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Questions and Answers
Which type of cell culture hood primarily protects the user from airborne contaminants?
Which type of cell culture hood primarily protects the user from airborne contaminants?
What is considered essential for a tissue culture lab?
What is considered essential for a tissue culture lab?
What equipment is specifically recommended for maintaining cell culture conditions?
What equipment is specifically recommended for maintaining cell culture conditions?
Which piece of equipment is beneficial but not essential for tissue culture labs?
Which piece of equipment is beneficial but not essential for tissue culture labs?
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What flow characteristic of a horizontal flow hood provides protection to the culture?
What flow characteristic of a horizontal flow hood provides protection to the culture?
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Which of the following is categorized as useful but not necessary equipment in a tissue culture lab?
Which of the following is categorized as useful but not necessary equipment in a tissue culture lab?
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Which equipment is critical for cell counting in a tissue culture lab?
Which equipment is critical for cell counting in a tissue culture lab?
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What role do HEPA filters play in cell culture hoods?
What role do HEPA filters play in cell culture hoods?
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What is the primary purpose of using sterile equipment when working with cell cultures?
What is the primary purpose of using sterile equipment when working with cell cultures?
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Why is it important to determine the specific growth requirements for different cell types?
Why is it important to determine the specific growth requirements for different cell types?
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What is a disadvantage of using HEPES as a buffering system in cell cultures?
What is a disadvantage of using HEPES as a buffering system in cell cultures?
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Which of the following statements about phenol red in culture media is true?
Which of the following statements about phenol red in culture media is true?
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What is the purpose of maintaining cell cultures in an atmosphere of 5-10% CO2 when using the bicarbonate buffering system?
What is the purpose of maintaining cell cultures in an atmosphere of 5-10% CO2 when using the bicarbonate buffering system?
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What impact does exposure to fluorescent light have on media containing HEPES?
What impact does exposure to fluorescent light have on media containing HEPES?
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In what scenario would serum-free media be particularly beneficial?
In what scenario would serum-free media be particularly beneficial?
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What is the primary function of a cell culture hood?
What is the primary function of a cell culture hood?
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What is the primary characteristic of adherent cells in animal cell culture?
What is the primary characteristic of adherent cells in animal cell culture?
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What distinguishes primary cell cultures from continuous cell cultures?
What distinguishes primary cell cultures from continuous cell cultures?
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Which type of media are primarily used for maintaining cell cultures without serum?
Which type of media are primarily used for maintaining cell cultures without serum?
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Which of the following is NOT considered a key principle of aseptic technique in cell culture?
Which of the following is NOT considered a key principle of aseptic technique in cell culture?
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Cell culture hoods primarily provide which of the following?
Cell culture hoods primarily provide which of the following?
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Growth factors in animal cell culture serve what primary purpose?
Growth factors in animal cell culture serve what primary purpose?
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What factors are crucial for optimizing cell culture conditions?
What factors are crucial for optimizing cell culture conditions?
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Which characteristic defines suspension cells in animal cell culture?
Which characteristic defines suspension cells in animal cell culture?
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Study Notes
Instrumentation in Medical Diagnostic, Laboratory and Blood Banking
- Course: BIO62004
- Topic 2: Maintenance of Mammalian Cell Culture
- Section 2.1 (L4): Aseptic Techniques in maintenance of cell culture in vitro
Learning Objectives
- Discuss the principles of animal cell culture
- Demonstrate animal cell culture techniques
- Relate animal cell culture to specialized applications
- Understand the importance of aseptic technique (Sterile work area, Personal hygiene, Sterile reagents and media)
- Discuss the principles of cryopreservation and cell banking
Animal Cell Culture
- Cell culture: Process of growing cells in a controlled artificial environment
- In vitro maintenance: Cells can be maintained outside their original body
- Simpler than organ and tissue culture
Primary Cell Culture
- Outgrowth of cells: From a piece of tissue or disaggregated tissue (enzymatic/mechanical)
- Morphology: Similar to the parent tissue
- Limited cell divisions: Enter senescence and die
- Proliferative capacity: Extended by introducing viral transforming genes (e.g., SV40)
- Phenotype: Intermediate between finite and continuous cultures
- Finite cultures: Limited lifespan
- Continuous cultures: Proliferate indefinitely (similar to senescent primary cells)
Two Main Types of Cells
- Adherent cells: Anchorage dependent; grow as a monolayer attached to the culture vessel.
- Most tissue-derived cells are anchorage-dependent
- Suspension cells: Can survive and proliferate without attachment to a substrate.
Handling Cell Cultures
- Good laboratory practice (essential): Reduce exposure to infectious agents in the culture; prevent contamination by microbes
- Aseptic techniques and lab equipment: Vital for working with cell cultures
- Sterile equipment and reagents; wash hands, reagent bottles, and work surfaces with biocide/70% ethanol.
Cell Culture Conditions
- Culture media and serum: Different cell types require specific growth requirements. Media composition must be experimentally determined.
- Common basal media: Eagle minimal essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), RPMI 1640, and Ham F10. Contains amino acids, glucose, salts, vitamins, and other nutrients (powder or liquid form from suppliers).
- Media types: Balanced salt solutions (PBS, Hanks' BSS, Earle's salts, DPBS, HBSS, EBSS); Basal media (MEM, DMEM, GMEM); Complex media (RPMI 1640, Iscoves DMEM, Leibovitz L-15, TC 100, Graces, Schneider's, CHO, HEK293, Ham F10, DMEM/F12); Serum free media (Serum free insect Medium 1)
Basic Components of Media
- Bicarbonate buffering system (low cost, non-toxic): CO2 balances with HCO3- content; cultures need 5-10% CO2 atmosphere (CO2 incubator).
- HEPES buffering system: Superior buffering capacity in 7.2-7.6 pH range. Relatively expensive and toxic at higher concentrations for some cell types; increases media's sensitivity to phototoxic effects from fluorescent light.
- Phenol red: pH indicator; yellow at low pH, purple at higher; disadvantages of using phenol red?
Serum
- Growth factors and hormones: Promotes growth and specialized cell function
- Binding proteins: Albumin, transferrin that carry molecules into cells (lipids, vitamins, hormones)
- Attachment promotion: Proteins such as fibronectin; spreading factors allowing cells to spread out before division
- Protease inhibitors: Protect from proteolysis
- Minerals (Na+, K+, Zn2+, Fe2+, etc): Essential nutrients
Serum Inhibitors
- Heat inactivation: Removes complement proteins; reduces cytotoxic actions of immunoglobulins without damaging other growth factors; usually incubated for 30 minutes at 56°C.
Serum Advantages and Disadvantages
- Advantages: Growth factors, hormone attachment, spreading factor, buffering agent, binding protein, minimizes damage.
- Disadvantages: Batch variation, quality control needed, growth inhibiting properties, risk of contamination, interfere with purification and isolation.
Serum Free Media
- Serum-free media (SFM): Replaces serum, thus avoiding issues with animal-derived sera; Formulation exists for primary cultures and cell lines and includes those producing recombinant proteins (CHO, hybridoma cell lines, insect lines Sf9 and Sf21, and others for viral production.
Requirements of a Tissue Culture Lab
- Essential equipment: Cell culture hood, incubator (humid CO2 recommended), water bath, centrifuge, refrigerator & freezer (-20°C), cell counter, inverted microscope, liquid nitrogen freezer or cryostorage, sterilizer (autoclave)
- Beneficial equipment: Aspiration pump, pH meter, tube racks, pipettes, plate reader
- Additional equipment: Glassware washing machine, confocal microscope, flow cytometer, sterile containers, syringes, and needles
Cell Culture Hoods
- Air flow characteristics: Protects working environment from dust, airborne contaminants, by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area.
- Types: Horizontal (air flows towards the user) or vertical (air flows from top to bottom).
Cell Culture Conditions
- Incubation conditions: Incubator with controlled temperature and CO2 concentration is needed for optimal growth; includes dry and humid CO2 incubators.
- Cell culture vessels: Sterile, treated dishes and flasks for cell attachment; available commercially.
- Substrates and matrices: Cells attach to acid-washed glass, polystyrene, and plastics with a net positive charge. Cells secrete matrix products, such as collagen type IV, fibronectin, or laminin, to help adhere to the substrate and interacting with matrix receptors (e.g., integrins).
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Description
Explore the essential principles of maintaining mammalian cell cultures with a focus on aseptic techniques. This quiz covers the importance of sterile environments, personal hygiene, and the role of sterile reagents in successful cell culture. Learn about applications and processes such as cryopreservation and cell banking.
