This presentation gives all the basic necessary details about cell culture and karyotyping in cell lines . It describes the important methods like subculture,confluency,cell count,passaging and karyotyping in cell culture.It also describes about BSL.It also gives a brief idea about types of cell culture.This presentation also includes the requirements in the cell culture lab

1. Cell Culture
Submitted by Nancy Singh
Submitted to MRU MAMC
Date- 04-07-2022

2. Introduction
Cell culture is a process of isolating a cell from an animal, plant and/or microbes by growing it in vitro in a
culture media by providing conditions required for it’s growth. Basic requirements are nutrients, antibiotics,
antifungals, pH indicator, etc
Uses:
● To study processes like protein synthesis, cell to cell interaction and drug action.
● Uses for implementations in the Pharmaceutical Industry
● Research

3. Types of Cell Lines :
Primary Cell Lines
● Cells removed directly from the tissue or by enzymatic dissociation and it is subcultured multiple times
and passaging date is mentioned.
● Have Limited lifespans and can be used over a limited period of time.
Continuous Cell Lines
● Cells have gained the ability for infinite growth by action of certain carcinogens or certain viruses like
EBV. Unlike primary cell lines they don’t have limited growth, they are immortalized. They are not
required to adhere to the surface and need less serum.

4. Cell Culture Laboratory and Equipment
Required
Maintaining a cell culture laboratory is not an easy tasks. Proper sterilization and checks are required while
working. The laboratory should be set up in a closed space to avoid contamination and in order to do so, the
following conditions are recommended:
● Remove shoes and wear shoe covers while entering the lab.
● Wear proper PPE which includes gloves, masks, apron and head covers.
● Sanitize hands with ethanol time to time.
● Sanitize any spillage in the hood immediately with ethanol and disinfect the floor and surfaces from
time to time.

5. Equipment required in Cell Culture Laboratory
● Cell culture hoods: Provide streamlined flow of sterile air which is filtered through High Efficiency Particulate
Filter (HEPA). This provides protection to the culture media as well as the user.
● CO2 Incubator: Strict Condition for cell growth is required which includes 5-10% CO2 maintained by CO2
cylinder,humidity which is provided by adding tray filled with water inside,pH of 7.2 - 7.4 and a temperature of
37 degrees Celsius.
● Inverted Phase Microscope: The cells are adherent to the walls so require inverted phase microscope which
contains light source above and objective lens below the stage.
● Centrifuge: For centrifuging the the cells
● Waterbath: For thawing frozen samples
● Freezer: For cold storage
● Pipettes: Electronic auto pipetter,micro autopipettes.
● CONSUMABLES: Petri dishes, multi-well plates (with either 96, 24, 12 or 6 wells per plate) and screwcap flasks
classified according to their surface areas: T-25, T-75, T-225 (cm2 of surface area),falcon tube (15ml and 50 ml)

6. Biosafety Levels
Source: BSL Autoclaves for Biosafety Sterilization - Tuttnauer, Information in table and pyramid concept from CDC

7. Composition of a Cell Culture Medium and
Techniques Used:
A culture medium must contain nutrients ( carbohydrates, vitamins, amino acids) , Broad spectrum
antibiotics ( example penicillin and streptomycin), anti mycotic agent example amphotericin, pH
indicator example phenol red, Foetal bovine serum for growth factor,nutrients and cell attachment
and a basal medium example DMEM
Media Preparation:
● 10% or 20 % depends upon the concentration of FBS in the media
● 10% 30 ml media preparation : *27ml DMEM, 3ml FBS,0.3 ml antibiotic and anti fungal, once
prepared can be stored at 4 degree Celsius
Subculture:
● Process by which cells are harvested.
● Passaging is the term coined for harvesting the cells,diluting it in fresh growth medium and
transferring it to a new cultures medium.
● It is required when media is used up by the cells which can be observed by change in the
color of the medium.After passaging the date is mentioned.
● Passaging is done when the growth is confluent(70%). Can be done by rubber spatula or
proteolytic enzymes.

8. Steps in passaging:
● Discard media
● Phosphate buffered solution wash two times and then discard
● Trypsin 0.25%+ EDTA , swirl around
● Add 10% media And do vigorous pipetting
Why Trypsin and EDTA: Trypsin breaks proteinaceous bond between cells and in between
cells and the flask and EDTA chelates calcium required by cells for adhesion.
Cell count
● Pre warm medium and trypsin in water bath
● Aspirate media in a waste container and give trypsin wash and aspirate trypsin
● Wash again with trypsin and incubate for 1 minute at 37 degree Celsius in CO2
incubator
● By gently tapping the base check if the cells are detached
● Prepare hemocytometer by cleaning with 70% ethanol
● Place cover slip over chamber and wait until Newton refraction ring is fixed
● Charge 20 microliter by capillary action
● Observe under 10 X magnification
● Formulae

9. Steps in Passaging Cont.

10. Cryopreservation:
● Suspended cells added to 20% media with 5%DMSO (Dimethyl Sulfoxide) , gently mix it.
● Transfer to cryovials
● -20 degrees Celsius overnight
● -80 degrees Celsius next day
● Followed by transfer vials to liquid nitrogen

11. Revive Culture:
● Take cryovials out of the freezer
● Keep at room temperature
● Prepare media with 20% FBs
● Pour sample dropwise in media with the help of pipette
● Mixed by pipetting to was cells and make if free from DMSO
● Centrifuge 2000-2500 RPM for 5 minutes
● Discard media and add 20% media to culture flask and incubate
● Replace 20% media with fresh media , only live cells adhere to the flask

12. Types of cell culture medias based on basal
medium:
1. Serum Based Cell Culture:
● Contains FBS (Foetal Bovine Serum). Obtained by foetus in second or third trimester with syringe injected in pumping heart. These
medias have various ethical concerns and majors are taken to produce an alternative method that will require less serum or no
serum at all.Example: DMEM, PB max, etc.
2. Chemical Based Culture Medias:
● Use of artificial components and chemicals like to prepare media.Example RPMI 1640 Which contains amino acids ( glycine,L
arginine, L asparagine,etc), vitamins (biotin,folic acid,etc), inorganic salts(calcium nitrate, magnesium sulphate, etc) source of
carbohydrate for energy and phenol red as an indicator.
3. Plant extract based culture:
● Contains vitamins (B1,B6), amino acids(L arginine and asparagine)carbohydrate, etc

13. Serum Based Cell Culture: Major Concerns
● FBS has a lot of advantages as it provide growth factors,nutrients and help cells got adhere but the way it is harvest is rather unethical. It is
obtained by foetus in second or third trimester with syringe injected in pumping heart.
● This causes pain, discomfort, growth abnormalities and death of the foetus.
● It is mentioned major threat by EU Directive 2010/63/EU on use of animal for scientific purpose.
● This has let to encourage the use of chemical based and plant based culture medium.
DISADVANTAGES:
● Non reproducibility: Many researches shows that the studies conducted on the same cell line,of same sample in the same environment
showed insignificant variation.
● Risks to the handler from prions,endotoxins,etc
● Low quality
● Animal welfare concerns
● Seasonal variability
● High molecular weight albumin and globulin not good for somatic cell growth
● Exact composition of FBS not known

14. Methods of Comparing Cell Lines
● Cell growth observed by inverted microscope
● Cell number(Hemocytometer)
● Multi well plate assay (Luminescence assay): Based on a luminescent signal that is
proportional to the metabolic activity of the cell
● Cell Imager
● Karyotyping

15. Procedure of Karyotyping in Cell Culture
Medium
● Draw blood, 1ml in heparin vial
● Add it to culture medium
● Store culture media in CO2 incubator at 37 degree Celsius for 72 hours
● Add Colchisin 20 microliter and incubate for 20 minutes in CO2 incubator
● Pour all to Centrifuge at 1000 RPM for 10 minutes
● Add 0.075 molar KCL till 8 ml mark
● Keep for 1 hour
● Centrifuge
● Discard supernatural
● Add chilled fixative (1:3 of acetic acid and ethanol)
● Centrifuge and discard supernatant
● Wash with fixative thrice till white cells appear.
● Prepare slides by try tonight wash and dipping in. N/10 HCL and store in freeze
● Make smears on the slide
● Stain with Giemsa stain.

16. End of Presentation
Thank you for your time