Contains everything about cell culture and cell culture laboratory. The data has been collected from various sources and piled up to make this presentation.

1. CELL CULTURE
SUBMITTED BY : RUPALI BHANWALA

2. CONTENT
 INTRODUCTION
 HISTORY
 ABOUT
 CELL TYPES
 CELL CULTURE REQUIREMENTS
 CONTACT INHIBITION
 LABORATORY SAFETY
 EQUIPMENTS
 CELL CULTURE REQUIREMENTS
 PROCESS
 CELL PASSAGE
 CELL GROWTH CURVE
 TRANSFORMATIONS
 MAINTENANCE OF CELL LINES
 CELL COUNTING
 TROUBLESHOOTING
 APPLICATIONS

3. INTRODUCTION
 Cell culture is the process by which cells are grown under controlled conditions, generally outside
their natural environment.
 The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical
means before cultivation, or they may be derived from a cell line or cell strain that has already been
established.
 Isolated cells of interest are maintained under controlled conditions. These conditions vary for each
cell type but generally consist of a suitable vessel with a substrate or medium that supplies the
essential nutrients : amino acid, carbohydrates, vitamins, minerals ; growth factors; hormones;
gases : CO2, O2 and regulates physio-chemical environment : pH buffer, osmotic pressure and
temperature.
 Most cells require a surface or an artificial substrate (i.e., adherent or monolayer culture) whereas
others can be grown free floating in culture medium (suspension culture) .

5. HISTORY
1907
First successful culture of
tissue in vitro by the hanging
drop technique by Ross
Granville.
1911
Alexis Carrel and Montrose
Burrows define the term
‘Tissue Culture’. They establish
the first ‘cell line’ and use
diluted blood plasma for
media.
1920s
Compositions of salt solutions
were formulated specifically
for cell culture.
1940s
Antibiotics were first used in
cell culture.
1960s
Carbon dioxide Incubators
become widely available
Present

6. ABOUT
 Primary Culture
Primary culture refers to the stage of the culture after the cells are isolated from the tissue and
proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach
confluence).
At this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with
fresh growth medium to provide more room for continued growth.
 Cell Line
After the first subculture, the primary culture becomes known as a cell line or subclone.
Cell lines derived from primary cultures have a limited life span (i.e., they are finite)
 Finite vs Continuous Cell Line
Cells (such as fibroblasts obtained from skin biopsies and hepatocytes isolated from liver explants) are
obtained directly from donor tissues. They are only able to divide a finite number of times due to
telomere loss. Primary cells or finite cell lines eventually reach senescence.
Whereas, when a finite cell line undergoes transformation and acquires the ability to divide
indefinitely, it becomes a continuous cell line or established cell line. They are used for long-term
research.

7. b
B
1. NORMAL 2. TRANSFORMED 3. STEM CELL
Taken from a tumor
tissue and cultured as
a single cell type.
Normal cells
underwent a genetic
change to be tumor
cells.
They are ‘master
cells’ that generate
other differentiated
cell types.
CELL LINE

8. Cell Types
 On the basis of morphology or on their functional characteristics, cells are divide into 3 parts:
1. EPITHELIAL LIKE : They are attached to a substrate and appears flattened and polygonal in
shape.
2. LYMPHOBLAST LIKE : Cells do not attach but remain in suspension with a spherical with a
spherical shape
3. FIBROBLAST LIKE : Cells attached to a substrate, appear elongated and bipolar.
EPITHELIAL-LIKE LYMPHOBLAST-LIKE FIBROBLAST LIKE

9. STEM CELLS
 Stem cells represent an exciting area in medicine because of their potential to regenerate and
repair damaged tissue.
 Some current therapies such as bone marrow transplantation, uses stem cells whereas other
therapies also involves transplanting stem cells into a damaged body part and directing them to
grow and differentiate into healthy tissue.
 CLASSIFICATION OF STEM CELLS based on their dividing capacity:
1. Pluripotent stem cells : Pluripotent stem cells can divide into most, or all, cell types in an
organism, but cannot develop into an entire organism on their own.
2. Totipotent stem cells : Totipotent stem cells can divide into all cell types in an organism.
3. Multipotent stem cells : Multipotent cells can develop into more than one cell type, but are more
limited than pluripotent cells
 MAJOR TYPES OF STEM CELLS
1. Embryonic stem cells (ES cells0
2. Adult stem cells.
3. Induced pluripotent stem cells (iPSCs)

10. Example of some cell lines used for in vitro culture
CELL LINE SPECIES of ORIGIN TISSUE of ORIGIN
CELL
MORPHOLOGY
1. 3T3 Mouse Connective Fibroblast
2. CHO Chinese hamster Ovary Epithelial
3. BHK21 Syrian hamster Kidney Fibroblast
4. HeLa Human Cervical carcinoma Epithelial
5. MRC-S Human Lung Fibroblast, finite
6. MDCK Dog Kidney Epithelial

11. CONTACT INHIBITION:
Contact inhibition is a process of arresting cell growth when cells come in contact with each other.
 NORMAL SOMATIC CELLS
When these cells are grown in culture will become growth inhibited when they encounter another cell.
The cells in our bodies are governed by growth control mechanisms and cellular senescence (i.e.,
aging).
 CANCEROUS CELLS
These cells typically lose this property and thus grow in an uncontrolled manner even when in contact
with neighboring cells. They pile up and grow over each other.

12. Cell Culture Laboratory Safety
 Before commencing any cell culture work, the reduced or eliminated exposure to potentially
hazardous agents therefore needs to be ensured to minimize infection, pathogenicity, allergic
reactions, and contact with released toxins.
 The cell culture lab must be kept tidy and cleaned routinely with a disinfectant (e.g., incubators,
laminar flow hoods, and work surfaces).
 Lab coats, gloves, and goggles create a barrier between the laboratory worker and potentially
hazardous sources.
 Wipe the surface of biosafety cabinet with 70% ethanol before placing anything into it.
 All equipment entering the biosafety cabinet also needs to be sprayed and wiped with 70%
ethanol.
 Always use sterile medium and equipments while working,
 Place every chemical or equipment in biosafety cabinet before starting.

13. Contd.
.  Hands should be washed before leaving the cell culture lab.
 The biosafety cabinet should only be turned off after its daily use has been completed and the
ultraviolet lamp may be turned on to sterilize the exposed surface areas over night.
 In addition, filter-sterilizing allows for the generation of cell culture media that are based on
nonsterile culture reagents, while autoclaving is conventionally used to sterilize equipment in
contact with cultured cells.
 Filter-sterilization of liquids can be achieved by forcing the liquid through a 0.22 μM
polyethersulfone low-binding filter system using a vacuum pump.
 Contamination
 Contaminants are most commonly of biological nature and can include bacteria, fungi, viruses, and
parasites.
 It is important to limit biological contaminants since they can alter the phenotype and genotype of
the cultured cell line through competition for nutrients; synthesis of alkaline, acidic or toxic by-
products; and the potential interference of viral components with the cell culture genome.

14. Contd.
.
 Other contaminants may include the introduction of undesired chemicals impurities (e.g.,
plasticizers in cell culture vessels) or other cell types cocultured in the lab.
 Addition of antibiotics (e.g., Penicillin/Streptomycin) further limits the risk of bacterial growth in
media bottles after opening and in cell culture vessels.
 However, some laboratories refrain from using antibiotics routinely since it can facilitate the
emergence of resistant bacteria strains, allow for low-level background contaminations, and may
lead to interference with cell metabolisms and experimental outcomes.

15. Equipment for the cell culture laboratory
Equipment Purpose
1. Biosafety cabinet To create sterile work surface; class II and III recommended
2. Humid CO2 Incubator To provide a physiological environment for cellular growth
3. Inverted light microscope to access cell morphology and count cells.
4. Fridge, freezers, liquid
nitrogen storage
To store cells, cell material, and culture components
5. Centrifuge To condense cells
6. pH meter To determine the correct pH of media components
7. Pipettes and pipettors To aliquot different volumes
8. Autoclave To sterilize pipettes and other equipment in contact with cells.
9. Vacuum pump To aspirate cell culture medium
10. Hemacytometer To count cells, determine growth kinetics and prepare suitable plating
densities
11. Water bath To warm up cell culture medium
12. Cell culture dishes To culture cells in different formats (e.g., flasks, Petri dishes, 96-well plates)
13. Containers for waste
(biohazardous)
To correctly dispose of waste

17. Cell Culture Requirements
 MEDIA (1) NATURAL MEDIA (2) ARTIFICIAL MEDIA
Media provides the necessary nutrients, growth factors, and hormones for cell growth, as well as
regulating the pH and the osmotic pressure of the culture.
The three basic classes of synthetic or artificial media are basal media, reduced-serum media, and
serum-free media, which differ in their requirement for supplementation with serum.
1. BASAL MEDIA : contains amino acids, vitamins, inorganic salts, and a carbon source such as
glucose, but these basal media formulations must be further supplemented with serum.
2. REDUCED-SERUM MEDIA : Reduced-serum media are basal media formulations enriched with
nutrients and animal-derived factors, which reduce the amount of serum that is needed. Serum in
the form of fetal bovine serum (FBS) is most commonly added to basal media.
2. SERUM-FREE MEDIA : Serum-free media (SFM) circumvents issues with using animal sera by
replacing the serum with appropriate nutritional and hormonal formulations.
**One of the major advantages of using serum-free media is the ability to make the medium selective
for specific cell types by choosing the appropriate combination of growth factors.**

18. Nutrients (culture media)
 Basal Media are used to maintain p. H and osmolarity and provide nutrients and energy
source. The components of basal media are as follows.
1. Inorganic Salts
Maintain osmolarity. • Regulate membrane potential (Na+, K+, Ca 2+). • Provide ions for cell
attachment and enzyme cofactors.
2. pH Indicator - Phenol Red
Optimum cell growth occurs at approx. p. H 7. 4 • Phenol red is used to monitor the changes from
red to yellow.
3. Buffers (Bicarbonate and HEPES)
Bicarbonate buffered media requires CO2 atmosphere. But, HEPES does not requires CO2.

19. Contd.
4. Keto acids (oxalacetate and pyruvate)
Intermediate in Glycolysis/Krebs cycle • Keto acids added to the media as additional energy
source • Maintain maximum cell metabolism
5. Carbohydrates
Energy source • Glucose and galactose • Low (1 g/L) and high (4. 5 g/L) concentrations of
sugars in basal media
6. Vitamins
Precursors for numerous co-factors • B group vitamins necessary for cell growth and
proliferation • Common vitamins found in basal media are riboflavin, thiamine and biotin
7. Trace Elements
Zinc, copper, selenium and tricarboxylic acid intermediates

20. Commonly used commercial media
1. Dulbecco’s Modified Eagle
Medium (DMEM)
2. Rosewell Park Memorial
Institute-1640 (RPMI)
3. Ham’s F12 Nutrient Mixture
(F12)

21. Contd..  pH
1. The pH level for most human and mammalian cell lines cultured in the lab should be tightly
controlled and kept at a physiological pH level of 7.2–7.4.
2. Fibroblast cell lines favor slightly more alkaline conditions between pH 7.4 and 7.7, while
transformed cell lines prefer more acidic environments between pH 7.0 and 7.4.
 CO2
1. Buffering is achieved by including an organic (e.g., HEPES) or CO2 -bicarbonate based buffer.
Because the pH of the medium is dependent on the delicate balance of dissolved carbon dioxide
(CO2 ) and bicarbonate, changes in the atmospheric CO2 can alter the pH of the medium.
2. 4–10% CO2 is common for most cell culture experiments.
 TEMPERATURE
1. Largely depends on the body temperature of host from which cells are isolated.
2. Most human and mammalian cell lines are maintained at 36°C to 37°C for optimal growth. Cell
lines derived from cold-blooded animals (e.g., amphibians, cold-water fish) tolerate a wide
temperature range between 15°C and 26°C.

22. What Cells to Choose for My Application?
• What species? Non-human and non-primate cell lines usually have less biosafety
restrictions, your experiments will dictate what cell line species to use.
• Which functional characteristics? For example, liver- and kidney-derived cell lines
may be more suitable for toxicity studies (ADMET).
• Finite or continuous? Choosing a primary (finite) cell may relate better to the in
vivo situation, continuous cell lines are often easier to clone and maintain.
• Normal or transformed? Genetically transformed cell lines usually have an increased
growth rate and higher plating efficiency, the counter part is they have undergone a
permanent change in their phenotype through a transformation, no longer an exact copy
of the original.
• Which growth conditions? For example, to express a recombinant protein in high
yields, you might want to choose a cell line with a fast growth rate and an ability to grow
in suspension.
• Other criteria? If you are using a finite cell line, be sure the cell line is well
characterized or you have to perform the validation yourself.

23. CELL CULTURE in vitro
Revive frozen cell population isolated from tissue
Isolate cells with the use of appropriate enzymes
Place the isolated cells on to an appropriate growth media in a culture dish
Culture cells by placing culture dish in an incubator
Verify the cultured cells are of interest
Cells are ready to be manipulated or modified for experimental procedures
Sub-culture to obtain
pure culture or to
bypass sone problems
(like senescence)

25. Cell Passage / Sub culturing
 Passaging a cell line is a cell culture technique where the cell culture medium is removed and
cells are transferred from a previous culture into fresh growth medium.
1. Check the confluency of the cells (70-80% confluency).
2. Remove the spent medium from the culture dish.
3. Wash cells with PBS (Phosphate Buffered Saline) to remove dead cells and serum. PBS is used
as a washing solution before disaggregation and as a diluent for trypsin.
4. Incubate the cells with trypsin/EDTA. This is done to digest protein-surface interactions to
release cells.
5. Resuspend the cells in serum (inactivates trypsin).
6. Transfer the dilute cell suspension to a new flask with fresh media.
NOTE : Most cell lines will adhere in approx. 3-4 hours.

26. Cell growth curve

27. TRANSFORMATION
 Transformation describes alterations in properties, including growth rate, mode of growth,
ability to grow indefinitely and tumorigenicity of cultured animal cells.
 Transformation also promotes genetic instability (i.e., mutations and chromosomal
aberrations).
 Transformation is associated with 3 major classes of phenotypic changes in cell lines :
1. IMMORTALIZATION : It signifies the events that make the cells capable of indefinite
survival and proliferation in vitro. This negatively regulate cell cycle and also repress the
expression of gene encoding telomerase.
• Immortalization can be induced by transfection with selected genes including viral genes
(SV40 large T antigen gene), telomerase gene htrt, etc.

28. Contd.
2. ABBERANT GROWTH CONTROL : It is characterized by ability to grow at higher cell
densities, lack of contact inhibition (so that they can grow on monolayers) reduced anchorage
dependence, ability to grow in suspension culture, etc.
• These cell lines have acquired these features either due to over-expression of some oncogenes or
by inactivation of growth suppressor genes.
3. TUMORIGENECITY : It signifies the ability of transformed cell to produce invasive tumors
when they are implanted in vivo. This is the end product of transformation process.
• Sometimes, changes that lead to aberrant growth control also contribute to malignancy.

29. MAINTENANCE OF CELL LINES
 American Type Culture Collection (ATCC) maintains a large number of cell lines acquired from
their originators; these cell lines are characterized before preservation or distribution on request to
research workers.
 Cell line is multiplied and divided into following 2 types of cultures:
1. Seed stock (cells are characterized and stored in frozen state i.e., -196°C)
2. A working or distribution of stock culture
 CRYOPRESERVATION
Why cryopreservation ?
• Reduced risk of microbial contamination
• Reduced risk of cross contamination with other cell lines
• Reduced risk of genetic drift

30. Contd.  Process
1. Harvest the cells from late log phase of growth (>90% viability).
2. Passage the cells in serum containing media.
3. Centrifuge and aspirate the supernatant.
4. Resuspend the cells in 10% DMSO (Dimethyl sulfoxide) (cryopreservant). Cryoprotective
agent is added to minimize injury to cells during freezing and thawing and also prevents ice
crystal formation.
5. Transfer the cryovial to freeze at -80°C.
6. Transfer the cryovials to liquid nitrogen storage tank.

31. CELL COUNTING
 Cell counting is generally done by hemacytometer.
 This protocol works well for either adherent mammalian cells that have been trypsinized or for
suspension cells including Sf9 insect cells.
 Red blood cells are typically too small and numerous for this protocol and utilize the middle square
instead.
Harvest cells by adding trypsin/EDTA
Resuspend cells in fresh medium
Under sterile conditions remove 100-200μl of cell suspension
Add an equal volume of Trypan Blue (dilution factor =2) and mix by gentle pipetting
Prepare hemacytometer with cover slip and fill chamber with cell suspension
Count cells and calculate concentration

32. Troubleshooting

33. APPLICATION OF CELL CULTURE
 Model System:
Cell culture are used as model system to study basic cell biology and biochemistry, to study the
interaction between cell and disease causing agents like bacteria, virus, to study the effect of drugs, to
study the process of aging and also it is used to study triggers for ageing.
 Cancer Research:
The basic difference between normal cell and cancer cell can be studied using animal cell culture
technique, as both cells can be cultured in laboratory. Normal cells can be converted into cancer cells by
using radiation, chemicals and viruses. Thus, the mechanism and cause of cancer can be studied. Cell
culture can be used to determine the effective drugs for selectively destroy only cancer cells.
 Virology:
Animal cell cultures are used to replicate the viruses instead of animals for the production of vaccine.
Cell culture can also be used to detect and isolate viruses, and also to study growth and development
cycle of viruses. It is also used to study the mode of infection.

34. Contd.
 Vaccine Production:
Cultured animal cells are used in the production of viruses and these viruses are used to produce
vaccines. For example vaccines for deadly diseases like polio, rabies, chicken pox, measles and
hepatitis B are produced using animal cell culture.
 Replacement Tissue or Organ:
Animal cell culture can be used as replacement tissue or organs. For example artificial skin can be
produced using this technique to treat patients with burns and ulcers.
 Genetic Engineering:
Cultured animal cells can be used to introduce new genetic material like DNA or RNA into the cell.
These can be used to study the expression of new genes and its effect on the health of the cell. Insect
cells are used to produce commercially important proteins by infecting them with genetically altered
baculoviruses.

35. Contd.
 Gene Therapy:
Cultured animal cells can be genetically altered and can be used in gene therapy technique.
First cells are removed from the patient lacking a functional gene or missing a functional gene.
These genes are replaced by functional genes and altered cells are culture and grown in
laboratory condition. Then these altered cells are introduced into the patient.
 Drug Screening and Development:
Animal cell cultures are used to study the cytotoxicity of new drug.