2.1 Preparation and treatment of traditional Chinese medicine
Eight types of TCM, which meet the standard of the 2015 edition of “the Pharmacopoeia of the People's Republic of China”, Bozhou, Anhui, China, were extracted by the same method. Briefly, 30 g of crude TCM was weighed and soaked in deionized water at 40°C for 1 h. The decoction was then boiled twice and concentrated to a final volume of 50 ml. The TCM components were divided into the alcohol-dissolved and alcohol-precipitated parts by the alcohol precipitation method (the alcohol content was more than 80%). The ethanol was then removed by heating the ethanol extract to 80℃ with a constant temperature magnetic stirrer. After evaporation, distilled water was added to make the final volume 6ml, that is, the crude drug concentration of all 8 kinds of traditional Chinese medicine was adjusted to 5g/ml. After filtering and sterilizing, the alcohol-soluble part was collected for subsequent experiments.
2.2 Cell preparation
A549 lung cancer cells were preserved in our laboratory. A stable A549-EGFP cell culture was established as follows. A549 cells at the logarithmic growth phase were prepared into a suspension of 5×104 cells/ml and seeded in six-well plates at 2 ml per well. Next, the supernatant was discarded after 24 h. After that, 10 µl lentivirus (Yeasen; Shanghai, China) at 1×108TU/ml titer was added to 1 ml of enhanced infection solution containing 5 µg/ml polybrene, and the mixture was added to cell well. The mixture was replaced with low-glucose Dulbecco’s modified Eagle medium (DMEM) (Gibco; Thermofisher, USA) containing 10% fetal bovine serum (Gemini; California, USA), 100 µg/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermofisher, USA) after 24 h of infection. On the third day, transfection was observed under a fluorescence microscope (Ts2R-FL; Nikon, Japan). After the cells had overgrown with monolayer, they were digested and seeded into six-well plates (Corning, USA) at a concentration of 1×105 cells/ml. Puromycin was added at a final concentration of 1 μg/ml for screening after incubation at 37°C for 24 h, and the medium was replaced with fresh screening medium every 2 days for a total of 7 days. The surviving cells were amplified and cultured as A549-EGFP cells.
2.3 Preparation of printing materials
Prepare 10 ml of the mixed printing material containing 5% gelatin (Sigma, USA) and 8% sodium alginate (low viscosity; Sigma). The materials were packed into airtight containers and pasteurized. The 3D printing materials were placed at 4°C until use. The study of printed material characterization, see references 1313.
2.4 Lung tumor model was constructed by 3D bio-printing (BioScaffolder, GeSiM, Germany)
A549-EGFP cells or A549 cells and printing materials were used as printing inks, respectively. The final concentration of the cells was 2×106 cells/ml. Under the control of computer-aided design and pneumatic drive, the printing inks were continuously printed out. The scaffold was crosslinked with 5% CaCl2 solution and transformed from sol to gel in a few minutes. Through 3D bio-printing, cells and extracellular matrix materials were arranged in a specific space; thus, lung tumor models were directly assembled and placed into six-well culture plates. Next, an appropriate amount of complete L-DMEM was added to the models, which were then cultured at 37°C in a 5% CO2 incubator (BB150; Thermo, USA).
Printing parameters: the printing needle had a diameter of 0.41 mm and extrusion pressure of 120 kPa. The side length of the scaffold was 5 mm, the layer height was 0.16 mm. Each scaffold had three layers with four bars per layer and 90° corner printing. The heating module is set to keep the printing cylinder at 37℃. The scaffold forming temperature was about 15℃.
The cytotoxicity of the printed scaffold (without cells) was studied in ref. 1313.
There were approximately 1.50×104 cells on each scaffold. On the first day after cell printing, the number of cells on the scaffold in one field of view was calculated by using the image acquisition and analysis software of ImageXpress Microcope (Molecular Devices, USA). The z-axis layer sweep, with one sheet per 60 μm, defines a particle with a fluorescence intensity between 20,000 and 25,000 and size between 30-50 μm as a cell, and calculates the total amount of cells per field. Nine fields of view were observed per scaffold, and the total number of cells on the scaffold = the number of cells per field × 9. The cell viability after printing = the total number of cells on the scaffold/initial number of cells planted × 100%.
2.5 Observation of cell growth on three-dimensional scaffold
To determine whether all cells show the same growth pattern on the scaffold and to provide experimental data for the construction of personalized models, we selected three lines of tumor cells and scaffold materials to construct 3D models, observed the growth of various cells on the scaffold, and compared them with the 2D culture. Three lines of tumor cells, namely A549 (lung cancer cell), HepG2 (hepatoma cell), and SSMC-7721 (hepatoma cell) at the same concentration (2×106 cells/ml) were mixed with the same ratio of sodium alginate/gelatin, printed, and then cultured in vitro in complete L-DMEM for 3 weeks. After staining with calcein-AM (Solarbio; Beijing, China), cell growth on the scaffold was observed under a fluorescence microscope and the effects of cell migration and invasiveness on the growth of the scaffold were preliminarily investigated.
2.6 Drug screening test grouping
Eight TCMs were divided into eight groups, which were then divided into two groups,a 3D-printed lung tumor model group and a 2D-cultured A549 cell group, and five concentrations were set for each group, respectively 1.00, 0.50, 0.10,0.05 and 0 g/ml. Each concentration had three replicate wells. Because of the complexity of TCM ingredients, we repeated the experiment at least three times. All cultures were performed in 96-well plates.
2.7 Screening and detection of A549 cell sensitivity in 2D and 3D cultures
Screening of A549 cell sensitivity to TCM in 2D culture
The MTT method was used to determine the growth and proliferation of A549 cells in a 2D culture. Briefly, A549 cells at 1×104 cells were inoculated in each well of 96-well plates and cultured for 1 day with different concentrations (1.00, 0.50, 0.10, and 0.05 g/ml) of Oldenlandia, Atractylodes, Mylabris, S. barbata, Zedoary, Nigrum, Cremastra, and Prunella solutions diluted in complete medium at 200 mL/well. Each concentration had three replicate wells. Changes in cell morphology were observed by microscopy after 48 h of drug treatment. At the same time, the supernatant was discarded, and 100 µL of 500 µg/ml MTT (Sigma, UAS) was added to each well, which was then incubated at 37°C in the dark for 4 h. The supernatant was discarded and 150 µL/well dimethyl sulfoxide was added to the well. The precipitated crystals were completely dissolved within 10 min by oscillating at room temperature, and the absorbance OD value of each well was detected at a wavelength of 490 nm by a microplate reader (Thermo, USA). From the absorbance, the cell proliferation inhibition rate of TCM at different concentrations and incubation time was calculated. The experiment was repeated four times, and the proliferation inhibition rate IR(%) = (OD value of the untreated group - OD value of the drug treatment group) / OD value of the untreated group × 100%.
Cell apoptosis and cell cycle were detected by flow cytometry (BD FACSCelesta, USA). A549 cells at 1×105cells/ml (without transfection with EGFP) were inoculated into six-well plates at 500 µl/well, and eight TCMs were added after 1 day of culture, with two concentrations for each TCM (1.00 and 0.10 g/ml) and three duplicate wells per concentration. The cells (including floating cells in the supernatant) were collected after 24 h of treatment. The cells were washed twice with Phosphate buffer saline (PBS), stained with annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Biovision, USA), and incubated in the dark for 5 min. The proportion of early apoptotic cells in PI-/FITC+ samples was detected on flow cytometry, and morphological changes after apoptosis were analyzed by the data of forward scatter (FSC) and side scatter (SSC). The data were analyzed by FlowJo (Version 10, BD).
The cells collected as described above were washed with precooled PBS, and then suspended in 1 ml PBS and vortexed. Next, 3 ml precooled anhydrous ethanol was slowly dropped on the suspension to a final concentration of 75%. The cells were fixed overnight at 4°C.The fixed cells were washed with precooled PBS, and an appropriate amount of RNase was added to its working concentration of 50μg/ml, then incubated at 37°C for 30 min. Subsequently, an appropriate amount of PI was added to a final concentration of 50μg/ml, and the cells were stained at 4°C for 30 min in the dark to determine the cell cycle.
Screening of sensitivity to TCM in three-dimensional culture of A549 cells:
The lung tumor model was printed to fit into a 96-well plate (5 mm side length, three layers, four strips in each layer, 0.16 mm in each layer). Each lung tumor model contained approximately 1.25×104 cells (owing to the large cell damage and high rate of cell death during the printing process, the initial concentration of the cells was slightly higher than that of the 2D culture). After 1 day of culture, different concentrations of Oldenlandia, Atractylodes, Mylabris, S. barbata, Zedoary, Nigrum, Cremastra, and Prunella were added to the cells. After 24 and 48 h of culture, changes in cell morphology after drug treatment were observed under a microscope. The state of A549-EGFP cells on the scaffold was observed under a fluorescence microscope and compared with that of cells grown in 2D culture. Surviving and dead cells on the scaffold were observed by Calcein-AM/PI staining of the A549 cells not transfected with EGFP. The inhibition rate of A549 cell proliferation in the 3D culture system was measured by the MTT assay or fluorescence intensity analysis.
2.8 Statistical methods
The SPSS13.0 statistical software package was used for statistical analysis. The experimental data were expressed as x̄±s. Comparisons between groups were conducted, with P<0.05 indicating statistical significance.