The Cellartis DEF-CS 500 Culture System is a complete system suitable for efficient generation and expansion of clonal populations from human-induced pluripotent stem (hiPS) cells in a feeder-free and defined environment. For many laboratories, the establishment of a clonal population is inefficient, challenging, and time consuming; it frequently results in cell death or premature differentiation. The solution to this problem may be the use of the DEF-CS culture system, a monolayer-based culture system which bypasses the challenges of colony-based culture by allowing single-cell passaging that promotes the survival and further expansion of plated single cells (Figure 1).

All procedures described in this protocol are optimized for Cellartis hiPS cell lines. If you want to use the Cellartis DEF-CS 500 Culture System for other hiPS cell lines or for Cellartis iPS cells grown in another system, please be aware that these cell lines will need to be adapted to the DEF-CS culture system before proceeding to single-cell culture (see General Considerations).

• 500 ml Cellartis DEF-CS 500 Basal Medium (Takara Bio, Cat. # Y30011; included)
• 4 ml Cellartis DEF-CS 500 COAT-1 (Takara Bio, Cat. # Y30012)
• Cellartis DEF-CS 500 Additives (Takara Bio, Cat. # Y30016; included)
  • 2 x 750 µl DEF-CS GF-1
  • 500 µl DEF-CS GF-2
  • 200 µl DEF-CS GF-3

The following tissue culture materials are required but not supplied:

• 96-well plates, flat bottom, cell-culture treated (Corning, Cat. # 3595 or equivalent)
• 48-well plates, flat bottom, cell-culture treated (Corning, Cat. # 3548 or equivalent)
• TrypLE Select Enzyme (1X), no phenol red (Thermo Fisher Scientific, Cat. # 12563011)
• PBS Dulbecco's with Ca²⁺ & Mg²⁺ (D-PBS +/+) (Sigma, Cat. # D8662 or equivalent)
• PBS Dulbecco's w/o Ca²⁺ & Mg²⁺ (D-PBS –/–) (Sigma, Cat. # D8537 or equivalent)

Storage and handling

Cellartis DEF-CS 500 Basal Medium and DEF-CS COAT-1 should be stored at 2–8°C; shelf life is specified on the product label. The Cellartis DEF-CS 500 Basal Medium formulation contains penicillin and streptomycin. Cellartis DEF-CS 500 Additives (DEF-CS GF-1, DEF-CS GF-2, and DEF-CS GF-3) should be stored at –20°C; shelf life is specified on the product label. At first use, thaw provided vials, mix each vial gently, and aliquot each component separately into appropriate volumes. Store at –20°C until the expiration date on the original vial. Thawed vials may be stored at 2–8°C for up to one week. Do not re-freeze aliquots after thawing. 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 recommends the use of media and reagents according to this manual. Takara Bio Europe cannot guarantee correct technical feedback on customer cultures unless the culture instructions below have been followed.

Transferring hiPS cells to the DEF-CS culture system

It is strongly recommended to transfer cells from other systems to the Cellartis DEF-CS 500 Culture System (Takara Bio, Cat. # Y30010) before single-cell cloning. hiPS cells maintained in other culture systems can be readily transferred: fresh cultures can be transferred at passage, and cryopreserved cultures can be thawed directly into the Cellartis DEF-CS 500 Culture System. Cells should be passaged at least three times in this system prior to performing single-cell cloning experiments using the Cellartis DEF-CS culture system.

Expected morphology of hiPS cells in the DEF-CS culture system

DEF-CS technology uses enzyme-based passaging in conjunction with a specific coating to promote single-cell survival, rapid expansion, and easier passaging. When transferring hiPS cells to this system, you may notice that some cell characteristics differ from those of hiPS cells cultured in your previous system. In contrast to commonly used colony-based culture systems, the DEF-CS culture system yields a monolayer of evenly spaced cells (see Figure 2). Newly passaged cells grown in the DEF-CS culture system tend to spread out (see Figure 3); however, as cells proliferate, the culture gets denser and cells display the typical undifferentiated stem cell morphology (i.e., high nucleus to cytoplasm ratio, defined borders, and prominent nucleoli).

Single-cell isolation to form a clonal cell line requires specific culture conditions enabled by the Cellartis DEF-CS 500 Culture System (Takara Bio, Cat. # Y30010). Table 1 describes a schedule of all media changes (volume and composition) necessary to create clonal lines in 96-well plates that are ready for culture with the Cellartis DEF-CS 500 Culture System.   

Preparation of 96-well plate and medium for single-cell seeding

Coating of a 96-well plate

1. Dilute the required volume of Cellartis DEF-CS 500 COAT-1 (Takara Bio, Cat. # Y30012) in D-PBS +/+ prior to use. Make a 1:5 dilution. Calculate the amount of diluted coating solution required depending on the number of wells to be used where 50 µl of diluted coating solution is used per well (see Table 2).
   
   Table 2. Preparation of coating solution for a 96-well plate.
   
   
   

   Number of wells (96-well plate)	Volume of diluted coating solution (µl)	Volume of COAT-1 (µl)	Volume of D-PBS +/+ (µl)
   1	50	10	40
   2	100	20	80
   96	960	192	768
   n	50 x n	(50 x n)/5	(50 x n) – (volume of COAT-1)

   
   
2. Mix the diluted COAT-1 solution gently and thoroughly by moving the pipette tip up and down while pipetting.
3. Add the diluted COAT-1 solution to a 96-well plate for single-cell seeding, making sure the entire surface of each well is covered.
4. Place the cell culture flasks for a minimum of 20 min in an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity or for 0.5–3 hr at room temperature (RT, 15–25°C).
5. Aspirate the diluted COAT-1 solution from the cell culture plate just before use. 

Preparing DEF-CS medium for single-cell seeding

1. Prepare the appropriate volume of DEF-CS medium by adding DEF-CS GF-1 (dilute 1:333), GF-2 (dilute 1:1,000), and GF-3 (dilute 1:1,000) to Cellartis DEF-CS 500 Basal Medium.
2. Prepare fresh medium on the day of intended use and warm it to 37°C ± 1°C immediately before using.
3. Discard any leftover, warmed medium. It is important to prepare enough DEF-CS medium for single-cell seeding to 1) dilute the TrypLE Select Enzyme (1X) used to dissociate cells from the initial culture vessel (1:10 dilution) and 2) seed the detached cells using 100 µl per well into a 96-well plate. Use Table 3 as a guide to ensure that there is sufficient medium for dissociation and seeding.
   
   Table 3. Required volumes of enzyme and medium for cell dissociation.
   
   
   

   Plate type	TrypLE Select Enzyme (1X) (µl per well)	Supplemented DEF-CS medium (µl per well)
   48 well	18	200
   24 well	38	400
   12 well	76	800
   6 well	190	2,000
   10 cm	1,100	12,000

   
   

Single-cell seeding

Single-cell seeding (Day 1)

1. Check cells under a phase contrast microscope. Take photographs for documentation as necessary.
2. Aspirate medium from the culture vessel and wash the cell layer once with D-PBS –/–.
3. Add TrypLE Select Enzyme (1X) to the culture vessel, using the amount indicated in Table 3. Place the vessel in an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity for 5 min, or until the cell layer has detached. Detachment can be aided by tapping the side of the vessel firmly but gently. It is not recommended to tilt or swirl the cell culture vessel.

   NOTE: If starting from multiple samples in the same plate, please make sure to replace the culture vessel lid after removing a sample from a well, then gently tap the side of the vessel. This redistributes the dissociation enzyme and minimizes the risk of the other samples drying out.

4. Resuspend the cells in DEF-CS medium for single-cell seeding with 200 µl per well and pipet up and down several times to ensure a single-cell suspension. The cells will aggregate if left too long in TrypLE enzyme.
5. Use your preferred method to isolate single cells (FACS or limiting dilution). For limiting dilution, we recommend using a final dilution of 0.5 cells per well.
6. Place the plate in an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity and leave the plate undisturbed for 48 hr. Continue culturing according to Table 1.

Adding media to wells containing single cells (Day 3)

Without discarding any medium, carefully add 100 µl of fresh, DEF-CS medium for single-cell seeding per well (see Table 1). There should now be a total of 200 µl per well.

Culturing single-cell colonies   

After single-cell cloning, cells will proliferate into emerging colonies (Figure 3) that will be passaged for further expansion during scale up.

Changing media on single-cell colonies in the 96-well plate

Preparing medium for media change on seeded single cells

1. Prepare 150 µl of DEF-CS medium for media change on seeded single cells per well by adding DEF-CS GF-1 (dilute 1:333), GF-2 (dilute 1:1,000), and GF-3 (dilute 1:1,000) to DEF-CS basal medium according to Table 1.
2. Prepare fresh medium on the day of intended use and warm it to 37°C ± 1°C immediately before using. Discard any leftover, warmed medium

Changing media

1. Check cells under a microscope. Take photographs for documentation as necessary.
2. Carefully discard 150 µl of the medium from each well and add 150 µl of newly warmed medium into each well of the plate. It is recommended to always use manual pipetting (not a vacuum pump). Avoid flushing medium directly onto the cell layer.
3. Place the cell culture plate in an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity.

   NOTE: The colonies will be ready to passage from the 96-well plate to a 48-well plate after 8–14 days, depending on the generation time of the specific hiPS cell line (Figure 4).

Passaging cells from the 96-well plate to a 48-well plate

Coating a 48-well plate

1. Dilute the required volume of COAT-1 in D-PBS +/+ prior to use. Make a 1:10 dilution. Calculate the amount of diluted coating solution required based on the number of wells to be used, where 200 µl of diluted coating solution is used per well (see Table 4).
2. Mix the diluted COAT-1 solution gently and thoroughly by moving the pipette tip up and down while pipetting.
3. Add the diluted COAT-1 solution to a 48-well plate (using 200 µl per well), ensuring that the entire surface of each well is covered.
   
   Table 4. Preparation of coating solution for a 48-well plate.
   
   
   

   Number of wells (48-well plate)	Volume of diluted coating solution (µl)	Volume of COAT-1 (µl)	Volume of D-PBS +/+ (µl)
   1	200	20	180
   2	400	40	360
   48	9,600	960	8,640
   n	200 x n	(200 x n)/10	(200 x n) – (volume of COAT-1)

   
   
4. Place the cell culture flasks for a minimum of 20 min in an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity or for 0.5–3 hr at room temperature (RT, 15–25°C).
5. Aspirate the diluted SSC-COAT-1 solution from the 48-well plate immediately before use.

Preparing medium for passaging

1. Prepare the appropriate volume of DEF-CS medium for thawing or passaging by adding DEF-CS GF-1 (dilute 1:333), GF-2 (dilute 1:1,000), and GF-3 (dilute 1:1,000) to DEF-CS basal medium according to Table 1. The volume of medium needed for each well of the 48-well plate is 500 µl. Calculate the amount of medium needed depending on the number of clonal lines to be expanded.
2. Prepare fresh medium on the day of intended use and warm it to 37°C ± 1°C immediately before use. Discard any leftover, warmed medium.

Passaging

1. Check the cells under a microscope. Take photographs for documentation as necessary.
2. Aspirate the media from the wells and wash the cell layer with D-PBS –/–.
3. Add 50 µl per well of room-temperature TrypLE Select Enzyme (1X) to the cells. Make sure the whole colony in the well is covered. Place the plate in an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity and incubate for 8-10 min or until all cells have detached.
4. Add 150 µl of pre-warmed DEF-CS for thawing or passaging to each well and mix. Transfer contents of each well to a freshly coated well of a 48-well plate containing 300 µl DEF-CS for thawing or passaging. These steps should provide a final volume of 500 µl.

   NOTE: To prevent cell loss, counting the cells at this stage is not recommended.

5. Gently tilt the dish backwards and forwards to ensure that the cell suspension is dispersed evenly over the surface, and then place the dish in an incubator at 37°C ± 1°C, 5% CO₂, and >90% humidity.