Hepatocellular carcinoma cell line (Huh-7) (Cell repository, National Centre for Cell Science, Pune, India), DMEM complete growth media (D5796, Sigma Aldrich, USA), fetal bovine serum (F2442, Sigma Aldrich, USA), trypsin-EDTA (T4049, Sigma Aldrich, USA), antibiotic-antimycotic (P4333, Sigma Aldrich, USA), gentamycin sulphate (G13970, Sigma Aldrich, USA), phosphate buffered saline (P3813, Sigma Aldrich, USA), acridine orange (A6014, Sigma Aldrich, USA), TRIzol reagent (162710, Invitrogen, USA), chloroform (AS039, Himedia, India), analytical grade ethanol (Hayman, UK), isopropanol (00270, Lobachemie, India), and quantitative real-time PCR kit (RR086A, TaKaRa, Japan) were the materials and reagents used in this study.
Huh-7 cells were grown in DMEM complete growth media supplemented with 10% fetal bovine serum, L-Glutamine, antibiotic-antimycotic solution and gentamycin sulphate. Cells were grown under optimum culture conditions as described previously . Cells were dissociated by using 0.25% Trypsin-EDTA solution and cell pellet was collected by centrifugation before seeding in 2D and 3D cultures. The Huh-7 cells were allowed to grow in a 5% CO2 incubator maintained at 37 °C.
3D cell culture
Three-dimensional culture of Huh-7 were prepared using alginate-CMC cryogel based scaffold. Briefly, the cells were re-suspended in DMEM complete growth media with a final cell count of 1×105 cells/ml. Meanwhile, the polymer solution was prepared by dissolving alginate and CMC in 1:1 ratio with double distilled water. The prepared solution was sterilized and stored at 4°C. Likewise, 2% calcium chloride was prepared in autoclaved water and syringe filtered (0.2 µm pore size) to pass through a nozzle. Using Nunc’s 35×10 mm cell culture dish, AL-CMC bioink solution was added and the lid was closed and tightly sealed with a parafilm (Bemis, USA). After freezing the polymer solution at -80°C for 24 h, instant thawing with calcium chloride was performed under sterile conditions. The fabricated scaffold was washed thrice with 1X PBS and DMEM complete growth media was added prior to incubation at 37°C for enhancing growth conditions of the spheroid .
Nucleic acid staining
The utilized DMEM media was aspirated from the Huh-7 cells grown in 2D culture and spheroid containing scaffold within the culture dish. The scaffold matrix was dissolved using algimatrix dissolving solution (A1134001-Gibco, USA). Live staining of Huh-7 cells grown in 2D and alginate-CMC based 3D cell culture was performed using acridine orange dye (Sigma A6014, USA). The utilized DMEM media was aspirated from the scaffold within the culture dish and stained with 500 µl of 30 µM acridine orange stain for 15-20 min. The spheroids were washed thrice with 1X PBS gently and observed under a fluorescent microscope (BX53-Olympus, Japan) [16, 17].
Total RNA was extracted from Huh-7 cells grown in a polystyrene flask (2D culture) and alginate-CMC (3D culture) at different time intervals (7, 14, and 21 days). In 3D culture, dissociation of the matrix was performed using algimatrix dissolving solution prior to RNA extraction. RNA extraction was performed using TRIzol reagent (Invitrogen, USA) following the manufacturer's instructions. The quality and quantity of RNA was estimated using nanophotometer (N60-Implen, USA) [18, 19].
Designing of primers
The primers for HKG’s were designed using real time PCR (TaqMan) primer designing software, GenScript (Piscataway, NJ) (https://www.genscript.com/tools/real-time-pcr-taqman-primer-design-tool). The sequences for all the gene sequences were retrieved from GenBank database (www.ncbi.nlm.nih.gov). The exon boundaries in eukaryotic gene code were defined and submitted in raw format. The software provided an advantage to pick primer/probe crossing exon junction. The criteria for choosing an appropriate primer were based on a length of 18-21 bases and an amplicon size of up to 200 base pairs. Further, the obtained primer design was matched with a model organism using BLAST analysis. The gene description, primer sequences and amplicon sizes used in this study are represented in Table 1.
Quantification of the housekeeping gene
Real time PCR was performed to quantify the level of housekeeping gene expression in hepatocellular carcinoma cell line. One-step RT-qPCR assay was performed with the final volume of 25 μl using PrimeScript one-step RT-qPCR kit (TaKaRa Biotech, Japan) as per the manufacturer’s instructions. The reaction mixture contained 12.5 µl of 2x SYBR green master mixes (RR0086A, TaKaRa, Japan), 300 nM forward and reverse primers, and 1µl of total RNA (50 ng/µl). The reverse transcription was conditioned at a temperature of 42°C for 5 minutes, and 95°C for 10 seconds during one cycle followed by a PCR reaction at 95°C for 5s, while the annealing temperature was ranging from 55-64°C for 20 seconds (40 cycles). A melting curve analysis was performed by heating at 95°C for 15 seconds, 60°C for 1 minute and finally 95°C for 15 seconds to verify the PCR product. The reaction was performed using CFX96- real time system (BioRad, USA). Based on the quantification/threshold cycle value (Ct) obtained, the expression stability of HKG’s was assessed.
The primer efficiency applied for the target amplification was evaluated in serial dilutions (1/10, 1/100, 1/1000, 1/10000, and 100000) using triplicates. Following quantitative real time PCR (RT-qPCR), the threshold cycle (Ct) values obtained at different RNA concentrations (0.01-100 ng/µl) was linearly plotted to determine the slope and correlation coefficient (R2) . After determining the corresponding values, primer efficiencies were calculated by the following equation, Eq. (1).
Analysis of gene expression
The stability of all the HKG’s obtained after RT-qPCR were analysed using two softwares: NormFinder software, version 0.9 (Trial version) and geNorm algorithm integrated in qbaseplus software, version 2.4 (Trial version). Both software integrated the raw threshold cycle values obtained specifically using CFX96-BIO-RAD real time system (BioRad, USA) in Microsoft excel format. The NormFinder software calculated the stability index by applying a statistical approach of analysis of variance (ANOVA) between the expression values of all housekeeping genes that were logarithmically transformed (Log Ct). The proportion of intra and inter group variation on overall variation was taken into consideration while deriving a normalization factor (NF). In situations where no optimal single reference gene was found, application of multiple reference genes comes into the scenario with a rationale: variation in the averaging of multiple genes was smaller than a variation among inter and intra subgroups of individual housekeeping genes. In this study, the geometric mean value of sample triplicate was applied for stability measure of candidate HKG during 2D, and 3D (7, 14, and 21 days) culture, as different time points were considered as grouping variables . The statistical framework ranked candidate housekeeping genes enabling the evaluation of systemic error introduced during the period of gene normalization .
For further evaluation of housekeeping genes, specifically during different time intervals of 3D culture, expression analysis software geNorm was applied, whose algorithm was based on the criterion that two ideal housekeeping genes have a minimal expression ratio across the investigated sample set regardless of cell type or treatment condition. The algorithm of the geNorm software was conditioned to calculate the standard deviation of the logarithmically transformed expression ratio across 3D culture samples for a particular gene relative to other genes under investigation. General specifications for the application of geNorm software required a minimum of three reference genes and two sample types. The software statistically applied a program to analyse the average stability measure (M) of each housekeeping gene with respect to the expression of other housekeeping genes and ranked them based on stepwise exclusion method. Further, geNorm software eliminated the genes with the highest M value from the panel and recalculated new M values for the remaining genes until reaching the last two genes with the smallest M value. The lower the M value, the higher stability was considered for the candidate gene [13, 22]. Hence, the most stable housekeeping gene was considered to be a gene with the lowest M value and the mathematical model, thus, evaluated the stability of the housekeeping genes by a robust measure for further confirmation.