Cell Culture Techniques

“A cross-border region where rivers connect, not divide” –
Interreg V-A Hungary-Croatia Co-operation Programme 2014-2020
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Cell Culture Basic Techniques
Ljubica Glavaš-Obrovac and Katarina Mišković Špoljarić
Introduction
Cell culture is a known technique applied all over the world. It is a powerful
investigating tool in the field of today’s life science and compiles knowledge and connects
research from cell biology, biochemistry, medicine and laboratory medicine. The technique is
applicable in diagnostic studies as well in the science research conducted in molecular,
cytogenetic or biochemical laboratories 1 (Slide 2).
The field of cell culture is now rapidly expanding from biopharmaceutical to the stem
cell investigation and regenerative medicine. Today, we have fully functional, specialized cells
which resulted from combination of selective culturing conditions and gene expression
manipulation.
History of cell culture started with Ross Harrison in 1907, who cultivated frog’s nerve
fiber followed by Carrel and Burrows in 1912, and their successful cultivation of chick embryo
tissue2. Major problem was culture sterility and a choice of media for the cultivation.
In the 1950’s major breakthrough happened3. It started with use of antibiotics (in 1948) by
Keilova, following with growth of viruses in cell culture by Enders (in 1949), polio virus in
monkey kidney cells by Kew (in 1952) and Dulbecco's use of trypsin for generation of replicate
culture (in 1952)3. In 1952 George Gey isolated, cultivated and established the first human
cancer cell line known as HeLa cells4. Final step in cell culture evolution was development of
first, defined medium or Eagles’s medium (1955)2. Eagles’s medium contained at least 13
essential amino acids, 8 vitamins, glucose as a source of energy, different salts for maintaining
osmolality3. After four years, Eagle modified the composition of the first medium and named
it “Minimal Essential Medium or MEM”3. Modification contained more nutritional
components but still required a protein supplement like serum, plasma or tissue extracts3. Till
today, MEM is the basics for new media formulations and special requirements due to cell lines
demands. From 1960’s till today every year was a new step in development and innovations in
cell culture methods and appropriate working techniques.
How do we define cell culture? Usually it is defined as a removal of the cells from organ
fragments, prior to, or during cultivation, thus disrupting their normal5,6 connection with
adjacent cells) and their maintenance in appropriate conditions. In term of tissue culture2 two
main types of cultures are designated: cell culture and organ culture2,5,6. Organ culture is
defined as a culture of entire organs and/or uncut organ fragments with the intent of studying

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their continued function or development5,6. It actually means studying of functions in
threedimensional form (Slide 3).
Cell types and culture characteristics (Slide 4)
Primary cell culture is obtained from the parental tissue of a solitary organs or blood and
prepared by combination of mechanical and/ or enzymatic methods6 to be cultivated in
adequate medium. Cells are considered primary culture until the first passage or
subcultivation2. It can be established from normal adult or embryonic tissue and from tumor
tissue. The alternative to establishing a culture by primary culture in the laboratory is to buy
established cell lines from organizations such as the America Type Culture Collection (ATCC),
European Collection of Cell Cultures (ECACC) or Coriel Institute for Medical Research7, 8.
Cells are divided in to two main types:
• Adherent or anchorage cells which require surface, usually treated, for their growth in
cell culture. These types of cells are derived from solitary organs like kidney, colon or
skin in which they are immobile and embedded in connective tissue.
• Suspension or non-adhesive cells are mostly derived from blood, like lymphocytes.
These types of cells do not need attachment surface for in vitro cultivation since they
are in vivo suspended in plasma.
After the first sub-cultivation secondary cell culture is obtained. Sub-cultivation or passaging
is obligatory step in cell culture maintenance. It refers to the change of growth medium and
detachment of cells from the surface of culture vessel usually by enzymes. The most frequently
applied enzyme is trypsin used by itself or in combination with cell scraper for mechanical
detachment.
Cell lines or cell strain can be designated as finite or continuous depending upon their life
span in culture. Finite cell line has a limited number of cell divisions while continuous cell line
is immortal and can be maintained in culture for an indefinite period of time. Immortality of
cells can be the result of from chemical or viral transformation2, 9. Some differences in
cultivation characteristics among finite and continues cell lines are shown in Table 1, (Slide
5).
Cells in culture grow as a monolayer or suspension cultures (Slide 6) Adherent cells mostly
grow as monolayer cultures usually one cell in thickness attached to vessel surface while
nonadherent cells grow in suspension mode sometimes creating small floating aggregates.
Healthy cells are required for reliable research and diagnostic analysis. Cell growth depends on
culture conditions and environment. This environment usually consists of a suitable glass or

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plastic culture vessel containing a liquid or semisolid medium as a source of essential nutrients6.
Cell growth is monitored during several days and is characterized with three specific phases,
lag, log, and plateau, as indicated in the Slide 7. Cells with deviation in growth curve usually
require modifications in culturing conditions: different culture vessel, change of medium,
additional growth supplements, and control of pH, temperature or gas environment. Number
of cells for starting a cell culture depends on cell type and differs among cell lines. Too small
or too high cell number can result in an abnormal growth curve. Seeding number for starting
and maintaining cell culture can usually be found in literature and specialized cell culture
collections’ web sites such are American Type Culture Collection (ATCC) and European
Collection of Cell Culture (ECACC). See Table 2, (Slide 8)
Types of Cell Culture Media
Culture media contain a mixture of different nutritiens such are glucose, amino acids,
salts, and vitamins, cofactors and can be purchased either as a powder or as liquids. The
requirements for the media composition vary among culturing cells, and these differences are
responsible for numbers of available medium formulations.
Cells growing in vitro can be cultured either using a natural or an artificial medium
supplemented with some natural products (Table 3, Slide 9).
Natural media consist of naturally occurring biological fluids and are convenient for a wide
range of animal cell culture. The major disadvantage of natural media is its poor reproducibility
due to lack of knowledge of the exact composition.
Artificial or synthetic media are prepared by adding organic and anorganic nutrients, vitamins,
salts, serum proteins, carbohydrates, and cofactors1,2. Based on the composition of artificial
media (Slide 10) are grouped into four categories:
Serum containing media. To provide an optimal culture medium a fetal bovine serum is, as a
low-cost supplement, very often used supplement in animal cell culture media. This serum
provides carriers or chelators for labile or water-insoluble nutrients, hormones and growth
factors, protease inhibitors, and neutralizes toxic compounds.
Serum-free media. Presence of serum in the media can have many drawbacks. A number of
serum-free media have been developed they are generally specifically formulated to support
the culture of a specific cell type, such as stem cells, and incorporate defined quantities of
purified growth factors, lipoproteins, and other proteins, which are otherwise usually provided

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by the serum. These media are also referred to as „defined culture media“ since their exact
composition is known.
Chemically defined media. These media contain contamination-free ultra-pure inorganic and
organic ingredients, and may also contain pure protein additives, like growth factors. Their
constituents are produced in bacteria or yeast by genetic engineering with the addition of
vitamins, cholesterol, specific amino acids, and fatty acids.
Protein-free media. Protein-free media do not contain any protein and only contain nonprotein
constituents. Compared to serum-supplemented media, use of protein-free media promotes
superior cell growth and protein expression and facilitates downstream purification of any
expressed product. Formulations like Minimal Eagle's medium (MEM) or RPMI-1640 are
protein-free and protein supplement is provided when required.
Cell culture laboratory
Cell culture laboratory does not require large space (minimum is 12 sq. m). When you design
laboratory for cell culture experiments, the main idea must be how to separate clean and aseptic
part from rest of the laboratory. Washing and sterilization should be in the separate room,
completely physically detached from the clean part of the laboratory. The central part of cell
culture room is a biosafety cabinet while rest of the equipment will be placed around it
according to space availability.
Cell culture laboratory equipment
Cell culture laboratory should be equipped with basic and specific equipment depending on the
type of research conducted or diagnostics and clinical applications (Slide 11).
In the basic equipment are included: cell culture hood (i.e. biosafety cabinet or laminar-flow
hood), CO2 incubator and a cylinder with CO2, centrifuge, refrigerator and freezer (-20°),
storage tanks with liquid nitrogen, inverted microscope, cell counter or hemocytometer,
equipment form sterilization (autoclave- steam sterilizer, sterilizing oven) 2,9,10 (Slide 12-14)
Additional equipment are pH meter, aspiration pump (peristaltic or vacuum), confocal
microscope, flow cytometer, orbital shaker, mini spin, vortex, camera for inverted microscope,
plate reader, water bath.
Cell culture hood (laminar or biosafety cabinet) is essential in every manipulating step
regarding cell culture (Figure 2, Slide 12). Hood is a ventilated cabinet with a set of HEPA

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filters and positive pressure. Its role is to provide clean and aseptic working area. Hoods are
classified as class I, II and III depending on the level of protection they provide. Cell culture
mainly requires class II laminar hood. Class II hood creates sterile environment and protects
from contamination using vertical circulating air within the three sides enclosed bench and
drawn through a HEPA filter2. They can be freestanding or bench top so you can choose one
that suites your accommodation.
CO2 incubator. Cell cultures can be grown in two main types of CO2 incubators: dry incubator
and humid CO2 incubator (Figure 3, Slide 13). Purpose of the incubator is to provide the
adequate environment by controlling the temperature, carbon dioxide concentration and
humidity. Atmosphere usually contains 5-10 % of CO2 to maintain optimal pH between 7.2-
7.4. Humidity prevents evaporation of media which can result with altered salt and nutrient
concentration.
Centrifuge is an obligatory part of every laboratory (Figure 4, Slide 14). Cell culture requires
centrifuge with ability of temperature control, emendable fix or swing rotor able to spin tubes
in a range 14000 – 20000 rpm. In a case of large-capacity suspension cell cultivation,
centrifuge must be able to rotate 4x1 or 6x1 L bottles in cold environment2.
Cooling and freezing. Cell culture supplements, media and other chemicals must be stored in
chilled space like a cold room or a refrigerator at + 4°C. Cold room is a luxury and usually a
classic refrigerator (+4°C) or combined cold/freezing (+4/-20°C) is a part of cell culture
laboratory. Another option for prolonged “shell life” is a -80°C freezer with CO2 backup.
Tanks with liquid nitrogen (4, 10 and 50-70L respectively) are an obligatory part of cell culture
lab intended for preservation of stock cell cultures (Figure 5, Slide 14).
Microscope. An inverted microscope is vital for cell culturing (Figure 6, Slide 14)).
Monitoring cells on daily basis is essential to detect every possible morphological change and
potential microbial or fungal contamination. It is good to have inverted microscope with
phototube for digital recording and viewing on the monitor. Camera makes educational part of
new staff and students much easier and detects changes in real time. In addition to inverted
microscope, a dissecting microscope is used for tissue dissection like small vertebrates and
embryonic tissue2.
Cell counting. For equalized and comparable research data, preparation of experiment and
evaluation of cell growth, number of cells must be known. For cell counting, hemocytometer
(Bürker-Türk, Neubauer) or electronic cell counter can be used.
Sterilizationequipment.Cell culture work requires sterilized supplies and laboratory materials.
Most of supplies are plastic, sterilized and prepared for one-time use. But, glassware is reusable

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and must be sterilized preferably by dry heat, while liquids (water, buffers) and dry items are
sterilized in the autoclave with a hot steam (Figure 7, Slide 14).
Necessary supplies are cell culture vessels (flasks, Petri dishes, multi-well plates), pipettes and
motorized pipette controller, syringe and needles, plastic centrifuge tubes, cryovials, cryo-
boxes, filters for syringes and bottles, waste containers, deionized water, laboratory glass
dishes, disinfectants (isopropyl or 70 % ethanol, Na-hypochlorite)2 (Slide 15) Additional
equipment is a matter of choice and market availability.
Work in aseptic conditions
To maintain sterility all materials that come in the direct contact with cell cultures must
be sterile while non-sterile surrounding must be restricted without direct contact with the cell
culture. An aseptic technique means practices and procedures involving strict working rules to
prevent contamination from pathogens11.To ensure aseptic condition it is necessary to wear
clean laboratory coat, surgical gloves, a mask, and sometimes protective goggles. Sterility and
purity of hood work surface is preserved by using an UV lamp. Furthermore, it is mandatory
wiping with 70% ethanol everything that comes in and goes out from the hood, including
working area. Air flow must be maintained during working time in conditions prescribed by
the manufacturer to sustain sterility of working area. While working in the hood, carry in only
necessary materials and equipment and arrange them in the in a manner that avoids crossing
paths in manipulation. Too crowded work area obstructs laminar flow and perturbs containment
and sterility (Figure 8, Slide 16).
A big problem in cell culturing is a microbial contamination by bacteria, mycoplasma, yeast or
fungal spores which can be introduced by operator, the atmosphere, work surface, solutions,
contaminated cell lines or working material like plastic and glassware, instruments and pipettes.
To minimize the risk of infection, aseptic technique applies. Maintaining good aseptic
techniques prevents most of contaminations which can arise in cell cultures. But, sometimes
infection occurs. To restrict damage, inspect cultures every day by eye and microscope,
maintain them without antibiotics to prevent cryptic contaminations, use sterile reagents, do
not share media and other reagents, and keep new cultures in quarantine. Fungal and yeast
contamination are easy to detect, bacterial contamination can be visually detected after culture
reaches confluence stage due to media blurring and change of color because of change in pH
value. The biggest problem is hidden, mycoplasma contamination. Undetected, can infect all
others cells. If something is unusual in cell morphology or growth rate, or contamination
continuously persists, do the mycoplasma test7.

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Keep personal hygiene by washing hands. Washing will moisten the skin and remove
dry skin that would very likely blow onto cell culture. Long hair needs to be tie in the back.
Talking is permissible, but keep glass barrier between you and your cultures. If you have cold,
avoid working with cell cultures, but if you have to, wear a face mask.
Reagents and media obtained in original packaging are sterile, but outside, those bottles
must be wiped with 70% ethanol. Also, wipe them when you take them from the water bath or
refrigerator to remove dew. Open them in the ventilated hood.
Culture vessels (plates, bottles) open only in the hood and keep them under angle to
protect from spillage. Open only to perform manipulating step.
Handling liquids is easiest with disposable or standard glass pipettes. Glass pipettes are
reusable and must be sterilized in house, while plastic, disposable pipettes are sterile, intended
for one time usage. At the top of every pipette, cotton plug should exist to prevent
contamination, keep pipette sterile during manipulation and prevent liquid entering motorized
pipette controller. Sometimes, small volumes are dispensed with a syringe, especially in a case
of filter sterilization. For larger volumes, adequate filters are at disposal.
Standard operating procedure – SOP12,13 is a part of good aseptic techniques and
standard good laboratory practices. Every laboratory develops its private SOP protocol
depending on type of research requirements considering basic principles of aseptic techniques.
Work in a cell culture laboratory is demanding, but easy. Compliance with a laboratory SOP
and proper equipment provides safe and reliable research data.
Basic protocols for cell subculturing1, 2, 10
Trypsinizing and subculturing cells growing in monolayer
Cells grown in monolayer proliferate to a confluent state in which the cells cover the growth
surface of the flask (Slide 17). Some cells can be maintained in this plateau phase of growth
for days to weeks, while others require trypsinization and subculture to survive. As cells reach
confluence, they must be subcultured or passaged. Failure to subculture confluent cells results
in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach
cells from the surface of the primary culture vessel by trypsinization or mechanical means. The
mechanical method involves physically removing the cells by cell scraping, which involves
using a spatula or scraper to gently remove the cells from the bottom, while enzymatic method
use proteolytic enzymes, usually a trypsin, to digest the proteins that adhere cells to the dish.
Cells are released from the dish by breaking the cell protein interactions with the surface of the

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dish. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures.
Secondary cultures are checked for growth and fed periodically, and may be subsequently
subcultured to produce tertiary cultures. The time between passaging of cells varies with the
cell line and depends on the growth rate.
Passaging cells growing in suspension
Subculturing suspension cells is less complicated than passaging adherent cells since that type
of cells is already suspended in growth medium (Slide 18) . Replacement of all growth medium
is not carried out in suspension cultures. This can be done by directly diluting the cells in the
culture flask and continue expanding them, or by withdrawing a portion of the cells from the
culture flask and diluting the remaining cells down to a seeding density appropriate for the cell
line. Usually, the lag period following the passaging is shorter than that observed with adherent
cultures. The cells are maintained by feeding them every 2 to 3 days until they reach
confluence. However, for cell lines that grow in clumps it may be necessary to bring the cells
into a single cell suspension by centrifugation and resuspension by pipetting in a smaller
volume before counting.
Comparison between subculturing of suspension and adherent cell cultures is shown inthe table
4 (Slide 19).
Freezing cells (Slide 20)
Best way for a long-term storage of the cell is to freeze them and keep in the liquid nitrogen. It
is important to freeze cells when they are at optimal density (80-90% confluent). The freezing
procedure begins with the trypsinization of the cells, following resuspension in medium,
transfer to a sterile glass tube, and cooling on ice for half to an hour. The cooling rate used to
freeze cultures must be just slow enough to allow the cells time to dehydrate, but fast enough
to prevent excessive dehydration damage. A cooling rate of -1°C to -3°C per minute is
satisfactory for most animal cell cultures. Larger cells or cells having less permeable
membranes may require a slower freezing rate since their dehydration will take longer. It is
essential to freeze the cells as soon as possible because of the toxicity cryoprotectant. A wide
variety of chemicals provide adequate cryoprotection, however, dimethylsulfoxide (DMSO)
and glycerol are the most convenient and widely used.

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Thawing and recovering cells (Slide 20)
It is essential to defrost the cells as soon as possible because of the toxicity cryoprotectant. In
brief, the frozen cells recover so that they are slightly heat up in hands and then medium with
cells rapidly pour into a tube with a fresh medium and centrifuge 3000g for 5 minutes. In this
way the cells are washed out of the cryoprotectant and placed on the bottom of the tube.
Immediately after centrifugation the supernatant is removed and the cell pellet resuspended in
5-10 mL of media and transferred to a cell culture dish and stored in the CO2 incubator.
Cell culture applications
Cell culture as an investigating technique has a wide range of applications in many different
science areas (Slide 21). It can be used as a model system to study cellular events on molecular
level (DNA synthesis, transcription, translation, hormone-receptor interactions, kinetics of a
cell division, and morphogenesis), processes and triggers of cellular aging as well. It is also
used to study the interaction between cells and disease causing agents like bacteria and viruses,
and the mode of infection too. Since cultured cells can be genetically altered and can be used
in gene therapy technique. Animal cell culture is used to study the effects of new drugs,
cosmetics and chemicals on the growth and proliferation capacity of a number of types of cells.
Furthermore, by using cell culture the mechanism and cause of cancer, and anticancer drugs
efficacy can be studied as well. 14Additionally, cultures of animal cells are used in the of
vaccines and in production of genetically engineered proteins such as monoclonal antibodies,
insulin, and hormones. To detect fetal disorders, fetal cell culture can be used to study or
examine the abnormalities of chromosomes and genes using karyotyping. Organ culture
techniques and research are being conducted on both embryonic and adult stem cell culture too.
Today, the cell culture is used to produce an artificial skin to treat patients with burns and ulcers
and current research is focused on the development of an artificial organ culture and drug
discovery to improve the health and quality of life of patients suffering from different life-
threating diseases15.
References:
1 a) Phelan, M. C. in Current protocols in cell biology Chapter 1, Unit 1.1 (John Wiley &
Sons, Inc., 2007).; b) Phelan, K. and May, K. M. in Current Protocols in Cell Biology 66,
1.1.1-1.1.22 (John Wiley & Sons, Inc., 2015).
2 Freshney, R. I. Culture of animal cells. (John Wiley & Sons, Inc., 2010). doi:10.1002/
9780470649367

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3 Jayme, D., Watanabe, T., Shimada, T. Basal medium development for serum-free culture: a
historical perspective. Cytotechnology 23, 95–101 (1997).
4 Lucey, B. P., Nelson-Rees, W. A. , Hutchins, G. M. Henrietta Lacks, HeLa cells, and cell
culture contamination. Arch. Pathol. Lab. Med. 133, 1463–7 (2009).
5 Introduction in: Culture of Animal Cells 1–10 (John Wiley & Sons, Inc., 2011).
doi:10.1002/9780470649367.ch1
6 Ryan, J.A. Introduction to animal cell culture technical bulletin. Life Sciences. 34, 1–8
(2008).
7 Geraghty, R. J. et al. Guidelines for the use of cell lines in biomedical research. Br. J. Cancer
111, 1021–1046 (2014).
8 Capes-Davis, A. et al. Check your cultures! A list of cross-contaminated or misidentified
cell lines. Int. J. Cancer 127, 1–8 (2010).
9 Invitrogen, Cell Culture Basics Handbook. ThermoFisher Scientific Inc., 1–61 (2010).
10 Nema, R.and Khare, S. An animal cell culture: Advance technology for modern research.
Adv. Biosci. Biotechnol. 3, 219–226 (2012).
11 Aseptic Technique: Uses, Benefits, and Complications. Available at:
https://www.healthline.com/health/aseptic-technique. (Accessed: 2nd January 2018) 12
http://www.sop-standard-operating-procedure.com/(Accessed: 2nd November 2017) 13 J. I.
Moss, M. B. Padua, P. J. Hansen, Standard Operating Procedures ( SOP ) for Cell Culture
Rooms (2010).
14
Lee MKK,Dilq. Drug Development in Cell Culture: Crosstalk from the Industrial Prospects. J
Bioequiv Availab. 6,96-114 (2014). doi: 10.4172/jbb.10000188
15
Yamamoto Y. and Ochiya T. Epithelial stem cell culture: modeling human disease and
applications for regenerative medicine. Inflammation and regeneration 37:3, (2017)

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