Cell culture is the process of growing cells outside their natural environment. Key events in the history of cell culture include Wilhelm Roux maintaining chicken tissue in saline in 1885, and Harrison establishing the first cell culture in 1907. Traditionally, cells were cultured in 2D monolayers, but 3D cell culture has emerged as a way to better mimic the in vivo microenvironment. 3D cultures can be scaffold-based, using matrices like collagen, or scaffold-free, allowing cell-cell interactions. Technologies like microfluidics and bio-MEMS now aid 3D cell culture research. Careful planning is required to properly design and safely operate a cell culture laboratory.

1. CELL CULTURE TECHNOLOGIES

2. INTRODUCTION TO CELL CULTURE
Cell culture is the process of isolating
cells/tissues from their natural environment(in
vivo) and subsequently growing them in a
controlled artificial environment (in vitro)
Cells from specific tissues/organs can be
cultured as short term culture – primary cell
culture & secondary cell culture OR as
established culture – cell lines.

3. HISTORY OF CELL CULTURE
• 19th century – Sydney Ringer developed a salt solution
containing chlorides of Na, K, Ca, Mg, etc,.to maintain the
beating of an animal heart outside the body.
• 1885 – Wilhelm Roux removed the neural plate of an
embryonic chicken and maintained it in a warm saline solution
for several days.
• 1907 – Ross Granville Harrison at John Hopkin’s University
made the first ever cell culture and his technique is known as
the Hanging Drop Method.
• He isolated frog embryonic nerve fragments and frog lymphoid
tissue, combined them and placed them on a glass coverslip.
When the lymph clotted, the coverslip was inverted on a glass
depression slide and observed for growth.

4. Contd.,
• To his surprise, Harrison was able to visualize the origination of new
neuronal processes coming out from the explant.
• Henceforth his experiment has broken down the previous hypotheses
that- cells outside the system cannot survive, thereby laying down the
foundation to a new field of science….CELL CULTURE.
• Initially, cell culture was exploited in virology.
• 1949 – John F. Enders, Thomas H. Weller, Fredreick Robbins –
reported the first successful in vitro culture of an animal virus,
poliovirus. Awarded the Noble Prize in 1954.
• 1952 - Jonas Salk applied Enders-Weller-Robbins technique to
culture large quantities of poliovirus on monkey kidney cell line (Vero
cell line) and developed polio vaccine, IPV which was the first product
to be mass produced using cell culture techniques.

5. NEXT GENERATION
CELL CULTURE TECHNOLOGY

6. 2D MONOLAYER
(VS)
3D SPHEROID

7. Comparison between 2D & 3D Cell culture
2D Cell culture
1.plastic is used as substrate.
2. Unable to mimic in vivo
microenvironment and Lack cell-
cell & cell-matrix interaction.
3.Cells in a 2D culture might lose
their morphology.
4. Economical
5. High throughput capacity:
feasible for mass screening of
Drugs.
3D Cell culture
1.the natural extracellular matrix is
used as the substrate.
2. Mimic the in vivo
microenvironment, as well as cell-
cell and cell-matrix interactions.
3.Physiologically, 3D culture
highly resembles its source tissue.
4.Expensive & laborious.
5. Relatively low throughput
capacity, but indispensable for
Tissue Engineering and
Regenerative Medicine.

8. 3D CELL CULTURE
• Cells are grown as cell spheroids/cell aggregates to form
a 3-dimensional structure of the original tissue.
• Since they highly resemble the original tissue, the
experimental outcomes of a 3D cell culture can be easily
extrapolated with that of an in vivo response.
• The additional dimensionality of 3D cultures influences
the spatial organization of cell surface receptors
• The 3D spatial arrangement affects a wide range of
cellular functions like:- cell proliferation & differentiation,
cell signaling, gene expression etc.,

9. TYPES OF 3D CULTURES
• While the traditional 2D culture usually grows as a
monolayer on glass/polystyrene substrate, 3D cultures
grow cells as 3D cell spheroids/ cell aggregates by:-
• 1.scaffold based 3D culture
• 2.scaffold free 3D culture
• Either scaffold based or scaffold free, both promotes cell
growth in a 3D pattern, allowing cell-cell interaction, cell-
ECM interaction and their microenvironment.

10. 1.SCAFFOLD BASED 3D CULTURE
• A scaffold based 3D culture is generated by:-
• (a).seeding cells on a 3D matrix of hydrogel
• (b).dispersing cells in a liquid matrix followed by
• polymerization
• Biological scaffold matrices are;-
• # collagen
• # hyaluronic acid
• # cellulose
• Synthetic scaffold matrices are;-
• # polyethylene glycol (PEG)
• # polyvinyl alcohol (PVA)

11. SCAFFOLD BASED 3D CULTURE

12. 2. SCAFFOLD FREE 3D CULTURE

13. TECHNOLOGIES THAT AID IN
3D CELL CULTURE

14. 1. MICROFLUIDICS TECHNOLOGY
A technology that mimics the blood vessels in our body.
Microfluidics is defined as:-
“the controlled/manipulated flow of fluids through
micron-sized channels.”
It is integrated with a 3D culture and ensures a steady blood
supply, providing the tissues with a constant supply of oxygen
and nutrients and withdrawal of waste products.
This technique is indispensable for tissue engineering and
Lab-on-a-chip (LoC) technology, drug testing, physiological
studies and characterization of tumors, etc,.
common fluids used in microfluidics are:-
1.whole blood sample, 2. bacterial cell suspension,
3.protein/antibody solution, 4. buffers

15. MICROFLUIDICS TECHNOLOGY

16. 2. Bio-MEMS
• Biological-Micro Electro Mechanical System.
• A technique of integrating embedded electronic devices
with a biological system.
• For example, bio-MEMS device can be coupled with
cultured neurons to study the electrical activities of
neurons.
• Bio-MEMS were widely used in biosensor technology
• Nowadays, bio-MEMS are utilized in Drug delivery
procedures – bio-MEMS have the potential to stimulate
controlled release of drugs to the cultured cells and also
acquire and analyse the molecules (proteins & hormones)
secreted by the cultured cells.

17. Bio-MEMS

18. DESIGN & LAYOUT OF
A CELL CULTURE FACILITY

19. SETTING UP A CELL CULTURE FACILITY
• First of all the objective of the lab must be determined
whether the lab will work on:-
• # drug screening
• # tissue engineering
• # cell line development
• # developmental biology
• # cell biology etc,.
• Second the level of sophistication must be decided, for
eg. if your lab works on cell biology, then you will need
high end microscopes such as fluorescent microscopes.
• Third the level of biosafety to be followed in the lab has to
be determined.

20. General precautions while setting up a cell
culture lab
• One should have a clear idea on what one wants to
achieve in the next 10 years, so set a target and work
towards it.
• Maintain a buffer space area, so that you can expand your
lab in the coming years.
• One should determine the goal of the lab, number of
users, the resources needed etc., and all these factors
are mainly determined by the BUDGET.
• Always maximise the floor area, use the corners to place
the equipments and the walls for storage- storage racks
build on walls to store chemicals.
• If your research demands in vivo studies then you have to
go for animal models which has to be sacrificed if needed.

21. Contd…
• For animal sacrifices you need Ethical clearances from a
Government recognised authority and Animal ethics committee.
• Fishes, Drosophila- no ethical clearances needed
• Guinea pigs, mice- ethical clearances needed.
• Entry & exit should be devoid of obstacles so as to enable
immediate evacuation in case of an accident.
• It is always advisable to have a specialized facility with
designated people in it, cause too many people working on a
single facility leads to a lot of contamination problems
• Avoid proximity of microbial facility with a mammalian cell
culture facility to avoid contamination.
• Always adopt aseptic practices in a lab

23. PRECAUTIONS WHILE HANDLING
LABORATORY EQUIPMENTS
• 1. CO2 INCUBATOR:-
• used to maintain the pH of cultured cells in medium.
• Generally 5% CO2 gas is supplied inside the CO2
incubator.
• Always place the co2 incubators on the corners to avoid
direct contact with the air current.
• The number of users of the incubator must be
determined, the depletion of co2 gas from the co2 cylinder
must be checked regularly.
• Allot specific trays for specific individuals.
• It is essential to have a UPS since if the incubator runs
out of electricity then the culture goes waste.

24. Contd…
• 2. LAMINAR AIR FLOW CHAMBER:-
• The size of the hood, the number of users, frequency of use
and the total working hours on the hood has to be determined.
• Only 2 people are allowed to work on it at a time.
• It is advisable to put minimum stuff inside the hood.
• If you are using the laminar hood for dissection of animals then
it is mandatory to keep a dissection microscope within the hood
• 3. MICROSCOPES:-
• Inverted microscopes are highly employed in cell culture
laboratories.
• The giants in microscope manufacture are:-
• Olympus, Zeiss, Leica and Nikon.