Takara Bio Europe AB
Cellartis® Definitive
Endoderm
Differentiation Kit with
DEF-CS Culture
System User Manual
Cat. No. Y30035
(121515)
Takara Bio Europe AB
A Takara Bio Company
Arvid Wallgrens backe 20, SE-413 46 Göteborg, Sweden
Europe Technical Support: [email protected]
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 21
Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
Table of Contents
I.
Introduction ............................................................................................................................................................... 4
II.
List of Components ................................................................................................................................................... 4
III.
Additional Materials Required .............................................................................................................................. 4
IV.
General Considerations ......................................................................................................................................... 5
A.
V.
Storage and Handling ............................................................................................................................................ 5
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 .................................................................................................... 10
G.
Start of Definitive Endoderm Differentiation ..................................................................................................... 10
VII.
Cellartis Definitive Endoderm Differentiation Kit ............................................................................................. 11
A.
Starting Material ................................................................................................................................................. 11
B.
Coating and Media Volumes............................................................................................................................... 11
C.
Day 0: Start Definitive Endoderm Differentiation .............................................................................................. 11
D.
Day 1: Change Medium ...................................................................................................................................... 12
E.
Day 2: Change Medium ...................................................................................................................................... 12
F.
Day 3: Change Medium ...................................................................................................................................... 12
G.
Day 4: Change Medium ...................................................................................................................................... 13
H.
Day 6: Change Medium ...................................................................................................................................... 13
I.
Day 7: Differentiation to Downstream Lineages, or Assays for Definitive Endoderm Formation ..................... 13
VIII.
Images of Cellartis Human iPS Cell Lines Maintained in the Cellartis DEF-CS Culture System ..................... 14
IX.
Images of hiPS Cells Differentiated Using the Cellartis Definitive Endoderm Differentiation Kit ................... 17
X.
RNA Profile of hiPS Cells Differentiated Using the Cellartis Definitive Endoderm Differentiation Kit .............. 19
Appendix A. Troubleshooting Guide Cellartis DEF-CS Culture System ....................................................................... 21
Appendix B. Troubleshooting Guide for the Cellartis Definitive Endoderm Differentiation Kit .................................. 21
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
Table of Figures
Figure 1. Schematic presentation of the Cellartis human iPS cell line work flow. ........................................................... 7
Figure 2. ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF-CS Culture System. ......................................... 14
Figure 3. ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF-CS Culture System. ......................................... 15
Figure 4. ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF-CS Culture System. ......................................... 16
Figure 5. Day 1–3 morphology of two human iPS cell lines differentiated using the Cellartis Definitive Endoderm
Differentiation Kit. .......................................................................................................................................................... 17
Figure 6. Day 4–7 morphology of two human iPS cell lines differentiated using the Cellartis Definitive Endoderm
Differentiation Kit. .......................................................................................................................................................... 18
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. ...................................................................................... 7
Table III. Media Volumes for Cellartis Definitive Endoderm Differentiation Kit ......................................................... 11
Table IV. Troubleshooting Guide for the Cellartis DEF-CS Culture System ................................................................. 21
Table V. Troubleshooting Guide Cellartis Definitive Endoderm Differentiation Kit .................................................... 21
Contact Us
Customer Service/Ordering
Technical Support
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tel: +46.(0)31.758.0900
fax: +33.(0)1.3904.6870
fax: +46.(0)31.758.0910
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e-mail: [email protected]
e-mail: [email protected]
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 +46 31 758 0900.
Your use of this product is also subject to compliance with any applicable licensing requirements as detailed in our catalogues, on our website at
http://www.clontech.com, 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. ©2015 Takara Bio Europe AB
This document has been reviewed and approved by the Takara Bio Europe AB Quality Assurance Department.
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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.
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)
III.
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 Ca2+ & Mg2+ (D-PBS +/+)
 PBS Dulbecco’s w/o Ca2+ & Mg2+ (D-PBS –/–)
 TrypLE Select Enzyme (1X), without phenol red
* Needed for DE cell dissociation
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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.
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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 106 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 cm2. 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
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Coating: Definitive Endoderm Differentiation Coating (0.1 ml/cm2)
Cell Seeding: Definitive Endoderm Differentiation Day 0 (0.2 ml/cm2)
Medium change: Definitive Endoderm Differentiation Day 1 (0.2 ml/cm2)
Medium change: Definitive Endoderm Differentiation Day 2 (0.2 ml/cm2)
Medium change: Definitive Endoderm Differentiation Day 3 (~0.27 ml/cm2)
Medium change: Definitive Endoderm Differentiation Day 4 (~0.52 ml/cm2)
Medium change: Definitive Endoderm Differentiation Day 6 (~0.52 ml/cm2)
DE cells ready to use
NOTE: Always work under aseptic conditions.
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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.
Medium Changes
(VI.F)
Thawing (VI.D) or
Transfer (VI.E)
•
•
• Medium Preparation
(VI.C.2)
Coating (VI.B)
Medium Preparation
(VI.C.1)
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 1.5–3.0 x 105
cells/cm2. A suggested weekly schedule is depicted in Table II.
Table II. Weekly schedule for medium changes and passaging.
Monday
Passage
Tuesday
Change
medium
Wednesday
Change
medium
Thursday
Passage
Friday
Change
medium
Saturday
-
Sunday
Change
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.
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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/cm2), 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.
D.
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.
Thawing Human iPS Cell Lines
When thawing human iPS cells in Cellartis DEF-CS Culture System, seed approximately 1.5–2.5 x 105
viable cells/cm2 in 0.3–0.4 ml medium/cm2.
1.
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.)
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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 106 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 1.5–2.5 x 105 cells/cm2 in 0.3–0.4
ml medium/cm2.
11. 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% CO2, and
≥90% humidity.
3.
E.
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.
Passaging Human iPS Cell Lines
As a general rule, cells should be seeded at a density of 4.0–5.0 x 104 cells/cm2 (use 4.0 x 104
cells/cm2 if leaving the cells four days between passages and 5.0 x 104 cells/cm2 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 1.5–3.0 x 105
cells/cm2 (see Figure 2–Figure 4 for images of a variety of Cellartis human iPS cell lines in culture).
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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/cm2 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.
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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 1:10.
6. 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 0.15–0.25 ml/cm2.
8. 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% CO2, and
≥90% humidity.
3.
F.
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.
Changing Medium for Human iPS Cell Lines
Medium change is recommended daily (except the day of passage). Use 0.25–0.4 ml/cm2 of medium.
If the medium turns yellow due to high metabolic activity, increase the medium volume.
G.
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 DEFCS 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% CO2, and ≥90% humidity.
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 1.5–3.0 x 105 cells/cm2 should be obtained at passage using the recommended passage
intervals.
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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 cm2. 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
6-well plate
T25 flask
T75 flask
C.
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Coating
1 ml
2.5 ml
7.5 ml
Days 0, 1 and 2
2 ml
5 ml
15 ml
Day 3
2.7 ml
7 ml
20 ml
Day 4 and 6
5 ml
13 ml
40 ml
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/cm2).
Make sure the entire surface of each vessel is covered.
2. Incubate at RT for 30–120 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 3.0–4.0 x 104 cells/cm2 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 (1.5–2.0 x 105 cells/ml) and seed in the coated cell culture vessels at a
density of 3.0–4.0 x 104 cells/cm2 in 0.2 ml medium/cm2.
6. Place the cell culture vessels in the incubator at 37°C ± 1°C, 5% CO2, and ≥90%
humidity.
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
D.
E.
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% CO2, and ≥90%
humidity).
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.
F.
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% CO2, and ≥90%
humidity).
Day 3: Change Medium
NOTE: Use nitrile gloves when changing medium, and discard the old medium in a closed container
as hazardous waste.
(121515)
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% CO2, and ≥90%
humidity).
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Page 12 of 21
Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
G.
H.
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% CO2, and ≥90%
humidity).
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% CO2, 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.


1.
(121515)
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.
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/cm2).
6. Incubate the cell culture vessels in the incubator at 37 ± 1°C and 5% CO2 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/cm2) 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.
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Page 13 of 21
Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
2.
Freezing of DE Cells
The DE cells can be frozen according to standard slow-freeze protocols, using STEMCELLBANKER® (Takara Clontech, Cat. No. CB041).
VIII. Images of Cellartis Human iPS Cell Lines Maintained in the Cellartis DEFCS 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 104 cells/cm2.
(121515)
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Page 14 of 21
Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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 105 cells/cm2.
(121515)
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Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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 105 cells/cm2.
(121515)
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Page 16 of 21
Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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
1
hiPSC line 1
hiPSC line 2
2
3
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.
(121515)
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Page 17 of 21
Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
Day
4
hiPSC line 1
hiPSC line 2
5
6
7
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.
(121515)
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Page 18 of 21
Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
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.
(121515)
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Page 19 of 21
Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
Figure 8. RNA profile of hiPS cell line 2 differentiated using the Cellartis Definitive Endoderm Differentiation Kit.
(121515)
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Page 20 of 21
Cellartis® Definitive Endoderm Differentiation Kit with DEF-CS Culture System User
Manual
Appendix A. Troubleshooting Guide Cellartis DEF-CS Culture System
Table IV. Troubleshooting Guide for the Cellartis DEF-CS Culture System
Problem
Cells do not detach at passage
Possible Explanation
TrypLE Select Enzyme is too cold.
Cell density too low at passage.
The cell density at passage varies
considerably
The cells seem to differentiate
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.
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
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.
Solution
Make sure TrypLE Select Enzyme is
at room temperature before use.
Cells are normally easier to detach
at higher densities.
Optimize the seeding density and
passage interval.
Make sure that the seeding density
is at least 4.0 x 104 cells/cm2; 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. 8x104 cells/cm2.
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 Mg2+
and Ca2+.
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
Cells die during DE
differentiation.
Possible Explanation
Cells are seeded too sparsely.
Differentiation to DE cells is not
efficient; low yield of DE cells.
Cells are seeded too densely.
(121515)
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Solution
Increase seeding density at start of
DE differentiation (referred to as day
0).
Decrease seeding density at start of
DE differentiation (day 0).
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