Animal Biotechnology

Models in Discovery and Translation

Ashish S. Verma,Anchal Singh

• Publisher: Academic Press
• Year: 2013
• ISBN:
  9780123914347
• Topic: Sciences biologiques
• Subtopic: Zoologie

Animal Biotechnology introduces applications of animal biotechnology and implications for human health and welfare. It begins with an introduction to animal cell cultures and genome sequencing analysis and provides readers with a review of available cell and molecular tools. Topics here include the use of transgenic animal models, tissue engineering, nanobiotechnology, and proteomics. The book then delivers in-depth examples of applications in human health and prospects for the future, including cytogenetics and molecular genetics, xenografts, and treatment of HIV and cancers. All this is complemented by a discussion of the ethical and safety considerations in the field.Animal biotechnology is a broad field encompassing the polarities of fundamental and applied research, including molecular modeling, gene manipulation, development of diagnostics and vaccines, and manipulation of tissue. Given the tools that are currently available and the translational potential for these studies, animal biotechnology has become one of the most essential subjects for those studying life sciences.

• Highlights the latest biomedical applications of genetically modified and cloned animals with a focus on cancer and infectious diseases
• Provides firsthand accounts of the use of biotechnology tools, including molecular markers, stem cells, and tissue engineering

Section II

Animal Biotechnology: Tools and Techniques

Outline

Chapter 11 Multicellular Spheroid

Chapter 12 Animal Tissue Culture

Chapter 13 Concepts of Tissue Engineering

Chapter 14 Nanotechnology and Its Applications to Animal Biotechnology

Chapter 15 Antibodies

Chapter 16 Molecular Markers

Chapter 17 Gene Expression

Chapter 18 Ribotyping

Chapter 19 Next Generation Sequencing and Its Applications

Chapter 20 Biomolecular Display Technology

Chapter 21 In Silico Models

Chapter 11

Multicellular Spheroid

3-D Tissue Culture Model for Cancer Research

Suchit Khanna, Anant Narayan Bhatt and Bilikere S. Dwarakanath, Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Delhi, India

Abstract

This section highlights the rationale, potential, and implementation of multicellular tumor spheroids in cancer research. A “multicellular tumor spheroid” (MCTS) is a three-dimensional in vitro model system that bridges the gap between two-dimensional monolayer cell cultures and an in vivo tumor tissue model systems. Compared to monolayer cultures, MCTS resembles tumor tissue in terms of structural and functional properties and is suitable for studying metastasis, invasion, and therapeutic screening of drugs. Spheroid models are helpful in accelerating translational research in cancer biology and tissue engineering.

Keywords

Spheroid; Multicellular Tumor Spheroid (MCTS); Radiation Response; Chemotherapeutics Drugs; Photodynamic Therapy (PDT); Anti-Angiogenesis Therapy Immunotherapy

Chapter Outline

Summary

What You Can Expect to Know

History and Methods

Introduction

Multicellular Tumor Spheroids

Historical Facts Towards the Development of Tissue Culture Technology from 2-D and 3-D Cultures

An Example where 3-D Culture is More Beneficial over 2-D Culture

Techniques for the Generation of Spheroids

Hanging-Drop Method

Liquid Overlay Method

Microfabricated Microstructures Method

Rotatory Flask Methods

Surface Modification-Based Methods

Chip-Based Spheroid Generation

Protocol for Tumor Spheroid Generation

Drug Treatment Protocol

Parameters to Monitor Drug Efficacy in 3-D Cultures

Radiation Response of Tumor Cells and its Modifications

Response to Anticancer Drugs

Response to Photodynamic Therapy

Response to Anti-Angiogenesis Therapeutics

Evaluation of Response to Immunotherapy

Application of 3-D Cultures in Other Diseases

Conclusions

Ethical Issues

Translational Significance

World Wide Web Resources

References

Further Reading

Glossary

Abbreviations

Long Answer Questions

Short Answer Questions

Answers to Short Answer Questions

Summary

This section highlights the rationale, potential and implementation of multicellular tumor spheroids in cancer research. A “multicellular tumor spheroid” (MCTS) is a three-dimensional in vitro model system that bridges the gap between two-dimensional monolayer cell cultures and an in vivo tumor tissue model system. Compared to monolayer cultures, MCTS resembles tumor tissue in terms of structural and functional properties and is suitable for studying metastasis, invasion, and therapeutic screening of drugs. Spheroid models are helpful in accelerating translational research in cancer biology and tissue engineering.

What You Can Expect to Know

Multi-cellular tumor spheroids (MCTS), the best described 3-D tumor models, offer an excellent in vitro screening system that mimics to a great extent the microenvironment prevailing in the tumor tissue, supporting studies on tumor-specific processes like angiogenesis, invasion and metastasis, as well as assessment of responses to various therapies and underlying mechanisms. Developed nearly 30 years ago as an alternative in vitro model to monolayer cultures, their role as a part of a high through-put, cell-based assay system in drug discovery is gaining considerable importance. Spheroids bridge the gap between monolayers and animal tumors, facilitating mechanistic studies and evaluation of anticancer therapies, particularly relevant to solid tumors. The distinct possibility of establishing MCTS from primary tumor cells and also co-culturing with different normal cells enhance the value of MCTS in cancer research and drug development. Recent developments in the generation of novel MCTS systems that allow analysis of a vast number of parameters can be conveniently employed in the high throughput screening system with cell-based assays, thereby significantly shortening the time required for translational research, bridging the gap between discovery and clinical application. Thus, spheroids have the potential to enhance predictability of clinical efficacy and may minimize, if not replace, animal studies to a large extent in the near future. Together with novel and emerging tools of biotechnology, this 3-D in vitro model is expected to substantially reduce the cost of new drug discovery.

History and Methods

Introduction

Model systems play an important role in biomedicine, as they form the backbone of translational research by bridging the gap between basic concepts and discovery, with applications in the management of diseases involving diagnosis, prognosis, and therapy. In the field of oncology, they are useful in therapeutic screening, preclinical evaluation, and to even study the basic biology of tumors. It is generally observed that the degree of complexity of various models available and deployed in experimental oncology bear an inverse relationship with the level of predictability either for diagnostic purposes or therapeutic evaluation (Khaitan et al., 2006b).

In vitro cell cultures are important experimental tools in understanding the biology of neoplastic cells, as well as for the evaluation of potential therapeutic agents, and understanding of the mechanisms underlying their actions. They can easily be created and manipulated, and hence help in the systematic studies of multicellular systems. Among the in vitro models of tumors, 2-D models such as monolayers and suspension cultures have been used widely to study various aspects of tumor biology. However, extrapolation of findings from these models has limited value in clinics, as only a fraction of the tumor cells generally develop fully in to a cell line, and do not necessarily reflect the primary tumors from which they were derived. Most importantly, they lack three-dimensional architecture and host tissue microenvironment, which are critical features of tumors (Khaitan et al., 2006b; Vinci et al., 2012). Therefore, there has been considerable amount of effort to develop and deploy various 3-D in vitro culture systems (organ culture, spheroid culture) that are associated with enhanced reliability and predictability for clinical efficacy, and to minimize studies with animal models (Khaitan et al., 2006b). It is very well realized that cell-based models used in experimental studies need to recapitulate both the 3-D organization and multicellular complexity of the tumor as well as the organs to translate findings from these studies into clinical applications. Three-dimensional cultures have been utilized in biomedical research since the first half of the 20th century to gain deeper insight into the mechanisms of organogenesis and expression of malignancy. However, only a small number of 3-D model systems are sufficiently well characterized to simulate the patho-physiological cellular microenvironment in a tumor or to reconstitute a tissue-like cyto-architecture, with cell-to-cell and cell-to-matrix interactions, growth, differentiation, and therapeutic responses similar to tumors in vivo.

Organ culture is one of the 3-D cultures, where small pieces of tumor explants are cultured in a moist gas or air phase on the surface of a relatively large volume of stationary nutrient medium (to retain the original structural relationships and differentiation of the tissue organization), and can be used to study the interactive function and the effect of drugs and other agents (Dwarakanath et al., 1985, 1987; Lasnitzki et al., 1992). However, lack of characterized reference stock, limited availability, and high variability, as well as ethical restrictions (at least with respect to human tissue), has limited the use of organ cultures.

MULTICELLULAR Tumor Spheroids

Spheroid models are sphere-shaped cell colonies that permit growth and functional studies of diverse normal and malignant tissues. The growth of spheroids from tumor cells mimics the growth of naturally occurring human tumors, as their extracellular matrix and network of cell-to-cell and cell-to-matrix interactions are similar to in vivo conditions and differ from the corresponding monolayer cultures (Khaitan et al., 2006b). In spheroids as in solid tumors, cells in contact with nutrients grow quickly, while the kinetics and activity of cells further inward depend on diffusion. Various possibilities of manipulating the spheroid environment using different techniques for their generation has not only provided insights into the complexity of tumor physiology, but has also facilitated research efforts in developing novel therapeutic agents (Khaitan et al., 2006b).

This model was adopted to cancer research several decades ago by Sutherland and his co-workers, and has since then considerably contributed to our knowledge regarding various biological mechanisms of tumors, as well as in studies related to cellular response ...