Organ culture involves maintaining small fragments of whole organs or tissues in culture media while retaining their three-dimensional structure and spatial distribution of cells. There are several methods of organ culture including culturing on plasma clots, agar, liquid media, or raft methods. Organ culture has various applications and allows studying cell interactions in a way that mimics the in vivo organ. It is currently being used to develop replacement organs and tissues for applications such as growing bladders, lungs, and heart patches. While progress is being made, developing fully functional human organs remains a challenge.
This document discusses in vitro transformation, which is the alteration of cells in culture that results in a continuous cell line. In vitro transformation can occur spontaneously or be induced by viruses, transfection, carcinogens, or radiation. Transformed cells exhibit immortalization, aberrant growth control, and malignancy. Immortalization involves infinite lifespan and loss of contact inhibition and density-dependent growth control. Aberrant growth control includes loss of serum dependence and anchorage independence. Malignancy is characterized by tumorigenicity, invasiveness, angiogenesis, and metastasis.
Stem cells have the ability to renew themselves and differentiate into specialized cell types. There are two main sources of stem cells: embryonic stem cells derived from blastocysts and adult stem cells found in adult tissues. Stem cell research aims to understand development and cell differentiation processes and develop therapies for diseases. Embryonic stem cells are pluripotent while adult stem cells are multipotent or unipotent. Stem cells are cultured in controlled conditions to maintain their undifferentiated state and are characterized based on gene expression and differentiation potential.
This document provides a history and overview of animal cell culture techniques. It discusses the development of cell culture media and reagents used to support cell growth in vitro. It also describes different techniques for culturing mammalian cells, tissues, and organs, including organ culture, explant culture, and cell culture. The goal of animal cell culture is to maintain cells, tissues, or organs outside of their natural environment for research purposes.
Types of animal cell culture, characterization and preservationSantosh Kumar Sahoo
Animal cell culture involves growing cells outside their natural environment under controlled conditions. There are two main types of cell culture: primary cell culture which uses cells directly from an animal, and secondary cell culture which uses cell lines that can be propagated repeatedly. Cells may be adherent, attaching to culture surfaces, or in suspension. Characterization of cell lines assesses identity, purity and suitability for use. Cryopreservation allows long-term storage of cells by freezing them at very low temperatures.
Dr. B. Victor presented on stem cell technology. He discussed different types of stem cells including embryonic stem cells, which are pluripotent and derived from embryos, and adult stem cells, which are multipotent and found in adult tissues. Stem cells can differentiate into specialized cell types and have potential applications in cell therapy and tissue engineering to treat diseases. However, the use of embryonic stem cells is considered ethically controversial as it requires embryo destruction.
This document discusses animal cell culture techniques. It describes primary cell culture which uses cells directly from tissue and is heterogeneous and finite. Secondary culture is produced by sub-culturing primary cells, which can become cell lines that are homogeneous and can divide indefinitely. Cell lines are categorized as finite, which senesce after limited divisions, or continuous, which can divide indefinitely. Common cell lines discussed include MCF-7, HL-60, and HeLa. Requirements for cell culture include media and lab equipment, and it aims to maintain pH between 7.2-7.4.
Stem cells are unspecialized cells that can differentiate into specialized cell types. There are two main types of stem cells: embryonic stem cells, which are derived from embryos and are pluripotent, and adult stem cells, which are multipotent and found in adult tissues. Stem cell research holds promise for developing new treatments for diseases by enabling cell regeneration and replacement. However, there are still challenges to overcome regarding isolating and delivering stem cells safely and effectively for clinical applications.
Organ culture involves maintaining small fragments of whole organs or tissues in culture media while retaining their three-dimensional structure and spatial distribution of cells. There are several methods of organ culture including culturing on plasma clots, agar, liquid media, or raft methods. Organ culture has various applications and allows studying cell interactions in a way that mimics the in vivo organ. It is currently being used to develop replacement organs and tissues for applications such as growing bladders, lungs, and heart patches. While progress is being made, developing fully functional human organs remains a challenge.
This document discusses in vitro transformation, which is the alteration of cells in culture that results in a continuous cell line. In vitro transformation can occur spontaneously or be induced by viruses, transfection, carcinogens, or radiation. Transformed cells exhibit immortalization, aberrant growth control, and malignancy. Immortalization involves infinite lifespan and loss of contact inhibition and density-dependent growth control. Aberrant growth control includes loss of serum dependence and anchorage independence. Malignancy is characterized by tumorigenicity, invasiveness, angiogenesis, and metastasis.
Stem cells have the ability to renew themselves and differentiate into specialized cell types. There are two main sources of stem cells: embryonic stem cells derived from blastocysts and adult stem cells found in adult tissues. Stem cell research aims to understand development and cell differentiation processes and develop therapies for diseases. Embryonic stem cells are pluripotent while adult stem cells are multipotent or unipotent. Stem cells are cultured in controlled conditions to maintain their undifferentiated state and are characterized based on gene expression and differentiation potential.
This document provides a history and overview of animal cell culture techniques. It discusses the development of cell culture media and reagents used to support cell growth in vitro. It also describes different techniques for culturing mammalian cells, tissues, and organs, including organ culture, explant culture, and cell culture. The goal of animal cell culture is to maintain cells, tissues, or organs outside of their natural environment for research purposes.
Types of animal cell culture, characterization and preservationSantosh Kumar Sahoo
Animal cell culture involves growing cells outside their natural environment under controlled conditions. There are two main types of cell culture: primary cell culture which uses cells directly from an animal, and secondary cell culture which uses cell lines that can be propagated repeatedly. Cells may be adherent, attaching to culture surfaces, or in suspension. Characterization of cell lines assesses identity, purity and suitability for use. Cryopreservation allows long-term storage of cells by freezing them at very low temperatures.
Dr. B. Victor presented on stem cell technology. He discussed different types of stem cells including embryonic stem cells, which are pluripotent and derived from embryos, and adult stem cells, which are multipotent and found in adult tissues. Stem cells can differentiate into specialized cell types and have potential applications in cell therapy and tissue engineering to treat diseases. However, the use of embryonic stem cells is considered ethically controversial as it requires embryo destruction.
This document discusses animal cell culture techniques. It describes primary cell culture which uses cells directly from tissue and is heterogeneous and finite. Secondary culture is produced by sub-culturing primary cells, which can become cell lines that are homogeneous and can divide indefinitely. Cell lines are categorized as finite, which senesce after limited divisions, or continuous, which can divide indefinitely. Common cell lines discussed include MCF-7, HL-60, and HeLa. Requirements for cell culture include media and lab equipment, and it aims to maintain pH between 7.2-7.4.
Stem cells are unspecialized cells that can differentiate into specialized cell types. There are two main types of stem cells: embryonic stem cells, which are derived from embryos and are pluripotent, and adult stem cells, which are multipotent and found in adult tissues. Stem cell research holds promise for developing new treatments for diseases by enabling cell regeneration and replacement. However, there are still challenges to overcome regarding isolating and delivering stem cells safely and effectively for clinical applications.
This document describes the morphology and growth characteristics of three types of cells in culture: fibroblast-like cells that are elongated and attach to the substrate, epithelial-like cells that are polygonal and attach in patches, and lymphoblast-like cells that are spherical and grow in suspension. It also discusses two types of cell culture based on attachment - anchorage-dependent cells that must attach to a substrate and anchorage-independent cells that can grow floating. The key factors that affect the cell culture environment are the growth media, pH, CO2 and temperature levels. Commonly used growth media include basal media, reduced-serum media and serum-free media.
This document discusses cell culture based vaccine production. It begins by introducing different types of vaccines and traditional egg-based vaccine production methods and their limitations. It then describes the importance and advantages of cell culture based methods, including types of cells used. The key steps of the cell culture based production process are outlined, including strain selection, bulk production, purification, virus inactivation, formulation, quality control testing, and lot release. Specific cell culture based vaccines for influenza, rabies, dengue, and Ebola are discussed. The conclusion emphasizes the potential for cell culture to replace egg-based methods by producing vaccines faster and in larger quantities to meet global demand.
Introduction
Terminologies
Types of tissue culture
Applications
Culturing
Sub-culturing
Cryopreservation
Detection of contaminants
In vitro transformation of cells
Cell viability
Rules for working in the Lab
Advantages
Limitations
This document summarizes the key aspects of maintaining current good manufacturing practices (cGMP) for cell banks. It discusses the design, development, and delivery of cell banks. It defines cGMP and describes the cell banking system including master and working cell banks. It outlines the characterization, documentation, storage, and validation requirements for cell banks including testing for identity, purity, and stability. The goals are to ensure consistent production of safe, pure, and potent biotherapeutic products through well-characterized and validated cell banks.
As opposed to common belief, the measurement of growth in cell culture is fairly simple. Most of the tecchniques that are applied for measurement of microbial growth can be applied to cell culture.Of course with some modification. This presentation exactly explains growth measurement techniques with respect to cell culture. At the end you will also find sample multiple choice questions for practice.
Introduction
History
Scale up in suspension:Stirred culture,Continuous flow culture,Air- lift culture,Nasa bioreactor
Scale up in monolayer culture: Roller bottle culture , multisurface culture,fixed -bed culture
Other type of culture for scaling up: HARV Vessels,STLV vessels
Monitoring of scale up
Conclusion
References
Bioinformatics on the internet provides many resources and benefits. It allows for easy access and sharing of vast biological databases and genomic data. The internet facilitates collaboration between researchers globally and provides tools for storing, organizing, and analyzing biological information. Key resources available online include biological databases, software for data analysis, educational courses, journals, and tools for sequence analysis, structure prediction, and more. This expands the scope of bioinformatics and allows research to advance more rapidly through improved access to information and resources.
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
Rasmol and Swiss-PDB viewer are molecular visualization tools that allow users to view and analyze protein structures. Rasmol can display molecules in various representations like wireframe, cylinders, or ribbons. It supports common file formats like PDB and can rotate, zoom, and translate structures. Swiss-PDB viewer is tightly integrated with homology modeling and allows users to build models, compare structures, and view electron density maps. It utilizes template structures from the PDB to generate models and assess their quality. Both tools provide publication-quality images and interactive visualization of biomolecular structures.
Stem cells can be derived from embryonic stem cells, adult stem cells, or induced pluripotent stem cells. Stem cells are undifferentiated cells that have the potential to differentiate into other cell types. There are several types of stem cells including totipotent, pluripotent, multipotent, oligopotent, and unipotent stem cells, which differ in their ability to differentiate. Stem cells offer potential for treating diseases but also raise ethical issues that require more research.
This document discusses stem cells, cloning, and bioethics. It provides background on influential figures in bioethics like Aldo Leopold and Van Rensselaer Potter. It covers current issues related to stem cells, cloning, genetic engineering, and environmental health. The document outlines the history of stem cell research and cloning, including the cloning of Dolly the sheep in 1997. It explains techniques for stem cell cultivation and nuclear transfer. Overall, the document aims to inform readers about modern bioethical issues emerging at the intersection of biology, biotechnology and medicine.
This document discusses various applications of tissue culture, including intracellular studies, elucidation of intracellular processes, studies of cell-cell interactions, and evaluation of environmental interactions. It also notes that animal cell culture can be used to produce medically important proteins like interferon, blood clotting factors, and monoclonal antibodies. Major developments in cell culture technology included the use of antibiotics, trypsin to subculture cells, and chemically defined culture media. Common cell culture media include Eagle's Minimum Essential Medium, Dulbecco's Modified Eagle's Medium, and RPMI-1640.
Immobilization of cells involves encapsulating cells in polymers to prevent division while maintaining viability. This allows increasing cell density in bioreactors for higher metabolite production. Methods include entrapment in gels, on fiber mats, or behind semi-permeable membranes. Alginate is commonly used for gel entrapment. Immobilized systems offer advantages like prolonged biomass use, higher product yields, and genetic stability. They also simplify downstream processing when products are extracellular. Various bioreactor designs are used depending on the immobilization method. Applications show enhanced production of metabolites like serpentine, anthraquinones, and capsaicin using immobilized plant cells.
Bioreactors for animal cell suspension cultureGrace Felciya
This document discusses bioreactors for animal cell suspension culture. It begins by introducing animal cell culture and some key developments that enabled it. There are two main types of culture: primary culture using explants or enzymes, and secondary culture which is derived from primary culture. Cells can be anchorage-dependent, growing in monolayers, or non-anchorage dependent, growing in suspension. Bioreactors provide conditions for mass cultivation of suspension cells. Properties of animal cells require gentle mixing and aeration in bioreactors. Common bioreactor types for suspension culture include stirred tank, continuous flow, and airlift fermentors. Perfusion culture allows continuous medium exchange to achieve high cell densities and productivity.
Cell culture types and application in vaccine productionAnkita Singh
Cell culture can be used to produce vaccines. There are three main types of cell culture: primary cell culture, secondary cell culture, and cell lines/strains. Cell lines like Vero and MDCK cells are often used to grow viruses that are then purified and tested for vaccine production. This includes vaccines for influenza, Japanese encephalitis, rabies, rotavirus, measles, smallpox, and polio. Cell culture methods allow high purity vaccines to be produced more quickly than egg-based methods and can be scaled up for pandemic response. However, not all viruses can be grown in cell culture.
This document discusses the airlift fermenter. It notes that fermenters must provide a controlled environment for microorganism or cell growth to produce desired products. An airlift fermenter circulates liquid using the density difference between the riser and downcomer columns caused by sparged air or gas. The main type discussed is the concentric draft tube airlift fermenter, which has an internal riser tube that introduces gas to lift liquid up the riser and down the surrounding downcomer tube. Tower loop and ICI deep shaft airlift fermenters are also mentioned. Airlift fermenters provide mixing without mechanical agitation and have high oxygen transfer rates, making them well-suited
This document discusses different types of mammalian cell culture. It describes primary cell culture, which uses cells directly from tissue that can undergo a limited number of divisions before senescing. Finite and continuous cell lines can proliferate for extended periods through transformation or immortalization. Common cell lines include HeLa cells and other tumor-derived lines. The document also covers techniques for attachment and suspension cell culture, and factors that influence cell growth in vitro.
Cell culture involves growing cells under controlled conditions outside of their natural environment. A cell line is a permanently established cell culture that will proliferate indefinitely. Primary cell cultures have a finite lifespan before senescence, while continuous cell lines are immortalized, aneuploid, and tumorigenic. When selecting a cell line, factors like species, growth characteristics, stability, and phenotypic expression should be considered depending on the experimental purpose. Common cell lines are derived from tissues like liver, kidney, lung, and ovary and are used for applications such as drug screening, bioassays, and production of vaccines and therapeutic proteins.
The document discusses various characteristics of cells in culture. It describes how primary cultures are established directly from animal tissue, while cell lines come from established cultures. Primary cultures have cells taken directly from tissue and placed in growth medium. The document also discusses how to isolate a single cell type, factors that allow differentiation, and how normal cells differ from transformed cells that can grow indefinitely.
This document describes the morphology and growth characteristics of three types of cells in culture: fibroblast-like cells that are elongated and attach to the substrate, epithelial-like cells that are polygonal and attach in patches, and lymphoblast-like cells that are spherical and grow in suspension. It also discusses two types of cell culture based on attachment - anchorage-dependent cells that must attach to a substrate and anchorage-independent cells that can grow floating. The key factors that affect the cell culture environment are the growth media, pH, CO2 and temperature levels. Commonly used growth media include basal media, reduced-serum media and serum-free media.
This document discusses cell culture based vaccine production. It begins by introducing different types of vaccines and traditional egg-based vaccine production methods and their limitations. It then describes the importance and advantages of cell culture based methods, including types of cells used. The key steps of the cell culture based production process are outlined, including strain selection, bulk production, purification, virus inactivation, formulation, quality control testing, and lot release. Specific cell culture based vaccines for influenza, rabies, dengue, and Ebola are discussed. The conclusion emphasizes the potential for cell culture to replace egg-based methods by producing vaccines faster and in larger quantities to meet global demand.
Introduction
Terminologies
Types of tissue culture
Applications
Culturing
Sub-culturing
Cryopreservation
Detection of contaminants
In vitro transformation of cells
Cell viability
Rules for working in the Lab
Advantages
Limitations
This document summarizes the key aspects of maintaining current good manufacturing practices (cGMP) for cell banks. It discusses the design, development, and delivery of cell banks. It defines cGMP and describes the cell banking system including master and working cell banks. It outlines the characterization, documentation, storage, and validation requirements for cell banks including testing for identity, purity, and stability. The goals are to ensure consistent production of safe, pure, and potent biotherapeutic products through well-characterized and validated cell banks.
As opposed to common belief, the measurement of growth in cell culture is fairly simple. Most of the tecchniques that are applied for measurement of microbial growth can be applied to cell culture.Of course with some modification. This presentation exactly explains growth measurement techniques with respect to cell culture. At the end you will also find sample multiple choice questions for practice.
Introduction
History
Scale up in suspension:Stirred culture,Continuous flow culture,Air- lift culture,Nasa bioreactor
Scale up in monolayer culture: Roller bottle culture , multisurface culture,fixed -bed culture
Other type of culture for scaling up: HARV Vessels,STLV vessels
Monitoring of scale up
Conclusion
References
Bioinformatics on the internet provides many resources and benefits. It allows for easy access and sharing of vast biological databases and genomic data. The internet facilitates collaboration between researchers globally and provides tools for storing, organizing, and analyzing biological information. Key resources available online include biological databases, software for data analysis, educational courses, journals, and tools for sequence analysis, structure prediction, and more. This expands the scope of bioinformatics and allows research to advance more rapidly through improved access to information and resources.
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
Rasmol and Swiss-PDB viewer are molecular visualization tools that allow users to view and analyze protein structures. Rasmol can display molecules in various representations like wireframe, cylinders, or ribbons. It supports common file formats like PDB and can rotate, zoom, and translate structures. Swiss-PDB viewer is tightly integrated with homology modeling and allows users to build models, compare structures, and view electron density maps. It utilizes template structures from the PDB to generate models and assess their quality. Both tools provide publication-quality images and interactive visualization of biomolecular structures.
Stem cells can be derived from embryonic stem cells, adult stem cells, or induced pluripotent stem cells. Stem cells are undifferentiated cells that have the potential to differentiate into other cell types. There are several types of stem cells including totipotent, pluripotent, multipotent, oligopotent, and unipotent stem cells, which differ in their ability to differentiate. Stem cells offer potential for treating diseases but also raise ethical issues that require more research.
This document discusses stem cells, cloning, and bioethics. It provides background on influential figures in bioethics like Aldo Leopold and Van Rensselaer Potter. It covers current issues related to stem cells, cloning, genetic engineering, and environmental health. The document outlines the history of stem cell research and cloning, including the cloning of Dolly the sheep in 1997. It explains techniques for stem cell cultivation and nuclear transfer. Overall, the document aims to inform readers about modern bioethical issues emerging at the intersection of biology, biotechnology and medicine.
This document discusses various applications of tissue culture, including intracellular studies, elucidation of intracellular processes, studies of cell-cell interactions, and evaluation of environmental interactions. It also notes that animal cell culture can be used to produce medically important proteins like interferon, blood clotting factors, and monoclonal antibodies. Major developments in cell culture technology included the use of antibiotics, trypsin to subculture cells, and chemically defined culture media. Common cell culture media include Eagle's Minimum Essential Medium, Dulbecco's Modified Eagle's Medium, and RPMI-1640.
Immobilization of cells involves encapsulating cells in polymers to prevent division while maintaining viability. This allows increasing cell density in bioreactors for higher metabolite production. Methods include entrapment in gels, on fiber mats, or behind semi-permeable membranes. Alginate is commonly used for gel entrapment. Immobilized systems offer advantages like prolonged biomass use, higher product yields, and genetic stability. They also simplify downstream processing when products are extracellular. Various bioreactor designs are used depending on the immobilization method. Applications show enhanced production of metabolites like serpentine, anthraquinones, and capsaicin using immobilized plant cells.
Bioreactors for animal cell suspension cultureGrace Felciya
This document discusses bioreactors for animal cell suspension culture. It begins by introducing animal cell culture and some key developments that enabled it. There are two main types of culture: primary culture using explants or enzymes, and secondary culture which is derived from primary culture. Cells can be anchorage-dependent, growing in monolayers, or non-anchorage dependent, growing in suspension. Bioreactors provide conditions for mass cultivation of suspension cells. Properties of animal cells require gentle mixing and aeration in bioreactors. Common bioreactor types for suspension culture include stirred tank, continuous flow, and airlift fermentors. Perfusion culture allows continuous medium exchange to achieve high cell densities and productivity.
Cell culture types and application in vaccine productionAnkita Singh
Cell culture can be used to produce vaccines. There are three main types of cell culture: primary cell culture, secondary cell culture, and cell lines/strains. Cell lines like Vero and MDCK cells are often used to grow viruses that are then purified and tested for vaccine production. This includes vaccines for influenza, Japanese encephalitis, rabies, rotavirus, measles, smallpox, and polio. Cell culture methods allow high purity vaccines to be produced more quickly than egg-based methods and can be scaled up for pandemic response. However, not all viruses can be grown in cell culture.
This document discusses the airlift fermenter. It notes that fermenters must provide a controlled environment for microorganism or cell growth to produce desired products. An airlift fermenter circulates liquid using the density difference between the riser and downcomer columns caused by sparged air or gas. The main type discussed is the concentric draft tube airlift fermenter, which has an internal riser tube that introduces gas to lift liquid up the riser and down the surrounding downcomer tube. Tower loop and ICI deep shaft airlift fermenters are also mentioned. Airlift fermenters provide mixing without mechanical agitation and have high oxygen transfer rates, making them well-suited
This document discusses different types of mammalian cell culture. It describes primary cell culture, which uses cells directly from tissue that can undergo a limited number of divisions before senescing. Finite and continuous cell lines can proliferate for extended periods through transformation or immortalization. Common cell lines include HeLa cells and other tumor-derived lines. The document also covers techniques for attachment and suspension cell culture, and factors that influence cell growth in vitro.
Cell culture involves growing cells under controlled conditions outside of their natural environment. A cell line is a permanently established cell culture that will proliferate indefinitely. Primary cell cultures have a finite lifespan before senescence, while continuous cell lines are immortalized, aneuploid, and tumorigenic. When selecting a cell line, factors like species, growth characteristics, stability, and phenotypic expression should be considered depending on the experimental purpose. Common cell lines are derived from tissues like liver, kidney, lung, and ovary and are used for applications such as drug screening, bioassays, and production of vaccines and therapeutic proteins.
The document discusses various characteristics of cells in culture. It describes how primary cultures are established directly from animal tissue, while cell lines come from established cultures. Primary cultures have cells taken directly from tissue and placed in growth medium. The document also discusses how to isolate a single cell type, factors that allow differentiation, and how normal cells differ from transformed cells that can grow indefinitely.
The term ‘Tissue culture ’ refers to the culture of whole organism, tissue fragments as well as dispersed cell on a suitable nutrient medium. Tissue culture is divided into the two broad groups, i) cultures that facilitates cell to cell interactions and signaling among cell and allow their study and ii) those in which cell to cell interactions and signaling are missing. The first group consist of three distinct types of culture system, viz., (i) Organ culture( in this, whole embryonic organs or small tissue fragments are cultured in vitro in such a manner that they retain their tissue architecture, i.e., the characteristic distribution of various cell types in the given organ) (ii) Histotypic cultures ( in this, individual cell lineages are first isolated from organ, purified and multiplied; they are grown separately to high density in three-dimensional matrix to study interactions and signaling among homologous cells. (iii) Organotypic cultures ( in this, cells of different lineages are mixed together in specific ratios and spatial relationships in order to recreate a component of concerned organ). The second group consists of cell culture either as monolayer (cells are obtained either by enzymatic or mechanical dispersal of tissues into individual cells or by spontaneous migration of cells from an explants or as suspension culture.
This lecture discusses characteristics of animal cell cultures. It begins by describing primary cultures established directly from tissue using mechanical and enzymatic methods. Specific cell types are then discussed, including fibroblasts, epithelial cells, muscle cells, neurons, and lymphocytes. The concepts of normal vs. transformed cells, anchorage dependence, passaging, contamination, cell differentiation, and stem cells are also covered. Common cell lines available from culture collections are listed at the end.
Cultured animal cells have many important applications. They can be used as (1) model systems to study basic cell biology and interactions between cells and pathogens, (2) for toxicity testing of new drugs and chemicals, and (3) in cancer research to study normal and cancerous cell differences. Animal cell culture is also used for virology research, manufacturing of vaccines and proteins, genetic counseling, genetic engineering of cells, and gene and drug screening and development. Proper growth media, aseptic techniques, cryopreservation, and applications in various fields make animal cell culture a valuable tool.
Cell culture involves growing cells under controlled conditions. Key developments include the use of antibiotics to prevent contamination, trypsin to detach adherent cells for subculturing, and defined culture media. Cells are typically maintained through serial passaging when confluent. Cells can be cryopreserved for long-term storage in liquid nitrogen. Common cell lines include HeLa, HEK293, MCF-7, and Vero cells. Contaminants include mycoplasma, bacteria, and cross-contamination between cell lines. Basic equipment includes laminar flow hoods, incubators, refrigerators, and microscopes.
This document provides information on cell culture and the establishment of cell lines. It discusses the history of cell culture, major developments in the technology, and common uses of cell culture. It also describes primary cell culture, finite and continuous cell lines, culture conditions, cryopreservation, cell morphology, and factors to consider when selecting an appropriate cell line.
This document provides information on cell culture and the establishment of cell lines. It discusses the history of cell culture, major developments in the technology, and common uses of cell culture. It also describes primary cell culture, finite and continuous cell lines, culture conditions, cryopreservation, cell morphology, and factors to consider when selecting an appropriate cell line.
This document provides information on cell culture and the establishment of cell lines. It discusses the history of cell culture, major developments in the technology, and common uses of cell culture. It also describes primary cell culture, finite and continuous cell lines, culture conditions, cryopreservation, cell morphology, and factors to consider when selecting an appropriate cell line.
cell and tissue culturing and cell lineMicrobiology
Cell culture involves growing cells under controlled conditions outside of their natural environment. The document discusses the history and major developments of cell culture, including the use of antibiotics, trypsin, and defined culture media. It also outlines common uses of cell culture such as in cancer research, virology, genetic engineering, and gene therapy. The document provides details on primary cell culture, continuous cell lines, morphology of cells in culture, and considerations for selecting an appropriate cell line.
Introduction to cell culture- concepts of cell culture part-1PHARMA IQ EDUCATION
Cell culture involves removing cells from an animal or plant and growing them in a favorable artificial environment. There are several key concepts in cell culture: primary culture refers to cells after initial isolation and proliferation; cell lines are derived from primary cultures and have a limited lifespan; cell strains are positively selected subpopulations from cell lines. Cell culture conditions provide nutrients, growth factors, hormones and appropriate gas/physical conditions. Cells can be grown adhered to a substrate or in suspension. Cryopreservation allows long-term storage of excess cells. Cells in culture exhibit fibroblastic, epithelial-like or lymphoblast-like morphologies and applications include physiological studies, drug screening and large-scale manufacturing.
The document discusses different types of cell culture used in bioreactors. It describes organ culture, tissue culture, and cell culture. Cell culture involves dispersing tissue enzymatically into a cell suspension that can be grown as a monolayer or in suspension. Continuous cell lines can be propagated indefinitely and have gained immortality through transformation. Bioreactors must provide a well-controlled environment for cell culture and can operate in batch, fed-batch or perfusion modes. Common bioreactor designs include stirred tank, airlift and wave bioreactors.
This presentation will help to understand the basics of mammalian cell culture. I have also covered the difference between adherent and suspension cell lines. I have also included the advantages and disadvantages of the cell line.
Types of animal cell culture; characterization & Their preservation.Santosh Kumar Sahoo
This document provides an overview of animal cell culture, including the different types (primary and secondary cell culture, cell lines), techniques used for primary culture, and characterization and preservation of animal cells. It discusses how primary cell culture involves separating cells directly from tissue and allowing them to grow under controlled conditions. Secondary cell culture refers to sub-culturing primary cells by transferring them to new vessels with fresh media. Cell lines can be propagated repeatedly and sometimes indefinitely. The document also describes cryopreservation as a method for preserving live cells and tissues at ultra-low temperatures in liquid nitrogen.
This document provides an overview of animal cell culture. It discusses what animal cell culture is, the applications of culturing animal cells, and some of the advantages and disadvantages. Key aspects covered include growing cells in vitro by providing proper nutrients and growth factors, maintaining homogeneous or heterogeneous cell populations, and using cell cultures to investigate cell physiology, test compound effects, and produce biological products. The document also reviews various techniques involved in animal cell culture like using appropriate growth substrates, culture media, supplements, and methods for tissue disaggregation.
This document provides information about the course CODE 320 on Virology/Parasitology. It discusses cell culture, including its history, terminology, techniques, types of cells, equipment used, and factors that affect cell growth. Key points include that cell culture involves growing cells outside their natural environment, the importance of subculture when cells reach confluence, and that cell growth follows distinct lag, log/exponential, and plateau phases.
Cell lines are established cell cultures that can proliferate indefinitely in vitro. They are used in basic research and early drug development studies. Cell lines are categorized as finite or continuous based on their lifespan. Finite cell lines have limited proliferation while continuous cell lines can proliferate indefinitely. Characterization of cell lines is important to determine their species of origin, tissue of origin, and genetic stability. Common cell lines used in research include human cell lines like HeLa, mouse cell lines like 3T3 fibroblasts, and rat cell lines. Cell lines are useful for investigating disease pathways and developing therapies.
Industrial Bioprocessing Simulation and Modelling
The document discusses industrial bioprocessing, simulation, and modeling. It provides an overview of bioprocessing history and applications. Process simulation and modeling tools are used to optimize efficiency without extensive experimentation. Downstream processing aims to purify products through techniques like filtration. Process design considers product properties and impurities. Scale-up requires maintaining parameters like bed height and velocity. Career opportunities exist in engineering and science roles in biopharmaceutical industries, with salary packages ranging from 3.25-8 LPA depending on level.
This presentation will help to freshers in the field of upstream process development. In UPD bioreactor operations are crucial and understanding various aspects also has the same importance. Hence, this PPT will brief the introduction about bioreactors followed by their classifications. Eventually, the body construction.
This presentation will help you to understand the growth of cells and their maintenance in the culture medium. The medium components play a major role in the survival and function of cells. The growth curve help to understand the growth pattern of specific cells along with characteristics.
It will be helpful for students who is having very less time to prepare for enzyme immobilization and coimmobilization of an enzyme. This presentation have covered various methods and its induatrial applicability.
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1. Animal cells: Basic Concepts
Shubham A. Chinchulkar (Regulatory Affairs)
M.Tech (Pharm.)
National Institute of Pharmaceutical education and
Research (NIPER)
shubhamchinchulkar007@gmail.com
2. Introduction
Cell Culture - cultivation of dispersed cells taken from an original tissue, a primary culture, or a cell line
Cell culture techniques allow in vitro propagation of various cell lines including those from insects, humans, mice,
rats, and other mammals
Animal cells: a. More delicate
b. vulnerable to mechanical damage
c. present lower growth rates
d. require more complex culture media and special substrates
Rigorous aseptic conditions - animal cells grow more slowly than most usual contaminants, such as bacteria and
fungi
4. Animal Cell
Components
Plasma
membrane
Cytoplasm
Endoplasmi
c reticulum
Ribosome
Golgi
complex
Mitochondria Lysosome Peroxisome Nucleus
Separates the
intracellular
components
thickness
around 7.5–
10 nm,
controls the
flux of
molecules
between the
cytosol and
the
extracellular
environment.
Cytoplasm
consists of the
cytosol.
Ectoplasm &
Endoplasm
Structures are
Enveloped by
a membrane
that separates
the
endoplasmic
reticulum
cavities or
cisternae.
Cisternae
also serve
as a storage
area.
Protein synthesis,
Lipid
biosynthesis,
relaxation by
reabsorbing Ca2+
Secrete steroid
hormones
Spherical
cell
structures
enriched
with
ribonucleop
rotein with
a diameter
of 15–20
nm.
Proteins
accumulate
in the
cisternae.
Arrangement
is a stack of
circular
flattened
vesicles
Golgi complex
receives
products from
the
endoplasmic
reticulum
and packs
them in
secretory
vesicles
Enzyme-rich
structures that
catalyze the
oxidation of
organic
nutrients
Generate
adenosine
triphosphate
It containd
DNA, RNA and
ribosomes
It can also
divide during
cell replication
Spherical
organelles,
enveloped by a
membrane and
containing
several
hydrolytic
enzymes that
present maximal
activity at acidic
pH
Reservoirs of
different
enzymes
Spherical
organelles
that are 0.3–
1.5 m
in diameter
Interior is
full of
proteins,
frequently in
crystalline
form
Presence of
various
oxidative
enzymes
Very
complex
structure
Two
membranes
of the
nuclear
envelope
form pores
with a
diameter of
around
90 nm
5. Establishing a Cell Line
Primary culture – culture up to its first subculture or passage & Cell line – after subculture or passage
Primary cultures – obtained directly from organ or tissue fragments, following enzymic (trypsin or collagenase and
may be mixed with EDTA) or mechanical disaggregation
Origin – Animal or Human tissue
Tissue
culture
Cell culture
Primary explant
Organ culture
7. Primary cultures
Initially heterogeneous
Cells can be maintained in vitro for only a limited period
During this period, cells generally maintain the differentiated characteristics of the original tissue from which they
were harvested
Cells may be subjected to considerable stress
a) Enzymatic dissociation of organ fragments
b) Adherent cells breaks cell–cell or cell–surface interactions
Dissociated cells generally change their shape, becoming rounded and losing their phenotypic polarity, modifying
protein distribution in cell membrane
Certainly not all cells survive cell manipulation but those that survive should be able to correct any injuries and adapt to
environmental changes
8. Culture adaptation is
a) Time-consuming
b) Influenced by culture conditions
Environmental conditioning = cell conditioning
Environmental conditioning can be achieved by following methods -
I. Release of growth factors into the medium promote cell adherence and proliferation – environment conditioning – cell
conditioning
II. Using feeder cells - 3T3 fibroblastic cells
Gamma radiation
Mitomicin C
Feeder cells Target cells seeding and growthTreatment
Target cells
Environmental conditioning3T3 fibroblastic cells
9. Feeder
cells
Releasing
growth factors
to the culture
media
Detoxifying the
culture medium
Synthetizing
extracellular
matrix proteins
needed to
control the
cultured cell
growth
Acting as a
substrate for the
attachment of
cells
Role of the feeder cells
10. Nonreplicating viable and metabolically active feeder cells capable of stable synthesis of ligands or cytokines
needed to support the selective expansion of cultured target cells
Purified growth factors and also the conditioned medium of other cell lines can be used in some instances to
make fastidious cells independent of the presence of feeder cell layers
3T3 feeder fibroblasts constitutively produce hyaluronic acid (HA)
Production of HA rises locally in a variety of processes involving cell motility
HA becomes hydrated, expanding the extracellular space
This process mechanically weakens intercellular binding facilitating cell detachment and also provides
hydrated tracks for the migration of detached cells, as observed in wound repair and embryonic
morphogenesis
11. After successive subcultures of a very heterogeneous primary culture, containing many cell types of the original
tissue, a more homogeneous cell line with a higher growth rate may arise
After successive subcultures of a very heterogeneous primary culture, containing many cell types of the original
tissue, a more homogeneous cell line with a higher growth rate may arise
Finite cell lines are generally diploid and maintain some degree of differentiation
These cell lines die after a limited number of generations, the Hayflick limit, which is usually about 30–50
division cycles depending on the origin of the cells
Immortalization of cell lines – by oncogene or virus or by chemical treatment
Transformed cell line - Is a cell line that acquired infinite growth after insertion of viral gene components into
the cell's genome
12. Characteristics
of
Transformed
cell lines
Higher
growth rate
Less
dependency
on blood
serum or
selected
growth
factors;
Increase in
cloning
efficiency
Increase in
heteroploidy
Increase in
heteroploidy
Altered cell
morphology
Characteristics of transformed cell lines
Unlimited cell supply
Maintain few
characteristics of their
original tissue
Advantage
Disadvantage
13. Periodic change of culture medium is necessary when cells are proliferating even in cultures showing no cell
proliferation
Intervals between medium changes and subcultures may vary depending on the cell line, growth rate, and
metabolism
Indication of need for culture medium replacement –
a. Increase in cell density
b. pH decrease
c. Nutrient depletion in the medium
d. Alteration in cell morphology
Animal cells may be anchorage-dependent
These cells normally proliferate in monolayers and show contact inhibition
Available surface of culture vessel plays role in cell yield
Cell Line Maintenance
14. MRC-5 Human Lung Fibroblast
Name
Cell type and
tissue origin
Morphology
HeLa Human cervix Epithelial
Vero
African green
monkey kidney
Epithelial
NIH 3T3 Mouse embryo Fibroblast
L929
Mouse connective
tissue
Fibroblast
CHO
Chinese hamster
ovary
Fibroblast
BHK-21
Syrian hamster
kidney
Fibroblast
Adherent Cell Lines
HEK-293 Human kidney Epithelial
15. Suspension Cell Lines
NS0 Mouse myeloma
Lymphoblastoid-
like
Name
Cell type and
tissue origin
Morphology
U937
Human hystiocytic
lymphoma
Lymphoblastoid
Namalwa Human lymphoma Lymphoblastoid
HL60 Human leukemia
Lymphoblastoid-
like
WEHI 231
Mouse B-cell
lymphoma
Lymphoblastoid
YAC 1 Mouse lymphoma Lymphoblastoid
U 266B1 Human myeloma Lymphoblastoid
SH-SY5Y
Human
neuroblastoma
Neuroblast
16. Blood cells - grow in suspension & Cells derived from solid tissues - adherent cells
Steps involved in adherent cells proliferation –
Adsorption of adhesion
factors to the
substratum
• Step 1
Contact of the cell with
the surface
• Step 2
Cells attach to the
covered surface
producing multivalent
heparin sulphate
proteoglycans, which
bind to cell membrane
glycoproteins
• Step 3
Cell spreading over the
solid surface occurs
•Step 4
17. Solid substrata with a hydrophilic surface are required, because vertebrate cells have negative charges non-
uniformly distributed over their external membrane surface
Subculture of adherent cells - removal of culture medium & detachment of cells from the monolayer
Trypsin plays role in a cell detachment, additionally other proteases such as a pronase, dispase, and collagenase
will be also be used
EDTA as a chelating agent added to capture the Ca+ ions involved in cell adhesion process
Gently tapping or hitting the culture flask by hand will be used to removed the weakly bind cell lines
Trypsin treatment is not required and subculture is faster and less traumatic for the cells
Advantages of suspension cell culture -
Large cell quantities can be attained without increasing the superficial area of the substratum
This can rarely be achieved with a monolayer culture
Cells in different modes (adherent or suspension) behave differently with regard to proliferation, enzymatic
activity, glycolysis, respiration, specialized product synthesis, and many other properties
18. Cell growth phases
Normal cells in culture show a sigmoid pattern of proliferative activity which reflects culture adaptation, environmental
conditioning, nutrient availability and, for adherent cells, available free adhesion surface
Cell growth phases –
i. Lag phase,
ii. Exponential or log growth phase,
iii. Stationary or plateau phase, and
iv. Senescence or death phase
Cell growth can be mathematically represented by
In lag phase adherent cells may resynthesize the glycocalyx elements lost during trypsinization, bind, and spread on the
substratum
During spreading, the cytoskeleton reappears and new structural proteins are synthesized
Inoculum concentration and the point in the growth phase from which cells were taken in the previous culture
Specific cell growth rate
Cell concentration
Culture time
19. Cells originating from an actively growing culture have a shorter lag phase than those from a quiescent culture
Cultures initiated at low cell densities - condition the culture medium more slowly - increase the duration of the lag
phase, which is not desirable
Cell division can reach 90-100% - cells are in best physiological state – ideal for cell function studies
The cell doubling time -
Inoculum concentration, cell growth rate, nutrient availability, and accumulation of inhibitory metabolites are the
crucial factors which influences the log phase
For adherent cells log phase may also occur - When cells cover all the available growth surface, at which point
contact inhibition restricts further growth
Stationary Phase - Low nutrient concentrations and accumulation of inhibitory metabolites – cell growth rate
reduced
20. Cell division is equilibrated with cell death - percentage of cells in division is at most 10%
Stationary phase - relative increase in specialized protein synthesis - change in cell surface composition - charge
modification may occur
If the culture medium is replenished with fresh – this phase may prolong
This is not a stable period for most cell lines, and they are susceptible to injuries
Decline phase – necrosis (result of an irreversible injury and normal homeostasis is lost) and apoptosis (activation of a
biochemical program involving a cascade of cell components, which is internally controlled, requiring energy)
Necrosis
Apoptosis
May result in inflammation No inflammation
21. Activation of hydrolases – Autodestruction – when lack of lack of nutrients and oxygen, followed by progressive
disorganization and complete disintegration of the cell
Apoptosis - affects the neighboring cells and may result in inflammation
During apoptosis comprise caspase activation, mitochondrial membrane permeation, leakage of diverse molecules
from the mitochondria, nuclease activation, cytoskeleton destabilization, externalization of phosphatidylserine to the
outer membrane, and protein interconversion
Determination of the cell growth profile is important to evaluate the specific characteristics of a cell line
Because of changes in cell behavior and biochemistry we need to study the growth curves for all the cell line which
will also help us to establishing the most adequate inoculum concentration, prediction of the length of an experiment,
and the most appropriate time intervals for sampling
22. Influence of environmental conditions on animal cell culture
Cell culture systems - survival and proliferation
This figure depicts the factor influencing on cell growth
Cell growth
pH Temperature
Osmolality Gas concentration
Available surface substrata State of the cells at inoculation
23. 1. pH
Most mammalian cell lines proliferate at pH 7.4 and some normal fibroblasts proliferate well at a pH range between
7.4 and 7.7
Insect cells show better proliferation at lower pH values, from 6.2 to 6.5
Acid indicator used to monitor the pH variation - phenol red - rose-colored at pH 7.8 - red at pH 7.4, orange at pH 7.0,
and yellow at pH 6.5
This compound can interfere with the interpretation of experimental data obtained by the use of fluorescence and
absorbance techniques
Culture medium needs to be buffered to compensate for CO2 and lactic acid derived from glucose metabolism
Culture medium buffered with CO2 originating from the gaseous phase, in equilibrium with sodium bicarbonate
(NaHCO3) added to the culture medium
24. An increase in CO2 tends to increase H+ and HCO3 – in reaction (3), consequently increasing medium acidity
In compensation, the increase in HCO3 – causes NaHCO3 formation through reaction (4), until an equilibrium is
reached at pH 7.4
Traditionally, culture media are buffered with sodium bicarbonate at a final concentration of 24 mM
Cells in lag phase – growing at low densities - not produce CO2 in sufficient quantities to maintain the pH at a
optimal value – decrease CO2 content - increase in the culture equilibrium pH
Hence, control of CO2 concentration allows appropriate maintenance of culture pH
The gas phase in contact with cell culture in an incubator is usually adjusted to 5–10% of CO2 in volume (90–95%
air)
Organic buffer - Hepes (N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid) - most effective at the 7.2–7.6 pH
range - this compound is more resistant to rapid alterations in culture conditions
For insect cells, culture medium is buffered with sodium phosphate, and the use of CO2 or pH indicators is not
required
25. 2. Osmolality
The optimal osmolality range for mammalian cells in culture is from 260 to 320 mOsm/kg and for insect cells higher
values are optimal, from 340 to 390 mOsm/kg
Important to verify the osmolality of all culture media after alterations addition of salt solutions, supplements,
pharmaceuticals, and hormones, or large quantities of buffering agents
Metabolic transformation also responsible for osmolality changes
The greater the concentration of the substance dissolved, the higher the osmolality, hence osmolality can also increase
due to evaporation
Culture flasks are generally not sealed so as to allow equilibrium between culture medium and the CO2 –air gas
mixture
For open cultures in multiple well plates or in Petri dishes slightly hypotonic culture medium can be more adequate
Relative humidity of culture environment should be near to saturation to avoid large variation in osmolality during
culture
26. 3. Temperature
It is having influence on cell growth and it affects the solubility of various medium components, especially of gases
such as CO2 and O2, which have low solubility
Most mammalian cells - 35–37, Insects cell – 26-28, and old-blooded vertebrate cells normally grow well at lower
temperatures
4. Oxygen Supply
CO2 and O2 are the most important components of gaseous phases and monitoring and controlling is mandatory
For most cells, oxygen partial pressure conditions slightly below atmospheric pressure are preferred
Because of that, maintenance of dissolved oxygen concentration at a range from 30 to 60% for mammalian cells is
important
Oxygen is first component to be limiting in high cell densities due to its having low solubility in aqueous medium
27. Aeration can be done in direct way or in a isolated vessel surface aeration, gas bubbling, diffusion through
membranes, increasing O2 partial pressure and atmospheric pressure
In case of static culture – surface aeration – in a closed system - a void volume 10 times larger than the culture
medium volume is necessary for adequate oxygen supply
In case of agitated culture – surface aeration – oxygen transfer is dependent on agitation rate and impeller
geometry
Gas bubbling is a simple and efficient way to transfer oxygen, this procedure can cause damage to animal cells due to
bubble formation
Alternatively, through hydrophobic membranes (silicon tubes), but this method is inconvenient, presents high cost,
and is difficult to scale up, since the tube length for adequate oxygen supply in high capacity reactors needs to be
very long
An increase in O2 partial pressure also increases oxygen solubility and its diffusion rate at high cell density,
otherwise oxygen toxicity effects could occur
28. Although oxygen is essential for cell growth, it can be toxic in high concentrations
For suspension cultures, cell injury can occur due to the disruption of ascending bubbles or of those associated with
the foam accumulated at the surface
Serum and the surfactant Pluronic F68, have been employed since the 1950s to protect cells from fluid-
mechanical forces
Serum – good cell protector agent and attributed to the increase in medium viscosity
Methylcellulose and carboxymethylcellulose as serum substitutes has shown very promising results
In bioreactors with intense agitation and bubbling aeration, the use of Pluronic1 F68 at 0.1% (v/v) concentration can
result in an efficient cell-protecting effect
29. 5. Composition and nature of the substratum for cell adhesion
Glass and plastic are the most common materials employed as a solid substratum, which help cell to for adhesion,
spreading, and proliferation
Glass (borosilicate Surfaces - have a high silica content ) was only used since long hence ‘‘in vitro’’ means ‘‘in glass’’
Glass is used at small scale and very rare for large scale
At the end of 1960s plastic material introduced for routine cell culture in a laboratory
Glass and plastic (Polystyrene) - hydrophobicity and negative charge, were maintained in these materials
Nowadays, polystyrene is mostly used plastic because of surface characteristics, low cost, transparency
For demanding cell line surface should be coated with proteins such as poly-lysine, poly-ornithine, or extracellular
matrixderived proteins such as fibronectin, laminin, and collagens
Microcarriers as substrates is recommended for adherent cell culture on larger scales, large effective area for cell
adhesion, it produced from glass, dextran, agarose, collagen, or modified polymers
30. 6. Cryopreservation and storage of cell lines
The cells are unstable when they are present in culture state, consequently changes like alterations in their
morphology, function, growth pattern, and karyotype
When cells are in frozen state then we can preserve it for long period without change in viability and characteristics
Cells stock also minimizes the risk of losing a culture due to accidents such as contamination by microorganisms or
other cell lines, or due to failure of equipment such as CO2 incubators
The cells should be in active growth phase with 90% viability and free of contaminants during storage in frozen
condition
The optimal cryopreservation conditions are different for each cell line
The cells should be treated with a cryoprotector to support the freezing and thawing processes
Optimal cell concentration in a suspension of cryoprotector medium generally within the range of 1x106 to 1x107
cells/ml (depends on the cell type and is determined empirically)
31. Cryoprotectors (concentrations between 5 and 10% (v/v)) such as glycerol or dimethylsulfoxide (DMSO) affect
membrane permeability, allowing water release from the cell interior during cooling
With cell dehydration, occurring at between –5oC and –15oC, ice crystals are formed around the cells and not
inside them (prevent lysis)
Cooling velocity is also critical and should be low (about 1oC/min)
For cell cultivated in serum medium, cryopreservation medium should also contain fetal bovine serum to
protect cells from the stress associated with the freezing and thawing processes
Serum is added at concentrations above 20%, and can attain 90% of volume of the cryopreservation medium
However, storage in vapor phase liquid nitrogen (at –140oC to –180oC) is recommended, avoiding possible
contaminations or cell death in case of cryotube rupture
During cell thawing, the heating rate should be high
Flasks containing frozen cells are immersed in water at 37oC, and thawing occurs in about 90 seconds
32. Fresh culture medium should be gradually added to the thawed cell suspension
For cell line storage, a cell bank is generally established, with initially three to five flasks. One of these flasks is then
thawed and the cell population is expanded to produce a master bank with about 10 to 20 flasks, depending on future
requirements
As a quality control, some flasks of this bank (about two or three) should be used to confirm that the cell bank
concentration and viability are satisfactory and free of contaminants
33. 7. Culture quality control and laboratory safety
The culture system should be totally free of compounds that can cause toxic or inhibitory effects
Contaminants in medium –
Sr. No. Components Effect
1 High concentrations of a culture medium
component in culture
Growth inhibitory
2 Contaminants in medium components and in the water
employed for culture media formulation
Toxic for medium
3 Flasks that have not been well rinsed Toxicity for cell culture
4 Light (particularly short wavelength) can interact with
certain culture medium components, such as riboflavin,
tyrosine, and tryptophan
Generate toxic products
34. Obtaining cell lines from known and safe sources is important to guarantee the use of well-characterized and authentic
cells with respect to DNA profile, species of origin, and contaminants
The main contaminant types are –
Sr.
No.
Type of contamination Observation
1 Bacterial and fungal contamination Rapid increase in medium turbidity or by a rapid pH change
2 Mycoplasma contamination Most difficult to detect and can cause a reduction in cell growth
rate, morphological alterations, chromosomal aberrations, and
changes in metabolism of amino acids and nucleic acids
3 Virus contamination Causes changes in cell growth rate
Fungi Mycoplasmas VirusesBacteria
Fetal bovine serum is usually the main source of viral contamination
35. Some cell lines are immortalized employing virus, but these cases are not considered as contaminations
When contamination occurs, it is recommended to discard the culture and continue working with contaminant-free
stocks
Antibiotics could be used to try to eradicate the contamination but viral contaminations cannot be treated and they
are intracellular parasites
If virus-free stocks do not exist, a risk evaluation should be performed before continuing to work with the infected
cell line
Major risks are related to potential injuries resulting from liquid nitrogen manipulation or glassware accidents
36. 8. Characteristics of the main cell lines employed industrially
The cell lines most commonly employed for production of biopharmaceuticals include CHO-K1, BHK-21, and Vero
cells, besides various antibody-secreting hybridomas
The CHO-K1 (Chinese hamster ovary) cell line was established by Puck et al. (1958), and was isolated from a
Chinese adult hamster
The CHO-K1 cell line consists of epithelial cells that can be adapted for suspension growth
The BHK-21 (baby hamster kidney) cell line consists of adherent fibroblasts, that can also be adapted to suspension
culture, and was isolated from five 1-day-old hamsters (McPherson and Stoker, 1962)
These cells are commonly used for virus propagation (polio, rabies, and foot-and mouth disease) for production of
veterinary vaccines
The NS0 cell line, derived from mouse myeloma cells, is one of the most
The popular for heterologous proteins expression on a large scale due to its capacity for incorporating exogenous
DNA and stably producing recombinant proteins
37. PER.C61 cells have been used extensively for the production of gene therapy vectors
HEK-293 cells, derived from human embryo kidney, were transformed with human type 5 adenovirus
The MRC-5 cell line, derived in 1966 from normal human lung tissue (Jacobs et al., 1970), is adherent and shows a
fibroblast morphology
These cells are well known owing to their susceptibility to several virus types, being employed in assays related to
viral transfection, in cytotoxicity evaluation, and in vaccine production