette manually (use a multichannel pipette for 96-well plates). Aspirate the remaining medium in 4 12 wells at a time and add 0.5 ml/cm 2 (i.e. 1 ml/well for 24-well plates) to the empty wells. Repeat until the media is changed in each well. It is possible to temporarily aspirate the medium prior photo imaging. Try to take the images within 1 2 minutes after medium is aspirated. Then immediately add medium back to the well. Always discard any leftover warmed medium. Page 5 of 12

Table I. Suggested schedule for Cellartis Hepatocyte Diff Kit. Corresponding sections of this user manual are referenced in parentheses. Day Suggested Weekday Coating (VI) Thawing or Dissociation and Seeding (0 or 0) Progenitor Medium (IX) Maturation Medium (0) Maintenance Medium (XI) Friday X Saturday X Sunday X 0 Monday X 1.25 1.50 x 10 5 cells/cm 2 0.5 ml/cm 2 1 Tuesday 2 Wednesday 0.5 ml/cm 2 3 Thursday 4 Friday 0.5 ml/cm 2 5 Saturday 6 Sunday 7 Monday 0.5 ml/cm 2 8 Tuesday 9 Wednesday 0.5 ml/cm 2 10 Thursday 11 Friday 0.5 ml/cm 2 12 Saturday 13 Sunday 14 Monday 0.5 ml/cm 2 15 Tuesday 16 Wednesday 0.5 ml/cm 2 17 Thursday 18 Friday 0.5 ml/cm 2 19 Saturday 20 Sunday 21 Monday 0.5 ml/cm 2 22 Tuesday 23 Wednesday 0.5 ml/cm 2 24 Thursday 25 Friday VI. Coating of Cell Culture Vessels 1. Thaw the frozen Hepatocyte Coating at RT. 2. Add the Hepatocyte Coating to the cell culture vessels (0.15 ml/cm 2 ). Make sure the entire surface is covered. 3. Incubate at 37 C ± 1 C for at least 30 min. 4. Remove excess Hepatocyte Coating from the cell culture vessels just before seeding. Do not let the surface dry out. Page 6 of 12

VII. Cellartis Hepatocyte Diff Kit User Manual Day 0: Thawing and Seeding of Cellartis Definitive Endoderm Cells Hepatocyte Thawing and Seeding Medium (1) and Hepatocyte Coating are used when thawing Cellartis Definitive Endoderm cells. (If starting with DE cells derived using Cellartis Definitive Endoderm Diff Kit, please see section 0.) NOTE: Thawed Cellartis Definitive Endoderm cells are fragile. Avoid using a pipette for mixing the cell suspension if possible. Only use the pipette to seed the cells. Avoid centrifugation of the thawed cells if possible. One vial of Cellartis Definitive Endoderm containing 6 x 10 6 viable cells is enough for 50 cm 2 of culture area (i.e., one 24-well plate, one 12-well plate, 5 wells of a 6-well plate, or 1.5 96-well plates). 0.5 ml cell suspension should be added per cm 2, giving a cell density of 1.25 x 10 5 cells/cm 2. If other formats than the ones listed above are to be used, please consider the following: o Frozen DE cells should be seeded at 1.25 1.50 x 10 5 cells/cm 2. It is not necessary to count the cells upon thawing, but might be useful if other concentrations are desired. o Try to keep the medium volume to 0.5 ml medium/cm 2 when seeding the cells. It is important to thaw the cells rapidly. Ideally, the vial of cells should be kept in liquid nitrogen or on dry ice prior thawing. A. Preparation Thaw and warm the appropriate volume of Hepatocyte Thawing and Seeding Medium (1) to 37 C ± 1 C. Coat the appropriate number of cell culture vessels with Hepatocyte Coating according to above. B. 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, as quickly as possible, the frozen vials from liquid nitrogen to a 37 C ± 1 C water bath using forceps. 2. Thaw the cells by gently pushing the vial under the surface of the water, without swirling the vial. Do not submerge the cap of the vial in the water bath as this could contaminate the cells. 3. After approximately 1 min, check if the cell suspension is thawed by carefully turning the tube upside down. 4. Decontaminate the external surface with an appropriate disinfectant and place into the biological safety cabinet. 5. Once thawed, pour the cell suspension into 24 ml of 37 C ± 1 C Hepatocyte Thawing and Seeding Medium (1). Wash the vial with 1 ml of 37 C ± 1 C Hepatocyte Thawing and Seeding Medium (1) and add to the cells. 6. Mix by turning the tube with cell suspension carefully approximately 3 times. Do not mix by pipetting. 7. Optional: Count the viable cells using a haemocytometer. Take 50 µl of the cell suspension, add 5 µl trypan blue, count cells, and multiply the counted viable cells with 1.1 to get the concentration of viable cells. 8. Remove the Hepatocyte Coating from the cell culture vessels just prior to seeding the cells. 9. Using a 1-ml pipette, seed 0.5 ml cell suspension per cm 2. Mix the cell suspension by swirling or turning the tube, or use a pipette gently. 10. Place the cell culture vessels in an incubator at 37 C ± 1 C, 5% CO 2, 90% humidity and leave untouched overnight. The first medium changed is performed on day two after thawing (see section IX). Page 7 of 12

VIII. Day 0: Passage and Seeding of Definitive Endoderm Cells DE cells derived using Cellartis Definitive Endoderm Diff Kit can be dissociated at day seven after start of differentiation and used as starting material for hepatic differentiation. Hepatocyte Thawing and Seeding Medium (1) and Hepatocyte Coating are used when passaging and seeding DE cells. (If starting with Cellartis Definitive Endoderm cells, please see section 0.) A. Preparation Thaw and warm the appropriate volume of Hepatocyte Thawing and Seeding Medium (1) to 37 C ± 1 C. Warm TrypLE Select Enzyme and D-PBS / to 37 C ± 1 C. Prepare appropriate volume of 10% FBS or KOSR in D-PBS / and warm to RT. Coat the appropriate number of cell culture vessels with Hepatocyte Coating according to above. IX. B. Dissociation and Seeding Cells 1. Aspirate the medium in the culture vessels by using pipette or vacuum pump. 2. Wash the cell culture vessels with warm D-PBS / (0.1 ml/cm 2 ). 3. Remove the D-PBS in the culture units by using pipette or vacuum pump. 4. Add warm TrypLE Select Enzyme to the cell culture vessels (0.1 ml/cm 2 ). 5. Incubate the cell culture vessels in the incubator at 37 C ± 1 C and 5% CO 2 for 3-5 min until the cells have detached. 6. Transfer the cell suspension to a 50-ml tube. 7. Rinse the cell culture vessels with 10 % FBS or KOSR in D-PBS / (0.1 ml/cm 2 ) and transfer to the 50-ml tube to achieve a 1:1 dilution of the cell suspension. 8. Count the cells. 9. Transfer a volume of the cell suspension corresponding to 6.5 x 10 6 cells to a centrifugation tube. 10. Centrifuge the cell suspension at 300 x g for 5 min at RT and resuspend the cell pellet in 25 ml Hepatocyte Thawing and Seeding Medium (1). 11. Using a 1-ml pipette, seed 0.5 ml cell suspension per cm 2, corresponding to 1.2 1.3 x 10 5 cells/cm 2. It is crucial to seed more than 1.0 x 10 5 cells/cm 2 in order to get a successful hepatic differentiation. 12. Place the cell culture vessels in an incubator at 37 C ± 1 C, 5% CO 2, 90% humidity and leave untouched overnight. First medium change is performed day two after thawing, see section IX. Day 2 and 4: Medium Changes On day two and four after thawing, Hepatocyte Progenitor Medium (2) is used for medium changes. A. Preparations Thaw the bottle with Hepatocyte Progenitor Medium (2) at RT, or overnight in 4 C. Transfer 25 ml to a flask or tube for use on day 2 and warm to 37 C ± 1 C. Keep the remaining 25 ml of Hepatocyte Progenitor Medium (2) at 4 C, for medium change on day 4. B. Medium Change Day 2 and 4 1. Warm appropriate volume of Hepatocyte Progenitor Medium (2) to 37 C ± 1 C. 2. Aspirate the medium from the cell culture vessels according to section V.C and discard. 3. Carefully add warm Hepatocyte Progenitor Medium (2) to the cell culture plate, 0.5 ml/cm 2. 4. Place the cells in an incubator at 37 C ± 1 C, 5% CO 2, 90 % humidity. Page 8 of 12

X. Day 7 and 9: Medium Changes On days seven and nine after thawing, Hepatocyte Maturation Medium Base (3A) and Hepatocyte Maturation Medium Supplement (3B) are used for medium changes. Hepatocyte Maturation Medium Base (3A) is light sensitive, therefore avoid unnecessary exposure to light. NOTE: Hepatocyte Maturation Medium Base (3A) contains DMSO. Therefore, use nitrile gloves when preparing and changing medium and discard old medium in a closed container as hazardous waste. Keep the Hepatocyte Maturation Medium Supplement (3B) cold until just prior to use. Only half the volume of the supplement should be used on day 7. Therefore, keep the remaining Hepatocyte Maturation Medium Supplement (3B) at 4 C for final use on day 9. A. Preparations Thaw Hepatocyte Maturation Medium Base (3A) at RT or overnight at 4 C. Thaw Hepatocyte Maturation Medium Supplement (3B) at 4 C. One vial contains supplement for both days 7 and 9, always keep cold and store the volume for day 9 at 4 C. Warm 24 ml of Hepatocyte Maturation Medium Base (3A) no warmer than RT. Add 1.3 ml Maturation Medium Supplement per 24 ml Hepatocyte Maturation Medium Base (3A) to get complete Hepatocyte Maturation Medium. Mix gently and use directly. B. Medium Change Day 7 1. Remove the medium from the cell culture vessels according to section V.C and discard. 2. Carefully add RT Hepatocyte Maturation Medium to the cell culture plate, 0.5 ml/cm 2. 3. Place the cells in an incubator at 37 C ±1 C, 5% CO 2, 90% humidity. XI. C. Medium Change Day 9 1. On day 9, some components of the Hepatocyte Maturation Medium from day 7 has gelatinised on top of the cell layer. Be careful when medium is changed; aspirate carefully as described in section V.C. Also, it is recommended that a small volume is left in the well when aspirating the medium prior to adding fresh medium. 2. Carefully add RT Hepatocyte Maturation Medium to the cell culture plate, 0.5 ml/cm 2. 3. Place the cells in an incubator at 37 C ± 1 C, 5% CO 2, 90% humidity. Day 11 and Onwards: Medium Changes From day 11 and onwards Hepatocyte Maintenance Medium (4) is used for medium changes. The recommended days for medium changes are Mondays, Wednesdays, and Fridays. No medium change is necessary during the weekend. Hepatocyte Maintenance Medium (4) is light sensitive, therefore avoid unnecessary exposure to light. NOTE: Hepatocyte Maintenance Medium (4) contains DMSO. Therefore, use nitrile gloves when preparing and changing medium and discard old medium in a closed container as hazardous waste. A. Preparations Thaw Hepatocyte Maintenance Medium (4) in RT. Hepatocyte Maintenance Medium (4) can be in kept 4 C for up to three days. One bottle of Hepatocyte Maintenance Medium (4) is enough for two medium changes. B. Medium Change 1. Remove the medium from the cell culture vessels according to section V.C and discard. 2. Carefully add warm Hepatocyte Maturation Medium to the cell culture plate, 0.5 ml/cm 2. 3. Place the cells in an incubator at 37 C ± 1 C, 5% CO2, 90% humidity. Page 9 of 12

XII. Cellartis Hepatocyte Diff Kit User Manual Images of Cells Differentiated using Cellartis Hepatocyte Diff Kit Figure 1 3 are examples of morphology of definitive endoderm cells differentiated using Cellartis Hepatocyte Diff Kit. The morphology may vary depending on the cell line used and cell density. 4X (Scale bar corresponds to 250 µm.) Figure 1. Day 2 after start of hepatocyte differentiation 10X (Scale bar corresponds to 100 µm.) 4X (Scale bar corresponds to 250 µm.) Figure 2. Day 7 after start of hepatocyte differentiation 10X (Scale bar corresponds to 100 µm.) 10X (Scale bar corresponds to 100 µm.) Figure 3. Day 21 after start of hepatocyte differentiation 20X (Scale bar corresponds to 50 µm.) Page 10 of 12

Appendix A. CYP Activity Assay After 16 days in culture, the differentiated hepatocytes can be used in Cytochrome P450 (CYP) activity assays. Samples can be analysed using LC/MS to measure the formation of the following specific metabolites: acetaminophen (CYP1A), 4-OH-Diclofenac (CYP2C9), 4-OH-Mephenytoin (CYP2C19), OH-Bufuralol (CYP2D6) and 1-OH-Midazolam (CYP3A). A. Additional Material Required HEPES Solution, 1 M L-Glutamine Solution, 200 mm Penicillin/Streptomycin (PEST) (10,000 units/ml of penicillin and 10,000 µg/ml of streptomycin) Pierce BCA Protein Assay Kit (Life Technologies Cat. No. 23225) Probe Substrate Cocktail: o Phenacetin (Sigma-Aldrich, Cat. No. 77440) o Bupropion (Sigma-Aldrich, Cat. No B102) o Mephenyotin (Santa Cruz, Cat. No. sc-200975a) o Diclofenac (Sigma-Aldrich, Cat. No. D6899) o Bufuralol (Becton Dickinson, Cat. No. 451034) o Midazolam (Loradan, Cat. No. MID-111-HC) Williams Medium E (WME) (Life Technologies, Cat. No. 32551) B. Preparation Prepare a stock solution for the CYP substrate cocktail. In the assay, use the final concentrations listed in Table II. Warm the appropriate volume of WME with 0.1% PEST to 37 C ± 1 C. Prepare and warm CYP activity assay medium: WME with 0.1% PEST, 25 mm HEPES, 2 mm L-Glutamine. Add the CYP substrate cocktail just prior to use. Table II. CYP Substrate Cocktail CYP Substrate Final Assay Concentration 1A Phenacetin 10 µm 2B6 Bupropion 10 µm 2C19 Mephenytoin 50 µm 2C9 Diclofenac 10 µm 2D6 Bufuralol 10 µm 3A Midazolam 5 µm C. Activity Assay 1. Wash the differentiated hepatocytes twice very gently with 0.5 ml/cm 2 warm WME (0.1% PEST). 2. Add 110 µl/cm 2 warm CYP activity assay medium to the cells. 3. Incubate for 2 hours at 37 C ± 1 C, 5% CO 2, 90% humidity. 4. Collect 100 µl supernatant and store at 80 C until LC/MS analysis. 5. Determine the amount of protein per well using the Pierce BCA Protein Assay Kit. 6. Normalize the metabolite concentrations measured by LC/MS to the amount of protein per well and the assay duration (120 min). Page 11 of 12

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