Laboratory Facilities of Tissue Culture:
Sterile Area: For processing animal tissues for culture, it is essential to work in a sterile or aseptic area to prevent contamination. This environment ensures that the cultures remain pure and free from any microbial or particulate contamination. Two commonly recommended types of sterile work areas include:
Laminar Flow Cabinet
Biosafety Cabinet
Laminar Flow Cabinet
Function: Provides an aseptic environment by filtering air through HEPA filters before it passes over the work surface.
Design: Open at the front, allowing the researcher to comfortably handle cultures and equipment within the cabinet.
Airflow: Air is drawn in, filtered through a coarse filter to remove large particles, and then through a 0.3 μm HEPA filter to eliminate smaller contaminants.
Limitation: While it protects the culture from contamination, it does not offer protection to the researcher from potential pathogens.
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Bio-safety Cabinet
Function: Similar to the laminar flow cabinet, but with additional features to protect both the culture and the researcher.
Design: Provides a sterile environment for tissue culture while ensuring the safety of the researcher against exposure to human pathogens.
Airflow: Incorporates HEPA filters and may include mechanisms to safely exhaust air, preventing the escape of potentially harmful organisms.
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These sterile environments are crucial for maintaining the integrity of animal tissue cultures.
Tissue Culture Equipment:
The following equipment is required for animal tissue culture -
Autoclave: For sterilising culture media and instruments.
Centrifuge: To separate cells and other components from the culture.
Incubator: Regulates temperature and CO2 levels for optimal cell growth.
Water Bath: To maintain a consistent temperature during experiments.
Refrigerator: For storing culture media and reagents.
Freezer (-20°C): For storing cells and sensitive reagents.
pH Metre: To measure and adjust the pH of culture media.
Chemical Balance: For precise measurement of chemicals.
Stirrer: To mix solutions and culture media.
Bunsen Burner/Spirit Lamp: For sterilising instruments and maintaining a sterile environment.
Culture Vessels with Screw Cap: For growing and storing cultures.
Pasteur Pipettes: For transferring small volumes of liquids.
Inverted Microscope: For observing cell cultures.
Liquid Nitrogen Freezer: For cryopreservation of cells.
Liquid Nitrogen Storage Flask: For long-term storage of cryopreserved cells.
Bench Centrifuge: For quick centrifugation tasks.
Soaking Bath: For cleaning culture vessels.
Deep Washing Sink: For washing and rinsing equipment.
Pipette Cylinder(s): For holding and sterilising pipettes.
Pipette Washer: For cleaning pipettes.
Water Purifier: For obtaining deionized or distilled water.
Animal Tissue Culture Media
Mammalian cells in the body receive essential nutrients through blood circulation, which provides a constant supply of oxygen, nutrients, and growth factors necessary for cell survival and function.
Culturing Cells In Vitro:
Nutrient Requirements: When culturing these cells in vitro (outside the body), it is essential to replicate the nutrient-rich environment provided by blood. The culture media used must contain components similar to those found in blood, including vitamins, minerals, amino acids, glucose, and growth factors.
Choosing the Culture Medium
Factors Influencing Choice:
Type of Cells: Different cells have varying nutritional and environmental needs. The medium must be tailored to the specific type of cells being cultured.
Purpose of Culture:
Growth: Media optimised for cell proliferation.
Differentiation: Media that promotes the specialisation of cells into specific types.
Production of Desired Products: Media designed to maximise the production of proteins, antibodies, or other cellular products.
Types of Culture Media
Natural Media
Source: Obtained from various biological fluids like plasma, serum, lymph, amniotic fluid, and tissue extracts.
Examples:
Body Fluids: Plasma, serum, lymph, amniotic fluid, ascitic and pleural fluids, and aqueous humour.
Tissue Extracts: Liver, spleen, bone marrow, and embryo extracts, particularly chick embryo extract.
Usage: Tested for sterility and toxicity before being used for culturing cells. Some researchers still prefer natural media for organ culture.
Artificial Media
Development: Partially defined artificial media have been used for cell culture since 1950.
Criteria for Selection:
Nutrient Supply: The medium must provide all necessary nutrients for cell growth.
pH Maintenance: It should maintain a physiological pH of around 7.0 with adequate buffering.
Sterility: The medium must be sterile and isotonic to the cells.
The foundation of cell culture media is the balanced salt solution, initially designed to create a physiological pH and osmolarity necessary for maintaining cells in vitro. To support the growth and proliferation of cells, various components such as glucose, amino acids, vitamins, growth factors, and antibiotics are added, leading to the development of multiple media formulations. The addition of serum to these media is a common practice, though, in recent years, some researchers have begun using serum-free media.
The physicochemical properties required for tissue culture media are briefly described. This includes a discussion on balanced salt solutions, commonly used culture media, and serum-free media.
Physicochemical Properties of Culture Media
Culture media must possess specific physicochemical properties (such as pH, O₂, CO₂, buffering, osmolarity, viscosity, and temperature) to ensure the proper growth and proliferation of cultured cells.
pH:
Most cells grow optimally at a pH range of 7.0 to 7.4, though there can be slight variations depending on the cell type (e.g., cell lines).
The pH of the media is often monitored using the indicator phenol red.
This indicator changes colour according to the pH level:
At pH 7.4: Red
At pH 7.0: Orange
At pH 6.5: Yellow
At pH 7.8: Purple
CO₂, Bicarbonate, and Buffering:
Carbon dioxide in the medium is dissolved, and its concentration depends on atmospheric CO₂ tension and temperature. Within the medium, CO₂ exists as carbonic acid (H₂CO₃), which dissociates into bicarbonate (HCO₃⁻) and hydrogen ions (H⁺), as illustrated below:
CO2+H2O↔H₂CO₃↔H⁺+HCO₃⁻
The concentrations of CO₂, HCO₃⁻, and pH are interrelated, as shown in the equation above. Increasing atmospheric CO₂ lowers the pH, making the medium more acidic.
Adding sodium bicarbonate (as a component of bicarbonate buffer) neutralises the bicarbonate ions:
NaHCO₃↔Na⁺+HCO₃⁻
Commercially available media generally include recommended concentrations of bicarbonate and appropriate CO₂ tension to maintain the required pH.
HEPES Buffer:
In recent years, HEPES (hydroxyethyl piperazine 2-sulfonic acid) buffer has been increasingly used in culture media because it is more efficient than bicarbonate buffers.
However, many researchers prefer bicarbonate buffers due to their lower cost, reduced toxicity, and nutritional benefits to the medium. HEPES, while effective, is expensive and can be toxic to cells.
Pyruvate in Medium:
Pyruvate in the medium boosts the endogenous production of CO₂ by cells, which reduces reliance on exogenous CO₂ and HCO₃⁻.
This allows for buffering through higher concentrations of amino acids.
Oxygen
A significant majority of cells in vivo depend on a continuous oxygen (O₂) supply for aerobic respiration, which is facilitated by haemoglobin delivering O₂ to tissues.
For cultured cells, they rely mainly on dissolved O₂ in the medium. However, high concentrations of dissolved O₂ can generate free radicals, leading to toxicity. Thus, it's essential to provide just enough O₂ to meet cellular needs while avoiding toxic effects. Some researchers add free-radical scavengers like glutathione or mercaptoethanol to neutralise this toxicity. Adding selenium to the medium is also beneficial, as it acts as a cofactor for glutathione synthesis and helps reduce O₂ toxicity.
In general, glycolysis in cultured cells tends to be more anaerobic compared to in vivo cells. The depth of the culture medium influences the rate of O₂ diffusion, so it's advised to maintain the medium depth between 2-5 mm to optimise oxygenation.
Temperature
The optimal temperature for cell culture is typically linked to the body temperature of the organism from which the cells are derived. For human cells and cells from other warm-blooded animals, the ideal culture temperature is 37°C. In vitro cells cannot withstand temperatures higher than 40°C, and most will die beyond this point. Therefore, maintaining a stable temperature (±0.5°C) is crucial for reproducibility in experiments.
For cells from birds, a slightly higher temperature (38.5°C) is ideal for culturing. For cold-blooded animals (poikilotherms), such as cold-water fish, which do not regulate their body temperature, the ideal culture temperature ranges between 15-25°C.
Besides directly affecting cell growth, temperature also influences the solubility of CO₂, with higher temperatures enhancing its solubility.
Osmolality
In general, the osmolality for most of the cultured cells (from different organisms) is in the range of 260-320 mOsm/kg. This is comparable to the osmolality of human plasma (290 mOsm/kg).
Once an osmolality is selected for a culture medium, it should be maintained at that level, with an allowance of ±10 mOsm/kg.
Components Of Cell Culture Media
Balanced Salt Solutions:
The Balanced salt solutions (BSS) are primarily composed of inorganic salts. Sometimes Sodium-Bicarbonate, Glucose and HEPES buffer may also be added to BSS. Phenol Red Serves as a pH indicator.
The important functions of balanced salt solutions are listed below:
Supply essential inorganic ions.
Provide the requisite pH.
Maintain a desired Osmolarity.
Supply energy from glucose.
In fact balanced salt solutions from the basis for the preparations of complete media with the requisite additions.
Further BSS is also useful for a short period of time (up to 4 hours) incubation of cells.
Complete Culture Media:
In the early years Balanced salt solutions were supplements with various nutrients (amino acids, vitamins, serum etc.) to promote proliferation of cells in culture. Eagle was a pioneer in media formulation. He determined (during 1950-60) the nutrient requirements for mammalian cell cultures. Many developments in media preparation have occured since then. There are more than a dozen media now available for different types of cultures.
Some of them are stated below:
EMEM: Eagle's minimal essential medium
DMEM: Dulbecco’s modification of Eagle's medium
GMEM: Glasgow’s modification of Eagle's medium
RPMI 1630 & RPMI 1640: Media from Rosewell Park Memorial Institute
The other important culture media are Ham’s F10 & F12, TC 199 & CMRL 1060.
The detailed comparison of three commonly used media namely Eagle's MEM, RPMI 1640 & Ham’s F12 is given below:
The complete media, in general, contains a large number of components: amino acids, vitamins, salts, glucose, other organic supplements, growth factors and hormones and antibiotics, besides serum.
Depending on the medium the quality and quantity of the ingredients vary. Some important aspects of the media ingredients are briefly described.
Amino Acids:
All the essential amino acids (which cannot be synthesised by the cells) have to be added to the medium. In addition, even the non-essential amino acids (that can be synthesised by the cells) are also usually added to avoid any limitation of their cellular synthesis. Among the non-essential amino acids, glutamine and/or glutamate are frequently added in good quantities to the media since these amino acids serve as good sources of energy and carbon.
Vitamins:
The quality and quantity of vitamins depends on the medium. For instance, Eagle's MEM contains only water soluble vitamins (e.g. B-complex, choline, inositol). The other vitamins are obtained from the serum added. The medium M 199 contains all the fat soluble vitamins (A, D, E and K) also. In general, for the media without serum, more vitamins in higher concentrations are required.
Salts
The salts present in the various media are basically those found in balanced salt solutions (Eagle's BSS and Hank's BSS). The salts contribute to cations ( Na+, K+, Mg2+, Ca2+ etc.) and anions (Cl-, HCO3-, SO42-, PO43- ) and are mainly responsible for the maintenance of osmolality. There are some other important functions of certain ions contributed by the salts.
Ca2+ ions are required for cell adhesion, in signal transduction, besides their involvement in cell proliferation and differentiation.
Na+, K+and Cl-ions regulate membrane potential.
HCO3-, SO42-, PO43-, ions are involved in the maintenance of intracellular charge, besides serving as precursors for the production of certain important compounds e.g. PO43- is required for ATP synthesis.
Glucose
Most culture media contain glucose, which is an important energy source. In glycolysis, glucose breaks down into pyruvate or lactate, which then enters the citric acid cycle and gets converted into CO2. However, experiments show that glucose contributes very little to the citric acid cycle in cultured cells compared to living organisms. Instead, glutamine provides the carbon needed for the citric acid cycle, which is why cultured cells need a lot of glutamine.
Hormones and growth factors
For the media with serum, addition of hormones and growth factors is usually not required. They are frequently added to serum-free media.
Other organic supplements
Several additional organic compounds are usually added to the media to support cultures. These include certain proteins, peptides, lipids, nucleosides and citric acid cycle intermediates. For serum-free media, supplementation with these compounds is very useful
Antibiotics
In the early years, culture media invariably contained antibiotics. The most commonly used antibiotics were ampicillin, penicillin, gentamicin, erythromycin, kanamycin, neomycin and tetracycline. Antibiotics were added to reduce contamination. However, with improved aseptic conditions in the present day tissue culture laboratories, the addition of antibiotics is not required. In fact, the use of antibiotics is associated with several disadvantages
Possibility of developing antibiotic-resistant cells in culture
May cause antimetabolic effects and hamper proliferation
Possibility of hiding several infections temporarily
May encourage poor aseptic conditions
Serum
Serum is a natural fluid that contains many components essential for supporting the growth and multiplication of cells. It is widely used in culture media to supplement and provide necessary nutrients. There are different types of sera, including calf serum (CS), fetal bovine serum (FBS), horse serum, and human serum. If human serum is used, it must be tested for viruses like hepatitis B and HIV.
Typically, 5-20% of serum is added to media to help cells grow.
The table lists the key components found in serum, which include proteins, amino acids, lipids, carbohydrates, vitamins, hormones, and various inorganic molecules.
Serum is rich in important components like proteins, amino acids, lipids, and growth factors that are vital for cell growth and metabolism. When using human serum, it needs to be screened for safety. The nutrients, energy sources, and growth regulators provided by serum are critical in supporting cells during experiments or in culture environments.
Serum Free Media
Adding serum to culture media has been a long-standing practice. However, in recent years, certain serum-free media have been developed. It's important to understand the disadvantages of using serum and the pros and cons of serum-free media.
Disadvantages of Serum in Media
Variable Composition: Serum composition is not consistent. It varies depending on factors like the source, batch, season, and how it's collected and processed. These differences can affect the way cells grow in culture.
Quality Control: To ensure serum is of good quality, special tests must be done for each batch before it can be used. This can be a time-consuming process.
Contamination: It is hard to completely remove pathogens (like viruses) from serum, making it difficult to ensure it's contamination-free.
Presence of Growth Inhibitors: Serum usually has more growth promoters than inhibitors, but sometimes inhibitors like TGF-β (Transforming Growth Factor Beta) can slow down or stop cell growth.
Availability and Cost: Serum production relies on animals like cattle. This means its availability can be affected by economic, political, or agricultural factors. Serum can also be expensive, which makes its use less desirable.
Downstream Processing: Serum in the culture medium can interfere with the isolation and purification of cell culture products, making it harder to get the desired product. This may require extra steps in the process, adding complexity.
Advantages of Serum-Free Media
Defined Media Composition: Serum-free media allow precise control over cell growth because you can choose exactly what nutrients the cells receive. This is different from using serum, where growth can be less predictable.
Control of Cell Differentiation: You can use specific factors in serum-free media to help cells grow into specialised types. This gives you better control over how the cells develop.
Disadvantages of Serum-Free Media
Slower Cell Growth: Serum-free media usually do not help cells grow as fast as media with serum.
Need for Different Media: You might need to create different serum-free media for each type of cell. This can be challenging, especially if you are working with many cell types in a lab. Sometimes, even one type of cell might need different media at different stages of growth.
Purity of Reagents: Serum contains substances that protect and detoxify cells. Without serum, you need to use very pure chemicals and sterile equipment to avoid contamination.
Cost and Availability: Serum-free media are generally more expensive than those with serum. This is because the pure chemicals needed are costly. Additionally, finding these chemicals can sometimes be difficult.
Advantages of Tissue Cultures:
Control of physico-chemical environment- pH, temperature, dissolved gases (O, and CO₂), osmolarity.
Regulation of physiological conditions, nutrient concentration, cell to cell interactions, hormonal control.
The cultured cell lines become homogeneous (i.e. cells are identical) after one or two subcultures. This is in contrast to the heterogeneous cells of tissue samples. The homogenous cells are highly useful for a wide range of purposes.
It is easy to characterise cells for cytological and immunological studies.
Cultured cells can be stored in liquid nitrogen for several years.
Due to direct access and contact to the cells, biological studies can be carried out more conveniently. The main advantage is the low quantities of the reagents required in contrast to in vivo studies where most of the reagents (more than 90% in some cases) are lost by distribution to various tissues, and excretion.
Utility of tissue cultures will drastically reduce the use of animals for various experiments.
Application of Animal Cell Culture
There is a widespread concern that extensive use of animals for laboratory experiments is not morally and ethically justifiable. Animal welfare groups worldover are increasingly criticising the use of animals. Some research workers these days prefer to utilise animal cell cultures wherever possible for various studies.
Studies on intracellular activity e.g. cell cycle and differentiation, metabolisms.
Elucidation of intracellular flux e.g. hormonal receptors, signal transduction.
Studies related to cell to cell interaction eg. cell adhesion and motility, metabolic cooperation.
Evaluation of environmental interactions eg. cytotoxicity, mutagenesis.
Studies dealing with genetics eg genetic analysis, immortalization, senescence
Laboratory production of medical/pharma- ceutical compounds for a wide range of applications e.g., vaccines, interferons, hormones.
VAccine:
Plasmogen Activators:
Interferons:
Blood Clotting Factors
Hormones
Monoclonal Antibodies
Others
