Cell culture is the process of growing cells under controlled conditions outside of their natural environment. Key developments in cell culture technology include the use of antibiotics to prevent contamination, trypsin to detach adherent cells for subculturing, and chemically defined culture media. Cell culture is used in a variety of areas including basic research, toxicity testing, cancer research, virology, genetic engineering, and gene therapy. Successful cryopreservation of cell lines involves slow freezing and quick thawing to minimize ice crystal formation and damage to cells.
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
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
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 .
Centrifugation principle and types by Dr. Anurag YadavDr Anurag Yadav
concept of cnetrifugation,
basic Principle
centrifugal force
types of centrifugation based on use and rotor type
application of the each type of centrifuge
Ultracentrifuge in detail
application in general
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
A brief presentation on cell counting and cell viability assays. For cell cytotoxicity assays, you can check my profile where I have uploaded a separate file.
Prepared in July 2015
Stem cells
Undifferentiated cells capable of self-renew and to differentiate into different cell types or tissues during embryonic development and throughout adulthood.
Have possibility to become a specialised cell.
Have the ability to divide continuously and develop into various other kinds of cells.
Have immune potential and can help to treat a wide range of medical problems.
Discovery of stem cells lead to a whole new branch of medicine known as Regenerative medicine.
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
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
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 .
Centrifugation principle and types by Dr. Anurag YadavDr Anurag Yadav
concept of cnetrifugation,
basic Principle
centrifugal force
types of centrifugation based on use and rotor type
application of the each type of centrifuge
Ultracentrifuge in detail
application in general
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
A brief presentation on cell counting and cell viability assays. For cell cytotoxicity assays, you can check my profile where I have uploaded a separate file.
Prepared in July 2015
Stem cells
Undifferentiated cells capable of self-renew and to differentiate into different cell types or tissues during embryonic development and throughout adulthood.
Have possibility to become a specialised cell.
Have the ability to divide continuously and develop into various other kinds of cells.
Have immune potential and can help to treat a wide range of medical problems.
Discovery of stem cells lead to a whole new branch of medicine known as Regenerative medicine.
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..
Presentation made at the PDA Advanced Therapy Medicinal Products introducing an integrated equipment based on the Closed Vial to scale-up the manufacturing of an allogeneic product or deploy/scale-out for an autologus.
SeedEZ 3D cell culture application notes - gel and drug embeddingLena Biosciences
SeedEZ 3D cell culture application notes - gel and drug embedding. Many inert polymers used as scaffolds for 3D cell cultures and colony formation are also used in drug delivery systems both in vitro and in vivo. Read this practical guide to learn how SeedEZ lets you merge these two worlds in order to integrate 3D cell cultures into standard drug delivery and testing applications.
By incorporating or adding drugs to SeedEZ, or in polymer matrices embedded in SeedEZ, dosage forms which release a drug over a period of time may be prepared in a desired shape and size. More importantly, all SeedEZ-based dosage forms may be tested in situ, with cells in a 3D cell culture. SeedEZ wicks most sol-state hydrogels, hydrogel precursors, semisolid media, excipient formulations, pharmaceuticals and test compounds. As a result, SeedEZ offers a novel 3D framework for (A) development of sustained release drug delivery systems that are simple to make and convenient to use in vitro; (B) localized or distributed drug delivery into 3D cell cultures using spot-a-culture and spot-a-drug approach, wick, dip or SeedEZ-stack method; (C) gradient formation and testing of drug combination strategies; (D) quality control testing and assurance; and (E) development of test platforms for quasi-steady drug release.
Notably, in most diffusion-driven drug delivery systems, a drug release rate declines in time. A degradable polymer matrix embedded in SeedEZ may enable quasi-steady drug release from a defined volume, defined by SeedEZ, when the matrix degradation rate is adjusted to compensate for this decline via increased drug permeability from the SeedEZ/polymer matrix system.
The application note covers use of common biomaterials, including extracellular matrix hydrogels (Collagen and Matrigel), gels from natural sources for spheroid cultures and controlled drug release (Agarose, Alginate, Methylcellulose, Gelatin), and synthetic materials such as Poloxamers (Pluronic - used for cell encapsulation, drug delivery and pharmaceutical formulations), and Carbomers used in ocular, transdermal, oral and nasal delivery systems.
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
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
263778731218 Abortion Clinic /Pills In Harare ,sisternakatoto
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2. ∗ Cell culture is the process by which prokaryotic,
eukaryotic or plant cells are grown under
controlled conditions. But in practice it refers to
the culturing of cells derived from animal cells.
∗ Cell culture was first successfully undertaken by
Ross Harrison in 1907
∗ Roux in 1885 for the first time maintained
embryonic chick cells in a cell culture
Introduction
3. ∗ First development was the use of antibiotics which
inhibits the growth of contaminants.
∗ Second was the use of trypsin to remove adherent
cells to subculture further from the culture vessel
∗ Third was the use of chemically defined culture
medium.
Major development’s in cell culture
technology
4. Areas where cell culture technology is currently
playing a major role.
∗ Model systems for
Studying basic cell biology, interactions between disease
causing agents and cells, effects of drugs on cells, process and
triggering of aging & nutritional studies
∗ Toxicity testing
Study the effects of new drugs
∗ Cancer research
Study the function of various chemicals, virus & radiation to
convert normal cultured cells to cancerous cells
Why is cell culture used for?
5. ∗ Virology
Cultivation of virus for vaccine production, also
used to study there infectious cycle.
∗ Genetic Engineering
Production of commercial proteins, large scale
production of viruses for use in vaccine production e.g.
polio, rabies, chicken pox, hepatitis B & measles
∗ Gene therapy
Cells having a functional gene can be replaced to
cells which are having non-functional gene
Contd….
6. ∗ In vitro cultivation of organs, tissues & cells at defined
temperature using an incubator & supplemented with a
medium containing cell nutrients & growth factors is
collectively known as tissue culture
∗ Different types of cell grown in culture includes
connective tissue elements such as fibroblasts, skeletal
tissue, cardiac, epithelial tissue (liver, breast, skin,
kidney) and many different types of tumor cells.
Tissue culture
7. ∗ Cells when surgically or enzymatically removed from
an organism and placed in suitable culture
environment will attach and grow are called as primary
culture
∗ Primary cells have a finite life span
∗ Primary culture contains a very heterogeneous
population of cells
∗ Sub culturing of primary cells leads to the generation
of cell lines
Primary culture
8. ∗ Cell lines have limited life span, they passage several
times before they become senescent
∗ Cells such as macrophages and neurons do not divide
in vitro so can be used as primary cultures
9. ∗ Most cell lines grow for a limited number of generations
after which they terminate division
∗ Cell lines which either occur spontaneously or induced virally
or chemically transformed into Continuous cell lines
Continous cell lines
10. ∗ -smaller, more rounded, less adherent with a higher
nucleus /cytoplasm ratio
∗ -Fast growth
∗ -reduced serum and anchorage dependence and grow
more in suspension conditions
∗ -ability to grow unto higher cell density
∗ -different in phenotypes from donor tissue
∗ -stop expressing tissue specific genes
Characteristics of continous cell
lines
11. On the basis of morphology (shape & appearance) or
on their functional characteristics. They are divided
into three.
∗ Epithelial like-attached to a substrate and appears
flattened and polygonal in shape
∗ Lymphoblast like- cells do not attach remain in
suspension with a spherical shape
∗ Fibroblast like- cells attached to an substrate appears
elongated and bipolar
Types of cells
12. ∗ Choice of media depends on
the type of cell being
cultured
∗ Commonly used Medium are
GMEM, EMEM,DMEM etc.
∗ Media is supplemented with
antibiotics viz. penicillin,
streptomycin etc.
∗ Prepared media is filtered
and incubated at 4 C
Culture media
13. Confluency
∗ Once the available substrate surface is covered by
cells (a confluent culture) growth slows & ceases.
17. Confluency
∗ Cells to be kept in healthy & in growing state have to
be sub-cultured or passaged , It’s the passage of cells
when they reach to 80-90% confluency in
flask/dishes/plates
∗ Enzyme such as trypsin, collagenase in combination
with EDTA breaks the cellular glue that attached the
cells to the surface
18. ∗ Once the available substrate surface is covered by
cells (a confluent culture) growth slows & ceases.
∗ Cells to be kept in healthy & in growing state
have to be sub-cultured or passaged
∗ It’s the passage of cells when they reach to 80-
90% confluency in flask/dishes/plates
∗ Enzyme such as trypsin, dipase, collagenase in
combination with EDTA breaks the cellular glue
that attached the cells to the surface
Why sub culturing.?
19. ∗ Cells are cultured as anchorage dependent
or independent
∗ Cell lines derived from normal tissues are
considered as anchorage-dependent
grows only on a suitable substrate e.g.
tissue cells
∗ Suspension cells are anchorage-
independent e.g. blood cells
∗ Transformed cell lines either grows as
monolayer or as suspension
Culturing of cells
20. ∗ Cells which are anchorage dependent
∗ Cells are washed with PBS (free of ca & mg ) solution.
∗ Add enough trypsin/EDTA to cover the monolayer
∗ Incubate the plate at 37 C for 1-2 mints
∗ Tap the vessel from the sides to dislodge the cells
∗ Add complete medium to dissociate and dislodge the
cells with the help of pipette which are remained to be
adherent
∗ Add complete medium depends on the subculture
requirement either to 75 cm or 175 cm flask
Adherent cells
21. ∗ Easier to passage as no need to detach them
∗ As the suspension cells reach to confluency
∗ Asceptically remove 1/3rd
of medium
∗ Replaced with the same amount of pre-warmed
medium
Suspension cells
23. Cryopreservation
Definition
Cryopreservation is a process where cells or whole
tissues are preserved by cooling to low sub-zero
temperatures, such as, −196 °C (the boiling point of
liquid nitrogen).
Liquid Nitrogen
24. ∗ Vial from liquid nitrogen is placed into 37 C water
bath, agitate vial continuously until medium is
thawed
∗ Centrifuge the vial for 10 mts at 1000 rpm at RT,
wipe top of vial with 70% ethanol and discard the
supernatant
∗ Resuspend the cell pellet in 1 ml of complete
medium with 20% FBS and transfer to properly
labeled culture plate containing the appropriate
amount of medium
∗ Check the cultures after 24 hrs to ensure that they
are attached to the plate
∗ Change medium as the colour changes, use 20%
FBS until the cells are established
Working with cryopreserved cells
25.
26. Principles of cryopreservation
∗ Water in cell: Around 90% of water is free (water)
while the remaining 10 % bounds to other molecular
components of the cell (proteins, lipids, nucleic
acids and other solutes). This water does not freeze
and called hydrated water
∗ Removal of water is necessary during freezing to avoid ice
crystal formation, dehydration is limited to the free water
∗ Removal of hydrated water could have adverse effect on
the cell viability and the molecular function (freezing
injuries)
27. Cryopreservation of Cell Lines
∗ The aim of cryopreservation is to enable stocks of cells to be
stored to prevent the need to have all cell lines in culture at
all times. It is invaluable when dealing with cells of limited
life span.
The other main advantages of cryopreservation are:
∗ Reduced risk of microbial contamination
∗ Reduced risk of cross contamination with other cell lines
∗ Reduced risk of genetic drift and morphological changes
∗ Work conducted using cells at a consistent passage number
(refer to cell banking section below)
∗ Reduced costs (consumables and staff time)
28. Successful Cryopreservation of cell lines
∗ There has been a large amount of developmental
work undertaken for successful cryopreservation of a
wide variety of cell lines of different cell types.
∗ The basic principle of successful cryopreservation is a
∗ Slow freeze
∗ Quick thaw.
∗ Cell lines should be cooled at a rate of –1ºC to –3ºC
per minute and thawed quickly by incubation in a
37ºC water bath for 3-5 minutes..
29. ∗ A high concentration of serum/protein (>20%)
should be used. In many cases serum is used at
90%.
∗ Use a cryoprotectant such as dimethyl sulphoxide
(DMSO) or glycerol to help protect the cells from
rupture by the formation of ice crystals.
∗ The most commonly used cryoprotectant is DMSO
at a final concentration of 10%.
Successful Cryopreservation of cell lines
30. ∗ Remove the growth medium, wash the cells by PBS
and remove the PBS by aspiration
∗ Dislodge the cells by trypsin-versene
∗ Dilute the cells with growth medium
∗ Transfer the cell suspension to a 15 ml conical tube,
centrifuge at 200g for 5 mts at RT and remove the
growth medium by aspiration
∗ Resuspend the cells in 1-2ml of freezing medium
∗ Transfer the cells to cryovials, incubate the cryovials at
-80 C overnight
∗ Next day transfer the cryovials to Liquid nitrogen
Freezing cells for storage
31. ∗ 1. Laboratory design- in a safe and efficient manner
∗ 2. safety cabinets
Design and Equipment for the Cell
Culture Laboratory
32. Cell Culture Room
Close Small AC/Heater
ROOM FOR ANIMAL CELL CULTURE
sterile conditions (disinfection of the work surfaces,
microbiological safety cabinets)
Hood
33. Before use
∗ Ultraviolet lights are used to sterilize the air and
exposed work surfaces in laminar flow cabinets
between use.
∗ Detergent
∗ 70% alcohol
34. ∗ Laminar cabinet
∗ Incubation- Temperature is 37 C for mammalian cells,
Co2 2-5% & 95% air at 99% relative humidity.
∗ Refrigerators- Liquid media kept at 4 C, enzymes (e.g.
trypsin) & media components (e.g. glutamine &
serum) at -20 C
∗ Microscope- An microscope with 10x to 100x
magnification
∗ Cell culture tubes
∗ Autoclave-
Basic equipments used in cell
culture
35.
36. ∗ The working environment is protected from dust and
contamination by a
∗ constant, stable flow of filtered air
∗ Two types:
Horizontal, airflow blow from the side facing you,
parallel to the work surface, and is not circulating;
Vertical, air blows down from the top of the cabinet
onto the work surface and is drawn through the work
surface and recalculated
Laminar- flow hood
39. ∗ Routine maintenance checks of the primary filters are
required (every 3-6 months).
∗ They might be removed and discarded or washed in
soap and water.
∗ Every 6 months the main high efficiency particulate
air (HEPA) filter above the work surface should be
checked for airflow and hole
Laminar- flow hood
40. Precaution Measure Inside The Hood
Incubator Gloves are
always worn
The pipettes are disposable
Lab coat
42. ∗ It requires a controlled atmosphere with high
humidity and super controlled of CO2.
• The incubator should be large enough, like 50-200 l
have forced air circulation
• Temperature should be + 0.5oC
• It should be stainless steel, and easily cleaned
Cell Culture Incubator
43. ∗ 4C
∗ -20C
∗ -80C
∗ Liquid N2 tank
Refrigerators and Freezers
44. ∗ Large stage so plates and
flasks can be used.
∗ Magnification; 5X, 10X, 20X,
40X
Microscope