animal cell culture and methodologies

Chapter X

Culturing and maintaining mammalian cell culture

X.1 Introduction

Mammalian cell culture was first developed in the early twentieth century where

initially the tissue cultures were made by the isolation of individual cells from

fragments of tissue or organs for the use in laboratory. In the early years of tissue

culture, yielded tissue had very limited growth capabilities and limited by a finite

. The discovery of 1 number of divisions, a phenomenon known as Hayflick’s Limit

HeLa, a unique cervical carcinoma cell lines which has limitless lifespan, harvested

from patient called Henrietta Lawson, at John Hopkins Hospital, Baltimore in the

1950s, embarked modern advancement in clinical research for drug development and

. 2 therapy, where cells can be easily grown in large number and stored indefinitely

Cell culture technology has established wide applications in the field of cell biology

such as the study of intra and intercellular activities and flux, genetic and phenotyping

analysis, proteomics, toxicology studies, drug development, recombinant protein

development of Significant advancement and . 3treatment production, diagnosis and

mammalian cell culture had enabled researchers to further understand the underlying

mechanism of diseases in the surge for cure and therapy.

The routine for cell culture maintenance for feeding and subculturing should be

conducted in a sterile and in a laminar flow hood condition. Understanding of cellular

mitosis and morphological changes is important in the observation and critical

examination of cellular behavior in the culture. This begin with newly plated cells

attachment to the flask/plate surface to the cellular division until they reach

confluency and form a monolayer. At this stage, the cells will stop dividing to enter

resting period due to lack of space, nutrient and oxygen, a phenomenon known as

density-dependent growth inhibition. However, cancerous cells will continue to divide

on the top of the monolayer to form multilayers, even with the lack of nutrients and

oxygen supplies.

Subculturing is performed when cells attachment and proliferation in a culture reach

more than 70% of the surface area. Subculturing involves the removal of the media,

detachment of cells from the surface by the use of dissociating enzymes such as

trypsin and collagenase and chelator, Ethylene diamine tetra acetic acid (EDTA) for

ions. Long term exposure to trypsin is harmful to the cells as the 2+ the removal of Ca

enzymes may affect the essential extracellular matrix proteins such as laminin and

fibronectin required for cellular attachment and proliferation.

X.1.1 Laboratory safety and Professional Conduct

Safety policies are important in the laboratory to ensure safety and sterility conditions.

Failure to comply with these procedures may lead to accidents, contaminations and

health complications. The following policies must be adhered while in the laboratory.

1. No drink or food is allowed in the laboratory.

2. Wear a laboratory coat, long pants, clean and closed-toe shoes during the

laboratory session.

3. Use goggles when handling with hazardous chemicals and fumes.

4. Disinfect all benches and laboratory working area with 70% ethanol prior

work.

5. Wash hands before and after working in the laboratory, Gloves should be wear

all the time in the laboratory.

6. Running and playing in the laboratory is not allowed.

7. All accidents involving spillage, injuries, contaminations and break of

glasswares and apparatus should be brought to the attention of the instructors

and lab technicians.

8. Dispose all chemicals, biological, biohazard, domestics and accordingly to its

container box and to the instruction of sterilization and disposal.

9. Cell phone is not allowed in the laboratory.

10. Place the aspirate liquid waste from the cell culture flask in a beaker and then

bleached for 10 minutes before discard into a sink.

11. All lab users must familiarize with the locations of emergency safety

equipment in the laboratory such as first aid kit, eye-wash station and fire

extinguisher

X.1.2 Lab contamination

Cell-culture contamination is a very common problem in all cell-culture facilities.

Contamination can be very frustrating and will lead to the loss of the culture, time,

effort and cost. The risk of contamination can be reduced and managed by careful and

aseptic handling of the cultures and reagent preparation, good laboratory practice and

systematic procedures. Cell culture contaminations can be divided into two major

categories; chemical and microbial. Chemical contamination occurs when unwanted

chemical substance is present in the media or any of the reagent by leakage, spillage

or in direct contact. Chemical contamination may be harmful to the cell culture and

may alter cellular behavior, morphologies and biological effect and is usually

undetectable due to the low concentration of contaminants. Water, media components,

unsterile surface and chemical reagents are the examples source of contaminants.

Therefore, it is very important to everyone to take precaution and care when handling,

measuring and preparing the media and other solutions for cell culture. Alcohol

contamination is a common chemical contamination problem in the laboratory due to

routine aseptic procedure that involve spraying alcohol on bench and media/reagent

bottles before working with cells in a fume hood.

Microbial contamination happens when bacteria, fungus, viruses, mycoplasma and

other microorganism are present in cell culture medium. Cell culture medium rich

with nutrients, growth factors and energy source and warm temperature of the

incubators provide an ideal environment for the growth of microbial contaminants.

Some microbial contaminations such as bacteria and fungus are visible can be

detected easily by the changes in media coloration. Phenol red is the component in

culture media that is a pH marker. A yellow coloration indicates of a low pH and

possible bacteria contamination as bacterial growth often acidify the culture media. In

the later stage of bacteria contamination, the culture media will turn cloudy and

visible to the naked eye. Confirmation of bacterial contamination can be done under

the microscope. As bacteria cells are much smaller than mammalian cells, their

presence will appear as tiny dots on the cell surface and cover the empty spaces of the

plate/flask in between the cells. The use of antibiotic such as penicillin and

streptomycin in the media can prevent the growth of some bacteria. However, the

practice is not recommended due to the risk of more aggressive, antibiotic-resistant

bacteria.

Mold contamination is easily detectable from the naked eye by the fuzzy-looking

hyphae strands and spores in the culture media. Another form of fungus which is

circular and smaller in size than mammalian cells, yeast cells is also another

contaminant to the cell culture media and easily detectable under the microscope.

Virus and mycoplasma contaminations are invisible to the naked eye and difficult to

determine without thorough screening of molecular test and investigations.

Mycoplasma are very small microorganisms that can grow rapidly inside the cells

without any visible appearance and do not cause any alteration to cellular

morphology. Virus, however is a very small particles that infect cells and causing cell

death. Cultures that are found with these contaminants should be destroyed

immediately and routine cleaning of incubator has to be done to ensure sterility and

safety of future cell culture works.

Cross-contamination is another form of cell culture contamination due to the presence

of two or more cell lines in the laboratory. Contamination of cell culture media with

different type of cell lines, laboratory accidents, human error that involve of

negligence and mismanagement of cell lines, could be the factors for cross-

contamination. This impose a serious problem to the research work and will

invalidate/compromise with the experimental results. Proper labeling of the

vials/flask/culture dish is critical and can significantly reduce the risk.

X.1.2.1 Prevention of contamination and aseptic techniques

Aseptic techniques are a series of techniques and practices used to lower the risk of

contamination and to protect lab users from health hazard by the contaminants and

other potentially hazardous materials. Common contaminant sources in the laboratory

are non-sterile solutions and supplies, air-borne dust, laboratory personnel and

unclean equipment such as water bath, incubator and laminar flow hood.

Below is a list of laboratory practices and aseptic techniques that can reduce the risk

of contamination:

Always maintain and clean the equipment routinely with 70% ethanol before

and after every use. Laminar flow hood, incubator and water bath should be

cleaned extensively once in every week, depending on the heavy usage. The

water in the incubator and water bath need to be replaced every week and low

concentration of surfactant (160 μl/1000 ml) should be added into the water to

prevent microbial growth and to ensure sterility. In case of spillage and

contamination, the equipment must be cleaned thoroughly and aseptically

accordingly to the procedure.

All equipment and supplies must be wiped with 70% ethanol before

commencing work on cell culture.

Never put your note books, papers and stationery inside the laminar flow hood

to avoid outside contamination.

Do not cough or sneeze in the direction of the hood.

All chemical solutions and reagents for cell culture works must be sterile and

aseptic prior work.

Hands should be washed before and after handling cell culture. Gloves and a

clean lab coat should be worn all the time. The lab coat use for cell culture

works should be kept hanged in the laboratory to avoid possible contamination

from the outside.

Users should have back up cultures in cryovials and cell stocks to avoid long

period of cultivation and lag in research.

Always aliquot (distribute) solutions and reagents into smaller volumes

accordingly to usage concentrations in several sterile containers to lower the

risk of contamination.

Open the wrapped and sterile serological pipettes inside the hood. Be careful

not to touch the tip of the pipette. If doing so, the direct contact of the pipette

tip with the culture media will possess high risk of contamination to the

culture work.

Micropipettes can be cleaned and wiped with 70% ethanol prior use for aseptic

procedure in the fume hood. For cell culture work, you should ensure that only

the tip of micropipette, which is autoclaved and sterile should touch the

interior of the containers.

Users should always read the instruction and read the protocols prior working

in the laboratory,

Disorganization and mismanagement increase the risk and changes of

contamination.

The chemicals and apparatus for cell culture works should be organized

properly to not blocking the flow of air in the hood.

Always observe your cells under the microscope prior working to ensure

sterility and destroy contaminated samples immediately accordingly to

procedure.

Never leave the top of cell culture medium vessel or reagent bottle open when

they are not in use (even in the fume hood).

Wipe and clean the spillage inside the fume hood with 70% ethanol

immediately after an accident.

Never share or use other people’s media and solutions. This will only increase

risk of cross contaminations.

Do not use suspicious chemicals, reagents, equipment or cell culture media for

any cell culture works. You can put aside the non-sterile solutions and pipettes

to be used for other procedures in the laboratory.

X.1.2.2 In case of microbial contamination

Contaminated cell culture should be destroyed and disposed immediately to prevent

further contamination. Below is the procedure of disinfection and decontamination of

a cell culture:

1. Sterilize the contaminated culture vessel with10% bleach and leave for

10 minutes.

2. Discard the bleach culture in the sink with a running tap water.

Dispose of the flask/plate in a domestic waste disposal container.

3. Clean your work area and equipment that has been in touch with the

contaminated culture.

4. Record and inform other lab/incubator users to raise awareness of

possible contamination and risk to the research work.

5. If the contamination problem is widespread among your work and cell

cultures, discard all media and reagents that you have been using and

start new.

X.2 Cell culture laboratory equipment

Equipment commonly used in a cell culture laboratory

X.2.1 Laminar-flow hood

Many of cell culture procedures are conducted inside laminar flow hoods or biological

safety cabinets (Figure X.1). The hood provides a clean working environment to

prevent contamination of cell culture by filtration of circulating air and particles

inside the hood. The flow of air inside the hood is in smooth parallel lines, creating

movement that separates the inside from the outside. Some laminar hoods are

equipped with a UV-germicidal lamp to sterilize the working bench and the content

inside while not in use. The UV lamp must be turned off prior working in the hood to

prevent exposure to hazardous UV light.

The following guidelines must be followed while working inside the hood:

1. Ensure that the UV-germicidal lamp is turn off before any work.

2. Open the glass shield to allowable level and switch on the blower. Wait for 10

minutes before starting your work to allow for air filtration and clean air

circulation.

3. Wipe the working surface with 70% ethanol.

4. Keep your media, reagents and equipment organized. Do not block the air

filter and blowers. Ensure that all apparatus are placed in an undisturbed area

in the fume hood to not interfere your work. This is to avoid risk of spillage

and cross contamination.

5. Wipe any spillage immediately with 70% ethanol.

6. Clean the working bench with 70% ethanol after work, turn off the blower,

close the glass shield and turn on the UV-germicidal lamp before you leave.

Figure X.1 Laminar flow hood for biological works on

cell culture.

X.2.2 Inverted microscope

Inverted microscopes are used to observe the cells in culture (Figure X.2). It is the

type of microscope with objective lenses below the stage and the light source and the

condenser above the stage. The microscope is suitable for the observations of cellular

attachment and morphologies at the bottom of the plates and/or flasks. The image can

be focused by turning the focus knob located at the right side of the microscope.

There are 3 types of objective lens for magnifications, 4X, 10X and 20X which are

located at the turret below the stage. The phase rings located above the stage allow the

the change of phase of light when going through different structures of cells in order

to make the transparent structures more visible to the eyes. It also can be placed in the

light path for a clearer image. Whereas the condenser above the stage, concentrates

and focuses on the light from the light source.

Figure X.2 Inverted microscope

X.2.3 Clinical centrifuge

Clinical centrifuge are used to concentrate the cells and to separate the cells from

culture media and other reagents. An optimum slow rotational speed should be used to

prevent damage to the cells. A gravitational force between 80-100g is sufficient for

the separation of cells and culture media.

Following are the general rules for using a clinical centrifuge:

1. Transfer the liquid suspension to the appropriate size of centrifugal tubes.

2. Weight your tube contents on a pan-balance and make sure that the tubes are

of equal weight.

3. In case of uneven number of samples, you can prepare a balancing tube of the

same size by filling it with tap water.

4. Place the balanced tubes into two opposing slots of the centrifuge.

5. Close the safety lid and set the centrifuge to the appropriate speed and time.

Then press on.

6. Stay close to the centrifuge at the first minutes to ensure the centrifuge is

running smoothly. If the centrifuge is not well-balanced, it will vibrate and

some centrifuge will turn off immediately. In case of older version of

centrifuge, you may have to turn off the machine manually.

7. Do not open the safety lid while the motor is running.

8. Wipe any spillage that might have occurred after the centrifugation process.

X.2.4 Incubator

Incubators provide appropriate environment for cell culture and growth by providing

sufficient air circulation, oxygen and carbon dioxide supplies and exchange, and

humidity accordingly to cellular physiological conditions. There are three functions of

cell culture incubators:

1. Constant temperature – the incubator can be set to a specific temperature

appropriate to specific type of cells. For mammalian cell culture, the

temperature is set to 37oC which is the optimal temperature for their growth.

2. Humidity – Humidity is required in the incubation of cell culture to prevent

the evaporation of the media. Distilled water is placed in the incubator to

provide humidity and needs to be replaced weekly/regularly to prevent growth

of microorganisms and to reduce the possibility of contamination.

3. pH balance – 5% CO2 is required to keep the pH balanced. The gas is injected

inside the incubator and distributed by a fan or natural convection. Interactions

of CO2 with the bicarbonate buffer in the cell culture medium stabilized the pH

at about 7.4. An uncorrected changes in the pH medium can easily damage the

cells.

The optimal temperature and humidity provides an environment suitable for the

growth of bacteria and other microorganisms. Therefore, it is necessary to clean the

incubators frequently to prevent the growth and spread of contamination.

Figure X.3 Incubator for cell culture

X.2.5 37oC water bath

Culture media and supplementations are usually kept in the 4oC refrigerator, whereas

the cells are kept in the 37oC incubator. In order to prevent abrupt temperature

elevation, the media and reagents are warmed up in the water bath prior use.

The warm water in the water bath is an ideal environment for the growth of

microorganism and other contaminants. Therefore the water bath need to be cleaned

and the water need to be replaced routinely. Bottles and containers that have been

warmed up in the water bath must be wiped down carefully with 70% ethanol before

being transferred into the hood.

X.2.6 Refrigerator and freezer

Most reagents and solutions used for cell culture are kept in the refrigerator for short

term storage. Some reagents are kept in the -20oC freezer for longer term storage. Cell

culture facilities often have -80oC freezer to store reagents and cryopreserved cells.

For long term storage, cells are kept in liquid nitrogen tanks of the temperature -

180oC. Cells can be kept frozen in liquid nitrogen for many years. Extreme cold

temperature of liquid nitrogen is hazardous and may cause burn to skin when exposed.

Therefore, thick gloves must be worn when handling the tank with great care.

X.2.7 Biohazard waste container

Hazardous and potentially hazardous materials must be disposed properly and

accordingly in biohazard waste container. The materials need to be sterilized before

the disposal. The biohazard waster must be handled accordingly to the procedures and

municipal council, federal and institutional law. It is very important to adhere with the

rules to prevent the spread of potentially hazardous materials to the environment.

X.3 Mammalian cell culture

X.3.1 Media Preparation

Cell culture is a method of multiplying cells under sterile and controlled laboratory

condition. It is used by the scientists to study cellular biological activities, functions,

morphologies and behavior of cells. The most important factor influencing the

mammalian cell culture is the choice of the culture medium as the cells required an

abundant source of easy to use nutrients for their viability and growth4. The

formulations for the medium should mimic natural conditions of systemic interactions

that normally occur in the body of an organism to support their biological activities

and viability. There are many different types of culture media are available in the

market, in which vary accordingly to their sugar, protein, minerals supplemented with

animal serums, growth factors and hormones as different cell types may favor

different formulations for optimum growth and differentiation5.

Most cells in are grown in a basal media containing nutrient, vitamins and mineral

supplemented with animal serums of different type such as horse, calf and fetal

bovine, This type of media is called ‘undefined media’ due to its unknown exact

components of the supplemental serum. Examples of readily available cell culture

medium are Dulbecco’s Modified Eagle’s Medium (DMEM)6, α-Eagle’s Minimal

Essential Medium (α –MEM)4, and RPMI 16407.

All basal media should contain the following components:

Source of energy/carbon – glucose/glutamine

The building blocks of proteins – amino acids

Supplementation to support cell survival and growth – vitamin

Isotonic mixture of ions to act as cofactors for enzymatic reactions and cellular

activities – balanced salt solution

pH indicator that changes from orange/red at pH 7-7.4 to yellow at acidic

lower pH and purple at basic higher pH environment,

Buffer – HEPES or bicarbonate to maintain a balanced pH in the media.

In the preparation of ‘complete media’, supplementations of serum is added into the

basal media. Antibiotics such as Penicillin and Streptomycin and fungicides (i.e.

Fungizone) may be added to prevent bacterial and fungi growth. This supplementation

is however not recommended due to increase susceptibility of antibiotic resistant

bacteria/fungi. Alternatively, glutamine, epithelial growth factor and cholera toxin can

be added into the basal media and is said to be ‘defined media’. This defined media

can be customized and selective for specific cell type and experimental conditions.

X.3 Thawing and recovering mammalian cells from cryopreservation

Cells thawing from cryopreservation is the most critical procedure in the cell culture

routine that has to be done quickly with care. Cryopreserved cells are kept in

complete media with 5-10% of dimethyl sulfoxide (DMSO) to prevent crystallization

and breakage of the frozen cellular components. Although DMSO is harmful to cells

in culture medium it helps to maintain cellular membrane during the freezing-thawing

process. From this procedure, it is estimated that only 50% of the cryopreserved cells

will survive. Therefore, higher number of cells (>1,000,000 cells/ml) in the

cryopreservation will have higher chance for cell survival.

Materials and Equipment

1. Cryopreserved cultures of cells

2. Complete media (Dulbecco’s Modified Eagle’s Medium, DMEM supplemented

with 10% fetal bovine serum, FBS), 37°C

3. 70% ethanol

4. 25cm2 T-flasks or 60-mm petri plates, sterile

5. Sterile centrifugal tube

6. Sterile serological pipettes

7. Incubator with 5% CO2 and 37°C

8. Cooling centrifuge, 4oC

9. Water bath, 37oC

Method

1. Remove the cryopreserved vials from the -80oC freezer or the liquid nitrogen

tank. Examine the labels – cell type, passage number and date of storage to

ensure that you have the correct cell line. Place the frozen vials in a small

container in the 37oC water bath and make sure that the caps do not get wet to

avoid possible contamination. The procedure should be done very quickly

(about 1-2 minutes) to prevent the breakage of cellular membrane due to the

formation of DMSO crystallization.

2. Remove the vials from the water bath and wipe down well with 70% ethanol.

The vials then should be quickly transferred into a fume hood and mixed well

with 9 ml of a complete cell culture medium in a 15 ml tube.

3. The tubes should be closed properly and labelled. Then using a balanced tube,

centrifuge the cells at about 120g (1000-1500 rpm) in a cooling centrifuge (4°C)

for about 3 minutes.

4. The heavy cells will form a pallet and are separated from the media

(supernatant)

5. Using a sterile pipette, remove the supernatant carefully without affecting the

pellet which contains the cells at the bottom side of the tube.

6. Then, using another sterile glass pipette, resuspend the cell pellet with 5 ml of

a complete medium and transfer the mixture into a culture plate or 25cm2 T-

flask containing 5 ml complete medium.

7. Swirl the flask gently and be careful not to wet the flask’s cap.

8. Observe the cells using an inverted microscope. You should observe plenty of

floating cells in the media.

9. Place the flask in an incubator with 5% CO2 at 37°C.

10. Cells may take up about 5-24 hours or more to attach to the bottom of the flask

and to start dividing. Many cells may not survive the freezing-thawing process

and will not attach onto the flask. Therefore, after 24 hours, the cell culture

media should be changed and replenish with a new culture media.

Notes and Tips

Protective clothing, gloves and goggles, should be worn while removing the vials

from -80oC freezer and liquid nitrogen tank.

X.4 Detachment and subculturing of mammalian cells from flask/plates.

When a cell line is cultured, a lag period after seeding usually followed by

exponential growth, called the log phase. When the cell density reaches a level such

that all the available cell growth area is occupied (100% confluence), or when the cell

concentration exceeds the capacity of the medium, the growth ceases or is greatly

decreases, Then either the medium should be changed or the culture must be divided

(subculture). X.4.1 and X.4.2, subculturing of two types of culture, monolayer and

suspension culture, respectively are discussed.

X.4.1 Subculture of cells in monolayer cultures

The first step in subculturing cells from monolayer cell culture is to detach cells from

the surface of the primary culture vessel by the use of accutase, trypsine or

mechanical means. This is followed by transferring fraction of the detached cells to

new culture medium to grow inside new culture vessel.

Materials and Equipment

1. 100% confluence cultures of cells

2. Complete media (DMEM supplemented with 10% FBS), 37°C


4. Incubator with 5% CO2, 37°C

5. Cooling centrifuge, 4oC

6. 25cm2 T-flasks or 60-mm petri plates, sterile

7. PBS (Phosphate Buffer Saline), 37°C

8. Accutase or trypsin

Method

1. Remove the medium from the 100% confluence cultures of cells with a sterile

Pasteur pipette. Wash the monolayer cells with 5 ml (for culture in 25cm2 flask)

of 37°C PBS to remove the residual FBS.

2. Add 1 ml of 37°C Accutase/trypsin solution to the culture which cover adhering

cell layer.

3. Place plate in incubator with 5% CO2 at 37°C for 2 to 3 minutes. Check culture

with microscope to make sure that cells are rounded up and detached from the

surface.

4. Add 8 ml of 37°C complete medium. Draw cell suspension into a Pasteur pipet

and rinse the cell layer three or four times to dissociate remaining adherent cells.

Transfer cell suspension to centrifuge tube (sterile) and centrifuge it for 5-6

minutes at 120g cooling centrifuge (4°C). Discard the supernatant and

resuspend the cell pellet with complete media.

5. Add an equal volume of cell suspension (1 ml) to fresh culture vessels that are

well labeled.

6. Add 4 ml fresh complete media to each culture and incubate them in a 37°C,

5% CO2 incubator.

7. In some cases, feed cultures after 3 or 4 days by removing old media and adding

fresh media.

8. Passage the culture when it becomes confluent by repeating steps 1 to 6.

Notes and Tips

- Cells can be counted using a hemacytometer then diluted to the desired density

so a specific number of cells can be added to each culture vessel. - For primary cultures and early subcultures, 60-mm petri plates or 25-cm2

flasks are generally used; larger vessels (e.g., 150-mm plates or 75-cm2 flasks)

may be used for the following subcultures. - Culture vessels should be labeled with the passage number and date of

subculture. - For culturing cells using 75 cm2 culture flasks, 9 ml medium should be added

per flask. - CO2 incubators with 5% CO2 and 4% O2 should be used because the low

oxygen concentration simulate the in vivo environment of cells and enhance

cell growth.

X.4.2 Subculture of cells in suspension cultures

A suspension culture requires the same incubation conditions as in the monolayer

culture. Fortunately, subculturing of suspension cultures is less complicated than

subculturing of monolayer cultures because the cells are not adhering to the vessel

surface. There is no necessity for the detachment or dispersion prior to subculturing.

Materials

1. 100% confluence cultures of cells



4. Incubator with 5% CO2 and 37°C

5. 25 cm2 T-flasks, sterile

6. 70% (v/v) ethanol

7. Isopropanol

8. Mr. Frosty container

Method

1. Cells should be fed every 2 to 3 days as follows until the cultures reaches

confluency:

a. Take out the flask of suspension cells from the CO2 incubator, making

sure not to disturb cells that have settled at the 25cm2 T-flasks bottom.

b. Remove and discard about one-third of the medium (under sterile

conditions) from flask and replace with an equal volume of (37°C)

medium. Gently stir the flask to homogenize cells with the media.

c. Return flask to the incubator. If there is <20 ml of medium in the flask,

the flask should be incubated in horizontal position to assist

cell/medium contact. On higher volume of media, vertical incubation

of flask can be applied.

2. Culture should be checked regularly. Gentle swirling and stirring should be

applied to resuspend cells.

3. When suspension cultures reaches confluency (∼2 × 106cells/ml), subculturing

should be performed as follows:

a. Take out the flask from incubator and gently swirl flask in order to

distribute the cells evenly in the media.

b. Remove half of the volume of cell suspension and place into new

sterile flask.

c. Add 7 to 10 ml medium to each flask and return flasks to incubator.

X.5 Cryopreservation of mammalian cells in cell suspension

Cryopreservation of cells involve cell suspension in 10% DMSO in FBS or complete

medium to prevent crystallization and damage to the cells. To maintain cellular

viability, the cells has to be placed in a freezing container called Mr. Frosty (Nalgene

Ltd, USA) before being placed in the -80oC freezer. The container is filled with

isopropanol to prevent rapid cooling and slowly decreasing at the rate of -1oC per

minute. As many of the cells die in the process of freezing and thawing, higher

concentrations of 1x106 cells/ml is preferred to ensure cell survival.

X.5.1 Cell cryopreservation from monolayer cultures

Materials

1. 100% confluence culture of cells


3. Freezing medium (FBS with 10% (v/v) DMSO)

4. Incubator with 5% CO2, 37°C

5. Cooling centrifuge, 4°C

6. 25 cm2 T-flasks or 60-mm petri plates, sterile


8. PBS (Phosphate Buffer Saline), 37°C

9. Accutase or trypsin

10. Sterile serological pipettes.

11. Isopropanol


Method

1. Remove the cell culture medium from the flask with a sterile pipette. Wash the

monolayer cells with PBS twice (5 ml for 25 cm flask) to remove the residual

FBS.

2. Add 3 ml of accutase/trypsin solution to the culture to cover the attached cell

surface.

3. Place the flask in an incubator with 5% CO2 (37°C) for 2 to 3 minutes. Check

the cellular detachment from the surface with an microscope. Tap the side of

the flask and observe under the microscope again to make sure that all of the

cells arerounded up and detached from the surface.

4. Quench trypsin by adding 8 ml of a complete cell culture medium and mix well

by pipetting up and down to dissociate remaining adherent cells. Transfer cell

suspension to 15 ml centrifuge tube (sterile) and centrifuge it for 5-6 minutes at

120g cooling centrifuge (4°C). Discard the supernatant that contains accutase

or trypsin. Resuspend the cell pellet with a complete cell culture media.

5. Count cells using a hemacytometer, dilute with media to get a final cell

concentration of 106 or 107 cells/ml and transfer into a 15 ml tube.

6. Centrifuge the cell suspension at 120g for 3 minutes (4oC) to separate the cell

pellet from the supernatant. Then remove the supernantant and add 4 ml of cell

cryopreserved medium containing 10% DMSO in FBS. Mix the medium and

the pellet well by pipetting up and down slowly. Transfer 1 ml of the mixture

into a labelled 2 ml cryovials. (freezing cells from 25 cm2 flask requires 2 ml

freezing solution).

7. Place the vials in Mr. Frosty for overnight in a −80°C freezer before storage in

liquid nitrogen tank or in a -140oC freezer.

X.5.2 Cell cryopreservation of suspension cultures

The method of freezing cells from suspension culture is almost similar to freezing

cells from monolayer cultures. The major difference is that suspension cultures do not

requires cellular detachment from the surface using accutase or trypsin.

Materials

1. Suspension cultures of cells


3. Freezing medium (FBS with 10% (v/v) DMSO

4. Cooling Benchtop clinical centrifuge (4°C)

5. 25 cm2 T-flasks or 60-mm petri plates, sterile


7. Sterile serological pipettes.

8. Isopropanol


Method

1. Transfer the cell suspension to 15 ml centrifuge tube (sterile) and centrifuge

it for 5-6 minutes at 120g cooling centrifuge (4°C). Discard the supernatant

and resuspend the cell pellet with a known volume of complete cell culture

media for cell counting.

2. Count cells using a hemacytometer, dilute with media to get a final cell

concentration of 106 or 107 cells/ml and transfer into a 15 ml tube.

3. Centrifuge the cell suspension at 120g for 3 minutes (4oC) to separate the

cell pellet from the supernatant. Then remove the supernantant and add 4 ml

of cell cryopreserved medium containing 10% DMSO in FBS. Mix the

medium and the pellet well by pipetting up and down slowly. Transfer 1 ml

of the mixture into a labelled 2 ml cryovials. (freezing cells from 25 cm2

flask requires 2 ml freezing solution).

4. Place the vials in Mr. Frosty for overnight in a −80°C freezer before storage

in liquid nitrogen tank or in a -140oC freezer.

Notes and tips

Styrofoam boxes can be used as substitute to Mr.Frosty (with isopropanol) for

freezing cells in a -80oC freezer for overnight. The cryovials should be

covered at all sides in wet wipes/tissues and placed in the container. Label

with your name, date, cell type and passage.

For routine freezing procedure, estimation of cell concentration can be

predicted. Normally, for 100% confluent T25 flask, >4x106 can be harvested

and placed in 4 cryovials with 4 ml of cryopreserved medium. (Note that the

cell concentration varies with cell types)

Freezing media of DMSO and FBS can be stored in a refrigerator.

DMSO is light sensitive. Cover the freezing media with aluminium foil to

keep it in the dark.

X.6 Mammalian cell counting

Cell counting is the most important step in standardization of cell culture.

Hemacytometer is a device invented by Louis-Charles Malasses 8 to measure cell

concentration. It is made of a glass slide with a grid on each half (Figure X.7). Each

grid is made of nine squares and each square is subdivided into smaller squares. The

grid is covered by a cover slip and creates a chamber which can hold up to 0.1 μl of

liquid. It is very important to use the hemacytometer cover which is thick and evenly

surfaced cover slip because ordinary cover slips have uneven surfaces that may cause

errors in cell counting. In order to count, a small sample (0.1 μl) of known volume of

trypsinized cells will be placed in the hemacytometer chamber. Since the volume in

each square is known, therefore, the concentration of cells can be calculated.

Materials

1. Suspension cultures of cells

2. 0.4% (w/v) trypan blue

3. Hemacytometer with coverslip.

4. Hand counter


Method

1. Prepare hemacytometer by cleaning surface of hemacytometer slide and

coverslip with 70% ethanol.

2. Prepare cell suspension (preparation of cell suspension depends on the type of

culture (monolayer or suspension) by following the protocols above).

3. Stain cells with trypan blue (to determine cell viability) by adding an equal

volume of trypan blue to cell suspension on an ordinary slide. Mix thoroughly

using pipette and let stand 5 min before loading hemacytometer.

4. Loading the hemacytometer using a sterile Pasteur pipette to transfer cell

suspension to the edge of hemacytometer counting chamber. Hold tip of pipette

under the coverslip and dispense one drop of cell suspension.

5. Allow the cells to settle for a 2-3 minutes before beginning to count and remove

excess liquid.

6. Using the hand counter, count cells in the four corners and central squares of

one counting chamber and also other counting chamber of the hemacytometer

(Figure 1).

7. Calculate cell number by determine cells per milliliter using the following

calculations:

cells/ml = average count per square *dilution factor *104.

Notes and Tips

The hemacytometer is a non sterile equipment. The pipette that was used to

transport cells to the hemacytometer should not be reused in the original

suspension.

Hemacytometers are fragile and expensive, please be careful not to drop or

break them.

Settlement of cells at the bottom of the flask and tubes tend to form clumps. It

is very important to mix the cells well by pipetting up and downwards to

dissociate the clumps and to ensure homogenous cell suspension.

Cells at outside incubator and trypsinized are under stress and its viability may

deteriorate over time. It is important to speed up the procedure rather than be

accurate in cell counting.

Observe your cells in the culture prior trypsinization to ensure higher number

of cells for calculation.

Trypan blue is a blue stain that can transverse inside the dead cells through

their porous membranes. Live cells do not allow the stain to get in, therefore

the dead cells will appear dark blue as opposed to the clear live cells.

Glossary:

Aseptic techniques: Series of techniques and practices used to reduce the chances of

contamination

Basal media: Basic media required for cellular growth and metabolism that contains

basic nutrients, vitamins and mineral necessary for cell culture. The media needs to be

supplemented with additional growth hormones, growth factors and proteins for

efficient cell growth.

Bicarbonate buffer: The buffer used in cell culture media to keep the pH balance at

7.4 by interaction with CO2

Chelator: Molecules like Ethylene diamine tetra acetic acid (EDTA) that bind to

certain metal ions and inactivate the ions to prevent their interactions with other

elements

Complete-media: Basal media that contains additional proteins, growth factors and

components necessary for efficient cell growth for specific cell type.

Confluency: The percentage of the cell culture surface that is covered by the cells.

Cytotoxic studies: The measurement of cellular viability and indicator of cellular

metabolism when interacting with a toxic factor.

Defined media: Basal media supplemented with additional growth factors, proteins

and other components without the use of serum.

Density-dependent growth inhibition: Cell growth arrest due to insufficient space,

nutrients or oxygen.

Differentiation: A process when cellular enters maturity and specialized into specific

cells.

Extracellular matrix: Chemical components and structural support for the cellular

attachment, proliferation and differentiation in a tissue.

Hayflick’s limit: The limit for normal cells in culture to divide.

Undefined media: A basal media with serum supplementation without

acknowledging the exact components of the serum.

References

1. Hayflick, L. & Moorhead, P. S. The serial cultivation of human diploid cell

strains. Exp. Cell Res. 25, 585–621 (1961).

2. Masters, J. R. HeLa cells 50 years on: the good, the bad and the ugly. Nat. Rev.

Cancer 2, 315–319 (2002).

3. Ozturk, S. & Hu, W.-S. Cell culture technology for pharmaceutical and cell-

based therapies. (CRC Press, 2005).

4. Eagle, H. Amino acid metabolism in mammalian cell cultures. Science (80-. ).

130, 432–437 (1959).

5. Freshney, R. I. Culture of specific cell types. (Wiley Online Library, 2005).

6. Dulbecco, R. & Freeman, G. Plaque production by the polyoma virus. Virology

8, 396–397 (1959).

7. Moore, G. E., Gerner, R. E. & Franklin, H. A. Culture of normal human

leukocytes. Jama 199, 519–524 (1967).

8. Verso, M. L. Some nineteenth-century pioneers of haematology. Med. Hist. 15,

55–67 (1971).

animal cell culture and methodologies

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