Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favourable artificial environment.

1. DR. SONAM PANDEY
Research Development Officer
Centre for Stem Cell Research
(A unit of inStem, Bengaluru),
Christian Medical College Campus,
Bagayam, Vellore, Tamil Nadu

3. History of animal cell culture

5. One of the major contribution of the cell culturing is the
Develop
ment of
Polio
Vaccines
Cell
Culturin
g
Researchers used
live animals to grow
poliovirus
Creation of the
polio vaccine
1930s
1970s
Development of
mammalian cell
culture techniques
Modern
Day
Widely used in
laboratory settings
Importance of Cell Culture

6. Model cell culture exposed by RG
Harrison in 1907

7. Cell culture refers to the removal of cells from
an animal or plant and their subsequent growth
in a favourable artificial environment.
What is Cell Culture?

8. Tissue Culture?
In vitro cultivation of organs, tissues & cells at defined
temperature using an incubator & supplemented with a
medium containing cell nutrients & growth factors is
collectively known as tissue culture
Different types of cell grown in culture includes connective
tissue elements such as fibroblasts, skeletal tissue,
cardiac, epithelial tissue (liver, breast, skin, kidney) and
many different types of tumor cells

9. On the basis of morphology (shape & appearance) or on their
functional characteristics. They are divided into three.
 Epithelial like-attached to a substrate and appears flattened
and polygonal in shape
 Lymphoblast like- cells do not attach remain in suspension
with a spherical shape
 Fibroblast like- cells attached to an substrate appears
elongated and bipolar
Types of cells

10. Types of tissue
culture
Primary Continuous
Finite Indefinite
Single cell type roughly
thirty times of division,
enhanced by growth factors
It is nearly the same as
finite but the cells here
can divide indefinitely by
transformation into tumor
cells, They are called cell
line
Normal cells cultured without any
change in their division rate
Tissue Culture?

11. Cell culture: Adherent monolayer on a
solid substrate (various cell types)
suspension in the culture medium (few
cell types)
Primary explant culture: A fragment
of tissue attachment and migration
occurs in the plane of the solid
substrate
Organ culture: A spherical or three-
dimensional shape specific histological
Cell
culture
explant
culture
Organ
culture
Explant: living cells, tissues, or organs from animals or plants that
Three major categories of tissue culture

12. Three predominant cell types used in cell biology research

13. Categories of Cells
Culture
HISTORY OF THE
CELL
CELL GROWTH
ENVIRONMENT

14. Two Categories of Cells
Primary
cells Establishe
d cell lines
Obtained directly
from donor tissues
only able to divide
a finite number of
times due to
telomere loss
Cells eventually
reach senescence
Able to grow
indefinitely
Useful for Long-
term research

15. Established cell lines
Establishe
d cell
lines
Obtained
from
Clinical tumors
Transforming primary
cells with viral
oncogenes or chemical
treatments
M
G1
G2
S
G0
I

16. M
G1
G2
S
G0
I
M
G1
G2
S
G0
I allows cells to
commit to cell
division
1
M
G1
G2
S
G0
I
Checks
quality of
replicated
DNA
2
M
G1
G2
S
G0
I
Checks quality of
replicated DNA
again 3
Ensure cell is in
proper position
for cell division
4
M
G1
G2
S
G0
I
Ensure cell is in
proper position
for cell division
4
M
G1
G2
S
G0
I
4
1
2
3
E7
E6
Established cell lin

17. CELL GROWTH
ENVIRONMENT
Categories of cell
culture
CELL culture
laboratory

18. Two main growth conditions
Monola
yer
free-floating
(adherent
cultures)
(suspension
cultures)

19. • Cells when surgically or enzymatically removed from an organism and
placed in suitable culture environment will attach and grow are called
as primary culture
• Primary cells have a finite life span
• Primary culture contains a very heterogeneous population of cells
• Sub culturing of primary cells leads to the generation of cell lines
• Cell lines have limited life span, they passage several times before
they become senescent
• Cells such as macrophages and neurons do not divide in vitro so can
be used as primary cultures
• Lineage of cells originating from the primary culture is called a cell
strain
Primary culture

20. • Once the available substrate surface is covered by cells (a
confluent culture) growth slows & ceases.
• Cells to be kept in healthy & in growing state have to be sub-
cultured or passaged
• It’s the passage of cells when they reach to 80-90%
confluency in flask/dishes/plates
• Enzyme such as trypsin, dipase, collagenase in combination
with EDTA breaks the cellular glue that attached the cells to
the surface
Why sub culturing??

21. • Cells are cultured as anchorage dependent or independent
• Cell lines derived from normal tissues are considered as
anchorage-dependent grows only on a suitable substrate e.g.
tissue cells
• Suspension cells are anchorage-independent e.g. blood cells
• Transformed cell lines either grows as monolayer or as
suspension
Culturing of cells

22. • Cells which are anchorage dependent
• Cells are washed with PBS (free of ca & mg ) solution
• Add enough trypsin/EDTA to cover the monolayer
• Incubate the plate at 370 C for 1-2 mins
• Tap the vessel from the sides to dislodge the cells
• Add complete medium to dissociate and dislodge the cells
• with the help of pipette which are remained to be adherent
• Add complete medium depends on the subculture
• requirement either to 75 cm or 175 cm flask
Adherent
cells

23. • Easier to passage as no need to detach
them
• As the suspension cells reach to confluency
• Aseptically remove 1/3rd of medium
• Replaced with the same amount of pre-
warmed medium
Transfection methods
•Calcium phosphate precipitation
•DEAE-dextran (dimethylaminoethyl-dextran)
•Lipid mediated lipofection
•Electroporation
•Retroviral Infection
•Microinjection
Suspension cells

24. • Most cell lines grow for a limited number of generations after which they
ceases
• Cell lines which either occur spontaneously or induced virally or
chemically transformed into Continuous cell lines
• Characteristics of continuous cell lines
 smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio
 Fast growth and have aneuploidy chromosome number
 reduced serum and anchorage dependence and grow more in suspension
conditions
 ability to grow up to higher cell density
 different in phenotypes from donor tissue
 stop expressing tissue specific genes
Continuous cells lines

25. Examples of Culture ware
Flask Plates
Roller Bottles
Amino acid
Inorganic
salts
Vitamins
Culture
media
Macromolecule
s
Lipid
Growth
Factors
Fetal Bovin
Serum
Collect from a bovin
fetus

26. Sodium bicarbonate
1. Keep the pH of the medium
between 7.2 and 7.4 with 5-10% gaseous Co2
2. Zwitterions
pH Indicator
Parameters for the
growth and maintenance
cell culture. The factors
that make successful
cell culture should be
maintained balanced
and just right to allow
the development of
experiments that yield
results in which cell
Common Buffering system

27. Commonly Used Commercial Media
Dulbecco’s Modified Eagle Medium (DMEM)
Roswell Park Memorial-1640 (RPMI)
Ham’s F12 Nutrient Mixture (F12)
1
2
3
Appropriate culture Media
Temperature
Mammalian Cells
cold-blooded animals

28. a)Organ from which the explant is obtained;
b)the organ is segmented into pieces of about 1
mm3;
c) the fragments are placed in specific areas,
growth factors, antibiotics and other supplements
are added;
d) the culture dish is placed in an atmosphere with
95% air flow and 5% CO2 and incubated at 37°C;
e)the culture is maintained and observed at inverted
microscope
Mammalian cell culture technique

29. Cell culture
laboratory
CELL GROWTH
ENVIRONMENT
Next generation cell
culture technology

30. Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of
disinfectants that are invisible
 Biological-cause visible effects on the culture they
are
(cross contamination)
 Yeast
 Fungi
 Viruses
 Bacteria
 mycoplasma
Cell culture contaminants of two types
Contaminant's of cell culture
• They competes for nutrients with host
cells
• Secreted acidic or alkaline by-products
ceses the growth of the host cells
• Degraded arginine & purine inhibits the
synthesis of histone and nucleic acid
• They also produces H2O2 which is
directly toxic to cells
Effects of Biological Contaminant's

31. Appropriate culture Media
Cell culture
laboratory
Correct Temperature
Sterile environment
Microbial
Contamination
Mycoplas
ma
Fungi Bacteri
a

32. Cell culture
laboratory Cross-
Contamination

33. Detection of
contaminants
• In general indicators of contamination are turbid culture media, change
in growth rates, abnormally high pH, poor attachment, multi-nucleated
cells, graining cellular appearance, vacuolization, inclusion bodies and
cell lysis
• Yeast, bacteria & fungi usually shows visible effect on the culture
(changes in medium turbidity or pH)
• Mycoplasma detected by direct DNA staining with intercalating
fluorescent substances e.g. Hoechst 33258
• Mycoplasma also detected by enzyme immunoassay by specific
antisera or monoclonal abs or by PCR amplification of mycoplasmal
RNA
• The best and the oldest way to eliminate contamination is to discard the
infected cell lines directly

34. •Cell viability is determined by staining the cells with
trypan blue
•As trypan blue dye is permeable to non-viable cells or
death cells whereas it is impermeable to this dye
•Stain the cells with trypan dye and load to
haemocytometer and calculate % of viable cells
- % of viable cells= Nu. of unstained cells x 100
total nu. of cells
Cell viability

35. • Laminar cabinet-Vertical are preferable
• Incubation facilities- Temperature of 25-30 C for insect & 370 C for mammalian
cells, Co2 2-5% & 95% air at 99% relative humidity. To prevent cell death incubators
set to cut out at approx. 38.5 C
• Refrigerators- Liquid media kept at 40C, enzymes (e.g. trypsin) & media
components (e.g. glutamine & serum) at -20 C
• Microscope- An inverted microscope with 10x to 100x magnification
• Tissue culture ware- Culture plastic ware treated by polystyrene
Basic equipments used in
cell culture

36. Never use contaminated material within a sterile area
Use the correct sequence when working with more than
one cell lines.
• Diploid cells (Primary cultures, lines for the production
of vaccines etc.)
• Diploid cells (Laboratory lines)
• Continous, slow growing line
• Continous, rapidly growing lines
• Lines which may be contaminated
• Virus producing lines
Rules for working with cell
culture

37. Areas where cell culture
technology is currently playing a
major role.

38. 1. 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.
2. 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.
3. 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.
4. Toxicity Testing: Animal cell culture is used to study the effects of new drugs, cosmetics and chemicals on survival and
growth of a number of types of cells. Especially liver and kidney cells. Cultured animal cells are also used to determine
the maximum permissible dosage of new drugs.
5. 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.
6. Genetically Engineered Protein: Animal cell cultures are used to produce commercially important genetically engineered
proteins such as monoclonal antibodies, insulin, hormones, and much more.
7. 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. However research is going on artificial
organ culture such as liver, kidney and pancreas. Organ culture techniques and research are being conducted on both
embryonic and adult stem cell culture. These cells have the capacity to differentiate into many different types of cells
and organs. It is belived that by learning to control the development and differentiation of these cells may be used to
treat variety of medical conditions.
8. Genetic Counseling: Fetal cell culture extracted from pregnant women can be used to study or examine the
abnormalities of chromosomes, genes using karyotyping, and these findings can be used in early detection of fetal
disorders.
9. Genetic Engineering: Cultured animal cells can be used to introduce new genetic material like DNA or RNA into the cell.

44. Next generation cell
culture technology

45. 2D Monolayer cell culture 3D cell culture
Cells grow naturally in three dimensions
Researchers want to mimic this outside the body

46. 2D Monolayer cell culture 3D cell culture system
Monolayer cells Cells aggregate/
Spheroid
Matrix for 3D
cell culture
What is 3D cell culture ??

47. Comparison of 2D Vs. 3D cell culture

48. FIGURE: Cells and their microenvironment. Tissue-specific cells (red) encounter a complex
microenvironment consisting of extracellular matrix (ECM) proteins and glycoproteins (green),
support cells that mediate cell-cell interactions (blue), immune cells (yellow), and soluble factors
(white spheres). The tissue microenvironment is further defined by physical factors such as ECM
stiffness (indicated by increasing density of ECM proteins), and oxygen (indicated by red shading
of tissue-specific cells) and nutrient and growth factor gradients (indicated by density of white
spheres).

49. Why we need 3D cell culture?
3D culture better mimic tissue-like structures
Able to exhibit differentiated cellular function
Possible to co-culture two or more different cell types
Can simulate microenvironment conditions such as hypoxia and nutrient gradients
Better predict in vivo responses to drug treatment

50. 3D cell culture
Models
3D Cell Culture
Specialized 3D
Culture platforms
Anchorage
dependent
(Scaffold based
Anchorage
Independent
(non-Scaffold based
Hanging drop
Low attachment
plates
Biological
Hydrogels
Synthetic
Hydrogels
Microfluidic
devices
Micropatterned
plates

51. Spheroids and Organoids-What is the difference??
SPHEROIDS
A spheroid is a 3D cellular
aggregate composed of 1 (or
more) cell types that grow
and proliferate, and may
exhibit enhanced
physiological responses but
do not undergo
differentiation or self-
organization (i.e. non-stem
cells)
Spheroid-HepG2
ORGANOIDS
“An ‘organoid is a 3D structure
derived from either PSCs, neonatal
tissues stem cells or AdSCs/adult
progenitors, in which cells
spontaneously self-organize into
properly differentiated functional
cell types and progenitors and
which resemble their in vivo
counter part and recapitulate at
least some function of the organ.”
M. Huch and B. Y. Koo Development; 2015
PSC-derived Organoid

52. What is a 3D cell culture model?
 3D cell culture is a culture environment that allows biological cells to grow and
interact with surrounding extracellular framework in three dimensions.
 An improvement over the previous method of growing cells in 2D cell cultures in
which cells are grown in a flat monolayer on a plate. Accurately mimics the cell
natural environment.

53. Established organoid methods

54. Cells that form organoids

55. 3D Cells culture is a spectrum of cell culture types

57. 3D Cells culture: cell spheroid applications

58. Using 3D in vitro cell culture models in drug discovery
The high failure rate in drug discovery remains a costly and time-consuming challenge.
Improving the odds of success in the early steps of drug development requires disease
models with high biological relevance for biomarker discovery and drug development. The
adoption of three-dimensional (3D) cell culture systems over traditional monolayers in cell-
based assays is considered a promising step toward improving the success rate in drug
discovery
3D cell culture models are better models than the traditional 2D monolayer culture
due to improved cell–cell interactions, cell–ECM interactions, and cell populations
and structures that resemble in vivo architecture.
While the 3D cell culture models are currently widely studied in academia with a
focus on creation of 3D systems with excellent biological relevance, there are still
many hurdles that must be overcome before these systems can be widely accepted
in industry.
Schematic diagrams of the traditional two-dimensional (2D) monolayer cell culture (A) and three typical three-dimensional (3D)
cell culture systems: cell spheroids/aggregates grown on matrix (B), cells embedded within matrix (C), or scaffold-free cell
spheroids in suspension (D).

59. 3D Cells culture and organoids
Near-physiological models to study human development and diseases

60. Complex organoids have been developed from stem cells
Pancreatic organoid

61. Complex organoids have been developed from stem cells
Lung organoid

63. If cell culture has been around for >100 years, why are we only now transitioning to 3D?

64. 3D Cells culture methods

65. Enabling drug discovery: spheroid microplates
Allow for cell culture and assay of 3D spheroids
in one plate

66. Enabling drug discovery: organ-on-a-chip
To better replicate a living tissue
organization and functioning,
microstructures made of collagen or
polymer-based membrane are built within
the chips micro-channels. Unlike traditional
3D cell culture, those structures
can actually recreate a function
observed in vivo. For instance, a human
breathing lung-on-a-chip is a model of the
alveolar–capillary. It integrates a flexible
polymer membrane allowing movements
just like inside a living human lung

67. Enabling drug discovery: 3D Bioprinting

68. Figure : These techniques include: A: Liquid overlay; B: Hanging drop; C: Hydrogel embedding; D: Spinner
flask bioreactor; E: Scaffold; F: Three-dimensional bioprinting.
Chaicharoenaudomrung N, Kunhorm P, Noisa P. Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling. World J Stem Cells 2019; 11(12): 1065-1083 [PMID: 31875869
DOI: 10.4252/wjsc.v11.i12.1065]
Different techniques used for three-dimensional cell cultures.

69. The promise of 3D cell culture

70. Potential applications of three-dimensional cell culture systems
The invention of three-dimensional cell
culture systems could be applied into
various aspects, for instance anticancer
drug screening, tissue engineering,
cancer biology, and clinical uses.
Chaicharoenaudomrung N, Kunhorm P, Noisa P. Three-dimensional cell culture systems as an in vitro
platform for cancer and stem cell modeling. World J Stem Cells 2019; 11(12): 1065-1083 [PMID:
31875869 DOI: 10.4252/wjsc.v11.i12.1065]

71. Molecular biology techniques

72. Nucleic acid Structure and
Function

73. Molecular biology techniques utilize DNA, RNA and enzymes that interact with nucleic
acids to understand biology at a molecular level.
Molecular biology techniques

74. Relationship to other biological sciences

75. Techniques of Molecular Biology
1.Expression Cloning
2.Polymerase chain reaction (PCR)
3.Get electrophoresis
4.Macromolecule blotting and probing techniques
• Southern blotting
• Northern blotting
• Western blotting
• Eastern blotting
5.Arrays
6.Allele specific oligonucleotides
7.Antiquated technologies

76. What is blotting?
Blots are techniques for
transferring DNA, RNA
and proteins onto a
carrier so they can be
separated, and often
follows the use of a gel
electrophoresis. The
Southern blot is used for
transferring DNA, the
Northern blot for RNA

77. Steps of DNA cloning:
DNA cloning is used for many purposes. As an example, let's see how
DNA cloning can be used to synthesize a protein (such as human
insulin) in bacteria. The basic steps are:
1.Cut open the plasmid and "paste" in the gene. This process relies on
restriction enzymes (which cut DNA) and DNA ligase (which joins
DNA).
2.Insert the plasmid into bacteria. Use antibiotic selection to identify
the bacteria that took up the plasmid.
3.Grow up lots of plasmid-carrying bacteria and use them as
"factories" to make the protein. Harvest the protein from the
1. Expression
cloning

78. 1. Cutting and pasting DNA 2. Bacterial transformation and selection
3. Protein production
Once the protein has been produced, the bacterial cells can be split open to
release it. There are many other proteins and macromolecules floating
around in bacteria besides the target protein (e.g., insulin). Because of this,
the target protein must be purified, or separated from the other contents of
the cells by biochemical techniques. The purified protein can be used for

79. 2. Polymerase Chain Reaction (PCR)
PCR is a simple and widely used process in which minute amounts of DNA can be
amplified into multiple copies.
WHAT PCR DOES
 PCR is a very sensitive technique that allows rapid
amplification of a specific segment of DNA.
 PCR makes billions of copies of a specific DNA fragment
or gene, which allows detection and identification of
gene sequences using visual techniques based on size and
charge.
 Modified versions of PCR have allowed quantitative
measurements of gene expression with techniques called
real-time PCR
LIMITATIONS
 The DNA polymerase used in the PCR reaction is prone to
errors and can lead to mutations in the fragment that is
generated
 The specificity of the generated PCR product may be

80. 3. Gel electrophoresis

81. 4.Western Blotting

82. 5. Southern Blotting
Principle, Procedure & Application

83. 6. Northern Blotting
It is a technique for specific identification of RNA molecules.
RNA molecules are subjected to electrophoresis, followed by blot transfer
hybridization and autoradiography.
RNA molecules do not easily bind to nitrocellulose paper or nylon membrane

84. 7. Eastern
blotting
• Eastern blot is a molecular biology technique
that is used to detect post-translational
modifications in proteins and the presence of
components like lipids and carbohydrates.
• Eastern blot was discovered as an extension of
the more popular western blotting technique.
• Eastern blot is primarily performed to detect
the presence of biomolecules in different
proteins, which can be used to study the
differences in the post-translational
modifications in different species.
Applications of Eastern Blot
• The most important application of eastern blotting is
the analysis of post-translational modifications in
proteins.
• The technique has been used to identify and purify
different plant products.
• Eastern blotting also allows the detection of
modifications in proteins of different origins.
• It also helps to study the nature of interactions
between different molecules by the use of ligands.
• Eastern blotting has been extensively used to
compare modifications in proteins obtained from

85. 8. Arrays

86. 9. Allele Specific Oligonucleotide
An allele-specific oligonucleotide
(ASO) is a short piece of synthetic
DNA complementary to the sequence
of a variable target DNA. It acts as a
probe for the presence of the target
in a Southern blot assay or, more
commonly, in the simpler Dot blot
assay.
• It is a technique that allows detection of single base
mutations without the need for PCR or gel electrophoresis.
• Short (20-25 nucleotides in length(, labeled probes are
exposed to the non-fragmented target DNA.
• Hybridization occurs with high specificity due to the short
length of the probes and even a single base change will
hinder hybridization.
• The target DNA is then washed and the labeled probes that
didn’t hybridize are removed.
• The target DNA is then analyzed for the presence of the
probe via radioactivity or fluorescence.
• The Illumina Methylation Assay technology takes advantage
of ASO technique to measure one base pair differences in
sequence.