CTS™ StemScale™ PSC Suspension Medium - FAQs

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The CTS StemScale PSC Suspension Medium protocol recommends passaging every 5 - 6 days, depending on spheroid size. We recommend passaging suspension cultures when the average spheroid diameter is between 300 - 400 µm, which occurs approximately after 5 – 6 days of growth. Passaging earlier is also an option, although the final cell yield will be lower than what is typically obtained.

Passaging protocols for other suspension culture media may offer less flexibility, depending upon whether they utilize a fed-batch or overlay strategy. Generally, these cultures require a strict passaging schedule with little room for flexibility on the weekend.

The CTS StemScale PSC Suspension Medium passaging protocol also does not require the use of cell strainers, while some other PSC suspension culture media protocols may specifically require these devices.

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The CTS StemScale PSC Suspension Medium feeding method involves replacing 50% of the spent medium with fresh medium. This feeding method prevents spheroids from being cultured in medium that is accumulating significant quantities of waste by-products.

Feeding methods which use fed-batch or overlay strategy do not remove the spent medium from suspension cultures. These methods reduce hands-on time when feeding cultures; however, the accumulation of waste by-products is likely to negatively impact the health of the cells.

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The CTS StemScale PSC Suspension Medium kit consists of only two components: a basal medium and a supplement. Following reconstitution, the complete medium is used for all steps, from initiating cultures to feeding cultures.

Other suspension culture medium kits sometimes consist of multiple components, which may be utilized at different points in the suspension culture process.

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Some cells may lyse and release genomic DNA into the suspension culture environment during dissociation with CTS TrypLE Select Enzyme. Without DNase I, the presence of small quantities of genomic DNA will cause some spheroids to aggregate into a single clump.

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This may result in insufficient agtation of cells in the culture vessel, causing aggregation of all the cells into a large clump.

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If the suspension culture is removed from agitation for an extended time period, spheroids will attempt to aggregate into large clumps.

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Spheroids will not form without the presence of a ROCK inhibitor on Day 0.

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A lower volume of medium may be a result of evaporation. Additional fresh medium may need to be added during the next feed day to account for the evaporated volume. To help minimize evaporation, keep the incubator water pan full.

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It is likely that spheroids were damaged during the dissociation step of the passaging protocol. First, ensure that the large spheroids themselves were never triturated with a P1000 pipette or serological pipette. The pipette should only be used once with large spheroids - to gently transfer the spheroids from a vessel to a conical tube. Further handling of large spheroids using a pipette will only damage the spheroids and reduce cell viability.

Additionally, ensure that the spheroids were not over-exposed to the dissociation enzyme. It is important to stop dissociation once the spheroids have broken down into single cells and the liquid suspension is visibly cloudy. Over-exposure to the dissociation enzyme risks further damaging the cells and reducing their viability.

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Yes, these 'small dots' are single cells that have not been incorporated into spheroids. It is normal for suspension cultures to still have numerous single cells visible in the background.

The presence of single cells after spheroid nucleation will not interfere with spheroid expansion over the culture duration.

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First, confirm that the suspension cultures have not been removed from agitation for extended periods of time. When imaging spheroids, cultures should not be removed from agitation for more than 15 mins.

For orbital shaker cultures, confirm that the culture volume has not increased since Day 0. Increasing the culture volume makes it more difficult for spheroids to be sufficiently agitated. If the culture volume has drifted, spheroids may no longer remain suspended at the current RPM.

Confirm that the orbital shaker was constantly powered. A power outage may have occurred and interrupted agitation temporarily. Spheroids require constant agitation to grow properly. Having the orbital shaker connected to a battery backup will ensure that the cultures will be constantly agitated even in the event of a power outage.

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First, confirm that non-tissue culture treated vessels are being used, which prevent spheroids from adhering to the bottom of the vessels.

Next, confirm that DNase I was added on the day of seeding the suspension culture vessels. Without DNase I, it is possible that genomic DNA from lysed cells is resulting in the clumping issue.

After confirming the above, if spheroids are still forming large clumps, then the RPM applied to the culture vessel may be too low. Increasing the RPM will prevent spheroids from aggregating into large clumps.

Alternatively, for orbital shaker cultures, the volume of media in the culture vessel may be too large for the applied RPM, preventing spheroids from remaining in suspension. Decreasing the volume of media will prevent spheroids from aggregating into large clumps.

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First, confirm that ROCK inhibitor (Y-27623) was added to the culture on Day 0. Spheroids will not form without the presence of a ROCK inhibitor. If a ROCK inhibitor was added, then reduce the RPM applied to the culture vessel. Spheroids are unable to form if the RPM is too high.

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No. We recommend that suspension cultures be cryopreserved as single cells.

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Yes. Cells that have been cryopreserved can be thawed directly into CTS StemScale PSC Suspension Medium. The same protocol that is used for thawing cells for adherent cultures can be utilized for suspension cultures. Once cell counts have been obtained, the thawed cells can then be seeded into suspension cultures using the recommended seeding conditions for CTS StemScale PSC Suspension Medium.

Users should expect cells to require one passage in suspension before fully recovering from cryopreservation. After this recovery passage, suspension cultures will perform similarly to those seeded from adherent cultures.

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No. The CTS StemScale PSC Suspension Medium feeding method involves replacing 50% of the spent medium with fresh medium. This feeding method prevents spheroids from being cultured in medium that is accumulating significant quantities of waste by-products.

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Yes. The CTS StemScale PSC Suspension Medium feeding schedule recommends daily feeding to maximize cell health. It is possible to skip one day on the weekend if desired, but otherwise CTS StemScale PSC Suspension Medium cultures should always be fed daily.

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The CTS StemScale PSC Suspension Medium protocol recommends passaging every 5 - 6 days, depending on spheroid size. We recommend passaging suspension cultures when the average spheroid diameter is between 300 - 400 µm.

Passaging cultures earlier is also an option but may result in a lower cell yield. Early passaging is an option to avoid passaging on a weekend.

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On average, we have observed 5-10X expansion per passage in CTS StemScale PSC Suspension Medium, but this fold expansion can vary by cell line.

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Yes. In suspension cultures grown using an orbital shaker, spheroids that are not perfectly round may be observed on Day 1. By Day 2, these spheroids will take a more uniform, rounded shape.

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No. CTS StemScale PSC Suspension Medium promotes the formation of spheroids through self-aggregation in the presence of a ROCK inhibitor.

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A variety of culture vessels are suitable for suspension cultures grown in CTS StemScale PSC Suspension Medium, including non-tissue culture treated well plates, plain-bottom shake flasks, spinner flasks, and liter-scale bioreactors.

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Yes. We have evaluated multiple iPSC and ESC cell lines, all of which were demonstrated to be compatible with CTS StemScale PSC Suspension Medium. Notably, growth in CTS StemScale PSC Suspension Medium is cell-line dependent, as some lines are observed to expand in suspension more effectively than other lines.

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Yes. CTS StemScale PSC Suspension Medium maintains trilineage differentiation potential of hPSCs as assessed by Scorecard analysis.

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Yes. Trypan Blue staining of single cells obtained from dissociated spheroids indicates that the viable cell count of suspension cultures remains high (>90%).

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Yes. hPSCs grown as spheroids in CTS StemScale PSC Suspension Medium are genomically stable as assessed by the KaryoStat Assay.

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Yes. CTS StemScale PSC Suspension Medium maintains the pluripotency of hPSCs grown as spheroids in suspension culture as assessed by both flow cytometric analysis and the PluriTest Assay.

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Culturing spheroids in bioreactors is similar to culturing spheroids in small-scale vessels. The seeding density can remain the same (200,000 cells/mL), along with the inclusion of Y-27632 and DNase I. By following these recommendations, the spheroids can be grown for approximately 5 days. For additional considerations, follow the recommendations below for different types of bioreactors:

Vertical wheel bioreactor:
Working volume: 2-3 L
RPM: 20-25 RPM
Spheroid sedimentation time: Up to 10 mins
CTS TrypLE Select Enzyme concentration: 0.5X-0.75X
CTS TrypLE Select Enzyme dissociation volume: 50-60 mL
CTS TrypLE Select Enzyme dissociation time: 30-40 mins

Horizontal blade impeller bioreactor:
Working volume: 2-3 L
RPM: 65 RPM (first day); 130 RPM final (ramp over next day)
Spheroid sedimentation time: Up to 10 mins
CTS TrypLE Select Enzyme concentration: 0.5X-0.75X
CTS TrypLE Select Enzyme dissociation volume: 50-60 mL
CTS TrypLE Select Enzyme dissociation time: 30-40 mins

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Yes. The suggestions listed below may be helpful when feeding your suspension cultures:

- Loosen the cap on the shake flask before preparing your serological pipet so that the cap can be easily removed with one hand. - After removing the spent CTS StemScale PSC Suspension Medium, be sure to add the fresh CTS StemScale PSC Suspension Medium down the side of the flask to avoid dispensing directly on top of the spheroids settled at the bottom of the flask. This technique helps prevent the formation of bubbles in the medium.

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Yes. Swirling the shake flask before letting the spheroids settle can help collect the spheroids in the center at the bottom of the flask. This placement may allow for easier removal of medium. However, be sure that the shake flask is not disturbed further once you are ready to remove the spent medium.

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We recommend removing 50% of the spent medium from each well and replacing it with an equal volume of fresh medium.

Depending on the cell line, or if the seeding density was higher than recommended, users may choose to replace greater than 50% of the medium.

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When first growing suspension cultures in well plates or other culture vessels using CTS StemScale PSC Suspension Medium, we recommend feeding these vessels one at a time. As users become more experienced and familiar with the performance of their cell lines in suspension, they can choose to increase the number of cultures that are fed simultaneously. Experienced users can become comfortable feeding multiple 6-well plates or 125 mL shake flasks simultaneously. As long as the medium is exchanged in all vessels in a timely fashion, the risk of settled spheroids aggregating into a large mass of cells will be minimized.

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No. The CTS StemScale PSC Suspension Medium passaging protocol does not require a cell strainer, as spheroids are sufficiently dissociated into single cells through exposure to diluted CTS TrypLE Select Enzyme.

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Multiple 50 mL conical tubes may be required for large scale cultures. Large volume conical tubes (i.e., 250 mL) can also be utilized if your centrifuge is able to accommodate these larger conical tubes.

Once the supernatant has been removed from the pelleted spheroids, we recommend combining all the spheroids into a single conical tube prior to dissociation with diluted CTS TrypLE Select Enzyme. This will reduce hands-on time during dissociation. However, multiple conical tubes can be used during dissociation if preferred by the user. For cell counting, the entire dissociated cell suspension should be combined into a single conical tube prior to centrifugation.

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Dissociation can be assessed by visual inspection. The longer that spheroids are exposed to diluted CTS TrypLE Select Enzyme, the more easily they will break apart with gentle agitation of the conical tube. Floating clumps should not be present. Dissociation is complete when the suspension in the conical tube is homogeneous and forms a cloudy single-cell suspension.

Typically, spheroids will need to be exposed to diluted CTS TrypLE Select Enzyme for at least half of the recommended dissociation time before they begin to noticeably break apart. By gently agitating the tube every 2 - 3 mins, it is possible to estimate whether the spheroids are at the point of breaking down into single cells. Toward the end of the recommended dissociation time, the spheroids can be more vigorously agitated to promote their full dissociation.

It is important to never triturate whole spheroids by using a P1000 pipette, as doing so will negatively impact their viability and lead to cell death. Instead, wait for the spheroids to have gently dissociated into single cells, and then proceed to use a P1000 pipette to ensure the remaining clumps are fully broken apart.

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Yes. Dissociation with diluted CTS TrypLE Select Enzyme occurs faster at 37 degrees C, which helps break apart the spheroids more rapidly.

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No. The spheroids should not be centrifuged during routine feeding. Centrifugation may damage the large spheroids and reduce cell viability.

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The volume of diluted CTS TrypLE Select Enzyme and incubation time will vary depending on the culture size and vessel. In general, larger vessels with greater numbers of spheroids will require longer incubation times combined with higher volumes and higher concentrations of diluted CTS TrypLE Select Enzyme to account for the increased cell mass. Guidance is provided below. Incubation times can vary and may be extended for longer than listed if the spheroids are not completely dissociated.

6-well plate; 0.25X CTS TrypLE Select Enzyme dilution; 1 mL per well; 5 min
24-well plate; 0.25X CTS TrypLE Select Enzyme dilution; 1 mL per well; 5 min
125 mL shake flask; 0.25X CTS TrypLE Select Enzyme dilution; 5 mL; 10 min
250 mL shake flask; 0.25X CTS TrypLE Select Enzyme dilution; 10 mL; 10-15 min
500 mL shake flask; 0.25X CTS TrypLE Select Enzyme dilution; 20 mL; 15-20 min
100 mL vertical wheel; 0.5X CTS TrypLE Select Enzyme dilution; 15 mL; 15-20 min
500 mL vertical wheel; 0.5X CTS TrypLE Select Enzyme dilution; 50 mL; 20-25 min
125 mL spinner flask; 0.25X CTS TrypLE Select Enzyme dilution; 10 mL; 10-15 min

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While it can be difficult to estimate the presence of a necrotic core, a good indication is the coloration of the spheroid. Spheroids become darker in color as they grow and begin to form a necrotic core. We recommend passaging spheroids when the average spheroid diameter is between 300 - 400 µm. Beyond this range, the likelihood of a necrotic core forming will increase.

Large spheroids that begin to appear dark are not unusual and should be passaged as they are likely near the point of starting to develop a necrotic core. Healthy spheroids tend to have a dark appearance as the diameter gets closer to 400 µm.

Small spheroids which appear dark are likely experiencing stress and may require more frequent medium exchanges or a lower initial seeding density. When spheroids are small, they should have a brighter appearance compared to larger spheroids.

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The equation for a hemocytometer is as follows:
Final cell concentration = (average cell count from one corner square) x (dilution factor) x (10,000)

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No. Washing with CTS DPBS (-/-) before adding the diluted CTS TrypLE Select Enzyme is not necessary.

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Yes, it is important to always add DNase I when working with CTS TrypLE Select Enzyme. Even when the CTS TrypLE Select Enzyme is diluted, lysed cells may release genomic DNA and cause spheroid aggregation issues. We recommend adding DNase I to suspension cultures at a concentration of 0.1 U/mL. Higher concentrations (such as 1 U/mL) may also be used in large-scale vessels.

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Cells can be very sensitive to dissociation with CTS TrypLE Select Enzyme, leading to some cells becoming stressed and/or lysing during dissociation. To minimize the sensitivity of these cells to the dissociation enzyme, we recommend diluting CTS TrypLE Select Enzyme to a lower concentration with CTS DPBS (-/-). Dilution of CTS TrypLE Select Enzyme will help maintain highly viable suspension cultures post-dissociation.

For orbital shaker cultures, we recommend diluting CTS TrypLE Select Enzyme to a final concentration of 0.25X CTS TrypLE Select Enzyme. This can be done by adding 3 mL of CTS DPBS (-/-) for every 1 mL of CTS TrypLE Select Enzyme.

For larger culture volumes (i.e., ≥ 100 mL), we recommend a range of 0.5 - 0.75X CTS TrypLE Select Enzyme concentration. This higher concentration accounts for the increased cell mass during dissociation.

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For a cell therapy workflow, utilization of products designed for clinical applications helps minimize risk. CTS TrypLE Select Enzyme, an animal origin-free product, is recommended for dissociating spheroids grown in CTS StemScale PSC Suspension Medium. StemPro Accutase Cell Dissociation Reagent is not recommended as it contains animal origin components.

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When utilizing well plates smaller than a 6-well format to grow suspension cultures, the culture volume and RPM will need to be adjusted to account for the smaller well size. In general, the culture volume will decrease while the RPM will increase. Recommendations are listed below for different well plate formats: 6-well plate: 2 mL per well; 70-80 RPM
12-well plate: 1 mL per well; 90-100 RPM
24-well plate: 350µL per well; 120-130 RPM
48-well plate: 200 µL per well; 150-160 RPM

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The recommended final volume of medium when seeding a 6-well plate is 2 mL per well at 70 RPM. At 70 RPM, we do not recommend using volumes less than 1.7 mL or volumes greater than 2.3 mL as these volumes risk spheroids fusing into a large mass of cells.

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Using our recommended seeding density of 200,000 cells/mL, a total of 2.4 x 10E6 viable cells are necessary to seed an entire 6-well plate.

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For best results, your adherent cultures should be 50-80% confluent. While all ranges of confluency are capable of forming spheroids, the overall growth and expansion of your suspension cultures may potentially decrease if overly confluent adherent cultures are used for seeding.

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We recommend continuing to use CTS RevitaCell Supplement for applications with your adherent cultures. For optimal spheroid nucleation in suspension culture, we specifically recommend using Y-27632.

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The steps for the recommended cleaning procedure are described below:
- Soak the glass vessels in a 1% solution of 7X Detergent, either for 2 -3 hours or overnight.
- After removing the detergent solution, lightly scrub the glass vessels with a brush and rinse 3 - 4 times with tap water. Vessels should air dry completely in preparation for the next step.
- Once the detergent cleaning has been completed, siliconize the glass vessels to prevent cells from adhering to their surfaces. Addition of Thermo Scientific Water-Soluble Siliconizing Fluid for 5 mins is sufficient to induce siliconization.
- After removal of Thermo Scientific Water-Soluble Siliconizing Fluid, rinse vessels 3 - 4 times with tap water and air dry.
- Place glass vessels into sterilization bags and autoclave to ensure they remain sterile for future experiments.

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A greater number of spheroids can be obtained by simply increasing the initial seeding density. It is not recommended to increase the seeding density too high (i.e., >300,000 cells/mL), as media will be consumed far more rapidly than at the recommended seeding density. Consequently, necrotic cores may develop more easily at higher seeding densities.

For orbital shaker cultures, an alternative is to adjust both RPM and culture volume simultaneously. By increasing both RPM and culture volume together, the number of cells used to seed a suspension culture can be increased while maintaining the recommended 200,000 cells/mL seeding density and ensuring that spheroids do not fuse into a large mass of cells. Increasing both the RPM and culture volume results in a greater number of spheroids harvested from the same type of orbital shaker culture vessel.

Example using a 125 mL shake flask:
Standard recommended conditions: 70 RPM and a 20 mL culture volume
To obtain greater numbers of spheroids: 90 RPM and a 40 mL culture volume
In general, shake flasks should have their culture volume doubled as the shaker speed increases from 70 RPM to 90 RPM.

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The RPM may need to be altered for other orbital shakers, depending on the orbit diameter. It is possible to estimate the RPM for other shakers by using the following equation:

〖RPM〗_2=√(〖〖RPM〗_1〗^2×d_1/d_2 ))

Where d1 = orbit diameter of the Thermo Scientific CO2 Resistant Shaker (19 mm / 0.75 in); d2 = orbit diameter of the alternative orbital shaker; RPM1 = recommended RPM for the Thermo Scientific CO2 Resistant Shaker; RPM2 = recommended RPM for the alternative orbital shaker.
Additional testing will be necessary to determine the optimal RPM based on this initial estimation.

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Yes. While spheroids will be smaller at higher RPM, they will also be more uniform in size.

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For orbital shaker cultures, the suspension culture volume is a factor that can influence spheroid size. When initiating cultures, a larger volume will lead to the nucleation and expansion of larger spheroids over the culture duration. Therefore, ensuring the orbital shaker suspension cultures maintain a constant volume after performing medium exchanges is critical. Fluctuations in volume can potentially cause undesirable changes in spheroid size. The volume in a 6-well plate, for example, should never exceed a total volume of 2.3 mL.

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The recommended method to control spheroid size is by changing the rotor speed (RPM). When initiating cultures, a higher RPM will lead to the nucleation and expansion of smaller spheroids over the culture duration. A lower RPM will result in the nucleation and expansion of larger spheroids over the culture duration.

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When spheroids are small and not easily visible by eye (i.e., Day 1 to 2), it is recommended to wait the full 5 min duration to ensure all spheroids will settle to the bottom of the vessel.

When spheroids are large and easily visible by eye (i.e., Day 3 to 6), the spheroids do not require the full 5 min of gravity sedimentation. The medium can be replaced once the user can visually confirm that all spheroids have settled at the bottom of the well.

Notably, larger vessels (i.e., ≥100 mL) may require some additional sedimentation time to avoid aspirating small spheroids within the first two days of culture. Consider extending the sedimentation time in larger vessels by a few minutes if spheroids still remain suspended. Extending the total sedimentation duration in larger vessels up to 10 min should not adversely affect spheroid growth.

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The amount of time taken by the spheroids to settle via gravity sedimentation should be minimized to avoid undesirable spheroid aggregation. The 5-minute gravity sedimentation recommended for orbital shaker cultures in the feeding protocol has no adverse effect on spheroids.

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For suspension cultures that are growing in shake flasks or spinner flasks, remove a small volume of media (~2-3 mL) containing spheroids from the flask. This aliquot of spheroids can be placed into a 6-well plate for imaging.

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Suspension cultures grown in well plates can easily be imaged under a microscope. For larger vessels, such as shake flasks and spinner flasks, a small sample of spheroids should be removed and placed into a 6-well plate for imaging. Once in a 6-well plate, the suspension culture can be gently swirled in a clockwise or counter-clockwise motion to draw spheroids toward the center of the well. Images should then be acquired in a timely fashion to avoid undesirable spheroid aggregation. After acquiring images, gently agitate the plate to redisperse spheroids throughout the well.

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When suspension cultures are removed from agitation, spheroids may begin to aggregate into large clumps if they remain settled for extended periods of time. To avoid this undesirable aggregation, ensure that suspension cultures are not removed from an agitation source for longer than 15 mins.

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The major differences between the RUO and CTS StemScale PSC Suspension Medium protocols are:
For RUO StemScale PSC Suspension Medium:
- Dissociation reagent: StemPro Accutase Cell Dissociation Reagent
- DNase I: not required
- Estimated dates of growth: 4-5 days for spheroids to reach 300-400 µm in diameter
- Feed Strategy: every other day
- Seeding density: 150,000 cells/mL

For CTS StemScale PSC Suspension Medium:
- Dissociation reagent: CTS TrypLE Select Enzyme (diluted)
- DNase I: should be added
- Estimated dates of growth: 5-6 days for spheroids to reach 300-400 µm in diameter
- Feed Strategy: daily
- Seeding density: 200,000 cells/mL

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The formulation of CTS StemScale PSC Suspension Medium is similar to that of RUO StemScale PSC Suspension Medium but with specific modifications in line with regulatory guidance. These changes necessitate a few minor protocol differences between the RUO and CTS versions of StemScale PSC Suspension Medium to provide similar cell yields.

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The CTS StemScale PSC Suspension Medium kit consists of only two components: a basal medium and a supplement. Following reconstitution, the complete medium is used for all steps, from initiating cultures to feeding cultures.

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No. Aliquots of CTS StemScale PSC Suspension Supplement show no significant decrease in performance after one freeze/thaw cycle.

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No. Each aliquot of CTS StemScale PSC Suspension Supplement should undergo only one freeze/thaw cycle. Once an aliquot is thawed, it should not be frozen a second time.

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Yes. The CTS StemScale PSC Suspension Supplement may be aliquoted into smaller volumes and stored at -5 degrees C to -20 degrees C for up to 6 months. Avoid repeat freeze/thaw cycles.

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Following reconstitution, complete medium can be stored at 2 to 8 degrees C for up to 2 weeks.

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