Cell Culture

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Cell Culture
Dr. Tarek Elbashiti Assoc. Prof. of Biotechnology
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• The term cell culture refers to the cultivation
  of dispersed (isolated) cells taken from an
  original tissue, a primary culture, or a cell
  line.
• The practice of cultivating cells started at the
  beginning of the 20th century and was developed
  from a simple exploratory phase to an expansion
  phase in the 1950s.
• For more than 50 years, the culture of cells
  derived from primary tissue explants has
  predominated, justifying the original name
  tissue culture.

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• Currently, cell culture is in a specialization
  phase. With the increase use of detached cells
  since the 1950s, the term tissue culture was
  substituted by cell culture.
• At the present time, cell culture techniques
  allow in vitro propagation (proliferation) of
  various cell lines including those from insects,
  humans, mice, rats, and other mammals.

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• Basically, animal cell culture techniques are
  similar to those employed for bacteria, fungi,
  and yeast, although there are some characteristic
  differences.
• In general, animal cells are more delicate,
  vulnerable to mechanical damage, present lower
  growth rates, and require more complex culture
  media and special substrates

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• cell culture has to be performed under rigorous
  aseptic conditions, since animal cells grow more
  slowly than most usual contaminants, such as
  bacteria and fungi.

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HISTORICAL BACKGROUND

• Tissue culture was first developed at the
  beginning of the twentieth century as a method
  for studying the behavior of animal cells free of
  systemic variations that might arise in vivo both
  during normal homeostasis and under the stress of
  an experiment.
• The technique was elaborated first with
  un-disaggregated fragments of tissue, and growth
  was restricted to the migration of cells from the
  tissue fragment, with occasional mitoses in the
  outgrowth.

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• Roux in 1885 for the first time maintained
  embryonic chicken cells in saline).
• Cell culture was first successfully undertaken by
  Ross Harrison in 1907( lymph)
• 1911 Lewis made the first liquid media consisted
  of sea water, serum, embryo extract, salts and
  peptones. They observed limited monolayer growth.

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• 1913 Carrel introduced strict aseptic techniques
  so that cells could be cultured for long periods.
• 1916 Rous and Jones introduced proteolytic
  enzyme trypsin for the subculture of adherent
  cells.
• 1940s The use of the antibiotics penicillin and
  streptomycin in culture medium decreased the
  problem of contamination in cell culture.

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• Sanford was the first cloned cell strain,
  isolated by capillary cloning from mouse L-cells
  Sanford et al., 1948.
• It was not until the 1950s that trypsin became
  more generally used for subculture, following
  procedures described by Dulbecco to obtain
  passaged monolayer cultures for viral plaque
  assays
• Dulbecco, 1952, and the generation of a single
  cell suspension by trypsinization, which
  facilitated the further development of single
  cell cloning.

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• 1955 Gey established the first continuous human
  cell line, HeLa this was subsequently cloned by
  Puck, 1955
• Tissue culture became more widely used at this
  time because of the introduction of antibiotics,
  which facilitated long-term cell line
  proliferation although many people were already
  warning against continuous use and the associated
  risk of harboring cryptic, or antibiotic-resistant
  , contaminations .

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• The 1950s were also the years of the development
  of defined media which led ultimately to the
  development of serum-free media 1966.

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MAJOR

• First was the use of chemically defined culture
  medium.
• Second was the development was the use of
  antibiotics which inhibits the growth of
  contaminants.
• Third was the use of trypsin to remove adherent
  cells to subculture further from the culture
  vessel

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Advantages

• Model systems for
• Studying basic cell biology, interactions
  between disease causing agents and cells, effects
  of drugs on cells, process and triggering of
  aging nutritional studies
• Toxicity testing
• Study the effects of new drugs
• Cancer research
• Study the function of various chemicals,
  virus radiation to convert normal cultured
  cells to cancerous cells

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Advantages

• Virology
• Cultivation of virus for vaccine production,
  also used to study there infectious cycle.
• Genetic Engineering
• Production of commercial proteins, large
  scale production of viruses for use in vaccine
  production e.g. polio, rabies, chicken pox,
  hepatitis B measles
• Gene therapy
• Cells having a functional gene can be
  replaced to cells which are having non-functional
  gene

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Advantages

• To produce artificial tissues ( skin)
• Use single cell ( impossible in vivo)

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Disadvantages

• Cell characteristics can be changed
• Cell adapts to different nutrients
• If mixed cells cultivated some types will
  disappeared.
• Activity of enzymes may altered by environment.

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Typical structure of an animal cell

• Various animal cell lines can be cultivated in
  vitro, such as cardiac cells, fibroblasts, smooth
  muscle cells, endocrine cells (such as pituitary,
  adrenal, and pancreatic cells), epithelial cells
  (such as liver, mammary, lung, and kidney cells),
  tumor cells (such as melanocytes), nervous system
  cells (such as glial cells and neurons), as well
  as hybridomas. Although these cells have
  different origins and functions, they all show a
  typical structure.

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Animal tissue Cultures Classification

• 1-classification a
• A-Organ culture
• B-Tissue culture
• C-Cell culture

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Types
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A- Organ Culture

• The term organ culture will always imply a
  three-dimensional culture of undisaggregated
  tissue retaining some or all of the histological
  features of the tissue in vivo.
• The entire embryos or organs are excised from the
  body and cultured

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• Disadvantages
• Scale-up is not recommended.
• Growth is slow.
• Fresh explanation is required for every
  experiment.

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B-Tissue Culture

• the term tissue culture is used as a general term
  to include organ culture and cell culture.
• Fragments of excised tissue are grown in culture
  media
• A fragment of tissue is placed at a glass (or
  plastic)liquid interface, where, after
  attachment, migration is promoted in the plane of
  the solid substrate

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• Advantages
• Some normal functions may be maintained.
• Better than organ culture for scale-up but not
  ideal.
• Disadvantages
• Original organization of tissue is lost.

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C- Cell Culture

• Cell culture refers to a culture derived from
  dispersed cells taken from original tissue, from
  a primary culture, or from a cell line or cell
  strain by enzymatic, mechanical, or chemical
  disaggregation into a cell suspension, which may
  then be cultured as an adherent monolayer on a
  solid substrate or as a suspension in the culture
  medium

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• Advantages
• Development of a cell line over several
  generations
• Scale-up is possible
• Disadvantages
• Cells may lose some differentiated
  characteristics.

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classification b

• Primary Cultures
• Derived directly from excised tissue and cultured
  either as
• Outgrowth of excised tissue in culture
• Dissociation into single cells (by enzymatic
  digestion or mechanical dispersion)
• Advantages
• usually retain many of the differentiated
  characteristics of the cell in vivo
• Disadvantages
• initially heterogeneous but later become
  dominated by fibroblasts.
• the preparation of primary cultures is labor
  intensive
• can be maintained in vitro only for a limited
  period of time.

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• Continuous Cultures
• derived from subculture (or passage, or transfer)
  of primary culture
• Subculture the process of dispersion and
  re-culture the cells after they have increased to
  occupy all of the available substrate in the
  culture
• can be serially propagated in culture for several
  passages
• There are two types of continuous cultures
• Cell lines
• Continuous cell lines

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• Cell lines
• finite life, senesce after approximately thirty
  cycles of division
• usually diploid and maintain some degree of
  differentiation.

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• Continuous cell lines
• can be propagated indefinitely
• generally have this ability because they have
  been transformed
• tumor cells.
• viral oncogenes
• chemical treatments.
• the disadvantage of having retained very little
  of the original in vivo characteristics

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Common cell lines

• Human cell lines
• -MCF-7 breast cancer
• HL 60 Leukemia
• HEK-293 Human embryonic kidney
• HeLa Henrietta lacks
• Primate cell lines
• Vero African green monkey kidney
  epithelial cells
• Cos-7 African green monkey kidney
  cells
• And others such as CHO from hamster, sf9 sf21
  from insect cells

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American Type Culture Collection.
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3- Culture Morphology

• Suspension (as single cells or small
  free-floating clumps)
• or as a monolayer that is attached to the tissue
  culture flask.
• The form taken by a cell line reflects the tissue
  from which it was derived
• from blood tend to grow in suspension
• from solid tissue (lungs, kidney) tend to grow as
  monolayer's.
• Attached cell lines can be classified as
  endothelial, epithelial, neuronal or fibroblasts
  and their morphology reflect the area within the
  tissue of origin

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Anchorage dependent or independent

• Cell lines derived from normal tissues are
  considered as anchorage-dependent grows only on a
  suitable substrate. ( Epith, CT)
• Suspension cells are anchorage-independent e.g.
  blood cells

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Adherent cells

• Cells which are anchorage dependent
• Add enough trypsin/EDTA to cover the monolayer
• Incubate the plate at 37 C for 5 mts
• Tap the vessel from the sides to dislodge the
  cells
• Add complete medium to dissociate and dislodge
  the cells with the help of pipette which are
  remained to be adherent
• Add complete medium depends on the subculture
  requirement either to 75 cm or 175 cm flask

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Suspension cells

• Easier to passage as no need to detach them
• As the suspension cells reach to confluence
  aseptically remove 1/3rd of medium
• Replace with the same amount of pre-warmed medium

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Confluence

• Once the available substrate surface is covered
  by cells growth slows ceases.

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hep 3B - 70 confluency
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100 confluence
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After 24 h
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Confluence

• Cells to be kept in healthy in growing state
  have to be sub-cultured or passaged , when they
  reach 80-90 confluence in flask/dishes/plates
• Enzyme such as trypsin, dipase, collagenase in
  combination with EDTA breaks the cellular glue
  that attached the cells to the surface

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Design and Equipment for the Cell Culture
Laboratory
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Design and Equipment for the Cell Culture
Laboratory

• 1. Laboratory design- in a safe and efficient
  manner
• 2. safety cabinets

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Laminar- flow hood
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Laminar- flow hood

• The working environment is protected from dust
  and contamination by a
• Constant, stable flow of filtered air
• Two types
• Horizontal, airflow blow from the side facing
  you, parallel to the work surface, and is not
  circulating
• Vertical, air blows down from the top of the
  cabinet onto the work surface and is drawn
  through the work surface and either re-circulated
  or vented.

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Laminar- flow hood

• The efficiency of the hood depends on a minimum
  pressure drop across the filter
• When the filter resistance builds up, the
  pressure drop increases and the flow rate of air
  falls.
• Below 0.4 m/s (80 ft/min), the stability of the
  laminar airflow is lost, and sterility can no
  longer be maintained. The pressure drop can be
  monitored with a manometer fitted to the cabinet

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Laminar- flow hood

• Routine maintenance checks of the primary filters
  are required (every 3-6 months).
• They might be removed and discarded or washed
  in soap and water.
• Every 6 months the main high efficiency
  particulate air (HEPA) filter above the work
  surface should be checked for airflow and hole

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Before use

• Ultraviolet lights are used to sterilize the air
  and exposed work surfaces in laminar flow
  cabinets between use.
• Detergent
• 70 alcohol

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Cell Culture Incubator
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• It requires a controlled atmosphere with high
  humidity and super controlled of CO2 tension.
• The incubator should be large enough, probably
  50-200 liter, have forced air circulation,
  temperature control and a safety thermostat that
  cuts out if the incubator overheats.

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• - It should be stainless steel, and easily
  cleaned.
• A double cabinet, one above the other,
  independently regulated, is preferable to one
  large cabinet.
• Incubators are supplied either with a heated
  water jacket as a method for distributing the
  heat evenly around the cabinet or with surface
  heater elements for heating.

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Humid CO2 Incubator

• CO2 incubators are more expensive, but the are
  ease of use and superior in the control of CO2
  tension and temperature
• A controlled atmosphere is achieved by using a
  humidifying tray and controlling the CO2 tension
  with a CO2-monitoring device, which draws air
  from the incubator into a sample chamber,
  determines the concentration of CO2, and injects
  pure CO2 into the incubator to make up any
  deficiency.

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• Air is circulated around the incubator by natural
  convection or by using a fan to keep both the CO2
  level and the temperature uniform.
• Dry, heated wall incubators also encourage less
  fungal contamination on the walls, as the walls
  tend to remain dry, even at high relative
  humidity.

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Sterlization Heat

• Moist heat denatures proteins by coagulation,
  caused by H bonds breakage, can happened more
  quickly in the presence of water
• Autoclave Steam under pressure
• Air should be Exhausted

Figure 7.2
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• Pasteurization reduces spoilage organisms and
  pathogens and can be safe if refregerated
• Equivalent treatments as the temprature increase
  the time decreased and the same no of mo killed
• 63C for 30 min
• High-temperature short-time P 72C for 15 sec
• Ultra-high-temperature P 140C for lt1 sec
• Thermoduric organisms survive

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Physical Methods of Microbial Control

• Dry Heat Sterilization kills by oxidation effect
• Flaming
• Incineration
• Hot-air sterilization temp 170C for 2 hrs

Hot-air Autoclave
Equivalent treatments 170C, 2 hr 121C, 15 min
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Steam Sterilizer (Autoclave)

• A simple bench-top autoclave that generates 100
  kPa (1 atm, 15 lb/in.2) may be sufficient, but a
  larger model with a timer and a choice of
  presterilization and poststerilization evacuation
  and temperature recording give more capacity and
  greater flexibility in use
• and offers the opportunity to comply with good
  laboratory practice (GLP).

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• The chamber should also be evacuated after
  sterilization, to remove steam and promote
• subsequent drying otherwise the articles will
  emerge wet, leaving a trace of contamination from
  the condensate on drying.
• To minimize this risk when a postvac cycle is
• not available, always use deionized or
  reverse-osmosis water to supply the autoclave.

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Cell culture contaminants

• two types
• 1- Chemicals-difficult to detect caused by
  endotoxins, plasticizers, metal ions or traces of
  disinfectants that are invisible
• 2- Contamination by microorganisms remains a
  major problem in tissue culture.
• Bacteria, mycoplasma, yeast, and fungal spores
  may be introduced via the operator, the
  atmosphere,work surfaces, solutions, and many
  other sources

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Contamination

• They competes for nutrients with host cells
• Secreted acidic or alkaline by-products ceases
  the growth of the host cells
• Degraded arginine purine inhibits the synthesis
  of histone and nucleic acid
• They also produces H2O2 which is directly toxic
  to cells

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Safety
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• For best results in tissue culture, we want to
  work to keep microbial (bacteria, yeast and
  molds) contamination to a minimum.
• Guidelines to follow
• Work in a culture hood set-aside for tissue
  culture purposes.
• Most have filtered air that blows across the
  surface to keep microbes from settling in the
  hood.
• Turn off the UV/antimicrobial light and turn on
  the hood 30 minutes prior to entering the hood.

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• Wear short sleeves or roll your sleeves up. Turn
  your baseball caps back if you MUST wear them,
  tie long hair back and remove rings and watches
• Wash hands with soap and water before beginning
  the procedure and rewash if you touch anything
  that is not sterile or within the hood.

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• Spray down your hands, work surface, and anything
  that will go into the hood with 70 ethanol.
  Re-wipe at intervals if you are working for a
  long time in the hood. This will reduce the
  numbers of bacteria and mold considerably.
• Do not breathe directly into your cultures,
  bottles of media, etc. This also means to keep
  talking to a minimum. No singing or chewing gum.

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• Work as quickly as you can within limits of your
  coordination. Also, keep bottles and flasks
  closed when you are not working with them. Avoid
  passing your arm or hand over an open bottle.
• Use only sterilized pipettes, plates, flasks and
  bottles in the hood for procedures.

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• Take special precautions with the sterile
  pipette. Remove them from the package just before
  use. Make certain to set up the numbers on the
  pipette so that they face you.
• Never mouth-pipette, use the pipetting aid.