12/15/2020

Animal Cell Culture: Growth of animal cells in culture.

 

Introduction:

  • Animal cell culture is a microbiological technique in which animal cells and tissues are obtained and maintained in a suitable environment.

  • It is further divided into,

    • Organotypic: Animal Organ Tissue Culture.

    • Histotypic: Animal Cell Culture.

  • Animal cells can be grown only upto certain generations.

  • Their growth requires typical conditions and a physicochemical environment.

  • Animal cell culture prepared from collecting tissue or cells from an organism is called a “Primary culture.”

  • The sequence of subcultures prepared from primary culture is called “Cell lines”.

  • History:

    • It  was Ross Harisson from America who first cultured animal cells successfully in the laboratory.

    • Campbell, Wilmut and colleagues at Roslin Institute in Scotland successfully cloned the first animal cell using nuclear transfer technique and gave birth to a sheep named “Dolly” in 1996.

Growth of Animal Cells in Culture.

  • Growing animal cells in cultures is a difficult task, it requires specially made culture media.

  • The culture media used for animal cell culture are classified as,

    • Natural,

    • Artificial,

    • Synthesized.

  • The selection of culture media depends on the objective of experiment and type of cell to be cultured.

  1. Natural Culture Media:

  • As the name indicates these media are obtained from natural resources like, tissue extracts, biological fluids, plasma clots or coagulants etc.

  1. Blood Plasma:

  • It provides good nutrition and support to many types of animal cell culture.

  • During subculturing it provides good protection against traumatic damage and better attachment of cells to glass surfaces.

  • Chicken plasma is most favored for its clarity even on dilution.

  • The plasma is obtained by centrifugation before coagulation.

  • This tissue or cells are placed in such plasma and then coagulation is initiated by addition of thrombin.

  1. Blood Serum:

  • Blood serum is plasma without fibrinogen.

  • Blood serum with or without nutritional substances is used as a culture media to grow animal cells.

  • The sera commonly used in animal culture are bovine, horse, and human.

  • Calf and fetal bovine serum are most widely used sera in animal cell culture.

  • Human serum is used sometimes but is needed to be screened for presence of hepatitis and HIV. 

  • The composition of a serum is highly complex as it is a mixture of various plasma proteins, peptides, lipids, carbs, enzymes and minerals.

  1. Tissue Extracts:

  • Commonly used tissue extracts in animal cell culture are, embryo, spleen, liver, bone marrow etc.

  • CHick embryo extract is the most commonly used tissue extract in animal cell culture, it is obtained from 10-12 days old embryos.

2) Artificial Media:

  • Synthetic or artificial media are prepared by adding organic or inorganic substances, nutrients, vitamins, proteins, carbs, gaseous phases.

  • Different artificial media are prepared for different types of cells as per their requirement.

  • Some common examples of artificial media are,

    • Minimal Essential Medium (MEM),

    • CMRL 1066,

    • RPMI 1640.

  • Synthetic media re classified as,

    • Serum Containing Media.

    • Serum Free Media.

  1. Serum Containing Media:

  • It is prepared by adding 5 to 20 % serum in serum free medium.

  • Serum is one of the best sources of many basic nutrients, contains hormones, growth factors, proteins, and minerals.

  • Serum also acts as a buffer.

  • Serum combines and neutralises many toxins.

  • Although its a best source of nutrients it has following disadvantages,

    • Most expensive of all ingredients of the culture medium.

    • It increases difficulties and cost of downstream processing.

    • Its content varies from batch to batch.

    • Extensive testing is required to check similarity on serum change.

    • Supply gets affected in drought or cattle disease spread.

  • Source of contamination: May contain viruses, bacteria, prions etc.

    • Some growth factors may be inadequate and can not be supplemented.

  1. Serum Free Media:

  • They are developed to overcome limitations of serum containing media.

  • These media can make the medium more selective for particular cells as per their requirements e.g. melanocytes can be cultivated in absence of keratinocytes.

  • As serum is not present there is no problem of toxicity, serum change tests.

  • Bioassays and other processes become easy due to absence of the serum.

  • However, they also has some disadvantages like,

    • Slower growth.

    • Useful only for a few generations.

    • Purity of ingredients.

    • pH control and temperature control required is more.

    • Most media are specific to a particular cell type.

    • Preparation time is more.

Physicochemical Parameters needed for growth animal cell culture:

  • Control on different physicochemical parameters is necessary during animal cell culture.

  • The temperature of the culture has to be the same as that of the body temperature of the animal from which cells are collected.

  • Osmolarity between 260 m Osm/kg and 320 m Osm/kg is good for many cell lines.

  • Buffers are incorporated for maintaining the pH as per requirement of the cell culture.

  • Most cell cultures grow well at a pH of 7.4, phenol red is commonly used indicator to detect the pH change in medium.

  • External Carbon dioxide addition is needed for some cell cultures to replenish CO2 loss.

  • Oxygen is a component of the gas phase as most cells require oxygen for respiration.

  • Cell damage in the culture is prevented by increasing viscosity of the medium by addition of CMC or PVP.

  • Balanced Salt Solution (BSS) is used as a diluent for vitamins, amino acids to make complete media.

Commonly Asked Questions:

  1. What is the difference between Blood Plasma and Blood Serum? (For Viva)

  2. Write in short about culture media used in animal cell culture.

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