A Complete Guide to hiPSC Induction and Culture

Reagents and Consumables for the Whole Workflow

Category	Cat. No.	Product Name	Specification
Stem Cells	abs90290	hiPSC Human Induced Pluripotent Stem Cells	1 mL
Pluripotent Stem Cell Medium	abs90487	hESC/iPSC Cell Culture Kit	500 mL
Coating Solution	abs9496	Matrigel (iPSC-qualified, Phenol Red-free)	1.5 mL ×4
Basal Medium	abs9560	DMEM/F-12 Medium	500 mL
Passage Dissociation Buffer (Colonies)	abs90493	hESC/iPSC Passaging Working Solution (Enzyme-free)	500 mL
DPBS	abs970	D-PBS Buffer (1×, Ca²⁺/Mg²⁺-free)	500 mL
Stem Cell Cryopreservation Medium	abs9412	ES/iPSC Cryopreservation Medium (Serum-free)	100 mL
Cell Consumables	abs7033	Cell Culture Plate (Standard 6-well, Clear)	1 case
	abs7034	Cell Culture Plate (Standard 12-well, Clear)	1 case
	abs7035	Cell Culture Plate (Standard 24-well, Clear)	1 case
	abs7053	10 mL Disposable Serological Pipette	1 case
	abs7054	25 mL Disposable Serological Pipette	1 case
	abs7164	2 mL Internal-thread Cryovial	1 case
	abs7289	2 mL Low-temperature Metal Ice Box (24-well, flat-bottom)	1 pc

I. Key Points for Recovery

1. Preparation of hESC/iPSC Complete Medium

Table 1: hESC/iPSC Cell Culture Kit Components

Product Information	Specification	Storage
hESC/iPSC Basal Medium	450 mL	2–8 °C, 12 months
hESC/iPSC Growth Supplements A	50 mL	–20 °C or –80 °C, 12 months
hESC/iPSC Supplement C (Y27632)	1 mL (5 mM)	–20 °C or –80 °C, 12 months

Table 2: hESC/iPSC Complete Medium Preparation

Component	500 mL	100 mL	50 mL
hESC/iPSC Basal Medium	450 mL	90 mL	45 mL
hESC/iPSC Growth Supplements A	50 mL	10 mL	5 mL

(1) Thaw Supplement A overnight at 4 °C; do NOT thaw at 37 °C in incubator or water bath. Limit freeze-thaw cycles of A to ≤2; aliquot into 5 mL tubes.

(2) Thaw Supplement C overnight at 4 °C; do NOT thaw at 37 °C. Limit freeze-thaw cycles of C to ≤2; aliquot into 100 µL tubes. Y27632 is used only on the first day of recovery/passaging and for cryopreservation.

(3) Combine components as in Table 2 under sterile conditions to prepare complete hESC/iPSC medium. Store at 4 °C and use within 2 weeks; aliquots may be kept at –20 °C for up to 6 months.

(4) Pre-warm required volume of complete medium to room temperature before use.

2. Matrigel Coating (6-well plate example)

abs9496 – 1.5 mL ×4

1. Thaw Matrigel overnight at 4 °C on ice. Pre-chill 200 µL and 1 mL pipette tips at –20 °C overnight.

2. Dilute Matrigel 1:100 in ice-cold DMEM/F-12. Example: add 1.5 mL Matrigel to 150 mL cold DMEM/F-12. Coating density: 300 µL/cm²; 6-well plate = 10 cm²/well → 3 mL per well. Spread evenly. Use remaining coating solution within 2 weeks when kept at 4 °C.

3. Place coated plates in incubator to pre-equilibrate.

4. Leave plates at room temperature for 1 h. Do not allow the coated surface to dry out.

5. Aspirate coating solution and seed cells immediately.

3. Recovery of hESC/iPSC (6-well plate example)

(1) Pre-heat water bath to 37 °C; place Matrigel-coated 6-well plate in biosafety cabinet for ≥1 h to equilibrate to 15–30 °C.

(2) Add 6 µL Supplement C to 3 mL complete hESC/iPSC medium (final 10 µM). Prepare 18 µL Supplement C in 9 mL DMEM/F-12 (10 µM). Bring both to 15–30 °C. Do NOT pre-warm media in 37 °C water bath.

(3) Immerse one vial of frozen cells in 37 °C water bath; gently swirl for ≤2 min until only a rice-grain-sized ice crystal remains.

(4) Wipe vial with 75 % ethanol; transfer to biosafety cabinet. Pool cell suspension into a 15 mL tube, add 10 mL Y27-containing DMEM/F-12 drop-wise while gently rocking. Centrifuge 160 ×g, 5 min.

(5) Aspirate coating solution from wells; gently add 2 mL complete hESC/iPSC medium + Supplement C per well.

(6) After centrifugation remove supernatant, leaving ≈50 µL. Flick tube 3–4× to resuspend. Add 1 mL complete medium + Supplement C drop-wise, flick again, and seed this 1 mL drop-wise into the 2 mL medium already in the well.

(7) Perform horizontal cross-shaped shaking (left-right twice + up-down twice = one cycle) three times. Incubate at 37 °C, 5 % CO₂, saturated humidity; repeat shaking after 5 min. Note: three cycles = 3×(2+2) movements.

(8) Replace with fresh complete medium at 18–24 h; change daily thereafter (2 mL/well). If confluency >50 %, increase volume to 3–4 mL/well.

II. Key Points for Passaging

1. Criteria and Split Ratio

Fig. 1. Morphology of hiPSC cultured in complete hESC/iPSC medium: (A,B) low-power view at day 2 and 4; (C,D) high-power view at day 2 and 4.

(1) Passage when:

① Colonies reach ≈85 % confluency (Fig. 1C-D), usually every 3–4 days;

② Colonies become overly dense or large;

③ Increased differentiation is observed.

Note: do not continuously culture >5 days even if confluency is low.

(2) Split ratios: 1:5–1:12 depending on colony density and experimental needs.

Normal colonies at 85 % confluency (Fig. 1B-D) → 1:10. Adjust ratio downward for lower density or upward for higher density.

Note: 1:10 means one well into ten wells (6-well plate).

2. Dissociation

Fig. 2. hiPSC morphology after dissociation: (A) 5 min; (B) 6 min.

(3) Equilibrate Matrigel-coated 6-well plate, hESC/iPSC Passage Solution, and DPBS to room temperature (~25 °C) in biosafety cabinet for ≈1 h.

(4) Prepare complete hESC/iPSC medium + 10 µM Supplement C at 2 mL/well + 1 mL extra; bring to ~25 °C.

(5) Aspirate spent medium; rinse with 1 mL DPBS (Ca²⁺/Mg²⁺-free) per well and remove.

(6) Add 1 mL hESC/iPSC Passage Solution per well to completely cover the surface.

(7) Incubate at 37 °C for 5–6 min.

① Inspect under microscope: when most colonies appear bright and round but have not yet detached, terminate dissociation. If most colonies are still compact, extend incubation (total <10 min). Do not exceed 10 min in Passage Solution.

② Keep plate in direct contact with incubator shelf to ensure uniform heating; do not stack plates.

(8) Gently return plate to biosafety cabinet without shaking; tilt and aspirate Passage Solution.

(9) Immediately add 2 mL pre-warmed complete medium + Supplement C per well. Gently triturate once with the same pipette tip, then once more after collecting the suspension to detach colonies.

① Limit trituration to 1–2 gentle strokes; 10–15 % colonies remaining attached is normal. If large clumps persist, extend dissociation (<10 min).

② Work quickly; do not process >4 wells at a time to keep total exposure <15 min.

(10) Aspirate residual Matrigel solution from new plate; add 2 mL pre-warmed complete medium + Supplement C per well.

(11) Label plates with cell line, passage number, split ratio, date, operator. Distribute cell suspension evenly according to predetermined ratio.

(12) Perform horizontal cross-shaped shaking three times; incubate at 37 °C, 5 % CO₂, saturated humidity; repeat shaking after 5 min. Culture overnight.

(13) Replace with fresh complete medium at 18–24 h; change daily thereafter. Passage or freeze when 85 % confluent (usually day 4–5).