A modified H9 pluripotent stem cell line where eGFP was expressed under the control of LMX1A promoters (LMX1A-eGFP) (Niclis et al., 2017 (link)) was used to track the expression of LMX1A. All plates or flasks were pre-coated with 0.5 µg/cm2 Laminin-521 (Life Technologies, Australia). Undifferentiated cells were grown using a slightly modified method of (Watmuff et al., 2015 (link)). Briefly, pluripotent stem cells were seeded at a density of 3,000 cells per cm2 and cultivated in Essential 8 Medium with Essential 8 supplement (Thermo Fisher Scientific, Australia) and Penicillin/Streptomycin (Life Technologies, Australia) at 37°C in a humidified incubator containing 5% CO2. Cells were passaged at ∼80% confluence (ReLeSR™, Stemcell technologies, Australia). To enhance the survival of cells seeded at low-density, the ROCK inhibitor (10 μM Y-27632) was added to the medium for the first 24 h after passaging. Cells were maintained for a maximum 10 passages.
Laminin 521
Manufactured by Thermo Fisher Scientific
Sourced in Australia, United States
Laminin-521 is a component of the extracellular matrix that plays a role in cell adhesion and survival. It is used in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Relesr, by STEMCELL (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
Cell tak, by Corning (1 mentions)
E cadherin, by R&D Systems (1 mentions)
Laminin 521, by BioLamina (1 mentions)
24 well insert, by Merck Group (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Growth factor reduced basement membrane matrix, by Corning (1 mentions)
E8 flex, by Thermo Fisher Scientific (1 mentions)
Chir99021, by Bio-Techne (1 mentions)
Imr90 4, by WiCell (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
5 protocols using laminin 521
1
Tracking Pluripotent Stem Cell Differentiation
2
Pluripotency Maintenance Protocol
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Laminin-521 (Biolamina, Sundbyberg, Sweden) / E-cadherin (R&D Systems) coating was performed as previously described [25 (link)]. Tissue culture plates were coated at a concentration of 13.5μg/ml Laminin-521 and 1.5μg/ml E-cadherin in DPBS containing Ca2+ and Mg2+ (Life Technologies) for 2h at 37°C. Next the plates were washed twice with DPBS containing Ca2+ and Mg2+ (Life Technologies) and used immediately.
Coating with Cell-Tak (Corning, Bedford, MA, USA) adhesive was performed as recommended by supplier. Stock solution was diluted in NaHCO3, pH 8.0 and 1N NaOH to obtain a concentration of 3.5 μg Cell-Tak/cm2. Incubation was performed at room temperature for 1h and then plates were washed twice with DPBS containing Ca2+ and Mg2+ (Life Technologies). Plates were used immediately or air-dried and stored at 4°C until use. The ICM was dissected from day 8 embryos cultured in either SOF, 3iL, 5iLA or NHSM as described above and placed in any one of the coated wells. Passaging of colonies was done mechanically using fine glass Pasteur’s pipettes each 7th day. Medium was replaced every 2–3 days.
Intact embryos in 5iLA medium were cultured until day 23pf in uncoated plates. From day 12pf, embryos were transferred to fresh medium every other day and from day 17pf, half of the culture medium was replaced by fresh medium every day.
Coating with Cell-Tak (Corning, Bedford, MA, USA) adhesive was performed as recommended by supplier. Stock solution was diluted in NaHCO3, pH 8.0 and 1N NaOH to obtain a concentration of 3.5 μg Cell-Tak/cm2. Incubation was performed at room temperature for 1h and then plates were washed twice with DPBS containing Ca2+ and Mg2+ (Life Technologies). Plates were used immediately or air-dried and stored at 4°C until use. The ICM was dissected from day 8 embryos cultured in either SOF, 3iL, 5iLA or NHSM as described above and placed in any one of the coated wells. Passaging of colonies was done mechanically using fine glass Pasteur’s pipettes each 7th day. Medium was replaced every 2–3 days.
Intact embryos in 5iLA medium were cultured until day 23pf in uncoated plates. From day 12pf, embryos were transferred to fresh medium every other day and from day 17pf, half of the culture medium was replaced by fresh medium every day.
3
Collagen Scaffold Fabrication and Functionalization
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To improve the collagen integrity, collagen was cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at pH 5, as previously described (68 (link)). Collagen solution (in 0.02 M acetic acid) was lyophilized for 72 hours to remove water and acetic acid. The lyophilized collagen was dissolved in MES buffer (0.1 M; pH 5) at a concentration of 5 mg/ml. A collagen scaffold was micromolded and cross-linked on the surface of a porous polytetrafluoroethylene (PTFE) membrane (0.4-μm pore size) held in a 24-well insert (Merck Millipore). A mixture of collagen (4 mg/ml), 60 mM EDC, and 15 mM NHS in MES buffer (0.1 M; pH 5) was prepared, and bubbles were removed by centrifugation at 3000g for 1 min. The collagen mixture (60 μl) was added to the center of the insert, and the PDMS stamp was placed on top of the mixture for 2 hours. The PDMS stamp was then removed from the collagen scaffold, and the scaffold was then soaked in PBS for 24 hours to remove unincorporated EDC and NHS. The following day, the collagen scaffold was coated overnight with laminin-521 (20 μg/ml), laminin-111 (BioLamina), fibronectin (50 μg/ml; Thermo Fisher Scientific), and 10% (v/v) Matrigel.
4
Rabbit Corneal Endothelial Cell Isolation
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Rabbit CECs(rCECs) were isolated from adult New Zealand white rabbits, with weights ranging from 1.8 to 2.2 kg (Orient Bio Inc., Seongnam-si, Gyeonggi-do, Korea). Corneal endothelium was scraped without removing the Descemet’s membrane. After incubation for five minutes at 37 °C in 0.25% TE digestion, rCEC suspension was collected, centrifuged (2000 rpm: 800× g) for five minutes, and cultured on a Laminin-521 (ThermoFisher Scientific, Cat. No.: A29249)-coated cell culture plate with CEC basal medium of DMEM containing 20% FBS and 1% AA. The rCECs were cultured in a humidified atmosphere at 37 °C under 5% CO2, and the rCEC culture medium was replaced every two days. When the cells were confluent, a serial passage was performed at a ratio of 1:3. The cell number for each passage was 2.1 × 104 cells/cm2. In the early stages of rCEC culture (passage 2), the cell number was confirmed by directly counting cells with trypan blue staining and a hemocytometer.
5
Directed Differentiation of hiPSCs into BMECs
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The hiPS cell line iMR90-4 (WiCell) 18) (link) was cultured in mTeSR1 medium (Stem Cell Technologies, Vancouver, Canada), and dissociated into clumps using 0.5 mM ethylenediamine tetraacetic acid (EDTA)/phosphate-buffered saline (PBS) and plated onto growth factor-reduced basement membrane matrix (Corning, Corning, NY, U.S.A.). The hiPS cell line 201B7 19) (link) was maintained in E8-Flex (Thermo Fisher Scientific, Waltham, MA, U.S.A.), which was changed daily, and dissociated into clumps using 0.5 mM EDTA/PBS and plated onto Laminin-521 (Thermo Fisher Scientific). The differentiation protocol for the induction of BMECs from hiPS cells was described previously. 11) (link) Chir99021 (Lot: 5B/198191) and BIO (Lot: 3A/178335) were purchased from TOCRIS. Chir99021 was reconstituted in dimethyl sulfoxide (DMSO) and included at concentrations of 3-6 µM depending on the experiments. BIO was reconstituted in DMSO and included at concentrations of 2-3 µM depending on the experiments. They were added into the culture medium from day 6 to day 8.
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