Human PSC Embryoid Body (EB) Induction Medium 

Human pluripotent stem cells (PSCs) can spontaneously form spherical three-dimensional structures known as embryoid bodies (Embryoid Bodies, EB, EBs) under defined in vitro culture conditions. Embryoid bodies comprise cell populations representing the three germ layers—endoderm, mesoderm, and ectoderm—and closely recapitulate key structural and developmental features of early mammalian embryogenesis. As such, EBs serve as an important in vitro model for studying early developmental processes and multilineage differentiation.

MCE Human PSC Embryoid Body (EB) Induction Medium is specifically designed to support the efficient and reproducible formation of uniform embryoid bodies from pluripotent stem cells under suspension culture conditions. This optimized culture system integrates microenvironmental regulation with metabolic adaptation strategies, significantly improving the stability and reproducibility of EB formation.

By optimizing nutritional composition and energy metabolism support, this medium minimizes cell state fluctuations caused by metabolic stress during EB formation. In combination with sustained IGF signaling modulation and CEPT Cocktail Plus, it effectively promotes PSC aggregation and early developmental progression. Under standard culture conditions, uniform and morphologically stable embryoid bodies can be generated within 48-72 h, providing a reliable starting material for downstream directed differentiation and organoid development.

1. Trypan Blue Staining Solution (0.4%) (MCE Cat. No.: HY-K2004)

2. DPBS (MCE Cat. No.: HY-K3008)

3. Basement Membrane Matrix IPSC-qualified (MCE Cat. No.: HY-K6006)

4. CEPT Cocktail Plus (1000×) (MCE Cat. No.: HY-K6021)

5. ECM Gentle Dissociation Solution (MCE Cat. No.: HY-K6022)

6. Human PSC Maintenance Medium (MCE Cat. No.: HY-K6501)

7. Human iPSC-Derived Brain Organoid Long-term Culture Medium (MCE Cat. No.: HY-K6502)

8. Equipment and Consumables: Horizontal plate centrifuge, 96-well low-attachment plates, and multichannel pipettes (8-12 channels), among other related equipment and consumables.

1. Preparation of PSCs

Revive PSCs following the standard pluripotent stem cell (PSC) culture SOP (MCE Cat. No.: HY-K6501). Passage the cells in a 6-well plate at a 1:6 ratio for three consecutive passages.

2. Preparation of P4 Cells for Induction

Continue culturing P4 cells for 3-5 d, with daily medium changes, to maintain optimal cell status. Allow the cell density in each well to reach 80-90%.

3. Cell Status Confirmation and Reagent Pre-warming

1) Cell status check: Confirm that PSCs show no edge differentiation. Cells should have large, clear nuclei and form compact colonies.

2) Pre-warm the following reagents to room temperature: Human PSC Embryoid Body (EB) Induction Medium 30 mL + CEPT Cocktail Plus (1000×) 60 μL, ECM Gentle Dissociation Solution (6 mL), DPBS (6 mL).

4. Cell Dissociation and Collection

1) Washing: Add 1 mL pre-warmed DPBS to each well, wash once, and aspirate DPBS.

2) Dissociation: Add 1 mL ECM Gentle Dissociation Solution per well and incubate at 37°C for 8-12 min.

3) Collection: Without removing the dissociation reagent, add 1 mL EB induction medium containing CEPT Cocktail Plus to each well. Gently pipette up and down twice to detach cells. Collect the suspension into a 15-mL tube and centrifuge at 150 g for 5 min. Discard the supernatant and retain the cell pellet.

5. Cell Counting and Viability Assessment

Count cells using trypan blue and assess viability. Cell number: > 2 × 107 cells/mL. Viability: > 80% required to proceed.

Note: If viability is low, verify the quality of trypan blue, dissociation duration, and incubation temperature.

6. EB Formation

1) Add 1 mL cell suspension to 8 mL EB induction medium containing CEPT Cocktail Plus, and pipette 10 times to mix thoroughly.

2) Seed 3 mL of the cell suspension into each well of a 6-well plate. Allow the plate to stand in the incubator for 30 min, then transfer to a horizontal shaker and culture overnight at 37°C, 110 rpm.

3) On the next day, add 2 mL EB induction medium (without CEPT Cocktail Plus) to each well.

4) On Day 3, evaluate EB formation. If the EB diameter is ≥ 100 μm, proceed to downstream differentiation. For PSC lines forming smaller EBs, aspirate 4 mL of the supernatant carefully (without disturbing EBs), add 2 mL EB induction medium (without CEPT Cocktail Plus), and continue culturing until 72 h before differentiation.

Note: EB formation using a 96-well plate

1) Add 1 mL cell suspension to 23 mL EB induction medium containing CEPT Cocktail Plus, mix thoroughly, and seed 100 μL per well into a 96-well plate.

2) Seal the plate with a sealing film and centrifuge at 1500 rpm for 1 min to promote aggregation.

3) Remove the seal and incubate overnight.

4) On the next day, add 100 μL EB induction medium (without CEPT Cocktail Plus) per well.

5) Typically, single cells aggregate within 24 h, and EBs formed after 48 h or longer are suitable for downstream differentiation.

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.