This document discusses the culture of specialized epithelial cells. It describes various types of epithelial cells and their functions. It discusses challenges in culturing epithelial cells, such as removing contaminating fibroblasts. Various methods are described for obtaining pure epithelial cell cultures, including selective attachment, feeder layers, selective media, cloning, and physical separation techniques. Characterization of epithelial cell cultures involves assessing markers like cytokeratins and junctional complexes. The document also discusses culturing of mammary epithelial cells and tumor cells from breast tissue, as well as mesenchymal stem cells from bone marrow.
Culture techniq and type of animal cell culturePankaj Nerkar
A primary culture refers to the initial culture of cells directly taken from an organism before the first subculture. A cell line refers to the propagation of cells after the first subculture. Primary cultures contain a variety of differentiated cell types and require higher cell quantities due to lower survival rates. Tissues are disaggregated into single cells using mechanical or enzymatic techniques for primary culture. Organ cultures involve culturing whole organs or tissues to preserve their structure and function in vitro. Various techniques like plasma clot, raft, and grid methods are used to culture different organ explants.
Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
Introduction
Primary Culture
Steps In Primary Culture
Isolation Of Tissue
Dissection And/Or Disaggregation
Types Of Primary Culture
Primary Explant Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
reference
This document discusses basic concepts of animal cell culture. It begins with an introduction and brief history of cell culture. Some key advancements include the use of antibiotics to reduce contamination, trypsin to subculture adherent cells, and chemically defined culture media. Current applications of cell culture include cancer research, genetic engineering, and gene therapy. The document then covers various cell culture types (primary vs cell line), requirements, techniques, and factors affecting growth. It distinguishes between adherent and suspension cultures as well as finite and continuous cell lines.
Blood production agency. all types of blood cellls are produced in it. to understand it is the need of this era. it also will help in the physiology of blood making mechanism.
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
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 cell immobilization techniques for cell culture. It describes three main types of cell culture: adherent cell culture, suspension cell culture, and immobilized cell culture. Immobilized cell culture involves encapsulating, adsorbing, or entrapping cells within a polymeric or porous matrix. The document then discusses types of cell immobilization including encapsulation, entrapment, adsorption, and cross-linking. Specific examples provided include immobilizing yeast cells in calcium alginate beads and using immobilized cells in bioreactors. Benefits of immobilized cell culture include higher cell densities, longer culture stability, and protection from shear forces.
Culture techniq and type of animal cell culturePankaj Nerkar
A primary culture refers to the initial culture of cells directly taken from an organism before the first subculture. A cell line refers to the propagation of cells after the first subculture. Primary cultures contain a variety of differentiated cell types and require higher cell quantities due to lower survival rates. Tissues are disaggregated into single cells using mechanical or enzymatic techniques for primary culture. Organ cultures involve culturing whole organs or tissues to preserve their structure and function in vitro. Various techniques like plasma clot, raft, and grid methods are used to culture different organ explants.
Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
Introduction
Primary Culture
Steps In Primary Culture
Isolation Of Tissue
Dissection And/Or Disaggregation
Types Of Primary Culture
Primary Explant Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
reference
This document discusses basic concepts of animal cell culture. It begins with an introduction and brief history of cell culture. Some key advancements include the use of antibiotics to reduce contamination, trypsin to subculture adherent cells, and chemically defined culture media. Current applications of cell culture include cancer research, genetic engineering, and gene therapy. The document then covers various cell culture types (primary vs cell line), requirements, techniques, and factors affecting growth. It distinguishes between adherent and suspension cultures as well as finite and continuous cell lines.
Blood production agency. all types of blood cellls are produced in it. to understand it is the need of this era. it also will help in the physiology of blood making mechanism.
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
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 cell immobilization techniques for cell culture. It describes three main types of cell culture: adherent cell culture, suspension cell culture, and immobilized cell culture. Immobilized cell culture involves encapsulating, adsorbing, or entrapping cells within a polymeric or porous matrix. The document then discusses types of cell immobilization including encapsulation, entrapment, adsorption, and cross-linking. Specific examples provided include immobilizing yeast cells in calcium alginate beads and using immobilized cells in bioreactors. Benefits of immobilized cell culture include higher cell densities, longer culture stability, and protection from shear forces.
Cell synchronization helps in obtaining distinct sub population of cells representing different stages of cell cycle.It helps in collecting population wide data of cells progressing through various stages of cell cycle. Immortalization, refers to cells having capability of undergoing cell division infinitely. Immortal cells are particularly preferred in cell culture to enable long time storage and use. This presentation teaches about cell synchronization, methods of cell synchronization, cellular transformation, immortalization and mechanism of immortalization.
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.
Biology and characterization of the cell cultureKAUSHAL SAHU
This document provides an overview of cell culture techniques. It discusses the history of cell culture beginning in the late 1800s. Key terms like cell line, normal cell line, and continuous cell line are defined. The document outlines the biology of cultured cells including adhesion, proliferation, differentiation, and energy metabolism. It also discusses the origin of cultured cells and characterization techniques for authenticating cell lines such as morphology, chromosome content, and DNA analysis. The applications of animal cell culture and conclusions are presented.
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
This document discusses stem cell culture and provides definitions, classifications, and methods for culturing different types of stem cells. It summarizes the history of stem cell research from 1981 to present. It describes embryonic stem cells, adult stem cells including bone marrow and umbilical cord stem cells. Methods are outlined for isolating and culturing stem cells from bone marrow and umbilical cord. Advantages and disadvantages of different stem cell sources are compared.
This document discusses various techniques for cell cloning and selection, including:
- Primary cell cultures are sub-cultured at a 1:2 ratio while continuous cell lines replicate at higher rates.
- Cloning involves isolating and growing cells with similar properties through dilution cloning or cloning on feeder layers.
- Conditions like growth medium, serum, hormones, and carbon dioxide levels can improve cloning efficiency.
- Colonies are selected through isolation with cloning rings or drug/growth factor resistance and further propagated.
- Proper examination, good laboratory practices, and new automated systems help optimize the cloning process.
Gene transfer methods in animals can be natural or artificial. Natural methods include conjugation, transformation, and transduction which transfer genes between bacteria. Artificial methods like microinjection, biolistics, liposome mediated transfer, calcium phosphate mediated transfer, and electroporation are used to directly insert genes into cells. These techniques transfer genes into organisms for genetic engineering applications such as producing transgenic animals, developing vaccines, and gene therapy to treat diseases.
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
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
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
Cell culture, Different type of cell culture media, types of mediaRajashekar Baldhu
This document provides information on different types of cell culture media. It discusses natural media including biological fluids and tissue extracts. It also discusses artificial/synthetic media including balanced salt solutions, basal media, and complex media. Specific components of media are outlined including amino acids, vitamins, inorganic salts, glucose, serum, proteins, growth factors, hormones, and antibiotics. Different types of specialized media are also mentioned such as serum-free, chemically defined, conditioned, and protein-free media. Criteria for selecting the appropriate media for specific cell lines is discussed.
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.
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.
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.
Cell culture involves growing cells from tissue or organ samples in artificial environments outside of the original organism. There are several stages of cell culture, beginning with isolating tissues through enzymatic or mechanical means. Primary cell cultures have a limited lifespan, while continuous cell lines can proliferate indefinitely. Proper culture conditions require appropriate media, substrates, gases, and temperature/humidity control. Cells may be grown as adherent monolayers or in suspension. Cell culture has many applications including drug development, cancer research, and production of therapeutic products.
1. Callus culture involves growing undifferentiated plant cells and tissues on a nutrient medium under sterile conditions. This allows for the production of genetically identical clones without seeds or pollination.
2. A callus is an unorganized mass of cells formed from injured or cultured plant tissue. Successful callus culture requires selecting an explant, preparing sterile culture media, and regulating hormone levels to induce cell proliferation.
3. Callus cultures are maintained through periodic sub-culturing to replenish nutrients and prevent toxicity. The growth and characteristics of callus tissue can provide insights into plant cell metabolism, differentiation, and pathways for genetic engineering applications.
This document discusses callus and suspension cultures. Callus culture involves culturing explants on agar medium to form an unorganized cell mass called callus. Suspension cultures involve culturing tissues or cells in liquid medium, producing single cells and clumps. There are three types of suspension cultures: batch, continuous, and immobilized. Batch cultures use a limited nutrient supply until growth declines. Continuous cultures drain out used medium and add fresh medium to maintain a steady state. Immobilized cultures encapsulate plant cells in gels like agarose.
Organ culture technique in synthetic media- animal tissue culture neeru02
Organ culture is a development from tissue culture that allows for the culture of pieces of organs on artificial media to accurately model organ functions in various states. Special culture methods are required as organs require high oxygen levels. Organ pieces can be cultured on plasma clots, agar, raft methods using lens paper or rayon, grid methods, or in liquid media using supports like gauze or rafts. Organ culture faces limitations as results may not match whole animal studies due to lack of in vivo drug metabolism.
It's about animal biotechnology Primary cell culture is the ex vivo culture of cells freshly obtained from a multicellular organism, as opposed to the culture of immortalized cell lines. ... When whole or partial tissues are isolated and maintained ex vivo, the procedure is termed primary tissue culture.
Histotypic culture involves growing cell lines in a three-dimensional matrix at high density to form tissue-like structures. Common techniques include using gels, sponges, hollow fibers, spheroids, and rotating chambers to provide a 3D environment that allows cells to organize similarly to how they would in tissues. This allows for the study of processes like drug penetration and cell differentiation that are not possible with traditional 2D cultures. While histotypic cultures provide a model for certain tissue functions, they also face challenges like loss of cell differentiation over time.
The document summarizes key aspects of culturing cells outside of their native biological environment. Cultured cells experience changes to their microenvironment, cell-cell interactions, and exposure to stimuli. Their growth is influenced by factors like the substrate, medium composition, temperature, and gas phase. Most cells require attachment to a substrate to proliferate. Adhesion molecules like integrins and cadherins mediate attachment and formation of intercellular junctions. The extracellular matrix and cytoskeleton also influence cell behavior. Control of the cell cycle, proliferation, differentiation, motility, and response to the culture environment are described at a high level. Challenges like dedifferentiation and evolution of cell lines over multiple passages are also covered.
Cell synchronization helps in obtaining distinct sub population of cells representing different stages of cell cycle.It helps in collecting population wide data of cells progressing through various stages of cell cycle. Immortalization, refers to cells having capability of undergoing cell division infinitely. Immortal cells are particularly preferred in cell culture to enable long time storage and use. This presentation teaches about cell synchronization, methods of cell synchronization, cellular transformation, immortalization and mechanism of immortalization.
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.
Biology and characterization of the cell cultureKAUSHAL SAHU
This document provides an overview of cell culture techniques. It discusses the history of cell culture beginning in the late 1800s. Key terms like cell line, normal cell line, and continuous cell line are defined. The document outlines the biology of cultured cells including adhesion, proliferation, differentiation, and energy metabolism. It also discusses the origin of cultured cells and characterization techniques for authenticating cell lines such as morphology, chromosome content, and DNA analysis. The applications of animal cell culture and conclusions are presented.
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
This document discusses stem cell culture and provides definitions, classifications, and methods for culturing different types of stem cells. It summarizes the history of stem cell research from 1981 to present. It describes embryonic stem cells, adult stem cells including bone marrow and umbilical cord stem cells. Methods are outlined for isolating and culturing stem cells from bone marrow and umbilical cord. Advantages and disadvantages of different stem cell sources are compared.
This document discusses various techniques for cell cloning and selection, including:
- Primary cell cultures are sub-cultured at a 1:2 ratio while continuous cell lines replicate at higher rates.
- Cloning involves isolating and growing cells with similar properties through dilution cloning or cloning on feeder layers.
- Conditions like growth medium, serum, hormones, and carbon dioxide levels can improve cloning efficiency.
- Colonies are selected through isolation with cloning rings or drug/growth factor resistance and further propagated.
- Proper examination, good laboratory practices, and new automated systems help optimize the cloning process.
Gene transfer methods in animals can be natural or artificial. Natural methods include conjugation, transformation, and transduction which transfer genes between bacteria. Artificial methods like microinjection, biolistics, liposome mediated transfer, calcium phosphate mediated transfer, and electroporation are used to directly insert genes into cells. These techniques transfer genes into organisms for genetic engineering applications such as producing transgenic animals, developing vaccines, and gene therapy to treat diseases.
Introduction
Components of binary vector
Development of binary vector system
Properties of binary vector
Types of binary vector
Plant transformation using binary vector
Advantage of using binary vector
Conclusion
References
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
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
Cell culture, Different type of cell culture media, types of mediaRajashekar Baldhu
This document provides information on different types of cell culture media. It discusses natural media including biological fluids and tissue extracts. It also discusses artificial/synthetic media including balanced salt solutions, basal media, and complex media. Specific components of media are outlined including amino acids, vitamins, inorganic salts, glucose, serum, proteins, growth factors, hormones, and antibiotics. Different types of specialized media are also mentioned such as serum-free, chemically defined, conditioned, and protein-free media. Criteria for selecting the appropriate media for specific cell lines is discussed.
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.
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.
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.
Cell culture involves growing cells from tissue or organ samples in artificial environments outside of the original organism. There are several stages of cell culture, beginning with isolating tissues through enzymatic or mechanical means. Primary cell cultures have a limited lifespan, while continuous cell lines can proliferate indefinitely. Proper culture conditions require appropriate media, substrates, gases, and temperature/humidity control. Cells may be grown as adherent monolayers or in suspension. Cell culture has many applications including drug development, cancer research, and production of therapeutic products.
1. Callus culture involves growing undifferentiated plant cells and tissues on a nutrient medium under sterile conditions. This allows for the production of genetically identical clones without seeds or pollination.
2. A callus is an unorganized mass of cells formed from injured or cultured plant tissue. Successful callus culture requires selecting an explant, preparing sterile culture media, and regulating hormone levels to induce cell proliferation.
3. Callus cultures are maintained through periodic sub-culturing to replenish nutrients and prevent toxicity. The growth and characteristics of callus tissue can provide insights into plant cell metabolism, differentiation, and pathways for genetic engineering applications.
This document discusses callus and suspension cultures. Callus culture involves culturing explants on agar medium to form an unorganized cell mass called callus. Suspension cultures involve culturing tissues or cells in liquid medium, producing single cells and clumps. There are three types of suspension cultures: batch, continuous, and immobilized. Batch cultures use a limited nutrient supply until growth declines. Continuous cultures drain out used medium and add fresh medium to maintain a steady state. Immobilized cultures encapsulate plant cells in gels like agarose.
Organ culture technique in synthetic media- animal tissue culture neeru02
Organ culture is a development from tissue culture that allows for the culture of pieces of organs on artificial media to accurately model organ functions in various states. Special culture methods are required as organs require high oxygen levels. Organ pieces can be cultured on plasma clots, agar, raft methods using lens paper or rayon, grid methods, or in liquid media using supports like gauze or rafts. Organ culture faces limitations as results may not match whole animal studies due to lack of in vivo drug metabolism.
It's about animal biotechnology Primary cell culture is the ex vivo culture of cells freshly obtained from a multicellular organism, as opposed to the culture of immortalized cell lines. ... When whole or partial tissues are isolated and maintained ex vivo, the procedure is termed primary tissue culture.
Histotypic culture involves growing cell lines in a three-dimensional matrix at high density to form tissue-like structures. Common techniques include using gels, sponges, hollow fibers, spheroids, and rotating chambers to provide a 3D environment that allows cells to organize similarly to how they would in tissues. This allows for the study of processes like drug penetration and cell differentiation that are not possible with traditional 2D cultures. While histotypic cultures provide a model for certain tissue functions, they also face challenges like loss of cell differentiation over time.
The document summarizes key aspects of culturing cells outside of their native biological environment. Cultured cells experience changes to their microenvironment, cell-cell interactions, and exposure to stimuli. Their growth is influenced by factors like the substrate, medium composition, temperature, and gas phase. Most cells require attachment to a substrate to proliferate. Adhesion molecules like integrins and cadherins mediate attachment and formation of intercellular junctions. The extracellular matrix and cytoskeleton also influence cell behavior. Control of the cell cycle, proliferation, differentiation, motility, and response to the culture environment are described at a high level. Challenges like dedifferentiation and evolution of cell lines over multiple passages are also covered.
Cell culture involves growing cells under controlled conditions outside of their natural environment. There are several types of culture, including organ culture, tissue culture, and cell culture. Cell culture allows cells to be studied without the complexity of an entire organism and provides a model for understanding cell behavior. Key factors that must be controlled include the culture surface, growth media formulation, temperature, gas exchange, and passaging of cells to maintain healthy growth.
This document summarizes the structure and functions of cell membranes. It describes the main components of cell membranes as lipids (54%), proteins (46%), and carbohydrates (5-10%). The main lipids include glycerophospholipids, sphingophospholipids, glycosphingolipids, galactolipids, sterols like cholesterol. Membrane proteins can be integral or peripheral and perform functions like selective transport, enzymatic reactions, cell signaling, cell recognition and adhesion. The fluid mosaic model proposes lipids and proteins move laterally within the membrane. Overall, the cell membrane outlines the cell, maintains its shape and integrity, controls transport across it, and allows various cellular functions.
This document provides information on the basic structures of animal cells, including the cell membrane, cytoplasm, and organelles. It discusses the components and functions of the cell membrane, cytoplasm, cytoskeleton, mitochondria, endoplasmic reticulum, ribosomes, Golgi bodies, and nucleus. The key structures and their roles in transport, synthesis, modification and packaging of molecules are summarized.
Bacteria have a variety of shapes and arrangements. Their cells are surrounded by a cell wall and cytoplasmic membrane. The cell wall provides shape and protection, and its structure differs between gram-positive and gram-negative bacteria. Bacteria may also have extra structures like a capsule outside the cell wall or fimbriae. These extra structures help bacteria attach to surfaces and sometimes contribute to virulence.
Microscopic anatomy of gingival epitheliumNishiMahapatra
This document provides a detailed overview of the microscopic anatomy of gingival epithelium. It begins by defining the gingiva and classifying gingival epithelium as either keratinized or non-keratinized. The key layers and cellular features of both keratinized and non-keratinized epithelium are then described in depth, including descriptions of the stratum basale, stratum spinosum, stratum granulosum, stratum corneum, and cell-cell attachments such as desmosomes. The functions and characteristics of gingival epithelium are also summarized.
The document discusses the structure and functions of the plasma membrane. It describes the plasma membrane as a lipid bilayer with proteins embedded within it. The lipid bilayer is composed primarily of phospholipids and cholesterol, which gives the membrane a fluid mosaic structure. Integral proteins pass through the membrane, while peripheral proteins are attached to either side. Together, the lipids and proteins selectively regulate what passes in and out of the cell and allow the membrane to perform critical functions for the cell.
The document discusses the structure and functions of the plasma membrane. It describes the plasma membrane as a lipid bilayer with proteins embedded within it. The lipid bilayer is composed primarily of phospholipids and cholesterol, which gives the membrane a fluid mosaic structure. Integral proteins pass through the membrane, while peripheral proteins are attached to either side. Together, the lipids and proteins selectively regulate what passes in and out of the cell and allow the membrane to perform critical functions for the cell.
This document provides an overview of key cell organelles:
- The cell membrane controls movement of substances in and out of cells. It consists of a lipid bilayer and embedded proteins.
- Mitochondria produce ATP through respiration and regulate metabolism. They have an outer and inner membrane.
- The Golgi apparatus packages and modifies proteins and lipids in the cell.
- The endoplasmic reticulum synthesizes lipids and proteins. It has rough and smooth regions.
- Lysosomes contain enzymes for breaking down biomolecules through autophagy and endocytosis.
- Ribosomes are the sites of protein synthesis in the cell.
The document discusses protoplasts, which are plant cells that have had their cell walls removed, leaving the cell membrane and organelles. It describes methods for isolating protoplasts from plant tissues using either mechanical or enzymatic methods. The enzymatic method uses enzymes like pectinase and cellulase to break down the cell wall. Protoplasts have various applications including isolating cell organelles and studying cell structures. The document also discusses immobilizing enzymes by binding them to inert matrices, which has benefits like reusability and stability. Methods of immobilization include adsorption, covalent binding, and entrapment in gels.
This document provides an overview of cell cloning techniques. It begins with definitions of cloning and describes the different types including cell cloning. The key techniques discussed are dilution cloning, which involves diluting cells to isolate colonies, and microtitration plate cloning. Factors that influence plating efficiency are also covered. The document then discusses methods to improve clonal growth like using conditioned medium or feeder layers. Suspension cloning in agar or methylcellulose is also introduced. Applications of cell cloning and conclusions about ongoing improvements in cloning techniques are presented at the end.
Bacteria are microscopic, unicellular organisms that lack nuclei and organelles. They display a diversity of shapes and sizes, and structures like flagella and pili enable motility and genetic exchange. The bacterial cell wall provides structural integrity and protection, and differs between gram-positive and gram-negative bacteria in its chemical composition and layers. Flagella are helical filaments that rotate to propel bacteria and allow for swimming movement in different directions.
Biology 1 for grade 12 SHS (cell structure)knip xin
The document discusses the key scientists who contributed to cell theory and lists the three main tenets of cell theory. It also provides an overview of the major organelles found in cells like the nucleus, mitochondria, chloroplasts, lysosomes, and describes their main functions. Additionally, it compares the structure and organization of plant and animal cells and provides an analogy between the parts of a cell and that of a fictional city called Grant City.
The document discusses the extracellular matrix (ECM), which was once considered an inert scaffold but is now recognized as a complex, interactive network that regulates cell gene expression. It describes the various components that make up the ECM, including collagens, elastin, fibrillin, fibronectin, and laminins. It also discusses the functions of the ECM, such as providing mechanical support and controlling cell proliferation, and describes some clinical correlations regarding genetic defects in collagen.
2 second lecture Structure of Bacterial cells Mohamed Hafez
1. Bacterial cells have a cell wall, cytoplasm, nuclear material, and cytoplasmic membrane. They may also have extracellular structures like capsules, flagella, pili, and spores.
2. The cell wall provides structure and protects the cell. It differs in Gram positive and negative bacteria.
3. The cell membrane is semi-permeable and transports nutrients, waste, and is the site of antibiotic action.
4. Bacteria may form intracellular inclusions or extracellular structures like capsules, flagella, pili or spores that help with functions like adherence, motility or survival.
This document discusses techniques for primary cell culture, including mechanical disaggregation, enzymatic disaggregation using trypsin or collagenase, and the primary explant technique. It provides details on each technique, such as how tissues are chopped or sliced, the use of enzymes at different temperatures, and factors that promote efficient development of primary cultures. The document also covers criteria for cell separation methods, cell differentiation, methods of cell transformation, and characteristics of transformed cells.
The document summarizes key aspects of prokaryotic and eukaryotic cell structure. It describes the three layered cell envelope of most prokaryotic cells, consisting of an outer glycocalyx, middle cell wall, and inner plasma membrane. It also discusses how bacteria can be classified as Gram-positive or Gram-negative based on differences in their cell envelopes and staining. The document then covers additional structures like flagella, pili, and inclusion bodies in prokaryotes, as well as organelles and cytoskeletal elements in eukaryotic cells. It concludes by outlining the fluid mosaic model of the cell membrane and different transport mechanisms like diffusion, osmosis, facilitated transport, and active transport
This document provides information on epithelial tissues. It defines epithelium and describes its structure and functions. Epithelial tissues are composed of cells that cover surfaces and line cavities. They are classified based on cell shape and number of layers. Epithelial cells exhibit polarity with distinct apical, lateral, and basal domains. Tight junctions between cells form a barrier and anchorages attach cells. The basement membrane anchors epithelium to connective tissue and regulates signaling. Epithelial tissues include simple and stratified types that vary in keratinization and serve protective, secretory, absorptive, and sensory roles.
The small intestine is composed of three parts - the duodenum, jejunum, and ileum. The duodenum is about 25cm long and has four parts. It is connected to the liver and contains the duodenal papilla through which the common bile duct and pancreatic duct empty. The jejunum is around 2.5m long and makes up the middle section of the small intestine. It contains circular folds and finger-like villi that increase its surface area for absorption. The ileum is around 3.5m long and contains Peyer's patches. It connects to the large intestine at the ileocecal valve.
The oesophagus connects the mouth to the stomach through the pharynx. It is approximately 25cm in length and has three parts - cervical, thoracic, and abdominal. Food passes through the upper esophageal sphincter into the oesophagus, triggering peristalsis to push the food bolus through in 6-15 seconds. The lower esophageal sphincter then opens to allow the food to enter the stomach and closes behind it to prevent acid reflux. The oesophagus wall has four layers but lacks a serosa, and receives parasympathetic and sympathetic innervation to control sphincters and peristalsis.
1) The gastrointestinal tract is approximately 9 meters long and runs from the mouth to the anus, mechanically and chemically breaking down food.
2) The mouth contains taste buds that detect the five basic tastes and contains salivary glands that produce saliva to moisten food for swallowing.
3) Chewing and swallowing propel food through the esophagus and into the stomach through peristalsis, where further digestion will occur.
Describes the secretion and functions of Antidiuretic hormone, abnormalities associated with ADH secretion, reasons of SIADH etc in details with figures.
This document discusses the three phases of detoxification. Phase III involves efflux transporters that transport substances transformed in Phase II out of cells. The best known transporter is P-glycoprotein, which eliminates conjugated metabolites from tissues. Transporters play a dual role in Phase III and in first pass metabolism by eliminating compounds before circulation. During first pass metabolism in the liver and intestines, compounds are biotransformed before reaching systemic circulation, resulting in lower bioavailability. Deconjugation by intestinal bacteria can recycle some compounds back through the liver via enterohepatic circulation.
Second ppt on endocrine system, describing hypothalamus, pituitary and thyroid glands.
This describes the hormones from these glands and their mode of action etc
This is on the basic details of the endocrine system including the different types of hormones. It describes the mechanisms of actions of hormones. The general control mechanisms of hormone production and release are also included.
This ppt is about the variations in metabolic processes between different types of cells in different organs of our body. The reasons for the variations are also descried. This is the first set of slides on the topic.
Describes the different types of chemical messengers in mammalian body. This explains their synthesis and mode of action also. A short account of neurohormones and neuroendocrine function is also included.
Heme synthesis is the biochemical pathway that produces heme, an iron-containing molecule that is an essential part of hemoglobin. The pathway has many steps that occur in both the cytosol and mitochondria of cells. A deficiency in any of the enzymes or substrates involved can cause a condition called porphyria. The first reaction is the rate-limiting step of condensing glycine and succinyl-CoA to form delta-aminolevulinic acid (ALA). Subsequent steps modify ALA and its derivatives to ultimately form protoporphyrin IX. The last step is the insertion of an iron ion into protoporphyrin IX by the enzyme ferrochelatase to complete heme synthesis.
This presentation is about bioenergetics. It talks about energy changes and equilibrium during different biological reactions, how exergonic and endergonic reactions are combined as sequential reactions in body, how the body system is following the law of thermodynamics etc. Role of enzymes in thermodynamics is also explained
Describes the different pathways involved in the synthesis of different eicosanoids like prostaglandins, leukotrienes, lipoxins etc along with different enzymes involved.
Describes the process of ageing in cells, factors affecting cells like telomere, free radicals, oxidative stress, DNA damage, environmental factors, proteostasis, mitochondrial disfunction etc are described
The document discusses the Ramachandran plot, which shows statistically probable combinations of the phi and psi backbone torsion angles in proteins. It describes how these two angles describe rotations around bonds in the polypeptide backbone and influence protein folding. The plot reveals allowed and disallowed regions based on steric clashes between atoms at different angle combinations. Common structures like alpha helices and beta sheets correspond to allowed regions in the plot.
The gastrointestinal tract is approximately 9 meters long and runs from the mouth to the anus. It mechanically and chemically breaks down food. The document discusses the different parts of the GI tract including the mouth, tongue, taste buds, salivary glands, and swallowing process. It describes the roles and functions of these parts in digesting and moving food through the body.
Lipoproteins are complexes of lipids and proteins that transport lipids through the water-based blood system. They consist of a nonpolar lipid core of triglycerides and cholesterol esters surrounded by a single layer of amphipathic phospholipids and cholesterol. Apolipoproteins attached to the surface act as proteins. Lipoproteins are classified based on their density, which is determined by ultracentrifugation. The five major groups are chylomicrons, VLDL, IDL, LDL, and HDL.
This document summarizes the regulation of blood glucose levels. It discusses how blood glucose levels fluctuate after meals and during fasting states. The pancreas plays a key role by secreting hormones like insulin and glucagon that work to maintain normal blood glucose levels. Insulin promotes glucose uptake by cells to lower blood glucose, while glucagon has the opposite effect of raising blood glucose levels. The body uses negative feedback loops and other hormones to precisely control blood glucose levels through processes like glycogenesis and glycogenolysis in the liver.
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...Leonel Morgado
Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
Sexuality - Issues, Attitude and Behaviour - Applied Social Psychology - Psyc...PsychoTech Services
A proprietary approach developed by bringing together the best of learning theories from Psychology, design principles from the world of visualization, and pedagogical methods from over a decade of training experience, that enables you to: Learn better, faster!
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
The debris of the ‘last major merger’ is dynamically young
Culture of specialised cells
1. Culture of specialised cells
Dr. Radhakrishna G Pillai
Department of Life Sciences
University of Calicut
2. Epithelial cells
• Various layers of cells that either
– coat surfaces on the exterior of the organism or
– line internal organs, ducts, or secretary acini
• They may act as a total barrier, such as
– the epidermis, with minimum permeation of polar
substances, or
– a regulated barrier
• for example, in the intestine and the lung, where
• selected substances are able to cross the plasma membrane
or the whole epithelium via specific transporters
• Basal to apical polarization is fundamental to the
normal function of all epithelia.
3. Epithelial cells
• Epithelia are associated with the major functional role
of many tissues, such as
– hepatocytes and liver metabolism
– epidermal keratinocytes and the barrier properties of skin
– pancreatic acinar cells and digestive enzyme secretion
• have been a focus of interest in the development of in
vitro models for many years
• Because most epithelia are renewable;
– they have proliferating precursor compartments and stem
cells capable of self-renewal and
– hence form attractive models for studying the regulation of
cell proliferation and differentiation
4. Epithelial cells- cancer
• Regenerative nature makes epithelial cells sites for
malignant transformation in vivo, and
• the most common solid tumors are the
– carcinomas of lung, breast, colon, prostate, and bladder,
– derived from the epithelial cells of these tissues
• Epithelial cell systems have therefore been adopted as
appropriate models for studies of
– Carcinogenesis and
– Differentiation
– on the assumption that malignancy results, at least in part, from
a failure to differentiate
• This has produced many excellent models for differentiation
and,
• some of the clearest examples of stem cell maturation,
5. Epithelial cells -Histology
• Epithelial cell layers are separated from other cellular compartments (e.g.,
connective tissue, capillaries) by a basement membrane made up of
collagen, laminin, fibronectin, and proteoglycans, and the reconstitution
of this basement membrane in vitro has been featured in many attempts
to grow functional epithelium
The basement membrane is
usually a joint product of the
epithelium and the underlying
stroma and, together with soluble
factors from the stroma, serves to
regulate the differentiated
function of the epithelium as
well as providing physical support
and a barrier separating epithelial
and stromal compartments
6. Functions of epithelial cells
• Epithelial cells are closely associated in vivo
• regulated permeability and transport functions; to maintain this structural
integrity,
• they are usually joined by desmosomes -the mechanical junctions connected to
the intermediate filament cytoskeleton that hold epithelium together and are
characteristic markers of epithelial identity
• Where barrier properties are particularly crucial (e.g., kidney ductal epithelium,
or secretory acini), the desmosomes are accompanied by tight junctions forming
a junctional complex that is quite specific to epithelium
• The presence of these junctional complexes, together with cytokeratin
intermediate filaments provide very useful and specific markers for recognizing
epithelial cells in vitro
7. Culturing epithelial cells
• As epithelial layers are closely associated in vivo and are strongly
self-adherent
• they tend to survive better in vitro as clusters or sheets of cells
• Dissociation techniques that have been found to be most
successful tend to exploit this observation and do not try to reduce
the population to a single cell suspension
• For this reason, cultures have been derived either by gentle
mechanical disaggregation or collagenase digestion in preference
to trypsinization
• Collagenase appears to give good survival as it does not
completely dissociate the epithelium but frees it from the
surrounding stroma
• The clusters of epithelium formed by this technique can be
collected by
– allowing them to sediment through the more finely dispersed
fibroblastic stromal cells or
– by filtration through nylon gauze
8. Removing fibroblasts
• it is very difficult to eliminate all the fibroblasts by
physical methods
– more rapid growth rate
– they will tend to overgrow the culture
• This is caused by the stimulation of the fibroblasts by
• platelet-derived growth factor (PDGF) and
• by the cytostatic effect on the epithelium of
transforming growth factor type (TGF-), released from
platelets during the preparation of serum.
• It is also due partly to the design of the media, many of
which were developed for fibroblastic cells
9. Removing fibroblasts
Selective Attachment
• seed the cell mixture into a flask for a short time (e.g., 30 min
or 1 h) and then transfer the unattached cells into a fresh
flask
• This is repeated at intervals of 1–3 h up to 24 h or 48 h
• it is often found that the fibroblasts tend to stick down first,
whereas the epithelial cells remain in suspension and attach
in the later-seeded flasks
• This is probably due to a combination of circumstances
– some mechanical (the size of the epithelial aggregates) and
– some physiologic (the greater need for extracellular matrix
regeneration for epithelial attachment)
• selective adhesion methods such as these have limited
success but may be of value in combination with other
techniques or for short periods of culture
10. Removal of fibroblasts
• Selective Detachment
• enzymes such as dispase can release epithelial
sheets before the fibroblasts [
• like selective attachment
– it is effective in certain conditions
– for example, releasing epithelial patches of colonic
epithelial cells
– it is not universally successful and may select or alter the
epithelium released
• EDTA can also be used to remove fibroblasts
selectively from mixed cultures of keratinocytes
11. Removing contaminating fibroblasts
• Substrate Modification
• To exploit the principle of selective attachment, some groups
have attempted to modify the substrate to favor epithelial
attachment
• Collagen coating, particularly native or undenatured collagen,
has been used to select epidermal cells and breast epithelium
• Collagen and laminin separately or combined, particularly in
Matrigel, have also been found to encourage the expression
of the differentiated phenotype in many epithelial cells
although this may limit their proliferative capacity
• Becton Dickinson produces a modified plastic, Primaria, that
has a net positive charge claimed to favor epithelial growth in
preference to fibroblasts
12. Removal of contaminating cells
Feeder Layers
• The most popular substrate modification is to preplate with a
monolayer of fibroblasts, or other cells, that can be irradiated to
prevent their further growth
• A preformed layer of irradiated or mitomycin C-treated 3T3 cells
enhances the survival of many epithelial cells, including
keratinocytes, cervical epithelium, and breast epithelium
• This appears to repress the further growth of fibroblasts
• This may be caused, partly, by the ability of epithelium to
– attach to the fibroblasts
– normal fibroblasts are unable to do so
– also probably caused by release by the feeder cells of paracrine
factors that enhance epithelial survival and may block the action of
TGF
• It is probably the most generally successful method of enhancing
epithelial growth and inhibiting fibroblastic overgrowth
13. Physical methods of separation
• It is possible to separate many cell types by physical methods,
such as density gradient centrifugation, centrifugal
elutriation, and flow cytometry
• Of these, flow cytometry has the greatest resolution, but it
has a relatively low yield;
• centrifugal elutriation gives the highest yield
– only effective when there are clear distinctions in cell size
– The purification achieved is seldom complete but may suffice for
the generation of a purified population for immediate use
– it seldom has a lasting effect, as the contaminating stromal cells
usually proliferate more rapidly in standard media with serum
supplementation
• Both of these methods are technically complex to use and
involve expensive equipment, but they have been used very
successfully with many different cell systems
14. Obtaining pure culture
• Magnetic separation has become increasingly effective
• Specific antibodies conjugated to iron-containing coated beads have been used to isolate
different cell types magnetically, either by a positive sort for epithelium [
• a negative sort for contaminating fibroblasts [
• A cell suspension, previously incubated with antibody-conjugated beads, is passed down a
glass cylinder,
• the cells bound to the beads are trapped at the side of the tube by placing an electromagnet
outside the cylinder
Turning off the current allows the beads to
be eluted with the attached cells, which
can be separated from the beads by
trypsinization
Some systems (e.g., Miltenyi) used
magnetizable microbeads that permit
subsequent culture without requiring
removal of the beads and have antibody
conjugations suitable for both positively
sorting epithelial cells and negatively
sorting fibroblasts
15. Selective Culture
• The ideal method of purifying a population of cells is by cloning
• Used in conjunction with a feeder layer, this method permits many
epithelial cells to be cloned quite successfully
• unfortunately the culture may senesce before sufficient cells are
generated
• If relatively few cells are required, if the clones can be pooled, or if the line
has been immortalized, cloning may prove to be the ideal method
• Selective media have become one of the principal methods for growing
epithelial cells preferentially
• Several media have been developed capable of supporting different types
of epithelial cells
• They are serum-free, eliminating TGF- and PDGF, which favor fibroblastic
growth, and they often incorporate hormones and growth factors, such as
hydrocortisone, isoproterenol (isoprenaline), and epidermal growth factor
(EGF), which stimulate epithelial proliferation
• They have the advantage that they do not depend on one selective event
but continue to exert a selective pressure in a nutritionally optimized
environment. Many of these media are available commercially
16. Characterisation
• Validating selected epithelial cell lines requires the adoption of specific
criteria for identifying the cells as epithelial
• The time honored method is by morphology, as most epithelial cultures
have a characteristic tight pavement like appearance with cells growing in
well-circumscribed patches
• However, not all epithelia grow in this manner; some show greater
plasticity in shape, particularly when derived from tumors
• some mesenchyme derived cells such as endothelium and mouse embryo
fibroblasts like 3T3 cells (which are probably primitive mesenchyme rather
than committed fibroblasts) can look quite epithelial at confluence
• reliable epithelial identification depend on the recognition of certain
specific markers
• The intermediate filament proteins have long been recognized for their
tissue specificity
• Among these the cytokeratin group are found predominately in epithelia
• cytokeratins are rarely seen in other cells
17. Characterisation
• the cytokeratins are a large group with considerable diversity
• different anti-cytokeratin antibodies may have differing specificities, enabling distinctions to
be made among different types of epithelia or between different stages of differentiation
• When the culture reaches confluence, desmosome junctions, which are also specific to
epithelia, can be detected by electron microscopy and desmosomal proteins, like
desmoplakin can be demonstrated by immunostaining
• Where cultures of ductal cells are able to differentiate, it may also be possible to see
junctional complexes with desmosomes and tight junctions in a characteristic association
• Because of this ability to form tight junctions, and their ability to transport water and ions,
epithelial monolayers sometime generate so-called domes, formed when the cell layer
blisters off the substrate because of fluid and ion transport from the medium to the
subcellular space . This activity is characteristic of ductal and secretory epithelia
• A number of cell surface antigens have been shown to be specific to epithelium. These are
often from one group of transmembrane mucin like glycoproteins and include epithelial
membrane antigen (EMA)
• human milk fat globule (HMFG)-1 and HMFG-2
• These antigens are most strongly expressed in differentiated ductal epithelium, where they
may become polarized to the apical surface, but present to varying degrees in many
different epithelia. There are also a number of more specific markers such as involucrin in
keratinocytes
18. Culturing mammalian mammary epithelial
cells
• Different types of epithelial cells at different stages of
differentiation
• Immunological markers are used against specific phenotypes
• Epithelial keratins are useful in immunological detection as they
are expressed in culture also
• Different mammalian epithelial cells express different keratins
• Luminal epithelial cells express 8 and 18
• Basal cells express keratins 5 and 14 – not express K 8 and 18
• Polymorphic epithelial mucin (PEM) expressed by luminal
epithelial cells
• Common leukocytic leukemia antigen expressed by basal epithelial
cells
• In vivo breast epithelial cells interact with other cell (fibroblasts,
adipose cells, matrix components) types; normal culture
conditions do not model this complexity
20. Culturing tumour cells from mammalian
breast
• Culturing mammary tumour epithelial cells were found
difficult than normal HMEC
• They do not lead to outgrowth of cells displaying
tumour -associate phenotypes
• Specialised culture conditions developed for the growth
of tumour derived HMEC that display phenotypes of
tumour cells
– Specialised media formulations
– Low Ca, nutrient and oxygen concentration
– Specialised methods of tissue digestion
• There is no one standard procedure allowing
demonstrable tumour cells from primary tumours
21. Tissue processing
• Tissue digestion with enzymes – break down stroma and free
epithelial organoids
• Filter to remove the digested stroma
• Fibroblasts cold be obtained from the filtrate
• MCDB 170 medium is used for primary culture
• Fibroblasts and blood vessel associated cells will not grow
well in MCDB 170 medium
• MCDB 170 medium developed for the clonal growth of
epithelial cells
• Sub cultured when large epithelial patches are present, but
before confluence
• Density of seeding and attachment will influence the time
required (aprox 7-14 days)
• To generate multiple secondary culture and to retain primary
culture – only 50% cells are removed by partial trypsinization
22. Mesenchymal stem cells
• Mesenchymal stem cells (MSCs) are multipotent stem cells
• also termed Mesenchymal Stromal Cells
• have the potential to self-renew and differentiate into a
variety of specialised cell types such as osteoblasts,
chondrocytes, adipocytes, and neurons
• MSCs are easily accessible, expandable, immunosuppressive
and they do not elicit immediate immune responses
• Therefore, MSCs are an attractive cell source for tissue
engineering and vehicles of cell therapy
• MSCs can be isolated from various sources such as adipose
tissue, tendon, peripheral blood, and cord blood
• Bone marrow (BM) is the most common source of MSCs
23. MSCs from Bone marrow
• MSCs have been successfully isolated and characterised
from many species including mouse, rat, rabbit, dog, sheep,
pig, and human MSCs isolated from bone marrow display
multilineage differentiation potentials
• Two main stem cell populations and their progenies,
haematopoietic stem cells and BM-MSCs, are the main
residents of bone marrow
• BM-MSCs are usually isolated and purified through their
physical adherence to the plastic cell culture plate
• Several techniques have been used to purify or enrich MSCs
including antibody-based cell sorting
• low and high-density culture techniques and
• positive and negative selection method
• frequent medium changes and, enzymatic digestion approach
24. Isolation and culture of MSCs
• All the available methods had some short falls:
• the standard MSCs culture method based on
plastic adherence has been confirmed to have
lower successful rate whereas
• the cell sorting approach reduced the osteogenic
potentials of MSCs
• Negative selection method leads to granulocyte–
monocyte lineage cells reappearing after 1 week
of culture
25. Human MSCs
• Low frequency of MSCs in primary tissue
• Expansion is critical in biological studies
• They could only be propagated a limited number of ties
• After that proliferation rate decreases
• Serum free or low serum media are used for expansion and culture
• Traditionally, two-dimensional (2D) adherent culture conditions
have been used as a standard technique for in vitro expansion of
MSCs
• In vitro culture of multicellular aggregates was originally described
for embryonic cells 70 years ago
• Because of their spherical shape, these multicellular aggregates
are now called multicellular spheroids, or spheroids
• Spheroids have been utilized in the field of oncology, stem cell
biology, and tissue engineering
26. Spheroid cell culture
• Different methods are used to develope spheroid culture and they include;
• The spinner flask method -constant agitation of high density cell suspension
to minimize cellular attachment to the solid surface and to maximize cell to
cell contact
• Liquid overlay technique uses agar to prevent attachment
• Early spinner flask and liquid overlay techniques result in a heterogeneous
population of spheroids
• Improved methods are developed to generate a more homogeneous
population of spheroids
• 96-well plates are now commercially available with low attachment surfaces
for single spheroid production per well (e.g., 96 Well Ultra-Low Attachment
Spheroid Plate from Corning in Corning,
• Another widely used technique for spheroid formation is the hanging drop
method, which eliminates surface attachment by placing the cell suspension
in a drop, allowing gravity to facilitate cellular aggregation at the bottom of
the drop
• These cells spontaneously attach to each other to form cell aggregates if the
possibility of surface attachment is abolished
27. Spheroid cell culture
• Another recent spheroid formation technique involves the use of chitosan
membranes to initiate the 2D to 3D transition
• Chitosan is a deacetylated derivative of a natural polysaccharide, chitin, and
is often paired with another glycosaminoglycan, hyaluronan, known to have
an impact on cell migration, proliferation, and matrix secretion
• Spheroidal cell culture has been used extensively in the field of oncology as
spheroidal cell culture exhibits both histological and physiological features
similar to those of solid tumors in the body
• Tumor spheroids synthesize ECM similar to original tumors in vivo, where
the capacity for ECM production is reduced in the same cells in 2D culture
conditions
• The response of cancer cells to therapeutic interventions in vivo is better
reproduced in in vitro spheroidal culture than in 2D adherent culture
• In evaluating the efficacy of radiation therapy, spheroid culture of cancer
cells produces a more comparable response to cells in vivo than cancer cells
in 2D culture
• Additionally, tumor spheroids might possibly mimic circulating tumor cell
aggregates
28. Spheroids in stem cell culture
• Spheroidal cell culture with pluripotent stem cells (PSCs), including embryonic stem
cells (ESCs), is specifically called embryoid body
• Utilization of embryoid bodies is a standard protocol to produce specific cell
lineages of interest in vitro, as the intercellular interactions of embryonic cells
occurring during embryogenesis are recapitulated in the 3D culture setting
• Similarly, spheroidal cell culture of neural stem cells (NSCs), or neurospheres, has
been used routinely for NSC isolation from embryonic and adult tissues and in
vitro expansion and differentiation of NSCs into neurons, oligodendrocytes, and
astrocytes
• Differentiation capability and potential of stem and progenitor cells are generally
enhanced in the 3D culture setting
• For example, salivary gland-derived progenitor cells can differentiate into
hepatocytic and pancreatic islet cell lineages, but these differentiations only take
place when the cells are cultured in 3D cell aggregates, not in 2D monolayer
• Neuronal differentiation of ESCs is enhanced in embryoid body culture compared
to 2D monolayer cell culture
• Moreover, in vitro reproduction of complex organ architecture, such as the optic
cup, is made possible only in 3D culture, in which the inherent tissue self-
organization capability of ESCs is maximized
29. Gonadal tissue
• Major players in growth and differentiation in the testis and
ovaries are different
• In severe cases of disorders of sex development (DSD), which
are due to mosaic sex chromosome aneuploidy, individuals
often present at birth with an uncertain phenotypic gender,
the differences are blurred
• Meiotic cell division is a unique feature of germ cell
development and an early morphological sign of sex
differentiation in the developing gonads
• initiation of meiosis involves the action of retinoic acid (RA),
which in fetal ovaries mediates the up-regulation of
stimulated by retinoic acid gene 8 (Stra8) that is required for
pre-meiotic DNA replication
• Such variation from somatic cells or tissues makes the
culturing of Gonadal tissue different
30. • elective termination of pregnancy during the
first trimester
• one of the terminations were for reasons of
fetal abnormality, and all fetuses appeared
morphologically normal
• used for hanging-drop culture
31. Culture techniques
• primary human germ cells are difficult to culture outside the somatic niche,
and
• the available testicular cancer cell lines are all isolated from fully developed
Testicular germ cell tumours (TGCTs) including a range of embryonal
carcinoma lines
• The lack of a suitable model system to investigate the early progression
from Carcinoma in situ (CIS) cells to invasive tumours has also made it
difficult to determine the role of specific pathways in the pathogenesis of
testicular cance
• Several different approaches to culture human testis tissue have previously
been employed, including ex vivo cultures of adult tissue and fetal testis
tissue on membranes
• xenografting of fetal, pre-pubertal and adult testis tissue into nude mice
• Xenografting of human testis cancer cell lines into nude mice, including JKT-
1
• Hanging drop cultures are the widely used culture approach for both
embryonic (including embryonic stem cells) and adult tissues, and have
been previously successfully applied to culture intact fetal mouse testes and
adult murine seminiferous tubules
32. Hanging drop culture
• Hanging drop cultures are the widely used culture approach for both
embryonic (including embryonic stem cells) and adult tissues,
• previously successfully applied to culture intact fetal mouse testes and
adult murine seminiferous tubules (
• This culture approach has multiple benefits, including
• three-dimensional tissue architecture maintenance,
• facilitation of efficient gas exchange and
• requirement for only small media and supplement volumes
• Importantly, these cultures are particularly effective at preserving ex
vivo functional integrity and signalling activity
• a suitable avenue through which to study the effects of specific
treatments on a range of human orchidectomy specimens, from relatively
normal testis tissue, to tissue containing CIS and seminoma tumours
33. Hanging drop culture
• cultures of normal testis tissue and CIS can be maintained for
up to 14 days without signs of increased apoptosis, while the
organisation of the seminiferous epithelium is preserved and
germ cells continued proliferation
• Cultures of seminoma samples can be maintained for up to 7
days, with histology indicating that samples remain consistent
with expected tumour morphology and that proliferating
seminoma cells are present for at least 3 days
• activin A treatment of hanging drop cultures induces specific
gene and protein alterations of relevance to TGCTs
• These outcomes illustrate the value of this approach for
investigating responses to growth factors or chemical
treatments that may ultimately be applied to alter the in
vivo development and growth of testicular germ cell tumours
34. Lymphocyte culture
• Isolation of Human T Lymphocytes
• Blood from a healthy donor is used
• Allow the blood to cool to room temperature (~30 min) before proceeding to the
next step.
• Density gradient centrifugation by layering blood over a density gradient media in a
round-bottom polystyrene tube
• Centrifuge the tubes at 500 x g for 45 minutes at room temperature
• Peripheral blood mononuclear cells (PBMC) are separated from the other cells in
blood
• The PBMC layer appears, from the top down, as the first cloudy band.
Carefully remove the clear yellow-colored upper
phase of the blood, above the PBMC layer, and
Recover the PBMC layer to a 15 mL or 50 mL conical
tube
Wash the PBMC twice with PBS, centrifuging cells at
500 x g for 5 minutes each time. The supernatant
will be somewhat cloudy after each wash
35. Purification of the cells
• PBMC transferred to a T-75 culture flask in 20 mL RPMI 1640 media
• The media will containing 10% foetal bovine serum (FBS), 1%
penicillin/streptomycin, and 1 μg/mL phytohemagglutinin (PHA)
• Incubate at 37°C and 5% CO2 for at least 1 hour, and up to 24 hours
• Allows monocytes, which will be adherent to the flask surface, to be separated
from the lymphocytes that remain in suspension.
• If a short incubation (1 hour) is used at this step, it is acceptable to use RPMI 1640
media containing 10% FBS and 1% penicillin/streptomycin without supplementing
with PHA
• Carefully remove all of the media from the flask, add it to a 50 mL conical tube, and
centrifuge at 500 x g for 5 minutes
Resuspend the cell pellet, which now primarily
contains lymphocytes, and transfer the cells to a
new T-75 flask containing 25 mL RPMI 1640 media
containing 10% FBS, 1% penicillin/streptomycin,
and 1 μg/mL PHA
The cells will grow as suspension culture