Cell culture media are designed to support the growth of cells outside their natural environment. They generally contain amino acids, salts, glucose, vitamins and other nutrients. Media can be natural (containing biological fluids) or artificial/synthetic. Artificial media are grouped into serum-containing, serum-free, chemically defined, and protein-free categories based on their ingredients. Key components of media include buffers, amino acids like glutamine, vitamins, inorganic salts, carbohydrates, proteins, lipids, trace elements, and supplements specific to cell lines. Selection of the appropriate medium depends on the cell type and purpose of culture. Primary cells especially benefit from ready-to-use conditioned media.
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
Introduction
Definition
History
Why are the transgenic animals being produced
Transgenic mice
Mice: as model organism
Methods of creation of transgenic mice
knock-out mice
Application of transgenic mice
Conclusion
References
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
Biology and characterization of the cell cultureKAUSHAL SAHU
Introduction
History
Important terminology
Biology of culture cell
Characterization of culture cell
Application of animal culture
Conclusion
References
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.
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
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
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
Introduction
Definition
History
Why are the transgenic animals being produced
Transgenic mice
Mice: as model organism
Methods of creation of transgenic mice
knock-out mice
Application of transgenic mice
Conclusion
References
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
Biology and characterization of the cell cultureKAUSHAL SAHU
Introduction
History
Important terminology
Biology of culture cell
Characterization of culture cell
Application of animal culture
Conclusion
References
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.
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
Equipments used , types of culture and media, subculturing, secondary culture, finite & continuous cell lines, cryopreservation and applications of cell culture
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
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
A growth medium or culture medium is a liquid or gel designated to support the growth of microorganisms,cells,or small plants.
Culture media generally comprise an appropriate sourcde of energy and compounds which regulate the cell cycle.
A typical culture media is composed of a complement of amino acids,vitamins,inorganic salts,glucose, and serum as a source of growth factors, hormones, and attachment factors.
In addition to nutrients, the medium also helps maintain pH and osmolarity.
Imporatant amino acids, trace elements, growth factor,hormone,transport protein and adhesion factor are added.
Adhesion factor added are main components of intercellular substance such as fibronectin,collagen and laminin.
Primary purpose of introducing SFM is to promote the specific growth of a particular type of cell.
Exapmple of one serum free media: DCMM-1 SFM. High protein serum free medium, designed for hybridoma cell growth and monoclonal antibody production.
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 .
How to Create Map Views in the Odoo 17 ERPCeline George
The map views are useful for providing a geographical representation of data. They allow users to visualize and analyze the data in a more intuitive manner.
This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
He discussed the concept of quality improvement, emphasizing its applicability to various aspects of life, including personal, project, and program improvements. He defined quality as doing the right thing at the right time in the right way to achieve the best possible results and discussed the concept of the "gap" between what we know and what we do, and how this gap represents the areas we need to improve. He explained the scientific approach to quality improvement, which involves systematic performance analysis, testing and learning, and implementing change ideas. He also highlighted the importance of client focus and a team approach to quality improvement.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptxEduSkills OECD
Andreas Schleicher presents at the OECD webinar ‘Digital devices in schools: detrimental distraction or secret to success?’ on 27 May 2024. The presentation was based on findings from PISA 2022 results and the webinar helped launch the PISA in Focus ‘Managing screen time: How to protect and equip students against distraction’ https://www.oecd-ilibrary.org/education/managing-screen-time_7c225af4-en and the OECD Education Policy Perspective ‘Students, digital devices and success’ can be found here - https://oe.cd/il/5yV
How to Split Bills in the Odoo 17 POS ModuleCeline George
Bills have a main role in point of sale procedure. It will help to track sales, handling payments and giving receipts to customers. Bill splitting also has an important role in POS. For example, If some friends come together for dinner and if they want to divide the bill then it is possible by POS bill splitting. This slide will show how to split bills in odoo 17 POS.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
We all have good and bad thoughts from time to time and situation to situation. We are bombarded daily with spiraling thoughts(both negative and positive) creating all-consuming feel , making us difficult to manage with associated suffering. Good thoughts are like our Mob Signal (Positive thought) amidst noise(negative thought) in the atmosphere. Negative thoughts like noise outweigh positive thoughts. These thoughts often create unwanted confusion, trouble, stress and frustration in our mind as well as chaos in our physical world. Negative thoughts are also known as “distorted thinking”.
2. What is cell culture media?
A growth medium or culture medium is a liquid or gel designed to
support the growth of microorganisms, cells, or small plants.
Cell culture media generally comprise an appropriate source of energy and
compounds which regulate the cell cycle.
A typical culture medium is composed of a complement of amino acids,
vitamins, inorganic salts, glucose, and serum as a source of growth factors,
hormones, and attachment factors.
In addition to nutrients, the medium also helps maintain pH and
osmolality.
2
3. Types of cell culture media
Culture media
Artificial media
Serum
containing media
Serum-free
media
Protein-free
media
Chemically
defined media
Natural media
3
5. Natural media
Natural media consist solely of naturally occurring biological fluids.
Natural media are very useful and convenient for a wide range of animal
cell culture.
The major disadvantage of natural media is its poor reproducibility due to
lack of knowledge of the exact composition of these natural media.
5
6. Artificial media
Artificial or synthetic media are prepared by adding nutrients (both
organic and inorganic), vitamins, salts, O2 and CO2 gas phases, serum
proteins, carbohydrates, cofactors.
Different artificial media have been devised to serve one or more of the
following purposes:
Immediate survival (a balanced salt solution, with specific pH and
osmotic pressure)
Prolonged survival (a balanced salt solution supplemented with
various formulation of organic compounds and/or serum)
Indefinite growth
Specialized functions.
6
7. Artificial media are grouped into four
categories:
Serum containing media
Fetal bovine serum is the most common supplement in animal cell
culture media. It is used as a low-cost supplement to provide an
optimal culture medium. Serum provides carriers or chelators for
labile or water-insoluble nutrients, hormones and growth factors,
protease inhibitors, and binds and neutralizes toxic moieties.
7
9. Serum-free media
Presence of serum in the media has many drawbacks and can lead to
serious misinterpretations in immunological studies. A number of
serum-free media have been developed. These media are generally
specifically formulated to support the culture of a single cell type, such
as Knockout Serum Replacement and Knockout DMEM from Thermo
Fisher Scientific for stem cells, and incorporate defined quantities of
purified growth factors, lipoproteins, and other proteins, which are
otherwise usually provided by the serum. These media are also referred
to as ‘defined culture media’ since the components in these media are
known.
9
11. Chemically defined media
These media contain contamination-free ultra pure inorganic and
organic ingredients, and may also contain pure protein additives, like
growth factors. Their constituents are produced in bacteria or yeast by
genetic engineering with the addition of vitamins, cholesterol, specific
amino acids, and fatty acids.
11
12. Protein-free media
Protein-free media do not contain any protein and only contain non-
protein constituents. Compared to serum-supplemented media, use of
protein-free media promotes superior cell growth and protein
expression and facilitates downstream purification of any expressed
product. Formulations like MEM, RPMI-1640 are protein-free and
protein supplement is provided when required.
12
14. Basic components of culture media
Culture media contain a mixture of amino acids, glucose, salts, vitamins,
and other nutrients, and available either as a powder or as a liquid form
from commercial suppliers.
The requirements for these components vary among cell lines, and these
differences are partly responsible for the extensive number of medium
formulations.
14
15. Buffering systems
Regulating pH is critical for optimum culture conditions and is generally
achieved by one of the two buffering systems:
Natural buffering system
In a natural buffering system, gaseous CO2 balances with the
CO3/HCO3 content of the culture medium. Cultures with a natural
buffering system need to be maintained in an air atmosphere with
5-10% CO2, usually maintained by an CO2 incubator. Natural
buffering system is low cost and non-toxic.
15
16. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
Chemical buffering using a zwitterion, HEPES, has a superior buffering
capacity in the pH range 7.2-7.4 and does not require a controlled
gaseous atmosphere. HEPES is relatively expensive and toxic at a
higher concentration for some cell types. HEPES has also been shown
to greatly increase the sensitivity of media to phototoxic effects
induced by exposure to fluorescent light.
16
17. Amino acids
Amino acids are the building blocks of proteins, and thus are obligatory
ingredients of all known cell culture media.
Essential amino acids must be included in the culture media as cells can
not synthesize these by themselves.
They ae required for the proliferation of cells and their concentration
determines the maximum achievable cell density.
L-glutamine, an essential amino acid, is particularly important.
L-glutamine provides nitrogen for NAD, NADPH and nucleotides and
serves as a secondary energy source for metabolism.
L-glutamine is an unstable amino acid, that, with time, converts to a form
that can not be used by cells, and should thus be added to media just
before use.
17
18. Caution should be used when adding more L-glutamine than needed for
the original medium formulation since its degradation results in the build-
up of ammonia, and ammonia can have deleterious effect on some cell
lines.
L-glutamine concentrations for mammalian cell culture media can vary
from 0.68 mM in Medium 199 to 4mM in Dulbecco’s Modified Eagles’s
Medium.
Invertebrate cell culture media can contain as much as 12.3 mM L-
glutamine.
Supplements like glutamax are more stable and can replace glutamine for
long term culturing of slow cells.
Nonessential amino acids may also be added to the medium to replace
those that have been depleted during growth.
Supplementation of media with non-essential amino acids stimulates
growth and prolongs the viability of the cells.
18
19. Vitamins
Many vitamins are essential for growth and proliferation of cells. Vitamins
cannot be synthesized in sufficient quantities by cells and are therefore
important supplements required in tissue culture.
Again serum is the major source of vitamins in cell culture, however, media
are also enriched with different vitamins making them suitable for a
particular cell line.
The B group vitamins are most commonly added for growth stimulation.
19
20. Inorganic salt
Inorganic salt in the media help to retain the osmotic balance and help
in regulating membrane potential by providing sodium, potassium,
and calcium ions.
Carbohydrates
Carbohydrates in the form of sugars are the major source of energy.
Most of the media contain glucose and galactose, however, some
contain maltose and fructose.
20
21. Proteins and peptides
The most commonly used proteins and peptides are albumin, transferrin,
and fibronectin.
They are particularly important in serum-free media.
Serum is a rich source of proteins and includes albumin, transferrin,
aprotinin, fetuin, and fibronectin.
Albumin is the main protein in blood acting to bind water, salts, free fatty
acids, hormones, and vitamins, and transport them between tissues and
cells.
The binding capacity of albumin makes it a suitable remover of toxic
substances from the cell culture media.
21
22. Aprotinin is a protective agent in cell culture systems, stable at neutral and
acidic pH and resistant to high temperatures and degradation by
proteolytic enzymes.
It has the ability to inhibit several serine proteases such as trypsin.
Fetuin is a glycoprotein found in fetal and newborn serum at larger
concentrations than in adult serum.
It is also an inhibitor of serine proteases.
Fibronectin is a key player in cell attachment.
Transferrin is an iron transport protein that acts to supply iron to the cell
membrane.
22
23. Fatty acids and lipids
They are particularly important in serum-free media as they are generally
present in serum.
23
24. Trace elements
Trace elements are often supplemented to serum-free media to replace
those normally found in serum.
Trace elements like copper, zinc, selenium and tricarboxylic acid
intermediates are chemical elements that are needed in minute amounts
for proper cell growth.
These micronutrients are essential for many biological processes, e.g. the
maintenance of the functionality of enzymes
24
25. Media supplements
The complete growth media recommended for certain cell lines requires
additional components which are not present in the basal media and
serum.
These components, supplements, help sustain proliferation and maintain
normal cell metabolism.
Although supplements like hormones, growth factors and signaling
substances are required for normal growth of some cell lines, it is always
best to take the following precautions: since the addition of supplement
can change the osmolality of the complete growth media which can
negatively affect the growth of cells, it is always best to recheck the
osmolality after supplements are added.
For most of the cell lines, optimal osmolality should be between 260
mOSM/kg and 320 mOSM/kg.
Shelf life of the growth media changes after the addition of supplements.
Complete media containing protein supplement tend to degrade faster
than basal media alone.
25
26. Antibiotics
Although not required for cell growth, antibiotics are often used to control
the growth of bacterial and fungal contaminants.
Routine use of antibiotics for cell culture is not recommended since
antibiotics can mask contamination by mycoplasma and resistant bacteria.
Moreover, antibiotics can also interfere with the metabolism of sensitive
cells.
26
27. Preparation of media
Culture medium is available in three forms from commercial suppliers:
Powdered form: it needs to be prepared and sterilized by the
investigator.
Concentrated form: to be diluted by the investigator.
Working solution: to be used directly without further manipulation.
Powdered medium is the least expensive but needs to be sterilized.
It is advisable to filter and sterilize it prior to the addition of serum as the
foaming that occurs in the presence of serum denatures the protein.
Fetal bovine or horse sera can be added after filtration. Media should
always be tested for sterility by placing it in a 37oC CO2 incubator for 72
hours prior to utilization to ensure that the lot is contamination-free.
Medium should be stored at 4oC.
Since several components of medium are light-sensitive, it should be
stored in dark.
27
28. Criteria for selecting media
CELL LINES
The choice of cell culture media is extremely important, and significantly
affects the success of cell culture experiments.
The selection of the media depends on the type of cells to be cultured and
also the purpose of the culture and resources available in the laboratory.
Different cell types have highly specific growth requirements, therefore,
the most suitable media for each cell type must be determined
experimentally.
In general, it’s always good to start with MEM for adherent cells and RPMI-
1640 for suspension cells.
28
30. PRIMARY CELL CULTURE
Primary cell culture provides unique and valuable research data, but
of the time cell number is the limitation.
For such critical samples, especially from diseased human biopsies, a
quality medium is required.
Most of the life sciences companies are providing complete and ready to
use, fully supplemented conditioned medium.
This reduces the risk of contamination as well as save time, labor and
money by eliminating the preparation steps and supplementation
required.
Moreover, all of these media are subjected to comprehensive quality
control tests and each lot is routinely tested for growth promotion,
absence of cytotoxicity, and physical parameters such as osmolality and
pH level.
30