types of cell culture and technology

1. INTRODUCTION ABOUT CELL CULTURE
• in-vitro culture (maintain and/or proliferate) of
cells ,tissue or organs
Types of tissue culture:
Organ culture
Tissue culture
Cell culture
Histotypic Culture
 Organotypic Culture
Primary culture
Cell line
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2. TYPES OF CELL CULTURE
• PRIMARY CULTURE
Adherent culture
Suspension culture
• SECONDARY CULTURE
• CELL LINES
Finite cell culture
Continous cell culture
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3. ORGAN CULTURE
• The entire embryos or organs are excised from
the body and culture
Advantages
• Normal physiological functions are maintained
• Cells remain fully differentiated
Disadvantages
• Scale up is not recommended
• Growth is alow
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4. TISSUE CULTURE
• Fragments of excised tissue are grown in
culture media
Advantages
• Some normal function may be maintained
• Better than organ culture for scale up but not
ideal
Disadvantages
• Original organization of tissue is lost
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5. CELL CULTURE
• Tissue from an explant is dispersed, mostly
enzymatically, into a cell suspension which may then
be cultured as a monolayer or suspension culture
Advantages
• Development of a cell line over several generations
• Scale-up is possible
Disadvantages
• Cells may lose some differentiated characteristics
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6. HISTOTYPIC CULTURE
• The culturing of the cells for their reaggregation to
form a tissue-like structure represents histotypic
culture
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7. ORGANO TYPIC CULTURE
• This culture techniques involves the
recombination of different cell types to form a
more defined tissue or an organ
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8. PRIMARY CULTURE
• The culture produced by the freshly isolated cells
or tissue taken from an organism is the primary
culture.
• These cells are heterogenous and slow gaining
and represents the tissue of their origin with
regard to their properties
• Isolation of tissues-Mechanical & Enzymatic
• Mechanical methods- sieving, syringing,
vigorous pipetting
• Enzymatic methods- warm trypsin, cold trypsin
& collagenase treatment
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9. TYPES OF ORGANS IN PRIMARY CELL
CULTURE
• Mouse embryos
• Chick embryos
• Human biopsy materials
• Transplantable animal
• Tumour Chick embryo organ
• Rudiments (brain, heart, lungs, liver, gizzard,
kidney, spinal cord, skin, muscle)
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10. ISOLATION OF TISSUES
• Must comply with local legislation and
medical ethical rules.
• Sterilize the site with 70% alcohol.
• Remove tissue aseptically.
• Transfer to the laboratory in transport medium
If delay in transporting to lab, keep at 4⁰C for
up to 72 hour.
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11. PRIMARY CULTURE
 Is a stage of the culture after first isolation of the cells but
before the first subculture. 4 stages:
1) acquisition of samples,
2) isolation of tissues,
3) disaggregation,
4) culture seeding into culture vessel.
 After isolation, a primary cell culture is obtained by allowing
cells to migrate out from the fragment of tissue adhering to a
suitable substrate by disaggregation.
PRIMARY EXPLANT
 Suitable for small amount of tissues example skin biopsy.
 Attachment on the substrate by using plasma clots, or
fibrinogen and trombin.
 Disaggregation by mechanical and enzymatic.
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14. Enzymatic disaggregation
 Warm trypsin, 37˚C for 30 mins, cell damaged if too long exposure.
 Cold pre exposure, soak at 4C overnight and 37C for less 30 mins.
Advantage:
 higher yield of viable cells,
 preserve more cell types Other enzyme -collagenase benefit for connective
tissues and muscle (fibrous tissue)
 dipase,DNase,hyaluronidase
Mechanical disaggregation (prevent proteolytic damage)
 Scrapping
 Spillage
 Sieving
 Syringes
 Trituration by pipette
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15. SECONDARY CULTURE
Derived from a primary cellculture.
Isolated by selection or cloning.
Becoming a more homogeneouscell population.
Finite life spanin vitro.
Retain differentiated phenotype.
Mainly anchorage dependant.
Exhibit contact inhibition.
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16. CONTINUOUS CULTURES
• Derived from a primary or secondaryculture
• Immortalised:
Spontaneously(e.g.:spontaneousgenetic
mutation)
By transformation vectors(e.g.:viruses&/or
plasmids)
• Serially propagated in culture showingan increased growth
rate
• Homogeneouscell population
• Lossof anchoragedependencyand contact inhibition
• Infinite life spanin vitro
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Genetically unstable
Characteristics of continous cell lines
-smaller, more rounded, lessadherent with a higher
nucleus/cytoplasm ratio
-Fast growth and have an euploidchromosome
number
-reduced serumand anchorage dependenceand grow
more in suspensionconditions
-ability to grow up to higher celldensity
-different in phenotypesfrom donor tissue
-stop expressingtissuespecific genes

18. CELL LINE
• The sub culturing of the primary culture gives
rise to cell lines. the term continuous cell lines
implies the indefinite growth of the cells in
the subsequent subculturing.
• Finite cell lines represent the death of cells
after several subcultures.
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20. ESTABLISHED CELL LINE CULTURE
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21. Subculturing
• Subculturing Subculturing or "splitting cells,"
is required to periodically provide fresh
nutrients and growing space for continuously
growing cell lines.
• The frequency of subculture and the split
ratio, or density of cells plated depend on the
characteristics of each cell line being carried.
• Subculturing - Adherent Cells Suspension
culture.
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24. Physical Environment for Culture:
• Physical Environment for Culture the aim is to provide
an environment that mimics, to the greatest extent
possible, the in vivo environment of that specific cell
type.
• The cell culture incubator, the culture dish or
apparatus, and the medium together create this
environment in vitro.
• Provides an appropriate temperature, pH, oxygen, and
CO 2 supply, surface for cell attachment, nutrient and
vitamin supply, protection from toxic agents, the
hormones and growth factors that control the cell's state
of growth and differentiation.
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25. CELL CULTURE MEDIA
• Cell culture media generally comprise an appropriate
source of energy and compounds which regulate the
cell cycle
• A typical culture medium is composed of;
Amino acids
Vitamins
Inorganic salts
Glucose and serum
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26. MEDIA
 Media Support survival and growth
 Natural Media
Clots-Plasma clots (male fowl)
Biological Fluids- Amniotic, Ascitic fluid, serum
Tissue extracts- Chicken and bovine embryo extract
 Artificial Media
 Defined & Complex media
Immediate Survival
Prolonged Survival
Indefinite growth
 Specialized function Classes
Serum containing media
Serum free media
Chemically defined Media
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27. Serum Containing Media:
Serum Containing Media
 MEM, DMEM, M199, F12, DMEM/F12,etc
 EMEM with 5-20% serum
 Provide plasma protein, peptides, lipids carbohydrates, minerals and
enzyme
 Hormones (cortisone, insulin and testosterone and prostaglandin)
 Growth factors (PDGF, TGF-p, epidermal growth factors etc)
 Supply protein ( fibronectin)
 spreading factor
 Binding factors (albumin, transferrin )
 Increase viscosity of medium Protease inhibitor Buffer Minerals
(Na, K, Fe, Zn, and Cu etc)
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28. Disadvantages of using serum media:
• Serum may inhibit growth of some cell types, e.g., epidermal
keratinocytes .
• Serum may contain some cytotoxic or potentially cytotoxic
constituents . For example, foetal calf serum contains the enzyme
polyamine oxidase which converts polyamines like spermidine and
sperrnine (secreted by fast growing cells) into cytotoxic
polyaminoaldehydes
• There is a large variation in serum quality from one batch to another
this requires costly and time consuming testing every time a new
batch has to be used .
• Some growth factors may be inadequate for specific cell types and
may need supplementation .
• It interferes with downstream processing when cell cultures are used
for production of biochemicals .
• The supply of serum is always lower than its demand.
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29. Serum Free Media:
 Analytical approach
Synthetic approach
 Limiting factor approach
Defined Media
 EMEM, DME, Ham,s F12, CMRL 1066,
RPMI 1640, Iscove,s modified Dulbecco,s
medium
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30. Serum-Free Media -advantages:
1. Improved reproducibility of results from different
laboratories and over time since variation due to batch
change of serum is avoided.
2. Easier downstream processing of products from
cultured cells.
3. Toxic effects of serum are avoided.
4. Bioassays are free from interference due to serum
proteins.
5. There is no danger of degradation of sensitive protein
by serum proteases.
6. They permit selective culture of differentiated and
producing cell types from the heterogenous cultures.
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31. Serum-Free Media - disadvantages:
1. Most serum-free media are specific to one cell type . Therefore,
different media may be required for different cell lines.
2. Reliable serum-free preparations, for most of the media
formulations are not available commercially. This necessitates time
consuming task of preparing the desired formulations in the
laboratory.
3. A greater control of pH , temperature etc. is necessary as compared
to that with serum containing media.
4. Growth rate and the maximum cell density attained are lower than
those with serum containing media.
5. Cells tend to become fragile during prolonged agitated cultures
unless biopolymers or synthetic polymers are added.
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32. Preparation and Sterilization of Medium
• The various media constituents and other reagents used in cell cultures
must be carefully sterilized either by autoclaving or by filtration.
• Heat stable constituents tike water, salts, supplements like peptone or
tryptose etc. are autoclaved at 121°C for 20 min.
• heat labile constituents like serum, trypsin, proteins, growth factors etc.
must be sterilized by filtration through a 0.2 mm porosity membrane
filter.
• Each filtrate should be tested for sterility to avoid failure due to
contamination.
• In case of soda glass, caps should be left slack to avoid breaking during
autoclaving.
• Autoclaving is preferred to filtration since it is cheaper, needs less
labour and is uniformly effective.
• Therefore wherever possible, autoclaving should be resorted to and
autoclavable versions of the media should be used.
• Most of the media, however, now available commercially are usually
pre sterilized. .
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35. • References:
1. Text book of biotechnology,
Dr.U.Satyanarayana, pg.no 415-437
2. https://www.slideshare.net/nadiamohdkp/ty
pes-of-cell-culture
3. www.biotechnology4u.com/animal_biotechn
ology_types_cell_cultures.html
4. https://microbeonline.com
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