Animal Cell Culture - Class 12 Biotechnology - Chapter 8 - Notes, NCERT Solutions & Extra Questions
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What is animal cell culture?
Animal cell culture refers to the in vitro maintenance and proliferation of animal cells, allowing them to grow outside their original biological context by providing the necessary nutrients and environmental conditions. This technique is fundamental in biological and medical research, enabling the study of cellular processes, drug development, and the production of vaccines and therapeutic proteins under controlled laboratory settings.
The key components of animal cell culture include selection of appropriate growth media, which supplies essential nutrients, and maintaining optimal physical conditions such as temperature, pH, and osmolality. It is vital for studies on cell behavior, drug testing, and genetic engineering, offering a simulated natural environment for various research and industrial applications.
Describe animal cell culture media and their types.
Animal cell culture media are essential for the in vitro growth and maintenance of animal cells. These media provide the necessary nutrients, such as vitamins, amino acids, glucose, and inorganic salts, while also maintaining the appropriate pH and osmolality. There are two primary types of culture media: Natural media and Synthetic (Artificial) media.
Natural media consist of naturally occurring biological substances like plasma, serum, and embryo extracts. However, their composition can be variable and non-uniform.
Synthetic media, on the other hand, are precisely formulated with defined amounts of organic and inorganic nutrients. They can be further categorized into serum-containing, serum-free, chemically defined, and protein-free media, each tailored to specific cell culture requirements. These media types offer more consistency and reproducibility compared to natural media.
Write the advantages and disadvantages of serum in the culture media.
Advantages of Serum in Culture Media:
- Rich Source: Serum is enriched with essential nutrients such as amino acids, proteins, vitamins, carbohydrates, lipids, and hormones that are vital for cell growth and proliferation.
- Growth Factors: It contains growth factors that are crucial for cell survival, growth, and differentiation.
- Binding Proteins: Serum offers various binding proteins like albumin and transferrin that help transport vital molecules into cells.
- Adhesion Factors: It provides adhesion factors necessary for cells to attach and spread on the culture substrate, facilitating proper growth as monolayers.
Disadvantages of Serum in Culture Media:
- Variability: The composition of serum can vary significantly between batches, which can affect experimental reproducibility.
- Contaminants: There is a risk of introducing contaminants such as viruses, fungi, and mycoplasma, which can compromise cell culture.
- Inhibition: Serum may contain inhibitors that can negatively impact cell growth and function.
- Interference: It can interfere with the purification and isolation of cell culture products, making downstream processing challenging.
- Ethical Concerns: The use of animal-derived serum raises ethical concerns regarding animal welfare.
Describe any two chemically synthesised media.
Chemically defined media are a type of synthetic or artificial media in which all the ingredients and their concentrations are clearly defined. This category lacks any complex additives such as serum, which makes the media exceptionally reproducible and contamination-free. Noteworthy examples include:
1. Phosphate Buffered Saline (PBS): PBS is a simple salt solution used primarily for maintaining osmotic balance and providing a stable pH environment. It contains inorganic salts like NaCl, KCl, and Na₂HPO₄, and lacks nutrients that support cell growth, typically used for washing cells during processing.
2. Dulbecco's Phosphate-Buffered Saline (DPBS): A more buffered version containing additional Ca²⁺ and Mg²⁺ ions, DPBS supports more sensitive applications where calcium and magnesium ions are crucial for processes like cell adherence and signaling.
Both media types are valued for their ability to maintain physicochemical stability during experimental procedures involving cell cultures.
What is primary cell culture? Also discuss as to how it is developed.
Primary cell culture is the initial culture of cells directly extracted from tissues of an organism. These cells retain the characteristics of the original tissue from which they were derived. To develop primary cell culture, a selected tissue undergoes sterile dissection followed by either mechanical disaggregation or enzymatic digestion using enzymes like trypsin. The resulting cell suspension is then placed in a suitable culture medium in a container. These cells may grow adherently forming a monolayer or remain in suspension, depending on their type. Primary cultures generally contain a heterogeneous mixture of cells and closely resemble the metabolic and physiological properties of the tissue of origin.
What is subculture or passaging of cell?
Subculture or passaging is the process of transferring cells from a crowded container to a new vessel with fresh growth medium to continue cell proliferation. This method is essential when cultured cells reach confluence or are overcrowded, which can lead to nutrient depletion and waste accumulation, inhibiting growth. During subculture, cells are typically detached from their growth surface using enzymatic (e.g., trypsin) or mechanical methods. The detached cells are then centrifuged, resuspended in fresh medium, and seeded into new culture vessels. This procedure, often referred to as "splitting" cells, is crucial for maintaining healthy cell lines and allows for the expansion and maintenance of cell cultures over extended periods.
Differentiate between finite and continuous cell lines.
Category | Finite Cell Lines | Continuous Cell Lines |
---|---|---|
Lifespan | Limited, undergo senescence after a certain number of divisions | Indefinite, capable of endless proliferation |
Genetic Stability | More stable, closer to original tissue phenotype | Less stable, may have significant genetic drift |
Origin | Derived directly from tissue | Often derived from finite lines through transformation |
Research Use | More suitable for studies close to in vivo conditions | Useful for high-volume production and less precise studies |
How is cell viability measurement performed?
Cell viability measurement is crucial to determine the proportion of living cells within a culture. It can be performed using dye exclusion viability assays and metabolic viability assays.
Dye exclusion viability assays involve using dyes like trypan blue, which are excluded by live cells but penetrate and stain dead cells, indicating compromised membrane integrity.
Metabolic viability assays measure cellular metabolism, such as the ability to convert substrates into products. One common method is the MTT assay, where the tetrazolium dye MTT is reduced by metabolic activity in live cells to form a colored formazan product. The intensity of the color, measurable by spectrophotometry, directly correlates with the number of viable cells.
Both methods provide essential data on cell health and function.
Write a detailed account of application of cell culture.
Cell culture technology is pivotal across various scientific domains, extending from basic research to practical applications in medicine and industry. Research and drug development heavily rely on cell culture techniques to analyze the effects of drugs on specific types of cells, facilitating the development of new medications and therapeutic strategies. Vaccine production is another crucial area, where cell cultures are used to grow viruses needed to create vaccines. Biopharmaceutical production uses cell cultures to produce complex proteins like antibodies and hormones that cannot be synthesized effectively through chemical methods. Additionally, gene therapy utilizes cell culture to cultivate cells into which genes are inserted to treat genetic disorders. Intersecting with advanced bioengineering, cell culture innovations contribute to tissue engineering and regenerative medicine, aiming to regenerate damaged tissues and organs.
The example of animal cell culture media is:
(a) DMEM
(b) MS media
(c) LB Media
(d) All of the above
(a) DMEM
DMEM (Dulbecco's Minimal Essential Medium) is an example of animal cell culture media. It is specifically designed to support the growth of mammalian cells. MS media is for plant cell cultures, and LB Media is for bacterial culture.
Name the type of culture which is prepared by inoculating
directly from the tissue of an organism to culture media.
(a) Primary cell culture
(b) Secondary cell culture
(c) Cell lines
(d) Transformed cell culture
The type of culture which is prepared by inoculating directly from the tissue of an organism to culture media is:
(a) Primary cell culture
Primary cell culture involves the direct transfer of cells from a tissue source to a suitable growth medium, where the cells dissociate and grow. This type of culture contains mostly heterogeneous cells that retain most of the genetic and phenotypic characteristics of the original tissue.
Sodium bicarbonate is added to animal cell culture media to
(a) keep cells stuck to the plastic
(b) promote the uptake of CO2 into animal cells
(c) maintain the correct pH when CO2 is present
(d) keep iron soluble
The correct answer is: (c) maintain the correct pH when CO2 is present
Sodium bicarbonate acts as a buffer in the culture medium. When CO2 levels increase in the incubator, it combines with water to form carbonic acid, which could lower the pH of the media. Sodium bicarbonate helps to stabilize the pH by neutralizing the acid, maintaining an environment suitable for the cells' growth.
Which of the following is NOT present in growth medium
for animal cell culture?
(a) Inorganic salts
(b) Bicarbonate
(c) Carbon source
(d) Starch
The correct answer is:
(d) Starch
Starch is not typically included in the growth media for animal cell culture. The common components of a typical culture medium, as mentioned in the chapter, include vitamins, amino acids, glucose (as a carbon source), inorganic salts, and serum. Starch is not a necessary or typical component of media used for culturing animal cells.
Disaggregating of cells can be performed by:
(a) Physical disruption
(b) Enzymatic digestion
(c) Treating with chelating agents
(d) All of the above
(d) All of the above
Disaggregating of cells can be performed by physical disruption, enzymatic digestion, and treating with chelating agents. These methods help in separating cells from each other and from the extracellular matrix to establish a cell culture.
The approach in which genes are transferred into animals to obtain a large scale production of the proteins encoded by these genes in the milk, blood, etc., is called
(a) In situ culture
(b) Molecular pharming
(c) Gene therapy
(d) Hybridoma technology
The correct answer is:
(b) Molecular pharming
Molecular pharming, also known as pharming, involves the genetic modification of animals to produce pharmaceutical proteins or compounds in their bodily fluids such as milk or blood. This biotechnological technique utilizes transgenic animals as bioreactors to generate high quantities of specific proteins for medical use.
Which of the following is a protein free animal cell culture media?
(a) RPMI-1640 Media
(b) MS media
(c) LB media
(d) None of the above
The correct answer is (d) None of the above.
None of the options listed (RPMI-1640 Media, MS media, LB media) are specifically categorized as protein free media. RPMI-1640 is a type of complex media typically supplemented with serum or other proteins. MS media (Murashige and Skoog media) is primarily used for plant cell culture, not animal cell culture. LB media is a bacterial growth medium, also not applicable for animal cell cultures. None of these are defined as protein-free media suitable for animal cell culture as described in the given text.
MTT assay is used for
(a) Cell viability test
(b) Monitoring of variation in pH of culture media
(c) Transformation screening
(d) Cell dissociation from substratum
(a) Cell viability test
The MTT assay is used to assess cell metabolic activity as an indirect measure of cell viability. This assay relies on the reduction of the MTT reagent by mitochondrial dehydrogenase enzymes in viable cells, resulting in a colorimetric change that is quantifiable.
Passaging of animal cells in animal cell culture is
(a) Sub-culturing of the cells
(b) Isolation of cells
(c) Passing the cells from culture tube to Petri dishes
(d) Counting of cells
Passaging of animal cells in animal cell culture refers to the process known as sub-culturing, where a portion of cells is transferred to a new vessel containing fresh growth medium to promote continued growth. Therefore, the correct answer is:
(a) Sub-culturing of the cells.
Which of the following is NOT the major function of serum?
(a) Enhance cell attachment
(b) Stimulate cell growth
(c) Promotion of tuber and bulb formation
(d) Provide transport proteins
(c) Promotion of tuber and bulb formation
Serum in cell culture media primarily enhances cell attachment, stimulates cell growth, and provides transport proteins, but it does not promote tuber and bulb formation which is unrelated to animal cell culture.
Assertion: Serum is the most important component of culture media.
Reason: Serum is a good source of nutrients and also helps in cell proliferation and cell-matrix attachment.
(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.
(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
Explanation:
- Assertion: True - Serum is indeed a critical component often added to cell culture media as it provides numerous essential growth factors, hormones, and nutrients necessary for cell growth.
- Reason: True - The reason is accurate in stating that serum supplies important nutrients, supports cell proliferation, and facilitates cell-matrix attachments, all of which are crucial for the growth and maintenance of cells in culture. These factors also properly explain why serum is considered an important component of the culture media.
Assertion: Cell lines derived from the primary culture of normal cells are finite cell line.
Reason: Some cells of the finite cell line undergoes transformation and retain the ability to divide indefinitely.
(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
Explanation: The assertion is true because cell lines derived from primary cultures of normal cells are indeed finite; they have a limited number of cell divisions before they senesce. However, the reason given relates to the transformation of some cells within finite cell lines which then may give rise to continuous cell lines. While it's true that some cells might undergo transformations and become continuous cell lines, this characteristic is not the explanation for why the original finite cell lines derived from primary cultures have a limited number of cell divisions. These are two distinct properties of different types of cell lines.
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Comprehensive Class 12 Notes on Animal Cell Culture
Introduction to Animal Cell Culture
Animal cell culture refers to the in vitro maintenance and proliferation of animal cells outside their natural environment. This technique enables cells to grow under controlled conditions if supplied with appropriate nutrients and growth factors. Cell culture plays a crucial role in various branches of life sciences, including molecular genetics, pharmaceutical research, and bioengineering.
Historical Perspective of Animal Cell Culture
Animal cell culture became a routine laboratory practice in the 1950s after George Gey established the first human cell line (HeLa) from the cervix cancer cells of Henrietta Lacks. This milestone opened the door to numerous significant discoveries in medical science. The following table highlights some notable events in the history of animal cell culture:
Year | Achievements |
---|---|
1882 | Sydney Ringer developed a balanced salt solution. |
1907 | Ross Harrison published experiments on frog embryo nerve fibre growth in vitro. |
1950s | Human cell lines were extensively used for vaccine production. |
Essential Components of Culture Media
Selecting the right culture medium is crucial for the successful growth of cells in vitro. The medium must provide all necessary nutrients, such as vitamins, amino acids, glucose, inorganic salts, and growth factors.
Significance of Serum
Serum is an important component of culture media, offering a rich source of growth factors, hormones, and binding proteins. However, it has disadvantages, including batch-to-batch variability and potential contamination risks.
Types of Culture Media
Culture media can be broadly categorised into natural and synthetic media.
Natural Media
These consist of naturally occurring biological fluids like plasma and embryonic extracts. While useful, they often lack reproducibility and consistency.
Synthetic Media
These are composed of a basal medium supplemented with essential nutrients and growth factors. They can be further classified based on their composition:
- Serum-containing media: Contains animal serum.
- Serum-free media: Uses protein fractions as supplements.
- Xeno-free media: Contains human-source components.
- Chemically defined media: Composed solely of known quantities of purified components.
Equipment Used in Animal Cell Culture
A well-equipped laboratory is essential for successful cell culture. The key equipment includes:
Laminar Flow Hoods
These provide a sterile environment, minimising contamination.
CO2 Incubators
These maintain the necessary temperature, CO2 levels, and humidity for optimal cell growth.
Inverted Microscopes
These are crucial for monitoring cell morphology and identifying any signs of contamination early.
graph TD;
A[Equipment for Cell Culture] --> B[Laminar Flow Hood]
A --> C[CO2 Incubator]
A --> D[Inverted Microscope]
Types of Animal Cell Cultures and Cell Lines
Primary Cell Cultures
Primary cultures are derived directly from host tissues and contain heterogeneous cells. These cells closely resemble the original tissue.
Secondary Cell Cultures
These are obtained by sub-culturing primary cells and often display greater uniformity.
Adherent Cells
These cells require attachment to a substrate, forming a monolayer. Examples include fibroblasts and epithelial cells.
Suspension Cells
These cells grow floating in the medium, requiring no attachment. Examples include blood and tumour cells.
Determining Cell Viability
Dye Exclusion Viability Assays
In this method, dyes like trypan blue are used to distinguish live cells, which exclude the dye, from dead cells, which absorb it.
Metabolic Viability Assays
These assays measure cellular biochemical activity. For example, the MTT assay involves the reduction of a tetrazolium compound by live cells, indicating their viability.
Cryopreservation of Animal Cells
Cryopreservation involves storing cells at ultra-low temperatures using liquid nitrogen. Cryoprotective agents like Dimethyl Sulfoxide (DMSO) are added to prevent ice crystal formation, which can damage cells. The cells are slowly cooled and then thawed rapidly for future use.
Advantages and Applications of Animal Cell Culture
Advantages
- Controlled Growth Conditions: Cells can be cultured under defined physico-chemical conditions.
- Genetic Uniformity: Homogeneous cell populations can be achieved.
- Ethical: No ethical clearance is typically required.
Applications
Animal cell culture is invaluable in many fields:
- Vaccine Production: Enables large-scale vaccine production.
- Drug Development: Facilitates the screening of potential drug compounds.
- Genetic Research: Offers insights into cellular mechanisms and gene functions.
graph LR;
A[Application of Cell Culture] --> B[Vaccine Production]
A --> C[Drug Development]
A --> D[Genetic Research]
Hybridoma Technology for Monoclonal Antibody Production
Monoclonal antibodies (MABs) have single specificity and are produced by hybridoma technology. This involves fusing B-cells with myeloma cells to create hybrid cells that can divide indefinitely and produce specific antibodies.
Process
- Immunisation: Animals are immunised with an antigen.
- Cell Fusion: B-cells are fused with myeloma cells.
- Selection: Hybrid cells are selected and cloned for producing antibodies.
- Screening: Clones are screened for specific antibody production.
graph TB;
A[Hybridoma Technology] --> B[Immunisation]
B --> C[Cell Fusion]
C --> D[Selection]
D --> E[Screening]
Conclusion
Animal cell culture is a versatile tool in scientific research. From vaccine production to genetic research, its applications are vast and significant. With continued advancements, this technology will keep contributing to various fields, improving health and quality of life.
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