This slide explains the various basic aspect of animal cell culture, cell line and cell strain, initiation and maintenance of primary cell culture, characteristic of primary cell culture and their applications. It also contains MCQs for practice.
Cellular coning refers to generation of genetically identical cells from parent cells. This presentation teaches differences between cell coning and molecular cloning and various methods of cell cloning. Sample questions are also provided for your review of concept learned
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
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
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
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
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
Cellular coning refers to generation of genetically identical cells from parent cells. This presentation teaches differences between cell coning and molecular cloning and various methods of cell cloning. Sample questions are also provided for your review of concept learned
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
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
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
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
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 presentation contains all the material regarding History of animal cell culture and different methods of organ and tissue culture.Hope it will be helpful..
Constituent of animal tissue culture media and their specific applicationKAUSHAL SAHU
INTRODUCTION
HISTORY
PHYSICOCHEMICAL PROPERTIES OF CULTURE MEDIA
pH
CO2, BICARBONATE AND BUFFERING
OXYGEN
TEMPERATURE
OSMOLALITY
BALANCED SALT SOLUTIONS
CONSTITUENTS OF CULTURE MEDIA
AMINO ACIDS
VITAMINS
SALTS
GLUCOSE
OTHER ORGANIC SUPPLEMENTS
ANTIBIOTICS
SERUM
PROTEINS
NUTRIENTS AND METABOLITES
HORMONES AND GROWTH FACTORS
LIPIDS
MINERALS
INHIBITORS
APPLICATIONS OF CULTURE MEDIA
CONCLUSION
REFERENCES
Role of serum and supplements in culture medium k.skailash saini
ROLE OF SERUM AND SUPPLEMENTS IN CULTURE MEDIA
Serum is a complex mix of albumins, growth factors and growth inhibitors.
Serum is one of the most important components of cell culture media and serves as a source for amino acids, proteins, vitamins (particularly fat-soluble vitamins such as A, D, E, and K), carbohydrates, lipids, hormones, growth factors, minerals, and trace elements.
Serum from fetal and calf bovine sources are commonly used to support the growth of cells in culture.
Fetal serum is a rich source of growth factors and is appropriate for cell cloning and for the growth of fastidious cells.
Calf serum is used in contact-inhibition studies because of its lower growth-promoting properties.
Normal growth media often contain 2-10% of serum.
Supplementation of media with serum serves the following functions :
Serum provides the basic nutrients (both in the solution as well as bound to the proteins) for cells.
Serum provides several growth factors and hormones involved in growth promotion and specialized cell function.
It provides several binding proteins like albumin, transferrin, which can carry other molecules into the cell. For example: albumin carries lipids, vitamins, hormones, etc. into cells.
It also supplies proteins, like fibronectin, which promote the attachment of cells to the substrate. It also provides spreading factors that help the cells to spread out before they begin to divide.
It provides protease inhibitors which protect cells from proteolysis.
It also provides minerals, like Na+, K+, Zn2+, Fe2+, etc.
It increases the viscosity of the medium and thus, protects cells from mechanical damages during agitation of suspension cultures.
It also acts a buffer.
Due to the presence of both growth factors and inhibitors, the role of serum in cell culture is very complex.
Unfortunately, in addition to serving various functions, the use of serum in tissue culture applications has several drawbacks .
Bioreactors are essential in tissue
engineering, not only because they provide an
in vitro environment mimicking in vivo conditions
for the growth of tissue substitutes, but also
because they enable systematic studies of the
responses of living tissues to various mechanical
and biochemical cues.
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).
Bioprocess development and technology-Introduction,History of bioprocess,Milestones of Bioprocess development,Bioprocess development,Impact on Biotechnology
German Scientist “Carl Vogt” was first to describe the principle of apoptosis in 1842. In 1885, Anatomist “Walther Flemming” gave more precise description of Programmed Cell Death. Apoptosis is a form of Programmed Cell Death that occurs in multicellular organisms. It is a Greek word which means falling off. It leads to breakdown and disposal of cells. Macrophages and other Phagocytic Cells remove them by Phagocytosis, without developing any type of inflammation. It is a biochemical event that leads to morphological changes and death. The average adult human looses 50-70 billion cells each day due to apoptosis.
This presentation contains all the material regarding History of animal cell culture and different methods of organ and tissue culture.Hope it will be helpful..
Constituent of animal tissue culture media and their specific applicationKAUSHAL SAHU
INTRODUCTION
HISTORY
PHYSICOCHEMICAL PROPERTIES OF CULTURE MEDIA
pH
CO2, BICARBONATE AND BUFFERING
OXYGEN
TEMPERATURE
OSMOLALITY
BALANCED SALT SOLUTIONS
CONSTITUENTS OF CULTURE MEDIA
AMINO ACIDS
VITAMINS
SALTS
GLUCOSE
OTHER ORGANIC SUPPLEMENTS
ANTIBIOTICS
SERUM
PROTEINS
NUTRIENTS AND METABOLITES
HORMONES AND GROWTH FACTORS
LIPIDS
MINERALS
INHIBITORS
APPLICATIONS OF CULTURE MEDIA
CONCLUSION
REFERENCES
Role of serum and supplements in culture medium k.skailash saini
ROLE OF SERUM AND SUPPLEMENTS IN CULTURE MEDIA
Serum is a complex mix of albumins, growth factors and growth inhibitors.
Serum is one of the most important components of cell culture media and serves as a source for amino acids, proteins, vitamins (particularly fat-soluble vitamins such as A, D, E, and K), carbohydrates, lipids, hormones, growth factors, minerals, and trace elements.
Serum from fetal and calf bovine sources are commonly used to support the growth of cells in culture.
Fetal serum is a rich source of growth factors and is appropriate for cell cloning and for the growth of fastidious cells.
Calf serum is used in contact-inhibition studies because of its lower growth-promoting properties.
Normal growth media often contain 2-10% of serum.
Supplementation of media with serum serves the following functions :
Serum provides the basic nutrients (both in the solution as well as bound to the proteins) for cells.
Serum provides several growth factors and hormones involved in growth promotion and specialized cell function.
It provides several binding proteins like albumin, transferrin, which can carry other molecules into the cell. For example: albumin carries lipids, vitamins, hormones, etc. into cells.
It also supplies proteins, like fibronectin, which promote the attachment of cells to the substrate. It also provides spreading factors that help the cells to spread out before they begin to divide.
It provides protease inhibitors which protect cells from proteolysis.
It also provides minerals, like Na+, K+, Zn2+, Fe2+, etc.
It increases the viscosity of the medium and thus, protects cells from mechanical damages during agitation of suspension cultures.
It also acts a buffer.
Due to the presence of both growth factors and inhibitors, the role of serum in cell culture is very complex.
Unfortunately, in addition to serving various functions, the use of serum in tissue culture applications has several drawbacks .
Bioreactors are essential in tissue
engineering, not only because they provide an
in vitro environment mimicking in vivo conditions
for the growth of tissue substitutes, but also
because they enable systematic studies of the
responses of living tissues to various mechanical
and biochemical cues.
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).
Bioprocess development and technology-Introduction,History of bioprocess,Milestones of Bioprocess development,Bioprocess development,Impact on Biotechnology
German Scientist “Carl Vogt” was first to describe the principle of apoptosis in 1842. In 1885, Anatomist “Walther Flemming” gave more precise description of Programmed Cell Death. Apoptosis is a form of Programmed Cell Death that occurs in multicellular organisms. It is a Greek word which means falling off. It leads to breakdown and disposal of cells. Macrophages and other Phagocytic Cells remove them by Phagocytosis, without developing any type of inflammation. It is a biochemical event that leads to morphological changes and death. The average adult human looses 50-70 billion cells each day due to apoptosis.
For decades, cell lines have played a critical role in scientific developments. In most cases, researchers just got data generated from cell lines. However, due to some weaknesses of cell lines, scientists become increasingly cautious about these generated results. But now the game has changed! Primary cells now are believed to be a more biologically relevant tool than cell lines for studying human and animal biology. And we design this primary cell culture guide aimed at showing new investigators the basic principles of primary cell and some practical culture skills.
This presentation consists of topics related to oncogene, proto oncogene, Tumor suppresor gene, Ras gene family and structure and functions of tumor suppressor gene.
Stem cells are the cells which have the capability to differentiate into any cells of the body when provided with right stimulus and environment. This presentation teaches about stem cells, characteristics, types and cultivation of stem cells in artificial environment. Sample practice questions are also provided in the end to review the concept learned from this presentation.
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.
8. Biology and characterization of cultured cellsShailendra shera
Immediate environment and environment of surrounding medium governs the various properties of cell. The in vitro condition markedly affects the cellular property of cultured cells. For e.g. Reduction in Cell–cell and cell-material interaction. Therefore, it is imperative to develop understanding of biology of cells in response to various environmental conditions. Characterization of cells helps to identify the origin, purity and authenticity of cells and cell lines.
Workplace safety is an important aspect to protect personnel against injury or serious accident.In case of animal cell culture safety takes a front seat due to nature of work i.e. handling of human cells and tissues, viruses with high potential to cause infections to humans and other adventitious micro organisms. This presentation presents various methods of safety to protect lab personnel from infectious biological agents.
Media is one of the important components for in vitro cultivation of animal cells. Every animal cells have specific requirements and media are designed by keeping in mind those requirements. However, the basic components and design principle remains the same. Every cell culture media contain carbon source, nitrogen source, trace elements, pH indicator, antibiotics ( although it is not recommended) for high value cell culture applications. While designing media various aspects are considered such as availability, cost effectiveness, types off cells to be grown and regulatory requirements. Tis slide also contains sample MCQs questions
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.
This presentation details the definition of cell cytotoxicity and cell viability, the difference between the two term and methods of assessment of cells in culture for presence and absence of cytotoxic chemicals or metabolites.
This presentation presents an overview of definition, equipment, various cell culturing methods,
characterization, and applications of animal cell culture. This presentation also contains MCQs to acquaint reader about types of question asked in various competitive examinations.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
1. Animal cell science And Technology
2. Basics of Animal Cell Culture
Shailendra Singh Shera, Ph.D
2. 2. Basic Techniques of Mammalian cell culture
Outline:
1. Primary Culture
2. Secondary culture
3. Established cell line culture
4. Disaggregation of Tissue
5. Initiation of primary culture
6. Characteristics of primary culture
7. Application of primary culture
8. Practice questions
3. What is primary culture?
Primary culture refers to culturing of cells in artificial environment by using tissue explants directly
isolated from animal/mammalian source.
•A primary culture starts with the biopsy (~1 cm3) from tissue or organ via dissection.
•The tissue or seeded cells grows until they occupy all of the available substrate (i.e., reach
confluence)
What is secondary culture?
The culture formed after sub-culturing of primary culture.
•Sub-culture refers to transfer of certain volume of primary culture in fresh growth medium for
continued growth.
•When the primary culture cells are overgrown and occupied all available substrate it needs to be
sub cultured.
4. Cell line and Cell strain
Cell line:
The very first sub culture of primary culture is known as cell line or sub clone.
•They have been continuously passaged over a long period of time and have acquired
homogenous genotypic and phenotypic characteristics.
Cell lines
Finite Cell lines Continuous cell line
5. Finite cell lines, Continuous cell line, Established cell line
Finite Cell line:
Finite cell line have limited cell division capacity.
•A finite cell line has been sub-cultured for 20-80 passages after which they senesce
•The maximum number of times a finite cell divide before losing ability to divide is known as
Hayflick limit
Continuous cell line :
Cells having indefinite cell division capacity and can be maintained indefinitely in cell culture.
•Primary cells undergo transformation leading to loss of cell cycle regulation and control, converts
normal cells into cells having unlimited division capacity just like tumourous cells.
Established cell line:
A cell line which has achieved ability proliferate indefinitely in an suitable artificial condition and
medium is called as established cell line.
•Cells of established cell lines have escaped the hayflick limit and have become immortalized.
6. Method of creating established cell line & examples
Modification of existing cell line using gene editing tools such as CRISPR/ Cas9
•A gene called human telomerase reverse transcriptase (hTERT) can impart immortality to human cell
lines.
•hTERT technique ( 1999) can yield cells that behave like primary cultures but propagate like
immortalized lines
Immortalization through viral infection. For example, the large T antigen from SV40 virus, or the E6
and E7 oncogenes from human papilloma virus, can quickly turn a primary cell culture into an
immortalized line .
•viral oncogenes essentially turn cells into a tumor, they tend to change the cells’ characteristics
The above two methods are suitable for establishing non-cancer type normal cell line.
Cell lines Origin
Vero cell line kidney of an African green monkey
HeLa cell line cervical cancer cells (human cervix)
Chinese hamster Ovary ( CHO) epithelial cell line derived from the ovary of the
Chinese hamster
HEK 293 cells Human Kidney
7. Cell strain:
cells derived from a primary culture or a single cell (clone) and possessing a specific feature such
as a marker chromosome, antigen, or resistance to a virus. ( Medical Dictionary).
•The specific cells are selected from primary culture by cloning or some other methods. These
specific cells give rise to cell strain.
•For example, non-immortalized cells from a cell line can be extracted, alter them with the help of
chemicals or a genetically modified virus, and watch them mutate or grow until senescence makes
it impossible for them to divide any longer.
Cell strain
8. Disaggregation of tissues
The whole purpose of disaggregation of excised tissues is to reduce size, remove cell to cell
connections and attachments and to convert cluster of cells into single cells.
Methods of Tissue disaggregation
Tissue disaggregation
Mechanical Methods
*Pressing the tissue pieces
through a series of sieves
*Forcing the tissue fragments
through a syringe and needle
Enzymatic methods
Enzymes such as Trypsin,
Collagenase, pronases are used
either alone or as cocktail for
dislodging intact tissues
•Mechanical methods are simple, inexpensive but it is harsh and has low cell yields.
•Enzymatic methods are tedious, expensive but is mild and has high cell yields.
9. Process flow for disaggregation of tissue using enzymatic method
Adapted without modification from:
https://biocyclopedia.com/index/biotechnology/animal_biotechnology/animal_cell_tissue_and_organ_culture/biotech_isolation_a
nimal_material.php
•It can be seen here that tissues were isolated,
chopped to reduce size and incubated with
collagenase enzymes to yield Single cells.
•Trypsin is also used routinely in two variant
process: (i) Cold trypsinization and,
(ii) Hot Trypsinization
10. Initiation of primary culture
Isolation of primary cells
from source tissues
Tissue disaggregation
to get single cells
Seeding into cell culture
flask or plate containing
appropriate media
Incubation at 37 °C and
sub-culture when cell
reach confluency
Cryopreservation after
characterization and
authentication of cells.
Conditions of primary culture
•Stringent and extreme sterility is required.
•Temperature and pH is dependent upon types of cultured cells.
For e.g. mammalian cells are incubated at 37 °C, pH 7.2-7.4; 5-
10 % CO2
•Primary cells can be grown as monolayer as adherent cells
and suspension culture as adherent independent cells.
•Adherent cells can be grown on flat surface tissue culture
dishes such as single and double coverslip method
•Cells in suspension culture grows floating in cell culture flask.
E.g. Flask culture method.
•Hanging drop method of primary culture initiation is used for
culturing 3D architecture of cells.
11. Characteristics of primary culture
•Primary cells have a finite lifespan i.e they can undergo cell division for limited number of time.
•Primary cells retain the true characteristics of original tissues from which they were isolated
i.e. they are genetically identical to tissue of source animal.
•Primary cell cultures are harsh, requiring optimized growth conditions, including the addition of
specific cytokines and growth factors for propagation in serum-free or low-serum growth media,
which is absolutely different from immortalized cell lines.
•The cultures are initially heterogeneous (represents a mixture of cell types present in the tissue)
•They have minimal chromosomal aberrations.
12. Application of primary cell culture
3D Cell Culture: These cells can act as a model system to study cell biology and biochemistry, to study the
interaction between cell and disease-causing agents (like bacteria, virus), to study the effect of drugs, to study the
process of aging, to study cell signaling and metabolic regulations.
Cancer Research: Primary cells can be exposed to radiation, chemicals and viruses to make them cancerous.
The side effects of cancer treatments (chemotherapy and irradiation) on normal cells can also be studied in this
context.
Virology: Detection, isolation, growth and development cycles of viruses can be studied. Primary cells are also
useful to study the mode of infection.
Drug Screening and Toxicity Testing: Primary cell cultures are used to study the cytotoxicity of new drugs (to
study the effect and safe dosage) and/or drug carriers (nanoparticles). They are also used to determine the
maximum permissible dosage of new drugs.
Vaccine Production: Primary animal cells are used in the production of viruses and these viruses are used to
produce vaccines (such as vaccines, for deadly diseases like polio, rabies, chicken pox, measles and hepatitis B
are produced using animal cell culture) thus avoiding the use of animal models.
Tissue or Organ Replacement: Primary cell cultures can be used as replacement tissue or organs
Stem Cell Therapy: Stem cells isolated from bone marrow, blood or embryo involve primary cell culture. This is
an area that is being explored to design therapies for genetic disorders, spinal cord injuries, degenerative
diseases and cancer.
13. Practice questions*
1. Cell culture refers to
A. In vitro culture of eukaryotic cells under controlled environment
B. In vivo culture of prokaryotic cells under controlled environment
C. In vitro culture of bacterial cells in controlled environment
D. In vivo culture of fungal cells in controlled environment
2. Primary cell lines are derived from
A. Secondary cell culture
B. Direct excision of tissues followed by in vitro culture
C. Stem cell lines
D. Subculture of continuous cell lines
3. In vitro cell culture requires which of the following essential component for successful cultivations?
A. A suitable growth medium supplemented with all the required nutrient and growth factors
B. A temperature suitable for culturing desired cells in vitro
C. 5-10 % CO2
D. All of these
4. Mechanical methods of disaggregation includes
A. Mincing the tissues with help of mortar and pestle
B. Forcing the intact tissues through gradual reduction in the mesh size
C. Forcing the tissue fragments through a syringe and needle.
D. All of the above are correct
*Questions adapted from : Practice and Learn Animal cell Science and Technology: Multiple choice question
for learning. Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
14. 1. A. Dove, “The art of culture: Developing cell lines,” Science (80-. )., vol. 346, no. 6212, pp. 1013–1015, 2014.
2. https://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-types-culture.html
3. https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/introduction-to-cell-culture.html
4. https://www.genetargeting.com/stem-cell/cell-strains/
5. https://biocyclopedia.com/index/biotechnology/animal_biotechnology/animal_cell_tissue_and_organ_
culture/biotech_isolation_animal_material.php
6. https://www.creative-bioarray.com/support/primary-cell-culture-guide.htm
MCQs practice questions
1. Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
References and Further Reading