In the last four decades, several researchers worldwide have routinely and meticulously exercised cell culture experiments in two-dimensional (2D) platforms. Using traditionally existing 2D models, the therapeutic efficacy of drugs has been inappropriately validated due to the failure in generating the precise therapeutic response. Fortunately, a 3D model addresses the foregoing limitations by recapitulating the in vivo environment. In this context, one has to contemplate the design of an appropriate scaffold for favoring the organization of cell microenvironment. Instituting pertinent model on the platter will pave way for a precise mimicking of in vivo conditions. It is because animal cells in scaffolds oblige spontaneous formation of 3D colonies that molecularly, phenotypically, and histologically resemble the native environment. The 3D culture provides insight into the biochemical aspects of cell-cell communication, plasticity, cell division, cytoskeletal reorganization, signaling mechanisms, differentiation, and cell death. Focusing on these criteria, this paper discusses in detail, the diversification of polymeric scaffolds based on their available resources. The paper also reviews the well-founded and latest techniques of scaffold fabrication, and their applications pertaining to tissue engineering, drug screening, and tumor model development.

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Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three-dimensional cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of recapitulation of in vivo tumors. In this aspect, expression of six housekeeping genes (HKG’s): glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13)) during the monolayer culture (two-dimensional), and alginate-carboxymethylcellulose scaffold based three-dimensional (3D) cell culture conditioned up to 21 days was analysed for hepatocellular carcinoma (Huh-7) cell line. The real-time gene expression using RT-qPCR of HCC spheroids in 3D culture were analyzed by determining the primer efficiency, melting curve and quantification cycle analysis of the selected candidate HKG’s. Further, RT-qPCR data were validated using analysis softwares i.e., geNorm and NormFinder for statistical significance. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates and considered them as potential internal controls during varying cell culture conditions.

Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three-dimensional cell culture. Especially, gene pro ling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of recapitulation of in vivo tumors. In this aspect, expression of six housekeeping genes (HKG's): glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13)) during the monolayer culture (two-dimensional), and alginatecarboxymethylcellulose scaffold based three-dimensional (3D) cell culture conditioned up to 21 days was analyzed for hepatocellular carcinoma (Huh-7) cell line. The real-time gene expression using RT-qPCR of HCC spheroids in 3D culture were analyzed by determining the primer e ciency, melting curve and quanti cation cycle analysis of the selected candidate HKG's. Further, RT-qPCR data were validated using analysis softwares i.e., geNorm and NormFinder for statistical signi cance. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates and considered them as potential internal controls during varying cell culture conditions.