Maddaly Ravi

3D cell culture systems: advantages and applications.

Cell cultures are important material of study for the variety of advantages that they offer. Both established continuous cell lines and primary cell cultures continue to be invaluable for basic research and for direct applications. Technological advancements are necessary to address emerging complex challenges and the way cells are cultured in vitro is an area of intense activity. One important advancement in cell culture techniques has been the introduction of three dimensional culture systems. This area is one of the fastest growing experimental approaches in life sciences. Augmented with advancements in cell imaging and analytical systems, as well as the applications of new scaffolds and matrices, cells have been increasingly grown as three dimensional models. Such cultures have proven to be closer to in vivo natural systems, thus proving to be useful material for many applications. Here, we review the three dimensional way of culturing cells, their advantages, the scaffolds and matrices currently available, and the applications of such cultures in major areas of life sciences. J. Cell. Physiol. 230: 16–26, 2015.

Contributions of 3D Cell Cultures for Cancer Research

Cancer cell lines have contributed immensely in understanding the complex physiology of cancers. They are excellent material for studies as they offer homogenous samples without individual variations and can be utilised with ease and flexibility. Also, the number of assays and end‐points one can study is almost limitless; with the advantage of improvising, modifying or altering several variables and methods. Literally, a new dimension to cancer research has been achieved by the advent of 3Dimensional (3D) cell culture techniques. This approach increased many folds the ways in which cancer cell lines can be utilised for understanding complex cancer biology. 3D cell culture techniques are now the preferred way of using cancer cell lines to bridge the gap between the ‘absolute in vitro’ and ‘true in vivo’. The aspects of cancer biology that 3D cell culture systems have contributed include morphology, microenvironment, gene and protein expression, invasion/migration/metastasis, angiogenesis, tumour metabolism and drug discovery, testing chemotherapeutic agents, adaptive responses and cancer stem cells. We present here, a comprehensive review on the applications of 3D cell culture systems for these aspects of cancers. J. Cell. Physiol. 232: 2679–2697, 2017.

Electrospun polycaprolactone matrices with tensile properties suitable for soft tissue engineering

The extracellular environment is a complex network of functional and structural components that impart chemical and mechanical stimuli that affect cellular function and fate. Cell differentiation on three dimensional scaffolds is also determined by the modulus of the substrate. Electrospun PCL nanofibers, which mimic the extra cellular matrix, have been developed with a wide variety of solvents and their combinations. The various studies have revealed that the solvents used influence the physical and mechanical properties, resulting in scaffolds with Youngs modulus in the range of 1.8–15.4 MPa, more suitable for engineering of hard tissue like bone. The current study describes the use of benign binary solvent-generated fibrous scaffolds with a Youngs modulus of 36.05 ± 13.08 kPa, which is almost 50 times lower than that of scaffolds derived from the commonly used solvents, characterized with myoblast, which can be further explored for applications in muscle and soft tissue engineering.

Differences of SiHa (human cancer of cervix) and BMG-1 (brain glioma) cell lines as 2D and 3D cultures.

Cell cultures have seen much progress in the numbers available cell lines, their applications and culture techniques. Three dimensional cultures and co‐cultures are examples of strategies that bring in vitro conditions closer to natural in vivo systems. We describe here, the formation of cell aggregates in three‐dimensional conditions for the cell lines SiHa and BMG‐1 utilizing agarose hydrogels. The optimal conditions for best aggregate formation were identified and the culture phases for the cell lines as monolayers and as aggregates were compared. Differences in protein profiles, susceptibility to a genotoxic drug and the antigenic properties of the protein extracts of the two cell lines, as can be induced by their aggregate formation were studied. The results from the four approaches indicate the usefulness of culturing cells as aggregates. Such systems using simple material and methods offer us an efficient way of utilizing cell lines for a variety of applications. J. Cell. Physiol. 229: 127–131, 2014.

A Rapid Biodosimetric Technique at the Human Glycophorin-A Locus

Abstract For risk assessment, it is necessary to evaluate the dose of human exposure to a mutagen. Environmental genotoxic factors such as Ionizing Radiations induce dose-dependent somatic mutations. Recent studies support the somatic-mutation theory of carcinogenesis. Mutations at the Glycophorin – A (GPA) loci have been studied for establishment of mutagen exposure particularly at a population level and also to estimate the cumulative dose responses. GPA assay provides lifetime biological dosimetry for exposure to radiation as the mutations are accumulated in the long lived hematopoietic stem cells of the bone marrow. Here we present a novel technique, the RS-1 Assay in quantifying the GPA mutations and its role in biodosimetry and risk assessment.

Culture phases, cytotoxicity and protein expressions of agarose hydrogel induced Sp2/0, A549, MCF-7 cell line 3D cultures

Advancements in cell cultures are occurring at a rapid pace, an important direction is culturing cells in 3D conditions. We demonstrate the usefulness of agarose hydrogels in obtaining 3 dimensional aggregates of three cell lines, A549, MCF-7 and Sp2/0. The differences in culture phases, susceptibility to cisplatin-induced cytotoxicity are studied. Also, the 3D aggregates of the three cell lines were reverted into 2D cultures and the protein profile differences among the 2D, 3D and revert cultures were studied. The analysis of protein profile differences using UniProt data base further augment the usefulness of agarose hydrogels for obtaining 3D cell cultures.

Optimizing Premature Chromosome Condensation (PCC) of Human Lymphocytes by Somatic Cell Hybridization to Study Primary DNA Damages

Abstract Cytogenetic tools are important in understanding the health effects of various chemical and physical agents implicated in human health. Several biodosimetric and cytogenetic techniques help in the assessment of the genotoxic effects of therapeutic drugs and these become essential for human health care management. There exist methods by which genotoxicity can be identified and quantified employing interphase condensed DNA. Premature Chromosome Condensation (PCC) can be achieved by Somatic Cell Fusions and can result in obtaining condensed DNA into “near chromosome like clarity” and the same can be utilized to check for common end-points such as DNA “breaks” and “gaps”, as can be induced by genotoxic drugs. PCC in conjunction with FISH can also be an added advantage for the same. We present here an efficient method to obtain high quality PCC spreads of human lymphocytes, which can be analyzed with relative simplicity for genotoxic monitoring. Mitotic CHO cells were used as the fusion partners followed by incubation and the processing of cells for analysis. Image capturing and analysis was done after fixing and casting cells. The key factors such as the ratio of the fusion partners, fusion and processing protocols are optimized and presented. Although PCC cannot replace conventional lymphocyte culture techniques completely, it can be efficiently utilized for genotoxic monitoring with advantages such as the considerable reduction in time required for processing and analysis of samples. This will also eliminate the chances of genotoxicity underestimation due to the inherent DNA damage repair capacity of human lymphocytes.

Human Brain Malignant Glioma (BMG-1) 3D Aggregate Morphology and Screening for Cytotoxicity and Anti-Proliferative Effects.

Two interesting aspects of cell lines grown in 3 Dimensional (3D) conditions are their distinct morphology and production of extracellular matrix (ECM). Also, it is known that 3D aggregates have different susceptibilities to damage‐inducing agents compared to their 2D monolayer counterparts. We describe the effect of ECM on 3D aggregate morphology, the effect of cisplatin, bleomycin, and UV on the 3D aggregates and 2 Dimensional (2D) monolayers of the BMG‐1 cell line. We also present a rapid method for analyzing cytotoxicity and anti‐proliferative effects of 3D aggregates in 96‐well plates. We utilized a single‐step protocol using the dye resazurin. BMG‐1 cells formed floating aggregates on 1% agarose hydrogels. The extent of ECM formed by them was dependent on number of cells seeded irrespective of the seeding density, which in turn directed the 3D aggregate compactness. The 3D aggregates were less susceptible to cisplatin and UV‐induced cytotoxicity compared to 2D counterparts. The IC50 value of cisplatin was elevated at 210 μg/ml for the aggregates compared to 170 μg/ml for the monolayers. Exposure to UV for 0, 10, 20, and 30 min gave inhibition values of 2.98%, 8%, 22.99%, and 31.8% for the aggregates as compared to 3.06%, 7.5%, 39.4%, and 46.7% for the monolayers. While bleomycin‐induced effects were unapparent when analyzed by vital staining for the doses used, the rapid, single‐step method in 96‐well plates was able to provide a dose‐response for cytotoxicity and anti‐proliferative effects. Also, comparative analysis of results obtained from vital staining and the single‐step method demonstrates the reliability of the assay described. J. Cell. Physiol. 232: 685–690, 2017.

Protein Expression Differences of 2‐Dimensional and Progressive 3‐Dimensional Cell Cultures of Non‐Small‐Cell‐Lung‐Cancer Cell Line H460

Non‐small‐cell‐lung‐cancer (NSCLC) constitutes about 75–80% of lung cancers. The challenge to tackle cancers is in early diagnosis and arriving at safer therapeutic options. In vitro studies using cancer cell lines continue to contribute significantly in understanding cancers. Cell culture methods have evolved and the recent developments in 3 dimensional (3D) cell cultures are inducing greater resemblance of the in vitro cultured cells with in vivo conditions. In this study, we established 3D aggregates of H460 cell line on agarose hydrogels and studied the protein expression differences among cells grown as monolayers (2D) and the progressively developing 3D aggregates from days 2 to 10. Analysis included matching of those proteins expressed by the developing aggregates and the available literature on progressing tumors in vivo. J. Cell. Biochem. 118: 1648–1652, 2017.

Glycophorin-A Allelic Distribution Frequency in South Indian Population

Abstract Lansteiner and Levine described a blood group system in 1927 called the MN system. According to thistheory, three possible genotypes MM, MN and NN occur. The antigenic determinant of human MN system onerythrocytes is a sialogylcoprotein with a chain of 151 amino acids. It is regarded that about 50% of the populationwill express the MN heterozygous genotype as this is based on the Mendalian inheritance. Studies on the distributionfrequencies of blood groups and subgroups of a population is one way to understand the population’s genetic makeupand susceptibility to disease. Blood samples were collected from 413 south Indian volunteers who were chosenrandomly for the blood grouping of MM, NN or MN types. The data was recorded and the gene frequencies for theMN, NN and MM types were calculated using the standard statistical analysis. The results show that approximately50% of the population is heterozygous for MN and fits into Hardy-Weinberg equilibrium.