Another simple differentiation protocol from AECs to hepatocytes was reported by Maymó et al[14 (link)]. In brief, the cells were cultured for 1 wk in Iscove’s modified Dulbecco’s medium containing epidermal growth factor (EGF) then cultured with EGF and dexamethasone. This method was named the two-step protocol.
Lipidure
Manufactured by NOF corporation
Sourced in Japan, United States
Lipidure is a laboratory equipment product designed for the analysis and separation of lipids. It is a high-performance liquid chromatography (HPLC) column that is specialized for the separation and purification of a wide range of lipid classes, including phospholipids, glycolipids, and neutral lipids. The core function of Lipidure is to provide efficient and reliable lipid separation, enabling researchers and scientists to accurately analyze the composition and structure of lipid samples.
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5 protocols using lipidure
1
Hepatic Differentiation of Alveolar Epithelial Cells
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2D culture: For the AECs, a custom culture medium was blended and named AEC basal medium (AECBM). The media and protocols are listed in Supplementary Figure 1. Primary AECs were used in most analyses. In the culture experiments, however, AECs were precultured with AECBM for 5-7 d then used for differentiation. In the present study, the multistep hepatic differentiation protocol of Nie et al[17 (link)] was applied. In that study, iPSCs were differentiated into hepatocytes. The AEC culture medium was changed step by step within 3 wk.
Another simple differentiation protocol from AECs to hepatocytes was reported by Maymó et al[14 (link)]. In brief, the cells were cultured for 1 wk in Iscove’s modified Dulbecco’s medium containing epidermal growth factor (EGF) then cultured with EGF and dexamethasone. This method was named the two-step protocol.
3D culture: A primary 2D culture was prepared to select a subpopulation of adherent AECs. Unattached cells were removed after 72 h. A 24-well 3D micropattern culture plate (Kuraray, Tokyo, Japan) was used for the next step. After coating the plate with lipidure (NOF Corporation, Tokyo, Japan), the precultured AECs were seeded at a density of 106/well. The AECs formed a sphere within 3-7 d. AECs, HUVECs, and MSCs were used for organoid propagation and they formed spheres within 1 d.
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Another simple differentiation protocol from AECs to hepatocytes was reported by Maymó et al[14 (link)]. In brief, the cells were cultured for 1 wk in Iscove’s modified Dulbecco’s medium containing epidermal growth factor (EGF) then cultured with EGF and dexamethasone. This method was named the two-step protocol.
2
Embryoid Body Formation from hESCs
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hESCs were disbursed into small clumps (200 μm diameter) by treatment with 0.25% trypsin and 0.1 mg/mL collagenase IV (Invitrogen, Carlsbad, CA) in phosphate-buffered saline (PBS) containing 20% Knockout™ Serum Replacement and 1 mM CaCl2 at 37°C for 5 min, followed by pipetting. Clumps of 2.5–3 × 105 cells were transferred to a non-adhesive 60-mm dish coated with Lipidure (NOF Corporation, Tokyo, Japan). Cells were cultured in 4 mL of EB medium: 80% knockout DMEM, 100 μM non-essential amino acids, 2 mM L-glutamine, 100 μM 2-mercaptoethanol, and 20% fetal bovine serum (Hyclone, Logan, UT, USA). Suspension cultures were maintained for 5 days.
3
Surface Functionalization of Devices via Coatings
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Devices were coated with mPEG (mPEG-silane, 5k Da, Creative PEGWorks, USA), Lipidure (NOF Corporation, Japan), or BSA (Fischer Scientific, USA). Coating concentrations and the modified coating protocol were based on previous reports [mPEG [39 ]; Lipidure [11 (link)]; BSA [21 (link)]].
For mPEG coated surfaces, 4 mM mPEG was prepared in 95% ethanol solution at room temperature. Then, devices were treated with handheld plasma (Electro Technic Products, USA) for approximately thirty seconds each. 120 μL of mPEG solution was immediately pipetted into the device and allowed to incubate at room temperature for two hours. All remaining solution was removed, and devices were stored at 4 °C until further use.
For Lipidure coated surfaces, Lipidure was prepared at 0.5 wt% in 200-proof ethanol. 120 μL of Lipidure solution was then pipetted into each device and allowed to incubate at room temperature for two hours. Afterward, all remaining solution was removed, and devices were stored at 4 °C until further use.
For BSA coated surfaces, devices were coated in a 3% BSA solution at 37 °C overnight. Afterward, the devices were washed with cell culture medium (3* 5 min) before cell-seeding. BSA had been diluted in DI water and sterile filtered before use.
For mPEG coated surfaces, 4 mM mPEG was prepared in 95% ethanol solution at room temperature. Then, devices were treated with handheld plasma (Electro Technic Products, USA) for approximately thirty seconds each. 120 μL of mPEG solution was immediately pipetted into the device and allowed to incubate at room temperature for two hours. All remaining solution was removed, and devices were stored at 4 °C until further use.
For Lipidure coated surfaces, Lipidure was prepared at 0.5 wt% in 200-proof ethanol. 120 μL of Lipidure solution was then pipetted into each device and allowed to incubate at room temperature for two hours. Afterward, all remaining solution was removed, and devices were stored at 4 °C until further use.
For BSA coated surfaces, devices were coated in a 3% BSA solution at 37 °C overnight. Afterward, the devices were washed with cell culture medium (3* 5 min) before cell-seeding. BSA had been diluted in DI water and sterile filtered before use.
4
Lipidure 3D Culture Plate Preparation
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Lipidure TM (NOF Corporation, Cat. #CMS206; 400 μL) solution (50 mg in 10 mL absolute ethanol) was placed into each well of the 3D culture plate (ElplasiaTM, Kuraray Co., Ltd., Cat #RB 500 400 NA 24) and was allowed to sit for 2 min. The Lipidure TM solution was aspirated out and the plate was dried for 3 h. After drying, 400 μL of PBS was placed in each well and the plate was centrifuged at 2000 g for 15 min at room temperature. The plate was observed under the microscope to ensure that there were no bubbles in the wells. The PBS was then discarded and the wells were washed twice with 400 μL of PBS. The plates were then stored in the cell culture incubator until use.
5
3D Culture of Amniotic Epithelial Cells with TCQA
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The hAECs were cultured in 3D system. Firstly, a solution of 50 mg Lipidure TM (NOF Corporation, Tokyo, Japan) dissolved in 10 ml 100% ethanol was added to the 3D culture plate (Kuraray Co, Tokyo, Japan) for 2 min. After removing the solution and a 3 h drying, 400 μl PBS was placed in each well and the plate was centrifuged for 15 min at Room Temperature (RT), and checked under the microscope to ensure that there was no bubble formation. The PBS was then discarded and a washing with new PBS solution was conducted twice and the plates were then ready for 3D culture.
The cells were seeded at a density of 1 × 106 in Placenta Basal Epithelial Cell Medium into each well of the previously prepared culture plates to ensure the spheroids formation. Day 0 control (D0 control) samples were collected before adding the treatment to the cells. The cells were maintained for one-week culture with changing the medium with 0 and 20 µM TCQA every 48 h. At the end of the week, we established two groups: the treatment (D7 TCQA) and control (D7 control).
The cells were seeded at a density of 1 × 106 in Placenta Basal Epithelial Cell Medium into each well of the previously prepared culture plates to ensure the spheroids formation. Day 0 control (D0 control) samples were collected before adding the treatment to the cells. The cells were maintained for one-week culture with changing the medium with 0 and 20 µM TCQA every 48 h. At the end of the week, we established two groups: the treatment (D7 TCQA) and control (D7 control).
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