Topic: instrumentation of

animal cell culture

Name : aisha
Roll no : 22204024-033
What is Animal cell culture?

 Animal cell culture is the removal of cell, tissue or organs from an animal
and its subsequent placement into an artificial medium conductive for
growth.
 Animal cell culture are used in the production of therapeutic proteins,
research and diagnosis of diseases.
 Animal cell culture is a common and widely used technique for the isolation
of cells and their culture under artificial conditions.
History of animal cell culture

 1885 – Wilhelm Roux first time maintained embryonic check cell in saline buffer.
 1903- Jolly first time showed that the cells can survive and divide in In vitro
condition.
 1907- Ross Harrison developed a nerve fiber from frog embryo tissue and culture it
in a blood clot.
 1912- Alexis Carrel evolved the cell culture by developing the first cell line.
 1940- Enders, Weller and Robbins grew the poliomyelitis virus
 1955- Eagle developed a defined medium for cell growth
 1964 – Little Field discovered the HAT medium for cell selection
 1965- Ham discovered the first serum free media
 1965- Harris and Watkins fused the first human and mouse cell by using a virus.
Essential equipment required for animal
cell culture
 Incubator
 Microscope
 Sterilizer
 Washing up instrument
 Sterilizing and drying oven
 Centrifuge
 Water purification
 Cell freezing
Beneficial Equipment's

 Laminar flow hood

 Cell counter
 Vacuum Pump
 CO2 incubator
 Preparation and quality control
 Temperature recording
 Bulk culture
 Pipette aids  and automatic pipetting
List of Basic Equipment's needed in animal cell
culture lab

 Sterile Work Area/Cell culture hood (i.e., laminar-flow hood or biosafety

cabinet)
 Incubator (humid CO2 incubator recommended)
 Water bath
 Centrifuge
 Refrigerator and freezer (–20°C)
 Cell counter (e.g. Automated Cell Counter or hemocytometer)
 Inverted microscope
 Liquid nitrogen (N2) freezer or cryo storage container
 Sterilizer (i.e., autoclave)
Biosafety cabinet

 The basic purpose of Biosafety Cabinet is to provide protection to the culture

from any organism such as fungi, virus, bacteria under aseptic conditions and
also protect the operator form the risk of infection.
 The purpose of this cabinet is to protect the product by use of HEPA filters
(High Efficiency Particulate Air).
 The level of contamination depends on the use of biosafety cabinet.
 1. BSC 1= Should have a biosafety cabinet, Efficiency of HEPA filters is 90%.
 2. BSC 2= Closed Biosafety cabinet, Efficiency of HEPA filters is 95%
 BSE 3= Closed Biosafety cabinet, High density, Radioactive sterilization,
Efficiency of HEPA filters is 99.5%.
Biosafety cabinets
In order to meet the diversified research and clinical needs, Three kinds of
laminar flow hoods, have been designated as Class I, II and III.
Co2 Incubator

 Co2 Incubator is a temporary chamber which provides suitable temperature, density,

moisture and pH for better growth and development of animal cells.
 They maintain sterility of the chamber by using HEPA filters.
 They maintain a constant temperature by using a silicon gasket on the inner door.
 Humidity is created which prevents desiccation of the media and maintain
osmolarity.
 pH is also maintained in the media.
 Humid CO2 incubators are relatively expensive, however it allows superior control of
culture conditions
 They can be used to incubate cells that are cultured in petri-dishes or multiwell
plates that needs a regulated atmosphere of high humidity and increased CO2
tension.
 Refrigerators and freezer (-20 °C) for specimen storage:

 Both refrigerators and freezer are very essential for storage of liquid media at
2–8°C and for enzymes (e.g. trypsin) and some media components (e.g.,
glutamine and serum) at –5°C to –20°C.
  To store medium and buffers, a refrigerator or cold room is needed. 
 A freezer is required for keeping pre-aliquoted stocks of serum, nutrients and
antibiotics.
  For large laboratories a cold room is restricted for the storage of cells.
Cryo-storage container

 There is high possibility for genetic instability in cell lines of continuous

culture as their passage number increases, hence, it is necessary to prepare
working stocks of the cells and preserve in cryogenic storage.
 It is to be noted that the cells should not be stored in 20oC or -80oC freezers
as their viability reduces when they are not stored at these temperatures.
 Liquid nitrogen freezers permit storage in the vapor phase just above the
liquid at temperature between -140oC and -180oC, or submerged in the liquid
at a temperature below -196oC.
 The possibility of leaky vials or ampules exploding during removal is highly
reduced by use of vapor phase storage, however, the liquid phase systems
generally have longer static holding times, and are thus, more cost-effective.
Liquid nitrogen container

 Liquid nitrogen container provide a long term storage with low liquid
nitrogen evaporation.
 There are two main types of liquid nitrogen storage system
 1. vapour phase- this system minimizes the risk of explosion with cryo-
storage tubes.
 2. liquid phase- This system usually have longer static holding time.
Hemocytometer

 It is an instrument used to estimate the cell number and viability.

 It has two parts
 1. Neubauer’s slide- it has counting scales on each quadrant. The depth of
the scales is 0.1mm.
 2. Coverslip – it is used to keep the liquid sample in a perfect shape into a
flat layer of even thickness
Water bath

 A water bath is a device that maintains water at a constant temperature.

 It is used for thawing of the cells directly taken out from the freezing media
or from liquid nitrogen to prevent the cell form any mechanical damage due
to fluctuation of temperature.
 The temperature of water bath is 5-100 degree centigrade
Flow cytometry

 Flow cytometry is a method for cell analysis that was first used in the 1950s
to determine the volume of cells in a fluid stream that circulated quickly as
they flowed in front of a viewing aperture.
 Flow cytometry is a laser based technique to analyses the characteristics of
the cells .
 This is mainly used for the analysis of the expression of the cell surface and
intracellular molecules.
 It is used to characterize different cells from a heterogenous mixture
Inverted microscope

 The inverted microscope is designed with the light source and the condenser
lens above the specimen.
 The objective and turret of the microscope is on the bottom.
 The objective focus the light to produce a real image.
 It is to observe any contamination in the cell culture
Dissecting microscope

 The dissecting microscope is also know as stereo microscope.

 It has a long working distance between 25 and 150mm.
 It has low magnification ability and one can manipulate the specimen even
performing a small dissection under the microscope.
 Light specimens can also be observed under this microscope.
Confocal microscope:

 Confocal microscopy is a specialized type of standard fluorescence

microscopy (also termed widefield fluorescence microscopy) that produces
high-resolution images of material stained with fluorescent probes using
specific optical components.
pH meter

 PH meter is an electrical instrument for calculating the activity of hydrogen

ions (acidity or alkalinity) in the solution.
 A pH meter comprises necessarily of a voltmeter connected to a pH-
responsive electrode and a reference (unvarying) electrode.
 The pH-responsive electrode is normally glass, and a mercury-mercurous
chloride (calomel) electrode is usually the reference, although sometimes a
silver-silver chloride electrode is used.
Cell culture vessels

 In order to shield cultures from the external environment while retaining the
correct internal environment, culture vessels provide a contamination barrier.
 The vessels have an effective and consistent cell attachment substrate for
anchorage-dependent cells.
 Simple access to cultures and optically transparent viewing surfaces are more
features of vessels.
 All cultural vessels were originally glass.
 In comparison to plastic, glass drawbacks include heavy weight, cost, labor-
intensive cleaning, and poor microscopic viewing.
Glassware's and plastic wares

 Beaker
 Pipette
 Measuring cylinder
 Horizontal and vertical flask
 1. T25
 2. T75
 3. T100
 Petri plate
 Microscopic slides
 Coverslips
 Cell culture plates.