CHAPTER III

Cell Culture and Asepsis

People's Democratic Republic of Algeria

Ministry of Higher Education and
Scientific Research University of 8 Mai
1945, Guelma. Algeria

Faculty of Life and Natural Sciences,

Earth and Universe Sciences Department
of Biology

Cell culture

Course for students of Master I Applied

Immunology

Prepared by Dr. Awatif BOUMAZA

PhD. Cellular and Molecular Toxicology
 CHAPTER III
Cell Culture and Asepsis
CHAPTER III Cell Culture and Asepsis

The concept of sterility in cell culture is sometimes difficult to understand. Indeed,

what is “sterile” does not contain any living organism. But a “sterile” cell culture in practice
means a culture containing only the cells that we want to find there, and no other living
organism.

1.Cell culture sterility

The biologist's fear is the contamination of cultures. Indeed, a cell whose culture is colonized
by another organism is very disturbed, because the contaminant proliferates more quickly and
consumes nutrients from the environment which profoundly modify the reactions of a cell.
The most common contaminants are bacteria from our skin: E.coli and S.aureus, as well as
certain yeasts. Contamination by the Mycoplasma genus is also common. It happens that a
cell culture becomes contaminated with another cell type. This is sometimes the case when
the same person works on many lines at the same time in the same place.

2.Aseptic techniques:

The aseptic techniques designed to provide a barrier between the microrganisms in the
environment and the sterile cell culture, depends upon a set of procedures to reduce the
probability of contamination from these sources. The elements of aseptic technique are a
sterile work area, good personal hygiene, sterile reagents and media, and sterile handling.

2.1.Sterile Work Area

The clean area must be as clean as possible, by regular decontamination using a surface
cleaner-decontaminant. As a precaution, it is also necessary to clean and decontaminate any
surface entering the clean zone: vials, bottles, gloves, hands, etc. The simplest and most
economical way to reduce contamination from airborne particles and aerosols (e.g., dust,
spores, shed skin, sneezing) is to use a cell culture hood.
 The cell culture hood should be properly set up and be located in an area that is
restricted to cell culture.
 The work surface should be uncluttered and contain only items required for a
particular procedure.
 Before and after use, the work surface should be disinfected thoroughly, and the
surrounding areas and equipment should be cleaned routinely.
 For routine cleaning, wipe the work surface with 70% ethanol before and during work,
especially after any spillage.
 You may use ultraviolet light to sterilize the air and exposed work surfaces in the cell
culture hood between uses.
 Using a Bunsen burner for flaming is not necessary nor recommended in a cell culture
hood.
CHAPTER III Cell Culture and Asepsis

 Leave the cell culture hood running at all times, turning them off only when they will
not be used for extended periods of time.

2.2.Good Personal Hygiene

Wash your hands before and after working with cell cultures. In addition to protecting you
from hazardous materials, wearing personal protective equipment also reduces the probability
of contamination from shed skin as well as dirt and dust from your clothes.

2.3.Sterile Reagents and Media

Commercial reagents and media undergo strict quality control to ensure their sterility, but
they can become contaminated while handling. Follow the guidelines below for sterile
handling to avoid contaminating them. Always sterilize any reagents, media, or solutions
prepared in the laboratory using the appropriate sterilization procedure (e.g., autoclave, sterile
filter).

2.4.Sterile Handling

 Always wipe your hands and your work area with 70% ethanol.
 Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before
placing them in the cell culture hood.
 Avoid pouring media and reagents directly from bottles or flasks.
 Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids, and
use each pipette only once to avoid cross contamination.
 Do not unwrap sterile pipettes until they are to be used. Keep your pipettes at your
work area.
 Always cap the bottles and flasks after use and seal multi-well plates with tape or
place them in resealable bags to prevent microorganisms and airborn contaminants
from gaining entry.
 Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to
use it and never leave it open to the environment. Return the cover as soon as you are
finished.
 If you remove a cap or cover, and have to put it down on the work surface, place the
cap with opening facing down.
 Use only sterile glassware and other equipment.
 Be careful not to talk, sing, or whistle when you are performing sterile procedures.
 Perform your experiments as rapidly as possible to minimize contamination.
3. Sterilization methods

3.1.Définition

Sterilization is defined as the process of destruction or elimination of all pathogenic

microorganisms and organisms capable of giving rise to infection such as all spores,
bacteria, fungi, etc., all disease-causing. It can be achieved by both physical, chemical, and
CHAPTER III Cell Culture and Asepsis

other effective methods that inhibit growth or are free from food products, fluids, objects, solid
materials, food packaging materials, raw materials, and other different products

3.2.Importance of Sterilization
Importance of sterilization are given below (9):
 Sterilization is used to prevent the transmission of certain pathogenic disease-causing
microorganisms into the body.
 It helps in sterile products to prevent contamination.
 Sterilization is an important process in research development laboratories.
 It prevents the contamination of instruments and areas in the pharmaceutical industry.
 It is used in the preparation process of cultures and other microbiology experiments.
 The sterilization process is used in the food industries such as canning, and high-
pressure methods.
 Sterilization is used for the preparation of sterile dosage forms and sterility testing.

3.3. Methods

In order to completely eradicate microorganisms, sterilization techniques involve

classification of sterilization which includes physical and chemical methods. Depending on the
material sensitivity and particular sterilizing requirements, each technique is used.

Figure 9: Sterilization methods [6]

3.3.1. Physical Sterilization

Physical sterilization is method to destroy the microorganism by using physical methods like
dry and wet heat, filteration and radiation. In these method wet or moist heat is considered to
be the most effective method to sterilize the glasswares.
CHAPTER III Cell Culture and Asepsis

 By Using Heat
There are two methods: dry and moist heat.
- Dry heat
 Flaming In this method heating instruments over the fire until they become hot in red.
Instruments that are used such as point of forceps, Spatulas, inoculating loops, and Wires.
 Incineration It is a process that involves the combustion of organic substances contained
in waste materials. Items such as contaminated cloth, animal carcasses, and pathological
material.
 Hot air oven Hot air ovens are the electrical devices used for sterilization. The oven uses
dry heat to sterilize items. Generally, they can be set at minimum to maximum
temperature from 50˚C to 300˚C. The thermostat is present to control the temperature.
This is the most widely used method of sterilization by dry heat.
- Moist Heat
Moist heat can be categorized into 3 groups (10):
 Temperature below 100 C Pasteurization of milk: This method is done by holding
period at 63˚C for 30 minutes or 72˚C for 15-20 minutes followed by cooling. This
method is not suitable for killing all spores.
 Temperature at 100 C Steam at atmospheric pressure is used to sterilize culture media.
This is an inexpensive method. The principle of this method is first exposure kills
vegetative bacteria and next exposure will kill vegetative bacteria that mature from the
spore. It is intermittent sterilization by holding at a temperature of 100˚C for 20 minutes
on three consecutive days.
 Temperature above 100 C An autoclave or steam sterilizer is an instrument that uses
steam to sterilize equipment and other objects. This implies that all microscopic
organisms, infections, parasites, and spores are inactivated. In any case, prions may not be
annihilated via autoclaving at the regular 134˚C for 3 minutes or 121˚C for 15 minutes. It
is suitable for the Items such as dressings, instruments, laboratory ware, media, and
medical products.
 Filtration
Filtration assists with eliminating microorganisms from heat-labile fluids such as sera and
solutions of antibiotics. Its working principle as viruses go through the normal filter channels,
filtration can be used to obtain bacteria-free filtrates of clinical samples for virus isolation.
Types of Filters
CHAPTER III Cell Culture and Asepsis

 Candle filters These filters are used for the purification of water for industrial and
drinking purposes. These are made under various grades of porosity.


 Asbestos filters are disposable and single usage. They tend to alkalinize filtered fluids.
Their usage is less because of their carcinogenic property.
 Sintered glass filters These filters have low absorptive properties. They are brittle and
costly.
 Membrane filters These filters are made of cellulose esters or other polymers. They are
usually used for water purification and analysis, sterility testing, and preparation of
solutions.
 Radiation
There are 2 types of radiation: Ionizing radiation & non-ionizing radiation
 Non-ionizing radiation In the non-ionizing method infrared is used for rapid mass
sterilization of prepacked items such as syringes, and catheters. UV is used for
disinfecting enclosed areas such as halls, operation theatres, and labs.
 Ionizing radiation In ionizing radiation, gamma rays and x-rays are used for sterilizing
plastics, syringes, swabs, catheters, animal feeds, cardboard, oils, and metal foils.

3.3.2. Chemical Sterilization

Chemical sterilization is the method of sterilization to make surface free from microorganisms
using certain chemicals like, chlorine, formaldehyde and ethanol. These chemicals can interfere
with the machinary of the microoganisms to terminate them. (10)

Using chemicals
 Chemical agents: The action of chemical agents are protein coagulation and Disruption
of cell membrane resulting in exposure, damage, and loss of contents.
 Chemical Alcohols: Commonly used are Ethyl alcohol, and Isopropyl alcohol and must
be utilized at concentrations of 60-90%. Isopropyl alcohol is used in the sanitization of
CHAPTER III Cell Culture and Asepsis

the clinical thermometer. Methyl alcohol is viable against contagious spores, treating
cabinets, and incubators. It is toxic and inflammable.
 Aldehyde Formaldehyde: It contains bactericidal and sporicidal and it has a great
effect on viruses. It is used to preserve anatomical specimens and destroy anthrax
spores on hair and wool.
 Phenols: These are acquired from the distillation of coal tar between 170˚ to 270˚C.
The deadly impacts include- it can cause cell membrane damage, releasing cell
contents, and causing lysis.

Gases

Types of gases used for sterilization are (7, 9, 10):

 Ethylene oxide works because of its alkylating of the amino, carboxyl, hydroxyl, and
sulfhydryl groups in protein molecules and furthermore on DNA and RNA. It very well
may be utilized on instruments such as heart-lung machines, respirators, dental
equipment, books, and clothing.

 Formaldehyde gas This is generally utilized for fumigation of operational theatres and
other rooms in clinics. Formaldehyde is formed by the addition of 150g of KMnO4 to
280ml of formalin for each 1000cu.ft of room volume, in the wake of shutting the
windows and different outlets. After fumigation, the doors ought to be sealed and left
unopened for 48 hours.

 Beta propiolactone (BPL) is a result of ketene and formaldehyde with a boiling point
of 163˚C. It has fat bactericidal activity yet is cancer-causing. It is equipped for killing
all microorganisms and it is exceptionally dynamic against infections.

4.Cell culture contamination

For the cell culturist, two types of contamination require careful monitoring and constant
vigilance:

 the contamination of cell cultures with microbiological organisms and

 the contamination of one cell line with another.
 Other types of contaminants, such as chemical contamination, may also
cause problems (e.g., deposits of disinfectants or detergents on glassware;
residues, impurities, and toxins in water, media or sera)
CHAPTER III Cell Culture and Asepsis

When a cell line is contaminated, procedures must be in place to dispose of the culture or
eradicate the problem in a manner that is safe to other cell cultures in the laboratory and to
the technical staff.
Where cell lines are being created de novo or being introduced into the laboratory from a
source without quality control procedures in place, then quarantine culture facilities must be
used.
4.1.Quarantine and receipt of animal cell lines
All new cell lines brought into a cell culture area should be introduced into a quarantine
laboratory specifically set aside and fully equipped for the purpose of handling unquail fied
cell lines, with appropriately trained personnel (11)

4.1.1.Accessioning scheme
All the major cell culture collections and research organizations, non-profit and
commercial, now have officers who are tasked with the sourcing, acquisition and addition of
new cell lines to their collection, in a process known as accessioning or acquisition.
The process of accessioning not only covers the shipment of a cell culture from one
organization to another but also includes obtaining, checking and archiving all attendant
documentation describing provenance, culture conditions, biohazard risk, ownership (of
intellectual property) and any restricted use.
Prior to dispatch of the cell line from the source laboratory or supplier, all cell lines
should be subject to a bio-hazard risk assessment by the accessioning representative.
4.1.2.Management of cell lines in quarantine
A flow diagram of the scheme used at European Collection of Authenticated Cell
Cultures (ECACC) for the introduction of new cell lines into the collection is given in Figure
7. The main points are listed below. (11)

- All cultures should be handled in a quarantine laboratory separate from the main
tissue culture area.
- Cultures should be handled in a Class II Mirobiological Safety Cabinet (MSC) unless
a higher level of containment is required. This will offer the operator protection, as
the exact source of the cell line and its contamination status may not be known.
CHAPTER III Cell Culture and Asepsis

Figure 10. Hierarchical banking scheme for incoming cell lines as used at ECACC
(10)
- An initial assessment of potential microbial contamination should be made
immediately, (broth for bacteria; PCR-based or other rapid methodology analysis for
mycoplasma).
- An assessment should be made of the cell type in the culture by examination and
photographic documentation.
- A token freeze of between three and five ampoules should be made as soon as
possible after receipt of the cell line.
- A complete set of quality control tests should be applied to the token freeze. These
include a cell count and viability assessment, sterility tests (bacteria, yeast, fungi,
mycoplasma), species identification and DNA characterization by restriction
enzyme analysis or short tandem repeat (STR) PCR analysis.
- After satisfying the above conditions, an ampoule from the token freeze can be
transferred to the main cell culture laboratory for the production of Master Cell Banks
(MCBs) and Working Cell Banks (WCBs).

It is recommended that this approach be adopted for all cell lines received into the laboratory,
irrespective of the source of the cell line.

Many laboratories routinely use antibiotics in their cell cultures. This practice is not
recommended for a number of reasons. From a contamination detection or surveillance
CHAPTER III Cell Culture and Asepsis

viewpoint, antibiotics can suppress bacterial contamination to a level that is undetectable by

microscopic examination and can lead to the development and spread of antibiotic resistant
microbial strains. In addition, although not eliminating any mycoplasma present, antibiotics,
particularly the amino-glycosides, can reduce the level of infection below the level of
detection of several currently used tests. It is essential that all quality control tests for
microbial contamination be performed on cell cultures subcultured for at least two passages
in antibiotic-free medium. (11)

4.1.3.Quarantine laboratory design

Optimal design criteria for the cell culture laboratory are described in Chapter I1. It is
worth emphasizing that when establishing a cell culture laboratory, either within an existing
facility or from new, an area (preferably an entire laboratory) should be set aside for
quarantine purposes.

The quarantine laboratory should have the following features:

(i) It should be as far away from the main cell culture clean area as possible.
(ii) The air-handling system of the laboratory should operate under negative pressure
with respect to the rest of cell culture area.
(iii) It should be self-contained with its own incubator, water bath, microscope, MSC,
and so on.
(iv) Staff should have separate personal protective equipment for working in the
quarantine laboratory.
(v) There should be an approved regimen in place for the routine and incident related
cleaning of the equipment and fabric (walls, floors, doors, etc.), and the fumigation of the
MSC. All staff working in the quarantine laboratory must be fully trained in the required
procedures.

(vi) Protocols for the disposal of contaminated cultures and associated reagents should
also be in place. (11, 10)

4.2.Microbial quality control

Microbial quality control is an essential part of all routine cell culture practice and should
not be neglected. It comprises the testing of cell cultures and associated reagents for a variety
of microorganisms.

4.2.1. Sources of contamination

CHAPTER III Cell Culture and Asepsis

Different microorganisms can contaminate cell cultures and come from different sources.
These sources can be divided into three main categories:
- poor laboratory conditions
- personnel (particularly inadequately trained personnel)
- Non-quality-controlled cell lines.

The various possible routes and likely sources of contamination are given in Table 1

Table 1. Common sources of microbial contamination (12)

4.2.2.Tests for bacteria, yeasts and fungi

 When cell lines are cultured in antibiotic-free media, contamination by bacteria,

yeasts or other fungi can frequently be detected by an increase in the turbidity of the
medium and/or by a change in pH.
 It is therefore good practice to set up quality control checks on culture media and
reagents prior to use.

4.2.3.Tests for mycoplasma

The incidence of mycoplasma-infected cultures has been found to vary between laboratories
and arises from poor aseptic technique and insufficient training in good cell culture practices.
(10)
CHAPTER III Cell Culture and Asepsis

It is crucial that mycoplasma-free cultures and cell lines are used because the presence of
mycoplasma can

- affect the rate of cell proliferation

- induce morphological changes
- cause chromosome aberrations
- influence amino acid and nucleic acid metabolism
- induce cell transformation.
4.2.4.Virus testing
There are three major concerns pertinent to the virus contamination of cell lines.
These are (11):
(i) The safety of laboratory staff handling the cell lines and performing the procedures
required for the reduction or elimination of the associated risk in experimental and other
procedures.
(ii) The medicinal safety of any products derived from cells and their clinical application.
This problem also impinges on regulatory matters.
(iii) The validity of any experimental data produced using contaminated cell lines.
4.2.4.1.Sources of viral contamination (11)

Potential sources of viral contamination are:

- the original tissue used to prepare the cell line

- other infected cultures
- growth medium
- serum
- other reagents (e.g. trypsin)
- laboratory personnel.

4.3.Eradication of contamination

 If a cell culture is found to be contaminated with bacteria, fungi, mycoplasma or virus

then the best method of elimination is to discard the culture. Fresh cultures can be
obtained from seed stock, from a bio-repository or from the originator of that cell line.
 It is important to locate the source of the contamination (media, culture reagents,
faulty safety cabinet, poor aseptic technique, etc.) to prevent a recurrence.
CHAPTER III Cell Culture and Asepsis

 As well as eliminating the contamination in any particular culture, it is important to

eliminate the source of contamination by using completely new and tested media and
reagents, cleaned and tested equipment and, where applicable, retrained staff.
 After any major contamination, the laboratory and MSC should, if possible, be
fumigated according to the manufacturer’s instructions (where applicable) and all
surfaces swabbed with a suitable disinfectant. (10)
 In the case of irreplaceable stocks it will be necessary to attempt to eliminate the
contamination using antibiotics. This approach is possible for bacteria, mycoplasma,
yeast and fungi. However, there are no reliable methods for the eradication of viruses
from infected cultures.
 Once the contaminant is identified and an antibiotic/antimycotic agent(s) has been
chosen, the eradication process should be rigorously challenged to ensure that it is
likely to be successful.
 Finally, all treated cultures must undergo a full regimen of quality control tests.
 Treated cultures must be put through a series of extended passages prior to retesting to
confirm the absence of the contaminant. (10)

5.Authentication

Authentication of a cell line is the sum of the processes by which a line's identity is verified
and shown to be free of contamination from other cell lines and microbes.

How are cell lines authenticated?

Identity verification with STR analysis (DNA fingerprinting) for human cell lines. DNA
fingerprinting is a powerful tool in determining the identity and uniqueness of a human line.
Short tandem repeat (STR) profiling establishes a DNA fingerprint for every human cell line
and may be used as a record of the line.

Why?

Using misidentified or contaminated cell lines will waste valuable time and resources on
experiments until the error is uncovered. The value of maintaining a complete record of each
cell line during its entire life cycle, including storage, passaging, and distribution of cells.
Ensuring you have the right cell line and that it is free from contamination avoids unreliable
research results that can lead to erroneous conclusions; wasted time, effort, and money; and
damaged reputations.
CHAPTER III Cell Culture and Asepsis