PROCEDURE FOR CELL CULTURE PREPARATION.pptx

BY : VAISHNAVI GOHRI
St. john institute of pharmacy & research 1

ELEMENTS
Preparation of cell culture
media and supplements
Sterilization of materials
and equipment
Passaging of cells
Seeding the cells
Thawing of cells
Cell growth and maintenance
Harvesting the cells Storage &
Disposal of materials
Record keeping

St. john institute of pharmacy & research
Cell culture media preparation significantly impacts mammalian cell growth and experimental outcomes.
Similarly, there are specific media formulated to support the cultivation of microorganisms in vitro.
Culture media provide a source of energy for cell growth and compounds that regulate cellular processes.
Both prokaryotic and eukaryotic cultures may have specific growth requirements.
Media selection depends on the culture type, purpose of cultivation, and cell density requirements.
MEDIA PREPARATION
• Commercial culture media are provided in dehydrated or powdered form, in
liquid concentrate, or as working solutions.
• Dehydrated media for broth cultures need to be prepared by dissolving in
distilled water and adjusting the final pH prior to sterilization.
• Powdered media for agar cultures must be dissolved in distilled water, stirred
then boiled or autoclaved prior to pouring into sterile petri dishes using aseptic
techniques.
• Culture media provided as liquid concentrates must be aseptically diluted in
distilled water, adjusted for pH, and dispensed into sterile containers for storage.
• Culture media provided as working solutions are ready to use after the addition
of any required serum or other supplements under sterile filtration, aseptic
conditions, and warming to the appropriate temperature for cell growth.
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CULTURE MEDIA STERILIZATION
• Culture media prepared from powders must be sterilized by filtration, heating or autoclaving prior to use.
• Although liquid media are often supplied sterile and at ready-to-use concentration.
• Sterile filtration is recommended, particularly if sera or other supplements are to be added before use.
• Following sterilization, aseptic techniques should be used when handling culture media to prevent cross-contamination.
• Also inhibit growth of unwanted microorganisms.
CULTURE MEDIA SUPPLEMENTS
• Certain supplements required for cell cultivation or regulation are unstable or heat-labile
• They must be added by the user to heat-sterilized basal culture medium prior to use.
• Serum typically derived from bovine sources is a common supplement used in animal cell culture media.
• Hormones, growth factors, and specific signaling molecules may be required for the growth of certain cell types.
• Additives such as IPTG are used to induce protein expression.
• Antibiotics may also be added to culture media to control the growth of bacterial and fungal contaminants
• Though contamination can be well-controlled with appropriate handling and storage of media.
CULTURE MEDIA STORAGE AND USAGE
• Commercial and prepared culture media in liquid form, poured agar form, or working solutions are typically stored at 4 °C in the dark.
• To suppress the growth of opportunistic microorganisms and to prevent light-induced degradation of culture media components.
• Prior to inoculation with cultures, media should be warmed to an appropriate temperature for cell growth.

•The constituents of culture media, water and containers contribute to the contamination by vegetative cells and spores.
•The media must be free from contamination before use in fermentation.
• Sterilization of the media is most commonly achieved by applying heat and to a lesser extent by other means
(physical methods, chemical treatment, and radiation).
Heat sterilization:
•Heat is the most widely and the most useful method for the sterilization technique of nutrient media
•A number of factors influence the success of heat sterilization.
•The number and type of microorganisms, the composition of the media and its pH and size of the suspended particles are
the important factors that influence the success of heat sterilization.
•Vegetative cells are rapidly eliminated at relatively low temperatures.
•In general, vegetative cells are destroyed at a lower temperature in a short time (around 60°C in 5-10 minutes).
•However, the destruction of spores requires higher temperature and relatively longer time (around 80°C for 15-20 minutes).
•Spores of Bacillus stearothermophilus are the most heat resistant.
•In fact, this organism is exploited for testing the sterility of fermentation equipment.
•Therefore they are used as assay organisms for testing the various procedures used to sterilize
Sterilization of Culture Media

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Chemical methods:
•Although a number of chemical disinfectants are known, they cannot be used to sterilize
nutrient media because there is a risk that inhibition of the fermentation organism could
occur from the residual chemical.
•Note: The chemical methods (by using disinfectants) and radiation procedures
(by using UV rays, y rays, X-rays) are not commonly used for media sterilization.
Mechanical removal of organisms (Physical methods):
•The physical methods such as filtration, centrifugation, and adsorption (to ion-exchangers
or activated carbon) are in use.
•Among these, filtration is the only method in practical use.
•Filter sterilization is often used for all components of nutrient solutions which are heat
sensitive and would thus be denatured through the steam sterilization process normally used
in industrial fermentations.
• Certain constituents (vitamins, blood components, antibiotics) of culture media are heat
labile and therefore, are destroyed by heat sterilization.
•Such components of the medium are completely dissolved (absolutely essential or else
they will be removed along with microorganisms) and then subjected to filter sterilization.
•Deep filters (plate filters) are sometimes used to filter complex nutrient solutions.

Two main limitations of filtration technique:
1.Application of high pressure in filtration is unsuitable or undesirable
for industrial practice.
2.Certain components of the media may be absorbed on filter material
from the media during filtration.
•One approach which is cost effective is the filtration of just the water
which is to be used in the preparation of the culture medium.
•Sometimes, a combination of filtration and heat sterilization is applied.
•For instance, the water used for media preparation is filtered while the
concentrated nutrient solution is subjected to heat sterilization.
• The filtered water is now added for appropriate dilution of the media.
•For example, in steroid bioconversion processes, a concentrated
nutrient solution is sterilized by heat in the fermenter and is then diluted
to the normal concentration with water which has been filter sterilized.
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st. john institute of pharmacy & research
• Thawing of cells refers to the process of warming frozen cells to room or body
temperature for the purpose of reviving them to their normal metabolic state.
• This process is critical in cell culture, where cells are often stored in the frozen
state for long periods of time for research or other applications.
• Thawing of cells should be done carefully and attentively to avoid damage or
contamination to the cells.
• Typically, the frozen cells are rapidly thawed by immersing the vial in a warm
water bath (37°C) for a short period (1-2 minutes).
• Once thawed, the cells are then carefully removed from the vial with a sterile
pipette and added to a culture plate containing pre-warmed growth media.
• The culture is then placed into a suitable incubator, at appropriate conditions such
as temperature, humidity and CO2 concentration, to ensure the cells recover and
grow to their normal, healthy state.
• It's important to note that thawing of cells can be different for each cell line, and it
is important to follow the specific instructions for thawing and culturing the
particular cell type to maintain cell viability and functionality.
• It is also essential to maintain sterile conditions throughout the process to avoid
contamination that could compromise the viability and quality of the cells.
Thawing of cells

Seeding the cells
• Seeding the cells in cell culture procedure is the process of transferring a known number of cells into a sterile culture vessel,
such as a petri dish or tissue culture plate, in order to initiate cell growth and maintain a cell culture.
• Cell seeding density has a major impact on cell behavior, metabolic rate, cell signaling, differentiation and gene expression.
• Hence, this step is considered critical in cell culture as density of cells seeded has an impact on the growth, differentiation and
other cellular functions.
• Seeding cells involves resuspending the cells in fresh growth media and then adding the cells onto a sterile culture dish or flask.
• The exact number of cells to be seeded and the volume of media to be used will depend on the specific cell line being cultured
and the density required to support optimal growth.
• Typically, an appropriate amount of the cell suspension is withdrawn and added to a pre-coated plate or flask and is placed in an
incubator with appropriate conditions such as temperature, humidity, and CO2 concentration to allow for growth.
• It is important to follow the specific protocols for each cell line and adhere to the standard operating procedures (SOPs) of the
laboratory to maintain the sterility, viability and functionality of the cells throughout the cell culture process.
• Proper cell seeding will allow for the optimal growth and proliferation of cultured cells, providing highly consistent and
reproducible cell culture experiments.

Here are the general steps involved in seeding the cells in cell culture:
1. Prepare the culture vessel: The culture vessel should be sterilized by
autoclaving or other methods to eliminate any potential contaminants.
2. Prepare the culture medium: The appropriate culture medium should be
prepared and warmed to the recommended temperature before starting the
procedure.
3. Count the cells: Count the cells using a hemocytometer or cell counter to
determine the accurate cell number.
4. Resuspend the cells: Once the cell count and viability are determined, the
cells are resuspended in the culture medium.
5. Add the cells to the vessel: The cells are carefully added to the culture
vessel using a pipette or other appropriate tool, being careful not to create
bubbles, overfill the vessel, or spill the suspension.
6. Incubate the cells: Once the cells are seeded in the culture vessel, the
culture vessel should be put into an incubator for cell growth, maintaining
the appropriate temperature, humidity and CO2 concentration required for
the specific cell line.
7. Monitor the cells: Cells should be monitored periodically to assess their
growth, morphology, and health to ensure that they are healthy and
proliferating well.

Cell growth and maintenance
• Cell growth and maintenance is a critical step in cell culture that involves ensuring the continued health and viability of the
cultured cells.
• In order to maintain appropriate cell function and accurate downstream analysis, the cells must be grown under optimal
conditions and maintained with regular attention and care.
Here is an overview of the typical steps involved in cell growth and maintenance in cell culture:
1. Provide Food for Cells: Cells are provided with appropriate nutrients which are essential for their survival and propagation.
Cell culture media contain growth factors, amino acids, and vitamins that are required to maintain cell growth and viability.
2. Manage Growth Conditions: Optimal growth conditions such as temperature, humidity, and gas exchange provisions such as
CO2 are maintained to preserve the health and viability of the cells. Temperature and pH are also closely monitored and
maintained within the range that supports the proper functioning of the cells.
3. Regular Media Changes: The culture media are periodically changed to maintain an appropriate nutrient supply and optimum
pH for good growth of cultured cells. Depending upon the cell type, the frequency of media change can vary from once a day
to once a week.
4. Subculture of Cells: When the cells reach a certain confluency, they need to be sub-cultured or passaged to a new surface area
or new dish. This process allows for continued cell growth and can prevent cell stress or death caused by overcrowding or
nutrient depletion.
5. Preventing Contamination: Contamination can become a big problem in cell culture, so it is important to maintain sterility
while working with your cells. Use of sterile techniques and proper disposal of contaminated materials are crucial.
6. Monitoring of Cell Health: Regular monitoring of cell health is important to ensure that the cells remain healthy and
responsive and able to maintain their specific functions.

By following the above steps to maintain the culture conditions and keeping regular check on the health of the cultured cells,
the growth and maintenance of the cells can be well-maintained, ensuring a healthy and high-quality cell culture experiment.

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Passaging of cells
• Passaging of cells is a process that refers to the transfer of cells from one cell culture
vessel to another to maintain healthy cell growth and prevent overcrowding.
• The goal of passaging is to ensure that the cells grow in an environment where there is
sufficient space, nutrients, and oxygen, allowing healthy confluency to be achieved.
• Over time, cells in a culture dish will grow and divide, eventually leading to
overcrowding, which can lead to reduced metabolic activity and impaired cell growth.
• This, in turn, can lead to cell death or differentiation, compromising the quality of
experimental result.
• Passaging of cells is necessary to prevent overcrowding, ensure consistent and healthy
growth, and maintain the viability of the cell culture.
• The process involves removing cells from the culture vessel using trypsin or other
dissociation reagents, washing them, resuspending them in fresh culture medium and
then seeding them into a new culture vessel with adequate space and nutrient availability.
• The interval at which cells are passaged varies depending on the cell type and the culture
conditions, but it is typically done once cells reach a suitable confluency level.
• The success of a culture depends on many factors, including the frequency of passaging,
the quality of the media and supplements, and the proper technique for handling cells.
• Thus, it is important to perform passaging of cells in cell culture in a controlled
environment that adheres to sterile techniques and establishes proper culture conditions.

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• Passaging of cells, also known as subculture, is the process of transferring cells from one culture vessel to another in order to
maintain healthy cell growth and prevent overcrowding.
• Overcrowding leads to nutrient deficiency which affects the viability and metabolic activity of the cells.
• Passaging of cells provides additional space for the cells to grow and offers an opportunity to discard undesirable phenotypes.
The general procedure for passaging cells in cell culture involves the following steps:
1. Aspirate the medium: The spent medium is removed, aspirated, or decanted from the culture vessel containing the cells.
2. Washing the cells: The cells are washed by adding an appropriate volume of a buffered saline solution or enzyme-free cell
dissociation reagent is added into the culture vessel, which dislodges cells from the culture surface by diffusion.
3. Cell detachment: The cells are usually dislodged by using a trypsin-EDTA or another mild dissociating enzyme, or by shaking the
culture vessel.
4. Neutralization of the Enzyme Treatment: Once the cells are gently detached, the enzyme treatment is neutralized using culture media
containing FBS or any other supplement that neutralizes the trypsin.
5. Cell suspension preparation: The cells should be resuspended in an appropriate fresh culture medium containing supplements and
other growth factors, and the concentration should be adjusted based on the required cell density or ratio for subculture.
6. Passage the cells: The cell suspension is then seeded into a new culture vessel containing fresh media in limited or high density as
per requirement and allowed to grow.

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Harvesting the cells
• Harvesting of cells is the process of removing cells from a culture vessel, either for further downstream analysis or for preservation.
• Harvesting cells in a cell culture setting requires technique, as cells must be removed without causing damage to the cells or
disrupting their biological properties.
• Cell harvest can be done in different ways based on the purpose and experimental goals.
The following are typical methods for harvesting cells from cell culture:
1. Enzymatic method: Enzymatic dissociation is commonly used to remove cells from culture dishes. Proteolytic enzymes such as
trypsin, dispase, or collagenase are added to the culture dish to cleave cell-cell and cell-substrate adhesions, making the cells
suspension. However, it is essential to neutralize the enzyme and stop the digestion process once cells have detached.
2. Mechanical method: The cells can also be harvested by physically removing them by scraping, which can be useful for cells that
resist enzymatic action.
3. Filtration method: Cells can be captured using filters of various pore sizes, which trap or select specific cell types based on filtration
criteria.
Once cells are harvested, they can be stored in appropriate storage solutions or reagents, preserved using cryoprotectants, or utilized
immediately for downstream analysis. The most common method of cell preservation is the cryopreservation of cells, which involves
freezing the cells in a low-temperature environment for later use.

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• Harvesting cells is a significant step in cell culture experiments as it involves the removal of the cells from the artificial environment
where they have been grown and stimulated to the downstream application.
• Therefore, it is essential to carry out the harvesting process carefully and to establish the appropriate specimen storage and handling
procedures to ensure the desired outcome and integrity of the cells.
Here are some common steps involved in harvesting cells in cell culture:
1. Preparing for cell harvest: Before the harvesting process, the cells must be assessed for their confluency and viability, and the media
should be aspirated.
2. Cell detachment: The cells are then removed from the culture surface using an appropriate detachment method, such as trypsin or
enzyme-free cell dissociation buffer, depending on the cell culture type and the downstream analysis desired.
3. Neutralization of detachment agents: Once the cells are detached, any enzyme treatment that was used should be neutralized using an
appropriate neutralizing reagent such as serum, which will prevent continued cleavage of adhesion molecules caused by the enzyme.
4. Collection of cells: The cell suspension is then collected by centrifugation or filtration.5. Storing harvested cells: Cells can be stored in
appropriate storage media or reagents for later use, or directly used for downstream applications depending on the experimental goals.

There are various methods available for preserving cells after harvesting from cell culture, including:
1. Cryopreservation: This method involves preservation of cells at temperatures below -80 °C. Cells are cryopreserved in a cryoprotective
solution such as dimethyl sulfoxide (DMSO) or glycerol, which protect the cells from damage during freezing and thawing.
2. Slow freezing: A widely used method for preserving cells is slow freezing. In this method, cells are first exposed to a freezing solution
containing cryoprotectant agents like DMSO and stored in a freezing container. The container slowly cools the cells at a controlled rate,
and once the temperature reaches -80 °C, the container is plunged into liquid nitrogen for long-term preservation.
3. Vitrification: Vitrification is a cryopreservation method that rapidly freezes cells in liquid nitrogen at a temperature of around -196 °C.
Unlike slow freezing, which can cause damage to cells, vitrification keeps cells' structural integrity in a dense amorphous glass-like state,
which can protect cells from damage.
4. Short-term storage: For short-term storage, cells can be suspended in a culture medium, including Fetal Bovine Serum (FBS) and stored
at 4 °C until needed for downstream applications.
5. Freeze-drying or lyophilization: This method involves dehydrating the cells after placing them under a vacuum. The cells are then
packaged under suitable conditions for storage. Freeze-drying can allow for long-term storage, but the process can be complicated, and
the cells may be vulnerable to damage.
The preservation of cells through these methods enables researchers to maintain valuable cell cultures, which can be used for further
experimentation, manipulation, or re-seeding. It is essential to choose the appropriate preservation method according to the different cell
types, storage suitability, and the specific requirements of downstream applications.

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Storage & Disposal of materials
Storage & disposal of materials are essential aspects of cell culture to ensure the integrity & safety of the culture to prevent contamination.
The following is a brief explanation of storage and disposal procedures carried out in the process of cell culture:
Storage:
1. Culture media and supplements: Culture media and supplements should be stored at the recommended temperature also protected from
light and humidity.
2. Virus stocks: Viruses should be stored at low temperatures either -80°C or -20°C with recommended cryoprotectants to maintain viability.
3. Cryopreserved cells: Cryopreserved cells should be stored in vapor phase nitrogen tanks at -196°C or in the liquid phase of liquid nitrogen.
4. Disposable equipment: Used pipettes, containers, and other disposable equipment should be discarded into appropriate biohazard waste
bins.

Disposal:
1. Culture media: Disposable culture media should be disposed of properly in a biohazard waste bin.
2. Used pipettes and containers: Used pipettes and containers should be discarded into appropriate biohazard waste bins.
3. Hazardous waste: Chemical hazards and other hazardous waste materials generated during cell culture procedures should be disposed of
in accordance with local regulations.
4. Biohazard waste: All biohazard waste materials, including used culture dishes, gloves, and other contaminated materials, should be
disposed of using appropriate biohazard waste bins.
It is essential to manage and dispose of the waste materials generated during cell culture procedures properly. Failure to properly dispose of
hazardous wastes, spill, or accidents during any of the cell culture procedures may pose serious threats to the researcher involved, the
laboratory workers, or to the environment. Therefore, understanding the proper protocols of material storage and disposal is critical for safe
and effective cell culture practices.

RECORD KEEPING
Record-keeping is an essential aspect of a laboratory experiment, including cell culture. Accurate and detailed records are critical for
maintaining the integrity of the experiment, for making informed decisions, and for ensuring the safety of laboratory personnel and the
environment.
The following are some fundamental aspects of record-keeping in cell culture:
1. Lab notebook: A lab notebook is a critical tool for recording the details of each cell culture experiment. The notebook should record the
date, the cell line used, the number of cells seeded, the culture medium composition and volume, and the passage number of the cells.
2. Sterilization logs: Record the dates, methods, and details of sterilization procedures for equipment, media, and solutions required for
cell culture, including solutions used for disinfection, autoclaving, and filtration.
3. Media inventory log: The Media inventory log should contain the names of all the media, solutions, and supplements used in cell
culture, including the date of receipt, expiry date, batch number, and storage conditions.
4. Cryopreserved cell inventory log: It is essential to maintain an inventory of all the frozen cell lines stored under -80°C, including the
cell line, the passage numbers, the date of cryopreservation, and the location of storage.
5. Waste disposal record: A waste disposal record should include the date, type, amount, and location of hazardous waste produced in the
laboratory following each cell culture experiment.
6. Equipment maintenance logs: Record the date and details of scheduled equipment maintenance, calibration, repair, and replacement.
Also, document any incident that may arise during routine maintenance or equipment use.

• Record-keeping in cell culture aims to promote efficient and safe laboratory practices, ensure the accuracy,
reliability and reproducibility of experimental results, safeguard the health and safety of personnel involved in the
experiments and comply with regulatory requirements.
• Therefore, it’s essential to treat the documentation and recordkeeping process with the utmost importance and to
maintain the records for an adequate period

PROCEDURE FOR CELL CULTURE PREPARATION.pptx

PROCEDURE FOR CELL CULTURE PREPARATION.pptx

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