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
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
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
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
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
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
NEED OF SYNCHRONIZATION
TYPES OF SYNCHRONIZATION
(I)PHYSICAL CELL SEPARATION
(II)BLOCKADE
PHYSICAL Vs BLOCKADE SYNCHRONIZATION
CONCLUSION
REFFERENCE
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.
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 .
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
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
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
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
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
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.
Introduction.
Properties of Stem Cells.
Key Research events.
Embryonic Stem Cell.
Stem cell Cultivation.
Stem cells are central to three processes in an organism.
Research & Clinical Application of stem cell.
Research patents.
Conclusion.
Reference.
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
TYPES OF SYNCHRONIZATION
(I)PHYSICAL CELL SEPARATION
(II)BLOCKADE
PHYSICAL Vs BLOCKADE SYNCHRONIZATION
CONCLUSION
REFFERENCE
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.
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 .
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
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
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
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
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
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.
Introduction.
Properties of Stem Cells.
Key Research events.
Embryonic Stem Cell.
Stem cell Cultivation.
Stem cells are central to three processes in an organism.
Research & Clinical Application of stem cell.
Research patents.
Conclusion.
Reference.
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
ATCC cell lines and hybridomas are shipped frozen on dry ice in cryopreservation vials or as growing cultures in flasks at ambient temperature. Upon receipt of frozen cells, it is important to immediately revive them by thawing and removing the DMSO and placing them into culture. If this is not possible, store the cells in liquid nitrogen vapor (below −130°C). Do not store frozen cells at temperatures above −130°C as their viability will decline rapidly
cell culture define as removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.
Today, it has large prospective, Used in cellular and molecular biology
Studying the normal physiology and biochemistry of cells(e.g., metabolic studies, aging)
Used in drug screening and development and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins) etc.
A tale of scale & speed: How the US Navy is enabling software delivery from l...sonjaschweigert1
Rapid and secure feature delivery is a goal across every application team and every branch of the DoD. The Navy’s DevSecOps platform, Party Barge, has achieved:
- Reduction in onboarding time from 5 weeks to 1 day
- Improved developer experience and productivity through actionable findings and reduction of false positives
- Maintenance of superior security standards and inherent policy enforcement with Authorization to Operate (ATO)
Development teams can ship efficiently and ensure applications are cyber ready for Navy Authorizing Officials (AOs). In this webinar, Sigma Defense and Anchore will give attendees a look behind the scenes and demo secure pipeline automation and security artifacts that speed up application ATO and time to production.
We will cover:
- How to remove silos in DevSecOps
- How to build efficient development pipeline roles and component templates
- How to deliver security artifacts that matter for ATO’s (SBOMs, vulnerability reports, and policy evidence)
- How to streamline operations with automated policy checks on container images
Removing Uninteresting Bytes in Software FuzzingAftab Hussain
Imagine a world where software fuzzing, the process of mutating bytes in test seeds to uncover hidden and erroneous program behaviors, becomes faster and more effective. A lot depends on the initial seeds, which can significantly dictate the trajectory of a fuzzing campaign, particularly in terms of how long it takes to uncover interesting behaviour in your code. We introduce DIAR, a technique designed to speedup fuzzing campaigns by pinpointing and eliminating those uninteresting bytes in the seeds. Picture this: instead of wasting valuable resources on meaningless mutations in large, bloated seeds, DIAR removes the unnecessary bytes, streamlining the entire process.
In this work, we equipped AFL, a popular fuzzer, with DIAR and examined two critical Linux libraries -- Libxml's xmllint, a tool for parsing xml documents, and Binutil's readelf, an essential debugging and security analysis command-line tool used to display detailed information about ELF (Executable and Linkable Format). Our preliminary results show that AFL+DIAR does not only discover new paths more quickly but also achieves higher coverage overall. This work thus showcases how starting with lean and optimized seeds can lead to faster, more comprehensive fuzzing campaigns -- and DIAR helps you find such seeds.
- These are slides of the talk given at IEEE International Conference on Software Testing Verification and Validation Workshop, ICSTW 2022.
DevOps and Testing slides at DASA ConnectKari Kakkonen
My and Rik Marselis slides at 30.5.2024 DASA Connect conference. We discuss about what is testing, then what is agile testing and finally what is Testing in DevOps. Finally we had lovely workshop with the participants trying to find out different ways to think about quality and testing in different parts of the DevOps infinity loop.
Welcome to the first live UiPath Community Day Dubai! Join us for this unique occasion to meet our local and global UiPath Community and leaders. You will get a full view of the MEA region's automation landscape and the AI Powered automation technology capabilities of UiPath. Also, hosted by our local partners Marc Ellis, you will enjoy a half-day packed with industry insights and automation peers networking.
📕 Curious on our agenda? Wait no more!
10:00 Welcome note - UiPath Community in Dubai
Lovely Sinha, UiPath Community Chapter Leader, UiPath MVPx3, Hyper-automation Consultant, First Abu Dhabi Bank
10:20 A UiPath cross-region MEA overview
Ashraf El Zarka, VP and Managing Director MEA, UiPath
10:35: Customer Success Journey
Deepthi Deepak, Head of Intelligent Automation CoE, First Abu Dhabi Bank
11:15 The UiPath approach to GenAI with our three principles: improve accuracy, supercharge productivity, and automate more
Boris Krumrey, Global VP, Automation Innovation, UiPath
12:15 To discover how Marc Ellis leverages tech-driven solutions in recruitment and managed services.
Brendan Lingam, Director of Sales and Business Development, Marc Ellis
The Metaverse and AI: how can decision-makers harness the Metaverse for their...Jen Stirrup
The Metaverse is popularized in science fiction, and now it is becoming closer to being a part of our daily lives through the use of social media and shopping companies. How can businesses survive in a world where Artificial Intelligence is becoming the present as well as the future of technology, and how does the Metaverse fit into business strategy when futurist ideas are developing into reality at accelerated rates? How do we do this when our data isn't up to scratch? How can we move towards success with our data so we are set up for the Metaverse when it arrives?
How can you help your company evolve, adapt, and succeed using Artificial Intelligence and the Metaverse to stay ahead of the competition? What are the potential issues, complications, and benefits that these technologies could bring to us and our organizations? In this session, Jen Stirrup will explain how to start thinking about these technologies as an organisation.
Epistemic Interaction - tuning interfaces to provide information for AI supportAlan Dix
Paper presented at SYNERGY workshop at AVI 2024, Genoa, Italy. 3rd June 2024
https://alandix.com/academic/papers/synergy2024-epistemic/
As machine learning integrates deeper into human-computer interactions, the concept of epistemic interaction emerges, aiming to refine these interactions to enhance system adaptability. This approach encourages minor, intentional adjustments in user behaviour to enrich the data available for system learning. This paper introduces epistemic interaction within the context of human-system communication, illustrating how deliberate interaction design can improve system understanding and adaptation. Through concrete examples, we demonstrate the potential of epistemic interaction to significantly advance human-computer interaction by leveraging intuitive human communication strategies to inform system design and functionality, offering a novel pathway for enriching user-system engagements.
Generative AI Deep Dive: Advancing from Proof of Concept to ProductionAggregage
Join Maher Hanafi, VP of Engineering at Betterworks, in this new session where he'll share a practical framework to transform Gen AI prototypes into impactful products! He'll delve into the complexities of data collection and management, model selection and optimization, and ensuring security, scalability, and responsible use.
Securing your Kubernetes cluster_ a step-by-step guide to success !KatiaHIMEUR1
Today, after several years of existence, an extremely active community and an ultra-dynamic ecosystem, Kubernetes has established itself as the de facto standard in container orchestration. Thanks to a wide range of managed services, it has never been so easy to set up a ready-to-use Kubernetes cluster.
However, this ease of use means that the subject of security in Kubernetes is often left for later, or even neglected. This exposes companies to significant risks.
In this talk, I'll show you step-by-step how to secure your Kubernetes cluster for greater peace of mind and reliability.
PHP Frameworks: I want to break free (IPC Berlin 2024)Ralf Eggert
In this presentation, we examine the challenges and limitations of relying too heavily on PHP frameworks in web development. We discuss the history of PHP and its frameworks to understand how this dependence has evolved. The focus will be on providing concrete tips and strategies to reduce reliance on these frameworks, based on real-world examples and practical considerations. The goal is to equip developers with the skills and knowledge to create more flexible and future-proof web applications. We'll explore the importance of maintaining autonomy in a rapidly changing tech landscape and how to make informed decisions in PHP development.
This talk is aimed at encouraging a more independent approach to using PHP frameworks, moving towards a more flexible and future-proof approach to PHP development.
State of ICS and IoT Cyber Threat Landscape Report 2024 previewPrayukth K V
The IoT and OT threat landscape report has been prepared by the Threat Research Team at Sectrio using data from Sectrio, cyber threat intelligence farming facilities spread across over 85 cities around the world. In addition, Sectrio also runs AI-based advanced threat and payload engagement facilities that serve as sinks to attract and engage sophisticated threat actors, and newer malware including new variants and latent threats that are at an earlier stage of development.
The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
Dev Dives: Train smarter, not harder – active learning and UiPath LLMs for do...UiPathCommunity
💥 Speed, accuracy, and scaling – discover the superpowers of GenAI in action with UiPath Document Understanding and Communications Mining™:
See how to accelerate model training and optimize model performance with active learning
Learn about the latest enhancements to out-of-the-box document processing – with little to no training required
Get an exclusive demo of the new family of UiPath LLMs – GenAI models specialized for processing different types of documents and messages
This is a hands-on session specifically designed for automation developers and AI enthusiasts seeking to enhance their knowledge in leveraging the latest intelligent document processing capabilities offered by UiPath.
Speakers:
👨🏫 Andras Palfi, Senior Product Manager, UiPath
👩🏫 Lenka Dulovicova, Product Program Manager, UiPath
Observability Concepts EVERY Developer Should Know -- DeveloperWeek Europe.pdfPaige Cruz
Monitoring and observability aren’t traditionally found in software curriculums and many of us cobble this knowledge together from whatever vendor or ecosystem we were first introduced to and whatever is a part of your current company’s observability stack.
While the dev and ops silo continues to crumble….many organizations still relegate monitoring & observability as the purview of ops, infra and SRE teams. This is a mistake - achieving a highly observable system requires collaboration up and down the stack.
I, a former op, would like to extend an invitation to all application developers to join the observability party will share these foundational concepts to build on:
5. First one is the use of Antibiotics that help in
reducing contaminations
Second one is use of Trypsin to remove
adherent cells from vessel
Third one is use of chemically defined medium
for enhancement in result
6. Cell culture refers to the removal of cells from an
animal or plant and their subsequent
growth in a favourable artificial environment. The
cells may be removed from the tissue
directly and disaggregated by enzymatic or
mechanical means before cultivation, or they
may be derived from a cell line or cell strain that
has already been already established
7. o Cell culture hood (i.e., laminar-flow hood or
biosafety cabinet)
o • Incubator (humid CO2 incubator recommended)
o • Water bath
o • Centrifuge
o • Refrigerator and freezer (–20°C)
o • Cell counter (e.g., Countess® Automated Cell
Counter or hemacytometer)
o • Inverted microscope
o • Liquid nitrogen (N2) freezer or cryostorage
container
o • Sterilizer (i.e., autoclave)
8.
9.
10. Two types of media used in culture technique:
1. Natural Media 2. Artificial Media
Media is generally defined as the medium
which contains all the necessary elements
requires for the growth of cell or tissue culture.
11. pH:
Most normal mammalian cell lines grow well at
pH 7.4, and there is very little variability
among different cell strains. However, some
transformed cell lines have been shown to
grow better at slightly more acidic environments
(pH 7.0–7.4), and some normal fibroblast
cell lines prefer slightly more basic environments
(pH 7.4–7.7). Insect cell lines such as Sf9
and Sf21 grow optimally at pH 6.2
12.
Carbon Dioxide
The growth medium controls the pH of the culture and
buffers the cells in culture against changes in the pH.
Usually, this buffering is achieved by including an
organic or CO2-bicarbonate based buffer. Because the
pH of the medium is dependent on the delicate balance
of dissolved carbon dioxide (CO2) and bicarbonate
changes in the atmospheric CO2 can alter the pH of the
medium. Therefore, it is necessary to use exogenous
CO2 when using media buffered with a CO2-
bicarbonate based buffer,especially if the cells are
cultured in open dishes or transformed cell lines are
cultured at high concentrations.
13.
TEMPERATURE
The optimal temperature for cell culture largely
depends on the body temperature of the host from
which the cells were isolated, and to a lesser
degree on the anatomical variation in temperature
(e.g., temperature of the skin may be lower than
the temperature of skeletal muscle). Overheating is
a more serious problem than underheating for cell
cultures; therefore, often the temperature in the
incubator is set slightly lower than the optimal
temperature. 36 -37’C optimal for growth.
14. (1) Acquisition of the sample
(2) Isolation of the Tissue
(3) Dissection and/or disaggregation
(4)Culture after seeding into the culture vessel
15. An attempt should be made to sterilize the site of the
resection with 70% alcohol if the site is likely to be
contaminated (e.g., skin). Remove the tissue aseptically
and transfer it to the tissue culture laboratory in dissection
BSS (DBSS) or transport medium (see Appendix I) as soon
as possible. Do not dissect animals in the tissue culture
laboratory, as the animals may carry microbial
contamination.
If a delay in transferring the tissue is unavoidable, it can be
held at 4◦C for up to 72 h, although a better yield will
usually
result from a quicker transfer.
16. Tissue can be isolated from any of these 3
means:
1. Mouse Embryo
2. Chick Embryo
3. Human Biopsy Material
17. Material Required:
Sterile:
DBSS: Dissection BSS (BSS with a high
concentration of antibiotics; see Appendix I) in 25-
to 50-mL screw-capped tube or universal container
BSS, 50 mL in a sterile beaker (used to cool
instruments after flaming)
Petri dishes, 9 cm
Pointed forceps
Pointed scissors
18. Kill the mouse by cervical dislocation and swab
the ventral surface liberally with 70% alcohol
Tear the ventral skin transversely at themedian
line just over the diaphragm and, grasping the
skin on both sides of the tear, pull in opposite
directions to expose the untouched ventral
surface of the abdominal wall
Cut longitudinally along the median line of the
exposed abdomen with sterile scissors, revealing
the viscera
19. Dissect out the uteri into a 25-mL or 50-mL screw-
capped vial containing 10 or 20 mL DBSS
Dissect out the embryos:
(a) Tear the uterus with two pairs of sterile forceps,
keeping the points of the forceps close together to
avoid distorting the uterus and bringing too much
pressure to bear on the embryos
(b) Free the embryos from the membranes and
placenta and place them to one side of the dish to
bleed.
Transfer the embryos to a fresh Petri dish.
20.
21.
22.
23. Cells when surgically or enzymatically remove from
an organism and placed in suitable culture
environment will attach and grow are called as
“Primary Culture”
These cells have a finite life span
These culture has a very heterogenous population of
cells
Subculturing of these cells leads to generation of cell
lines
Cell lines have limited life span,they passage several
times before they becomes senescent
Lineage of cells obtained from primary culture are
called as CELL STRAIN
24. Several techniques are employed for this
culture:
1. Fine dissection (Primary Explant)
2. Mechanical Disaggregation
3. Enzymatic Disaggregation
25.
26.
27. It can be achieved by any one of these 3
methods:
1. Warm trypsin
2. Cold Trypsin
3. Collagenase
28.
29.
30.
31.
32. The need to subculture a monolayer is
determined by the following criteria:
1. Density of Culture
2. Exhaustion of Medium
3. Time since last subculture
4. Requirment for other procedures
33.
34. The best method for cryopreserving cultured cells
is storing them in liquid nitrogen in complete
medium in the presence of a cryoprotective agent
such as dimethylsulfoxide (DMSO).
Cryoprotective agents reduce the freezing point of
the medium and also allow a slower cooling rate,
greatly reducing the risk of ice crystal formation,
which can damage cells and cause cell death.
DMSO is known to facilitate the entry of organic
molecules into tissues. Handle reagents containing
DMSO using equipment and practices appropriate
for the hazards posed by such materials. Dispose
of the reagents in compliance with local
regulations.
35. Freeze your cultured cells at a high conc. and at
as low a passage number as possible. Make
sure that the cells are at least 90% viable before
freezing. Note that the optimal freezing
conditions depend on the cell line in use.
Freeze the cells slowly by reducing the temp. at
approx. 1°C /minute using a controlled rate
cryo-freezer. Always use the recommended
freezing medium. The freezing medium should
contain a cryoprotective agent such as DMSO
or glycerol. Store the frozen cells below –70°C;
frozen cells begin to deteriorate above –50°C
36.
37.
38. Demonstration of the absence of cross-contamination
Confirmation of the species of origin
Correlation with the tissue of origin, which comprises the
following characteristics:
a) Identification of the lineage to which the cell belongs
b) Position of the cells within that lineage
Determination of whether the cell line is transformed or
not
Identification of specific cell lines within a group from
the same origin, selected cell strains, or hybrid cell lines,
all of which require demonstration of features unique to
that cell line or cell strain
39. Areas where this technique is playing a major
role:
Model systems for studying basic cell biology,
interaction between diseases causing agents
and cells
Toxicity testing
Cancer research
Gene therapy and genetic engineering
Virology