ANIMAL CELL

CULTURE
EQUIPMENT
LINDA K.
 INTRODUCTION

Equipment required for animal cell culturing can be divided into

subcategories:
 Equipment for the Aseptic Area
 Equipment for Incubation
 Equipment for Preparation & Sterilization
 Equipment for Storage
 Laboratory Backup
 Specialized Equipment
 Consumable Equipment
 EQUIPMENT FOR THE ASEPTIC AREA

 Laminar Airflow Hood

 Pipette Cylinders
 Aspiration Pump
 Service Carts
 Inverted Microscope
 Centrifuge
 LAMINAR AIR FLOW HOOD
 Laminar airflow hood or laminar airflow cabinet is also known as biological safety cabinet.
 A LAF hood is an enclosed system that uses filters (HEPA filter: High Efficiency Particulate Air
filter) and directional air flow to provide a contaminant-free work area.
 It ensures that the sample being manipulated inside of it does not have any particulate matter
added to it.
 Laminar airflow is defined as air moving at the same speed and in the same direction with
minimal crossover of air streams (lamina).
 Two types of LAF hoods: Horizontal & Vertical
 Horizontal LAF hood: It pulls air from the environment; the air goes through the filter and then
blows smoothly out of the front of the hood back into the working room. The laminar airflow
is forward from a filter positioned behind the work surface.
 Vertical LAF hood: It blows the air from the top of the hood straight down from a filter
positioned above the work surface. This prevents hazardous material from being directed at
the person working at the hood.
 HORIZONTAL VS. VERTICAL LAF HOOD

 A horizontal air flow hood is cheaper and provides the best sterile protection for
cultures, but is only suitable for media preparation and for culturing nonprimate cells.
 In animal cell culture, for handling hazardous materials such as primate/human cell
lines, virus producing cultures, radioisotopes and carcinogenic/toxic drugs, a
biohazard cabinet is used.
 In contrast to a LAF hood, a biohazard cabinet also filters the exhaust air (HEPA) to
protect both the lab worker and the surrounding environment from pathogens.
HORIZONTAL VS. VERTICAL LAF HOOD
 PIPETTE CYLINDER

 A pipette cylinder is also called pipette hod and is a cylindrical storage jar for pipettes
and is made up of polypropylene. It may be filled with disinfectant.
 They are free-standing and are distributed around the lab, one per workstation.
 There should be sufficient numbers of pipette cylinders in reserve, so that cylinders
are allowed to stand for 2 hours in disinfectant before washing.
PIPETTE CYLINDER
 ASPIRATION PUMP

 An aspiration pump uses suction from a peristaltic pump to remove spent medium or
other reagents from culture flasks. The effluent is collected directly through a suction
line (pump tubing) into a vented container filled with a disinfectant (hypochlorite).
 There is minimal risk of discharging aerosol into the atmosphere if the vent carries a
cotton plug or micropore filter.
 The effluent should be left to stand for at least 2h before the reservoir is emptied.
 Instead of a peristaltic pump, a vacuum pump may also be used.
ASPIRATION PUMP
 SERVICE CARTS

 Service carts are moveable carts to place items that are to be used at the laminar flow hoods.
 They can conveniently fit into the lower space between adjacent hoods and are easily
removed for maintenance of the hoods.
 They are also used to carry materials to and from the hoods and larger carts are useful for
clearing soiled glassware and used items from the aseptic area to the washup.
 INVERTED MICROSCOPE

 Inverted microscopes have the light source and condenser on the top, above the
stage pointing down, while the turret with the objective lenses are below the stage
pointing up.
 Inverted microscopes are useful for observing living cells at the bottom of a tissue
culture flask.
 The stage should be large enough or removable to accommodate large roller bottles
between it and the condenser.
 A microscope with a phototube and camera is useful for digital recording and viewing
of cultures.
INVERTED MICROSCOPE
 CENTRIFUGE

 A centrifuge is required as cell suspensions periodically need centrifugation to increase

the concentration of cells or to wash off a reagent.
 A small bench-top centrifuge is sufficient for most purposes.
 A large capacity refrigerated centrifuge will be required of large-scale suspension cultures
are used.
 EQUIPMENT FOR STERILE LIQUID
DISPENSING – Pipetting & Dispensing

 Pipetting aids
 Pipettors
 Graduated Bottle Dispenser (large volume dispensing)
 Cornwall syringe (for repetitive dispensing)
 PIPETTING AID

 A pipetting aid is a motorized pipetting device and has enhanced speed, accuracy and
reproducibility compared to simple pipetting methods (with a rubber bulb for
example).
 It may be obtained with a separate or built-in pump and can be operated with
rechargeable batteries or is mains operated.
 Pipetting aids usually have a filter at the pipette insert to minimize the transfer of
contaminants.
PIPETTING AID
 PIPETTORS
 Pipettor, Pipette or Pipetman™ are used to dispense fluid and enable sterile and
accurate liquid handling.
 Pipettes that dispense between 1 and 1000µl are termed micropipettes, while
macropipettes dispense a greater volume.
 Routine subculture, which should be rapid and secure from contamination, but need
not be very accurate is best performed with a conventional glass or disposable plastic
pipette.
 The tip of the pipette always needs to be sterile.
 Tips can be brought loose and can be packaged and sterilized in the lab.
 GRADUATED BOTTLE DISPENSER

 For dispensing 100ml, a graduated bottle dispenser is used.

 The dispenser is inserted into any bottle containing the required liquid (medium).
 The dellivery line (tube) is inserted into the neck of the graduated bottle and
connected to a dispensing bell, which protects the delivery of the liquid to the
receiving vessel.
 An inlet line is present for balancing air .
 The delivery tubes and the bottle can be sterilized separately.
GRADUATED BOTTLE DISPENSER
 CORNWALL SYRINGE

 Traditionally used for repetitive dispensing, the cornwall syringe takes the liquid via
one tube and expells it via another tube using a simple two-way valve.
 CELL COUNTER

 The cell counter is used when more than 2 or 3 cell lines are carried and are tools for
counting live and dead cells in a culture.
 Essential for precise growth kinetics.
 Both manual and automated cell counters are available:
 Manual cell counting is generally accomplished using a hemocytometer, a glass slide
etched with a counting grid.
 Automated cell counters operate either via electrical impedence ("Electrical Sensing
Zone" method), direct imaging (either on a disposable slide or in a flow chamber), or
flow cytometry.
CELL COUNTER

Video
 CCD CAMERA & MONITOR

 High resolution charge-coupled device (CCD) video cameras can be used to record and
digitize images for subsequent analysis & publication.
 The addition of a video recorder will allow real-time or time-lapse recordings.
 To discuss cultures with colleagues, a 300 or 400mm monitor is adequate.
CCD CAMERA & MONITOR
 DISSECTING MICROSCOPE

 A dissecting microscope is used for the dissection of small pieces of tissue (e.g.:
embryonic organs or tissue from smaller invertebrates).
 A dissecting microscope is also useful for counting monolayer colonies and for
counting and picking small colonies in agar.
 EQUIPMENTS FOR INCUBATION

 Incubator
 Humid CO2 Incubator
 Temperature Recorder
 Roller Racks
 Magnetic Stirrer
 INCUBATOR

 The incubator should be 50 to 200l or 1.5 to 6ft³ per person and should have forced
air circulation, temperature control to within +/- 0.2°C and a safety thermostat that
cuts off if the incubator overheats.
 It should be resistant to corrosion (stainless steel) and easily cleaned.
 A double chamber or two incubators stacked is preferable to one large incubator,
because it can accommodate more cultures with better temperature control.
 Many incubators have a heated water jacket to distribute heat evenly around the
cabinet.
 Incubator shelving is usually perforated to facilitate the air circulation.
INCUBATOR
 HUMID CO2 INCUBATOR

 Petridishes or multiwell plates require a controlled atmosphere with high humidity an

elevated CO2 tension.
 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.
 Air is circulated around the incubator by natural convection or by a fan to keep both
the CO2 level and the temperature uniform.
HUMID CO2 INCUBATOR
 TEMPERATURE RECORDER

 A temperature recorder is a recording thermometer with ranges from below -50°C to

about 200°C.
 It enables monitoring of frozen storage, the freezing of cells, incubators and sterilizing
ovens.
 It has one instrument fitted with a resistance thermometer or thermocouple with a
long teflon-coated lead.
 ROLLER RACKS

 Roller racks are used to scale up monolayer culture.

 The length of roller bottles will determine the maximum yield, but is limited by the
size of the rack.
 The height of the rack will determine the number of tiers (rows) of bottles.
 MAGNETIC STIRRER

 A magnetic stirrer is a lab device that employs a rotating magnetic field to cause a stir
bar immersed in a liquid to spin very quickly, hence stirring it.
 For the stirrer to be used in enzymatic tissue disaggregation or suspension culture,
the motor should not heat the culture and the speed must be controlled to 50 rpm.
 It should be capable of stirring up to 10l of fluid and should maintain several cultures
simultaneously.
EQUIPMENTS FOR PREPARATION &
STERILIZATION

STERILIZATION PREPARATION
Soaking Baths or Sinks Balances
Pipette Washer pH meter
Pipette Drier Hot Plate Magnetic Stirrer
Water Purifier Automatic Dispensers
Sterilizing & Drying Ovens Conductivity Meter
Steam Sterilizer (Autoclave) Osmometer
Glassware Washing Machine
 SOAKING BATH/SINK

 Soaking baths/sinks should be deep enough so that all glasswares (except pipettes
and the largest bottles) can be totally immersed in detergent during soaking, but not
so deep that the weight of the glass is sufficient to break smaller items at the bottom.
  400mm wide, 600mm long and 300mm deep
 PIPETTE WASHER

 Reusable glass pipettes are washed in a standard siphon-type washer.

 The washer should be placed just above floor level and connected to the deionized
water supply and the cold water supply.
 PIPETTE DRIER

 If a stainless steel basket is used in the washer, pipettes may subsequently be

transferred directly to an electric drier.
 WATER PURIFIER

 Purified water is required for rinsing glassware, dissolving powdered media and
diluting concentrates.
 Purified water should not be stored but should be recycled through the apparatus
continually to minimize infection with algae or other microbes.
 STERILIZING & DRYING OVENS

 It is preferable to sterilize pipettes and other glassware by dry heat to avoid the
chemical contamination by steam condensate and the corrosion of pipette cans.
 It requires a high-temperature (160-180°C) fan powered oven to ensure even heating.
 STEAM STERILIZER (AUTOCLAVE)

 This is a pressure chamber used to sterilize equipment and supplies by subjecting

them to high-pressure saturated steam at 121°C for around 20min.
 A wet cycle (water, salt solutions) is performed without evacuation of the chamber
before or after sterilization.
 Dry items (instruments, swabs etc.) require that the chamber be evacuated before or
after sterilization to remove steam and promote subsequent drying.
 GLASSWARE WASHING MACHINE

 When the amount of glasswares gets too great, the purchase of an automatic washing
machine is considered.
 BALANCES

 To prepare reagents in house, a balance is required, preferably an electronic one with

automatic tare.
 It is capable of weighing items from 10mg up to 100g or even 1kg depending on the
scale of the operation.
 pH METER

 The pH meter is used to measure the pH during media preparation, stock solution
preparation etc.
 HOT PLATE MAGNETIC STIRRER

 Magnetic stirrer with a hot plate is needed to accelerate the dissolution of some
reagents.
 AUTOMATIC DISPENSER

 Automatic dispenser or bottle-top dispenser is used for accurate dispensing of liquid

volumes above 50ml.
 It has a spring-loaded piston connected to a two-way valve and draws a preset volume
from the bottle with the liquid, which is then dispensed through a side arm.
 This device is useful for repetitive dispensing of non-sterile solutions such as PBS
(Phosphate Buffered Saline) for cell counting.
 CONDUCTIVITY METER

 A simple check of ionic concentration can be made with a conductivity meter against
a known standard such as normal saline (0.15M).
 OSMOMETER

 An osmometer is a device used to check the osmolality of solutions, to adjust new

formulations or to compensate for the addition of reagents to the medium.
 Osmometers work by depressing the freezing point of a medium or elevating its vapor
pressure.
 EQUIPMENT USED FOR STORAGE

 Refrigerators & Freezers

 Cryostorage Containers/ Cryofreezer
 Controlled Rate Freezer
 REFRIGERATORS & FREEZERS

 Domestic refrigerators are available without a freezer compartment giving more

space and eliminating the need for defrosting.
 If 400l (12ft³) or more storage is required, a large hospital, blood bank or catering
fridge is more adequate.
 A cold room can also be set up, which is easier to access.
 The walls of the room must be smooth so that cleaning is easier.
 Most tissue culture reagents keep at -20°C, so an ultradeep freeze is not essential.
 CRYOSTORAGE CONTAINER/CRYOFREEZER

 Storage in liquid nitrogen freezer is the best method for preservation of cultured cells.
 The frozen cells are transferred rapidly to the cryofreezer when they are at or below -
70°C.
 Cryofreezers differ in design depending on the size of the access neck, storage system
employed and location of liquid nitrogen.
 For a small lab, a 35l freezer with a narrow neck & storage in canes and canisters or in
drawers in a rack system should hold about 500-1000 ampoules.
 An appropriate storage vessel is also needed to hold a backup supply of liquid
nitrogen.
CRYOFREEZER
 CONTROLLED RATE FREEZER

 Some cells may need different cooling rates or complex, programmed cooling curves.
 A programmable freezer (e.g.: Cryomed Planer) enables cooling rates to be varied by
controlling the rate of injecting liquid nitrogen into the freezing chamber, under the
control of a preset program.
 LABORATORY BACKUP

 Computers & Networks

 Upright Microscope
 Confocal Microscope
 PCR Cycler
 COMPUTERS & NETWORKS

 Most labs have one or more computers, which may be networked (connected to each
other).
 They are used for entering records for cell line maintenance, primary culture and
experiments and make later retrieval and analysis easier.

UPRIGHT MICROSCOPE
 An upright microscope is needed for chromosome analysis, mycoplasma detection
and autoradiography.
 CONFOCAL MICROSCOPE

 Cytological investigations of fluorescently labelled cells often benefit from improved

resolution when viewed by a confocal microscope.
 It allows the microscope to view an optical section through the specimen presenting
the image in one focal plane and avoiding the interference caused by adjacent cells
not in the same focal plane.
 The data is stored digitally.

PCR CYCLER
 Techniques in cell line validation such as mycoplasma detection & DNA profiling rely
on amplification and detection of specific DNA sequences.
 They utilize the polymerase chain reaction and need a thermal cycler.
 SPECIALIZED EQUIPMENT

 Microinjection Facilities: Micromanipulators are used to inject directly into a cell.

E.g.: for a nuclear transplantation or dye injection.
 Colony Counter: Used to count colonies if many plates are to be counted.
 The simplest colony counter uses an electrode-tipped marker pen, which counts when
touched down on a colony.
 A programmable electronic counter with a preset program counts using image analyis
software (e.g.: Optomax).
 Centrifugal elutriator: It‘s a specially adapted centrifuge suitable for separating cells
of different sizes.
 Flow cytometer: This instrument can analyze cell populations according to a wide
range of parameters including light scatter, absorbance and fluorescence.
 CONSUMABLE EQUIPMENT
 Pipettes: Pipettes should be wide-tipped for fast delivery and graduated to the tip
with the maximum point of the scale at the top.
 Reusable pipettes are collected in pipette cylinders or hods.
 Pasteur pipettes should be discarded into secure glassware waste.
 Culture Vessels: The choice of culture vessels is determined by the yield required,
whether the cell is grown in monolayer or suspension and the sampling regime. Care
should be taken to label sterile, non-sterile, tissue culture and non-tissue culture
vessels clearly and to store them separately.
 Sterile Containers: Petridishes are required for dissection;
 5ml bijou bottles, 30ml universal containers or 50ml sample pots for storage;
 15 and 50ml centrifuge tubes and
 1.2ml plastic cryostorage vials for freezing in liquid nitrogen.
 CONSUMABLE EQUIPMENT

 Syringes & Needles: Syringes are required for filtration in conjunction with syringe
filter adapters and needles may also be needed for extraction of reagents from sealed
vials.
 Sterilization Filters: Most labs use disposable filters. Commonly 25mm syringe
adapters and 47mm bottle top adapters or filter flasks are used.
 Paper Towels & Swabs: Provided in a central location and beside each workstation or
hood for easy access.
 Disinfectants: All discarded biological material should go into a disinfectant to prevent
growth of potential contaminating organisms in waste containers.
 Chlorine-based disinfectants are used in liquid concentrate form (Clorox, Chloros) or
as tablets (Precept).