iPSC/ESC Dissociation Solution (Enzyme-Free) 

MCE iPSC/ESC Dissociation Solution (Enzyme-Free) is a chelation-based, enzyme-free cell dissociation reagent specifically designed for the culture and manipulation of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). Its core mechanism relies on ethylene glycol–based chelating agents that gently disrupt intercellular junctions, enabling efficient separation of cell clusters.

The formulation is supplemented with bicarbonate, glucose, and HEPES to establish a stable buffering system and maintain physiological pH. In addition, potassium chloride and sodium chloride are included to regulate osmolarity, creating a microenvironment that closely mimics embryonic physiological conditions.

This optimized solution enables gentle and efficient dissociation of iPSCs/ESCs within 5-8 min, making it suitable for routine passaging, dissociation, and cell-cluster handling. It provides superior stability and better preservation of cell state compared with traditional methods.

RT, 1 year.

1. Timing of PSC Passaging

Cells are ready for passaging when they reach approximately 85% confluency. Under routine culture conditions, passaging are typically performed every 4-8 d. If cell density is low or the distribution is uneven, the decision to passage should be made based on an overall assessment of cell morphology and growth status.

Note: Even when colony size is small or confluency appears suboptimal, continuous culture beyond 10 days is not recommended.

2. Adjustment of Split Ratio

Depending on cell growth status and experimental requirements, PSCs may be passaged at a 1:6-1:12 split ratio. When cells exhibit good morphology with uniform colony size and reach 85% confluency, a 1:12 split ratio is recommended.

Note: A “1:12” split means that cells from one well/flask are distributed into 12 wells (e.g., using a 6-well plate).

3. Preparation of Culture Plates

Place the Basement Membrane Matrix (Cat. No.: HY-K6006) –coated 6-well plates in a 37°C incubator for 1 h prior to use.

4. Preparation of Complete Medium

Prepare 2 mL of complete stem cell culture medium per well according to the number of inoculation wells. To prepare the complete stem cell culture medium, add CEPT Cocktail Plus (1000×) (Cat. No.: HY-K6021) to the stem cell culture medium (Cat. No.: HY-K6501) at a ratio of 1:1000. Mix thoroughly and equilibrate to room temperature (25°C) before use.

5. Cell Washing

Aspirate the spent medium and gently rinse each well with 2 mL DPBS (calcium- and magnesium-free, Cat. No.: HY-K3008). Aspirate the wash solution completely.

6. Cell Dissociation

Add 2 mL of iPSC/ESC Dissociation Solution (Enzyme-Free) to each well, ensuring full coverage of the well surface. Incubate at 37°C for 5-7 min.

Notes: a. Observe cells under the microscope after 5 min. When most cells appear round and bright but have not yet detached, dissociation should be stopped. If no apparent change is observed, extend the incubation time appropriately.

b. For uniform heating, ensure the plate is in direct contact with the metal shelf of the incubator; do not stack plates.

7. Termination of Dissociation

Remove the plate from the incubator carefully, avoiding shaking, and aspirate the dissociation solution.

8. Cell Collection

Add 1-2 mL of pre-warmed complete medium to each well. Gently pipette up and down no more than 3 times, then mix using a gentle horizontal cross motion to dislodge cell aggregates.

Notes: a. A small portion of cells (approximately 10-15%) may remain attached, which is normal.

b. For improved seeding uniformity and reduced mechanical stress, it is recommended to transfer the collected cell suspension into a 1.5 mL microcentrifuge tube for gentle mixing.

9. Preparation of Pre-coated Plates

Aspirate the coating solution from the 6-well plate and add 2 mL of complete medium to each well.

10. Cell Seeding

Label the plate with cell name, passage number, split ratio, and date. Gently mix the dissociated cell suspension and distribute it evenly into the designated wells according to the predetermined split ratio.

11. Plating and Culture

After seeding, gently rock the plate using a horizontal cross pattern 3 times. Place the plate in the incubator for overnight culture.

12. Medium Change

Replace with fresh complete medium 18-24 h after seeding. Subsequently, change the medium once daily.

13. Continued Culture

After 4-8 d, depending on cell condition, proceed with the next round of passaging or cryopreservation.

1. This product is sterile and should be handled using aseptic techniques. It is recommended to aliquot and store to avoid contamination.

2. This product is for R&D use only, not for drug, household, or other uses.

3. For your safety and health, please wear a lab coat and disposable gloves to operate.