Preparation of the 96 pillar/well plates
The 96-pillar/well plate was manufactured by plastic injection molding and is a robust and flexible platform for mammalian cell cultures, enzymatic reactions, viral infections, and compound screenings (Fig. 1). The 96-pillar plate is made of polystyrene (PS) and contains 96-pillars (with a 2 mm pillar diameter and 9 mm pillar-to-pillar distance). The 96-pillar plate was coated with poly-L-lysine (PLL) solution to support Matrigel attachment on the hydrophilic pillar surface. The 96-well plate has 96 complementary wells (with a 7 mm well diameter and 9 mm well-to-well distance) and it can be combined (or “stamped”) with a 96-pillar plate to conduct the 3D cell culture and the drug efficacy test. Plastic molding was performed with an injection molder (Sodic Plustech Inc., IL).
Experimental Procedure of High-Throughput Drug Screening
For high-throughput screening and implementation of 3D cell culture models, 1.5 µL volume of mixtures containing approximately 4000 cells and 50% Matrigel were automatically dispensed onto a 96-pillar plate surface using an ASFA™ Spotter ST (Medical & Bio Device, South Korea). The ASFA™ Spotter ST uses a solenoid valve (The Lee Company, USA) to dispense 1.5 µL droplets of the cell–Matrigel mixture on the 96-pillar plate surface.
After dispensing the cells, two types of 3D cell culture models were implemented (DSM and ASM), as shown in (Fig. 2-a). To implement a DSM model, the icing and gelation steps continued with the pillar surface facing upward (Figs. 2-b and -c). To implement the ASM model, the icing and gelation steps continued with the pillar surface facing down (Figs. 2-d and -e). The 96-pillar plate was combined with the pre-warming culture medium and subjected to an additional stabilization for 10 minutes (Fig. 2-f). The 96-pillar plate containing HCC cells in Matrigel was sandwiched (or “stamped”) with the 96-well plate for 3D cell culture (Fig. 2-g). Each 96 well plate was comprised of 96 wells, each 7 mm in diameter, which contained 200uL of cell culture medium. After the cell-Matrigel mixture was stabilized using each gelation method, 3D cells were observed with an optical microscope and appeared to be evenly spread over the pillar surface in the DSM model. In the ASM model, it was confirmed that cells were aggregated in the center of the pillar surface, as shown in (Fig. 2-k).
The cells needed to be stabilized in the Matrigel spots before being treated with drugs. After stabilizing cells, cell viability was checked by 3d live cell staining using Calcein AM dye (Invitrogen, Carlsbad, CA). Majority of cells were alive based on the level of green fluorescence as shown in (Fig. 2-j). To analyze the efficacy of all six drugs, we designed a dose-response curve (DRC). The 96-well plate was divided into 7 regions. Each region was composed of a 3 × 7 well array corresponding to six different drug doses (including one control) and their triplicates. After treatment with the six different drugs for a period of six days, drug response was analyzed through viability quantification of 3d live cell staining images. As shown in (Fig. 2-j), the area value of 3D live cells differed depending on the drug response.
Experimental Procedure for Image Scanning and Data analysis
An automatic optical fluorescence scanner (ASFA™ Scanner HE, Medical & Bio Device, South Korea) was used to measure green fluorescence intensities (excitation/emission, 494/517 nm to lasers) on the 96-pillar surface using an 8-bit code among the RGB codes (0–255), as shown in (Fig. 2-j). The ASFA Ez SW (Medical & Bio Device) was used to calculate the total and average green areas of the scanned images of the 3D cultured cells from each 3D cell culture model. To determine drug efficacy, the dose-response curves were plotted using the normalized 3D cell viability values according to the dose of the drugs (GraphPad Prism 8). The half-maximal inhibitory concentration (IC50) values were calculated automatically in the XY analysis completed with the GraphPad Prism 8 software. (GraphPad Software, CA)
Human hepatocellular carcinoma cell lines Hep3B and HepG2 were purchased from the Korean Cell Line Bank (Seoul, South Korea). Both Hep3B and HepG2 were cultured in DMEM medium (Gibco, Grand Island, NY) with 1 µg/mL of puromycin; both media were supplemented with 100 µg/mL of streptomycin, 100 units/mL of penicillin, 250 ng/mL of amphotericin B, and 10% fetal bovine serum. Cell lines were maintained at 37°C in a 5% CO2-humidified atmosphere and passaged every four days. Typically, we use the Hep3B and HepG2 cell lines under 20 passages after thawing the frozen cell stock. Under 20 passages, we observed that the Hep3B and HepG2 cell lines easily formed 3D cells in 50% Matrigel on the chip platform.
Two 3D-HCC cancer in-vitro models (DSM and ASM) were used in drug sensitivity assays to identify drug sensitivity change according to each model. The drugs most commonly used for HCC were selected. Sorafenib (S7397), Cabozantinib (S1119), Lenvatinib (S1164), Regorafenib (S1178), 5-Fluorouracil (5-FU) (S1209), and Doxorubicin (DOX) (S1208) were purchased from Selleckchem (Houston, TX). Drugs were solved in a stock solution of dimethyl sulfoxide (DMSO, 100 mM).
3D-Cell Viability Assay
Hep3B and HepG2 cells were seeded in a 96-pillar tray at a density of 4000 cells per pillar in triplicate for each treatment. One day after seeding, cells were treated with drugs in a two-fold and seven-dose point serial dilution from 100 µM to 3.12 µM. After 6 days of incubation at 37°C in a 5%-CO2 humidified incubator, cell viability was determined using an adenosine triphosphate (ATP) monitoring system based on firefly luciferase (CellTiter-Glo® 3D Cell Viability Assay, Promega, Madison, WI) and 3D-live cell staining (Calcein AM, Invitrogen, Carlsbad, CA), according to manufacturer’s protocol. Briefly, the assay mixture was prepared in an ATP monitoring system by adding 20 µL of CellTiter-Glo® 3D reagent into 60 µL of medium per well. Cells were lysed with an assay mixture by shaking for 5 min, followed by incubation for 25 min at room temperature. Viable cells were estimated using the SpectraMax iD3 Reader (Molecular Devices LLC, San Jose, CA). In the 3D live-cell staining, the staining solution was prepared by adding 1 µL of Calcein AM into 7 mL of DMEM medium. Cells were incubated with staining solution for 1 hour at 37°C in a 5% CO2-humidified atmosphere. Live cell images were acquired using an automatic fluorescence microscope scanner (ASTA Scanner™, Medical & Bio Decision, South Korea)
Western blot analysis
Total cell lysates were prepared using a cOmplete™ Lysis-M buffer solution (Roche Life Science, Germany) from the Hep3B and HepG2 hepatocellular carcinoma cell lines. Total protein in lysates was quantified using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA), 10 µg of total protein were loaded onto 4–20% Mini-PROTEAN TGX™ Precast Protein Gels (Bio-Rad, Hercules, CA) and transferred onto iBlot® PVDF gel Transfer Stack membranes (Thermofisher Scientific, Korea). The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) at room temperature, and incubated with antibodies against AKT (1:1000, Cell Signaling Technology (CST), #4970), phospho-AKT (p-AKT) (Ser473) (1:1000, CST, #4060), Erk 1/2 (1:1000, CST, #4695), phosphor-Erk (p-Erk) 1/2 (Thr202/Tyr204) (1:1000, CST, #4370), mTOR (1:1000, CST, #2983), phospho-mTOR (p-mTOR) (Ser2448) (1:1000, CST, #5536), E-cadherin (1:1000, CST, #3195), Vimentin (1:1000, Abcam, ab137321), ZO-1 (1:1000, Thermofisher, 339188), Occulidin (1:1000, Thermofisher, 331594), and β-actin (1:5000, CST, #4970) at 4°C overnight. After exposing to horseradish peroxidase (HRP)-conjugated secondary antibodies, proteins were visualized using SuperSignalTM West Pico PLUS (Thermofisher Scientific). Protein bands were visualized using a Chemidoc chemiluminescence imaging system (Bio-rad, Hercules, CA) and quantitative densitometry analysis was performed on the bands of protein detected by immunoblot using Image Lab software (Bio-rad, Hercules, CA) and normalized to β-actin which served as a loading control.
High Content Image Analysis
Cells were fixed in 2% formaldehyde for 40 min and further incubated with 0.25mg/ml NaBH4 for an additional 40 min. Exposure to primary antibodies against E-cadherin (1:250), Fibronectin (1:250), and ZO-1 (1:200) was done at 4°C for 2 days. After blocking non-specific binding using 5% BSA for 1 h, visualization was done using secondary antibodies conjugated with Alexa Fluor 594 (1:1000, Thermo Scientific, A27016). F-actin was stained with rhodamine phalloidin (1:1000, Invitrogen, R415). Samples were counterstained with DAPI (1:1000, Thermo Scientific, H3570) and subjected to confocal microscopy (LSM 780, Carl Zeiss, Oberkochen, Germany).
The expression levels of cytokines and chemokines were analyzed using a Human Cytokine Antibody Array (C5) (RayBiotech, GA). According to the manufacturer’s instructions, antibody-embedded membranes were incubated with 1 ml of conditioned media (CM) at 4°C overnight, followed by incubation with HRP-streptavidin at room temperature. Proteins were then visualized using a Chemidoc chemiluminescence imaging system (Bio-rad, Hercules, CA) and chemiluminescent substrate reagent. The signal intensities were quantified using Image lab software (Bio-rad, Hercules, CA).
Drug Penetration Assay
DOX was used to evaluate drug accumulation in spheroids because of its fluorescent properties. After 1 d of culture, media was replaced with DOX containing media (300 µM). After 12 h of exposure, the 3D cultured cells were washed with PBS before imaging to remove background fluorescence noise. Optical sections were acquired at 10 µm intervals and stacked into a Z-projection from which fluorescence intensity was calculated.