Three-dimensional culture of Huh7 cells.
Huh7 cells (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Biological Industries), 100 u/ml penicillin, 100 u/ml streptomycin (Thermo Fisher Scientific, Cat.No.10378016). All cells were cultured at 37℃ in a humidified atmosphere with 5% CO2 and were in the logarithmic growth phase upon initiation of the experiments. 0.25% trypsin-EDTA solution were used for digestion, and cell suspension was centrifuged at 350 × g for 5 min. For 3D culture, cells were culture in ultra-low attachment plates Corning® Spheroid Microplates (Cat. No. 4515) to form spheroids, and five thousand cells were seeded in per well. Changed the medium every other day (using the pipette tip to lightly stick to the inner wall of the hole, so as not to destroy the complete structure of spheroids).
Scanning electron microscope (SEM) analysis of 3D spheroids and monolayer cells.
The protocol of SEM sample preparations reported by Murtey et al was used in this study . Briefly, (1) dehydration and drying: 3D spheroids and cells grown on coverslips (2D) were fixed with 2.5% glutaraldehyde. Continuously change the ethanol from low to high concentrations (35%, 50%, 75%, 95%, 100%) in order to replace the water in samples. For further dehydration and drying, samples were transferred to hexamethyldisilazane (HMDS) in a fume hood overnight. (2) Metal sputtering: samples were mounted onto an SEM sample stub with double-sided sticky tape. Sputter the sample with about 10 nm of gold before viewing it in the SEM. The microscopic examination was performed under the scanning electron microscope (JEOL, HITACHI-S-4800).
Spheroids and cells grown on coverslips were fixed with paraformaldehyde (4%) for 15 min and then washed with PBS. Cells grown on coverslips and spheroids were blocked in buffer (1ⅹ PBS / 5% regular serum / 0.3% Triton™ⅹ-100) at room temperature for 60 mins. Primary antibody E-Cadherin (Cell Signaling Technology, (4A2) Mouse mAb #14472, 1:50) was incubated at 4°C overnight. The next day, fluorescent substance labeled secondary antibody (Abcam, ab150116, 1:200) was incubated for 2 h at room temperature in dark according to the manufacturer’s protocol. Nuclei were counterstained with DAPI (SIGMA, 1:100). 3D spheroids were put into OCT (optimal cutting temperature compound) embedding agent for frozen section processing after immunofluorescence staining. Confocal fluorescence images were recorded on a Leica-SP8 with a 63× immersion objective.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
Cells were cultured for 10 days and collected to extract total RNA with an RNAprep Pure Cell Kit (TIANGEN, Beijing, China) according to the manufacturer's instructions. To determine the mRNA expression level in different culture methods, cDNA was synthesized with a PrimeScript RT reagent Kit (TAKARA, Dalian, China). Quantitative RT-PCR was performed using a SYBRB® Premix Ex TaqTM Kit (TAKARA, Dalian, China) on a CFX96TM Real-Time System (BIO-RAD, United States). The sequence of qPCR primer was shown in Table 1. The gene expression level relative to β-actin was calculated by the 2-ΔΔCt method.
After the cells were collected, RIPA buffer was used to prepare cell lysis buffer, then cells were incubated on ice for 30 min to fully lyse. protein concentration was measured using the BCA assay kit from Thermo Fisher Scientific. 30 ug protein sample were loaded on sodium dodecyl sulfate polyacrylamide gels and started to electrophoresis, then transferred to PVDF membrane (ice bath during electrophoresis). The blotted PVDF membrane was incubated and blocked with 5% BSA solution at room temperature for 1 h. Incubated the E-cadherin (Cell Signaling Technology, 1:1000) with the PVDF membrane at 4°C overnight. The peroxidase-labeled secondary antibody was diluted with PBST, the PVDF membrane was incubated for 1 h at room temperature, and then washed with PBST. Added super-sensitive developer dropwise to the PVDF membrane for protein development. The relative protein expression levels were normalized to that of β‐actin.
Drug sensitivity test.
Huh7 cells were seeded into 96-well spheroid microplates (Cat.No.4515) and 96-well monolayer plates at 5000 cells/well. For 3D culture, cells were cultured for 24-48 h and were observed under a phase-contrast microscope (OLYMPUS, Cat. No. IX71) to confirm that they had gathered into spheroids in each well. Sorafenib was prepared as a storage solution with DMSO (SIGMA, Cat. No. D2650-100ML) and stored at -20℃. The storage solution was diluted with medium, and the final concentration of DMSO in the working solution was 0.1%. Different concentrations of Sorafenib (650 umol/l, 280 umol/l, 140 umol/l, 70 umol/l, 35 umol/l, 17.5 umol/l, 8.75 umol/L) were added to 3D spheroids and 2D monolayers. A blank control without cells (complete medium with 0.1% DMSO), a positive control (doxorubicin, 900 umol/l), and a negative control without the drug were set to control error range. After 48 h, added CCK8 detection reagent (Yeasen Biotechnology, Shanghai) and measured cell viability according to the manufacturer’s instructions. The absorbance of each well was measured at the wavelength D (450-620) nm. In this study, the concept of Z’-factor was used to ensure the detection efficiency within the same number of test repetitions, and data can be used only if 0.5 ≤ Z’- factor ≤ 1. The formula for cell viability and Z’-factor were as follows:
Where δ is the standard deviation of the average, μ is the average, p is positive, n is negative.
All data were statistically analyzed using GraphPad Prism 7.0 software, and the statistical results were expressed as mean ± standard deviation (x ± s). In the drug sensitivity experiment, the dose curve was fitted using the 3PLFT model (when the top value ≥ 120%, fixed the top value to 100%; when the bottoms value was ≤ -20%, fix the bottom to 0%). Comparison between 2D monolayers and 3D spheroids were calculated using independent samples two-tailed t-test. The results of Western blot were analyzed by using Adobe Photoshop CC image analysis software to analyze the gray value. Multiple comparisons were used for comparison of given two groups after two-way ANOVA test. P < 0.05 was considered as significant difference.