BioProtocols--C4

Establishment and characterization of human embryonic stem cell line (1)

Blastocyst culture

Following in vitro fertilization (IVF) treatment, human pronuclear (PN) stage embryos were first cultured to the eight cell stage. At day 3, embryos were graded according to the scale of Gardner et al.After assessment of number of divisions and cell morphology, good quality embryos were reserved for IVF treatment. The surplus embryos donated to this study with informed consent were transferred to droplets of medium G2.3 for further culturing up to the blastocyst stage. Culture medium G-2.3 (Vitrolife, Sweden) was supplemented with 5 mg/ml human serum albumin (Vitrolife) and 1000 U/ml leukaemia inhibitory factor (LIF, Chemicon, USA). The protocols used in this research follow the stem cell guidelines issued by the Ministry of Science and Technology and the Ministry of Health of China and were approved by the Ethics Committee of the Sun Yat-sen University.

Preparation of feeder layer

Mouse embryonic feeder cells (MEF, Cyagen Biosciences, USA) were isolated from 13-14 dpc of KM strain mice and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco Invitrogen, USA); supplemented with 2 mmol/L glutamine, 0.1 mmol/L β-mercaptoethanol and 1% nonessential amino acid (Sigma, USA); 10% foetal calf serum (HyClone, USA). Within 5 passages, the MEF cells were treated with 55 Gy γ-ray irradiation and washed by phosphate buffered saline (PBS) five times before transference to dishes treated with 0.1% gelatin coated, tissue culture of suitable density.

Isolation and primary culture of inner cell mass

Inner cell mass (ICM) was isolated from blastocysts by immunosurgery.In brief, zona pellucida was treated with 0.5% pronase (Sigma, USA) for 1-2 minutes at 37?C in 5% CO₂ atmosphere. Then zona free, blastocysts were incubated in DMEM containing rabbit antihuman serum (Sigma, USA) diluted 1:10 for 30 minutes at 37?C, followed by an additional 30 minutes treatment in 1:5 dilution of guinea pig complement (Sigma) at 37?C in 5% CO₂. Lysed trophectoderm cells were removed by gently pipetting and the ICMs were isolated from trophectoderm. The intact ICMs were plated immediately onto γ-ray irradiated and inactivated mouse embryonic fibroblast feeder layer in gelatin coated tissue culture dishes containing hES cell culture medium (80% knockout DMEM, supplemented with 15% knockout serum replacement, 1% nonessential amino acids, 8 ng/ml human basic fibroblast growth factor (bFGF, Gibco Invitrogen); 5% foetal bovine serum (HyClone, USA); 0.1 mmol/L β-mercaptoethanol, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma); 1000 U/ml recombinant human leukaemia inhibitory factor (hLIF, Chemicon)).

Culture of hES cells

Eight to ten days after initial plating, the ICM cell clumps that resembled stem cells were mechanically dispersed from differentiated cell outgrowths using a micropipette, placed on a fresh feeder layer and cultured under the same conditions as described above. Every seven or eight days, colonies were propagated using mechanical dissociation, or collagenase IV (1 mg/ml, Gibco Invitrogen). Culture media were changed every day. Colonies were also periodically selected by mechanical dissociation and cryopreserved in liquid nitrogen with the frozen medium containing 90% FBS/10% DMSO (Sigma).

After 10 passages, standard medium for hES cells culture was used. The medium comprised of 80% knockout DMEM supplemented with 20% knockout serum replacement, 2 mmol/L L-glutamine, 0.1 mmol/L β-mercaptoethanol, 1% nonessential amino acids, 100 U/ml penicillin, 100 μg /ml streptomycin, 1000 U/ml recombinant human leukaemia, inhibitory factor and basic fibroblast growth factor (bFGF, 4 ng/ml).

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