Cell Culture Techniques

Question 1

Cell culture

Answer 1

▪ Cells are building blocks of living organisms.
▪Smallest unit capable of performing all the functions necessary for life.
▪ Human body is composed of over 200 different types of cells, including white blood cells, red blood cells, nerve cells, heart muscle cells, and epithelial cells.
▪ Each type of cell has a specific role in the body,
▪ All cells work together to maintain the overall health and proper function of the organism.

Question 2

Primary cells

Answer 2

-cells freshly isolated from a living tissue
-finite life span in culture

Question 3

Transformed cells

Answer 3

-derived from clinical tumours or transforming primary cells

-immortalised or continuous cell lines

Question 4

Stem cells

Answer 4

-derived from embryonic tissues,feral tissues and induced pluripotent stem cells(iPSCs)

-self-renew or differentiate into various cell types

Question 5

Primary cells

Answer 5

▪ Derived directly from excised tissue by dissociation into a single cell suspension by enzyme digestion.
▪ Divide only a limited number of times and can be maintained in vitro only for a limited period of time. This number is known as the Hayflick limit.
▪ Senescence due to shortening of telomere length with each cell division- Replicative senescence
▪ Loss of their ability to proliferate after a certain number of cell divisions, these cell lines are known as finite.

Question 6

Advantages of primary cells

Answer 6

-thought to represent the best experimental models for in Vivo situations

-have the same karyotype as the parent tissues

Question 7

Disadvantages of primary cells

Answer 7

-difficult to obtain
-short life span In culture
-considerable variation in population and between preparations

Question 8

Immortalised cell lines

Answer 8

▪ Transformed cell lines or Continuous immortalized cell lines that can be propagated indefinitely.
▪ Escape the normal constraints of the cell cycle and grow indefinitely, making them extremely useful for long-
term research
▪ Tumor cell lines are often derived from actual clinical tumors, but transformation may also be induced using
viral oncogenes or by chemical treatments.
▪ Viral oncogenes such as the large T antigen from the SV40 virus suppress tumor suppressor genes in cells (e.g. p53 and retinoblastoma protein )

Question 9

Advantages of immortalised cell lines

Answer 9

-easy to maintain in culture
-easy to obtain large population of cells
-typically easy to manipulate gene expression

Question 10

Disadvanteges of immortalised cells

Answer 10

-the more aggressive the cell line the more it changes over time in culture
-not clear how the function of these cells relates to that of other cells,healthy or diseased

Question 11

HeLa cells

Answer 11

1) HeLa cells: isolated from tissue sample of a cancer patient, Henrietta Lacks in 1951.
▪ First immortal human cells in culture and one of the most commonly used for research purpose for cancer studies.
▪ HeLa cells were used by Jonas Salk to test the first polio vaccine in the 1950s.

Question 12

Jurkat T lymphocytes cell line(suspension)

Answer 12

Origin: late 1970s isolated from blood sample of a
14yr old boy with leukaemia.
Use: studies of acute T cell leukaemia, T cell signaling and the expression of various chemokine receptors susceptible to viral entry, particularly HIV.

Question 13

Stem cells

Answer 13

▪ Unlimited self-renewal capabilities.
▪ they are non-differentiated cells with unspecialized functions.
▪ Can differentiate into specific cell types under the right condition.
▪ Given their unique regenerative abilities, human stem cells are being used in biomedical research and therapeutics development, particularly in regenerative medicine.

Question 14

Type of stem cells

Answer 14

-multipotent
-pluripotent

Question 15

Multi potent stem cells

Answer 15

▪ Adult stem cells that can self-renew or to differentiate into specialized, tissue-specific cell types.
Examples:
Hematopoietic stem cells (HSCs): differentiate into various blood cells;
Mesenchymal stem cells (MSCs): differentiate into osteoblasts, myocytes, chondrocytes and adipocytes; Neural stem cells (NSCs) that differentiate into neurons, astrocytes and oligodendrocytes.

Question 16

Pluripotent stem cells

Answer 16

-can differentiate into any cell lineage

▪ Embryonic stem cells (ESCs): derived from embryos and
▪ Perinatal stem cells: placental blood or tissue
▪ Induced pluripotent stem cells (iPSCs): adult cells that are reprogrammed or induced to behave like stem cells.

Question 17

Classification of cells by the attachment to the surface

Answer 17

-adherent cells

-suspension cells

Question 18

Adherent cells

Answer 18

Also known as anchorage-dependent cells
▪ Must be cultured on a substrate that permits cell adhesion.
▪ Cells are grown while attached to a substrate as monolayers.
▪ Most human primary cells fall into this category.
▪ The growth is limited by the area of the adherent surface.

Question 19

Suspension cells

Answer 19

Also known as anchorage-independent cells
▪ Cells are suspended/free floating in the culture medium.
▪ The medium must be continuously agitated (e.g., stirred) to ensure proper cell growth.
▪ Cell lines derived from the blood such as lymphocytes, megakaryocyte, and neutrophils grow in suspension. Example: Jurkat cells, a line of lymphocytes derived from human T-cell leukemia

Question 20

Adherent cells

Answer 20

Picture

Question 21

Suspension cells

Answer 21

Picture

Question 22

Huh-7 cell line

Answer 22

from liver tumor of a 57-year-old patient is
▪ Used for the in vitro production of infectious HCV particles
▪ anti-HCVtherapeuticassays

Question 23

Caco-2 cells

Answer 23

from a colorectal adenocarcinoma patient.
Expresses the receptor required for SARS-CoV-2 viral entry into the cell.

Question 24

K-562 cell line

Answer 24

lymphoblast cells isolated from the bone marrow of a 53-year-old chronic myelogenous leukemia (cancers of the blood cells) patient

Question 25

Cell culture workflow

Answer 25

Aseptic techniques are used to prevent contamination of cells grown in vitro. provide a barrier between the microorganisms in the environment and the sterile cell culture
➢ Biosafety cabinets
➢ Cells are grown in sterile petridishes/tissue culture flasks (T-flasks) ➢ Media with required nutrients
➢ Incubators for stable growth environment
Mammalian cell cultures require passaging or subculturing – which involves dilution of cells that have reached confluence and replacement of depleted culture media.
Adherent cell cultures, cells must be dissociated from culture surfaces using enzymatic methods such as trypsinization or mechanical methods.
Cell counting methods using hemacytometers and automated cell counters assess cell viability and help to determine seeding densities for passaging.
Cultured cells can be cryopreserved by storage in liquid nitrogen.

Question 26

Requirements for growing mammalian cells in culture

Answer 26

▪ Cell culture media:
Cell growth requires a complex mixture of nutrients including sugars, amino acids, albumin, vitamins, minerals, and growth factors.
▪ Incubators: Stable environment for cell growth, i.e. Controlled
➢ pH
➢ Temperature: usually 37oC ➢ Gas: usually 5% CO2 in air
▪ Sterile environment free of contaminations with microbes or other cell lines.
▪ Facilities for long-term storage of cells (cryopreservation).

Question 27

CO2 incubator

Answer 27

1) Mammalian cells will grow best at their native temperature (37 °C/98.6 °F).
2) CO2 gas serves to maintain in vivo pH, similar to CO2 tension in the bloodstream.
➢ CO2 gas works with sodium bicarbonate in the growth medium to maintain pH of 7.4. This mimics bloodstream biochemistry.
➢ Varying pH, cells to stop growing and lose viability.
3) Humidity of 85- 95% limits evaporation of water from the growth media. ➢ Evaporation leaves too-high concentrations of salts, minerals, etc.,
resulting in toxicity and cell death

Question 28

Requirements for growing mammalian cells in culture

Answer 28

1)Cell culture hood
Also known as the Laminar flow hood, which provides an ultra-clean working place for aseptic handling of cell culture.

2) Incubator
Cell culture incubators enable optimal growth conditions by controlling the temperature, humidity and
CO2 level.

3) Personal protective equipment (i.e., lab coat, gloves, eyewear, mask) is required according to the biosafety levels.

4) Inverted microscope
An inverted microscope (4X, 10X, 20X, and 40X objective lenses) is essential to monitor the healthiness and confluence of cells.

5) Pipettes are used to dispense small volumes of liquid (usually, ranging from 1μL to 1mL) using single-use plastic pipette tips.

6) Liquid nitrogen tank
For the long-term cryopreservation of cells at temperatures below -130oC.

7) Fridge and Freezer:
For the storage of cell culture media (2 – 8oC) and other cell culture reagents (-20oC).

8) Culture-grade plasticware treated for adherent cell attachment. There are options in a variety of sizes and shapes

Question 29

Hazard groups in a lab

Answer 29

1)Hazard group 1 (HG 1): Biological agent that is unlikely to cause human disease. E.g. non-pathogenic strains
of Escherichia coli (e.g. E. coli K12)

2)Hazard group 2 (HG 2): Biological agent that can cause human disease and may be a hazard to employees but is unlikely to spread to the community and there is usually effective prophylaxis or treatment available. E.g. staphylococcus aureus (staph infections), Virus for Measles, Mumps.

3)Hazard group 3 (HG 3): Biological agent that can cause severe human disease and may be a serious hazard to laboratory workers and it may spread to the community but there is usually effective prophylaxis or treatment available. Mycobacterium tuberculosis, Anthrax

4)Hazard group 4 (HG 4): Biological agent that causes severe human disease and is a serious hazard to laboratory workers and it is likely to spread to the community and there is usually no effective prophylaxis or treatment available. Ebola virus, Lassa fever,

Question 30

Bio safety levels in a lab

Answer 30

1)Biosafety Level 1 (BSL-1) is the basic level of protection and is appropriate for agents that do not cause disease in normal, healthy humans. E.g. non-pathogenic strains
of Escherichia coli

2)Biosafety Level 2 (BSL-2) is appropriate for moderate-risk agents that can cause human disease through ingestion, inhalation, or injection. Most cell culture labs should be at least BSL-2.

3)Biosafety Level 3 (BSL-3) is appropriate for agents that have the potential for aerosol transmission and may cause serious and potentially lethal infections. Mycobacterium tuberculosis, SARS-CoV-2.

4)Biosafety Level 4 (BSL-4) is the highest level of containment and is appropriate for agents that pose a high individual risk of life-threatening disease through infectious aerosols and for which no treatment is available. These agents are restricted to high-containment laboratories. Ebola virus, Lassa fever.

Question 31

BSL-4: Extreme Risk

Answer 31

BSL-4 is the highest biosafety level and is reserved for work with dangerous and exotic microbes that pose an extreme risk to laboratory workers and the environment.

These microbes are typically fatal and have no available vaccines or treatments.

▪ BSL-4 laboratories are highly specialized and isolated facilities, often located in separate buildings with dedicated air supply and exhaust systems.

▪ Personnel working in BSL-4 labs wear full-body, air-supplied suits and must follow strict decontamination procedures, including showering before exiting the facility.

•Microbes Handled: Ebola virus, Marburg virus, Lassa fever virus

•Safety Measures: Complete isolation, full-body suits, decontamination protocols, dedicated air systems

Question 32

Bio safety cabinet

Answer 32

▪ A biosafety cabinet (BSC)—also called a biological safety cabinet or laminar air flow cabinet —a
contamination-free work environment through filters to capture all the particles entering the cabinet. ▪ For protection of environment and the personnel.
HEPA filter
• The High-efficiency particulate air (HEPA) filter is present within the cabinet that makes the environment sterile for the operation.
• Filtering out 99.97% of particles sized at 0.3 μm.
• The pre-filtered air passes through the filter which traps fungi, bacteria and
other dust particles.
• The filter ensures a sterile condition inside the cabinet, thus reducing the chances of contamination.

Question 33

Laminar air flow

Answer 33

The main objective of a laminar air flow cabinet is to provide a sterile and filtered air environment.
• Work surface
• Enclosure
• Blower/fan: suck in room air from the top or front of the device.
• Prefilter/filter pad
• HEPA filter
• UV lamp
These parts work together to construct a laminar air flow cabinet.
The air goes through the Prefilters pad that collects large dust particles.
HEPA filter: filters out the microbes and other particles. UV lamp to sterilize the work area.

▪ The primary purpose of a BSC is to serve as a means to protect the laboratory worker and the surrounding environment from pathogens/biohazardous/infectious agents .
▪ All exhaust air is HEPA-filtered as it exits the biosafety cabinet, removing harmful bacteria and viruses.
▪ Filters both the inflow and exhaust air.
▪ The U.S. Centers for Disease Control and Prevention (CDC) classifies BSCs into three classes.
▪ Depending on the level of personnel and environmental protection provided and the level of product protection provided.

Question 34

Class I microbiological safety cabinets

Answer 34

offer protection for the person using the cabinet and the environment surrounding them but doesn’t protect the material that they’re working with.
▪ Do not keep the sample safe from contamination which can limit their usage depending on what you for.
▪ Can be used to handle BSL-1 organism.
▪ Class I BSCs are designed with an open front with inward airflow (personnel protection) and HEPA-filtered exhaust air (environmental protection).
▪ They pull room air through the front of the cabinet and across the worksurface, away from the operator (similar to a fume hood), and use a HEPA filter at the exhaust outlet.
They commonly recirculate air back to the laboratory, but can be externally exhausted if needed.

Question 35

A Class II cabinet

Answer 35

is defined as a ventilated cabinet for personnel, product and environmental protection, often used for microbiological work to maintain sterile conditions.
▪ Cell culture or tissue culture hoods.
▪ BSL 2 and BSL 3 in conjunction with other protective measures required for these biosafety levels.

▪ Class II BSCs are designed with an open front with inward airflow (personnel protection), downward HEPA-filtered laminar airflow (product protection) and HEPA-filtered exhaust air (environmental protection).
▪ All Class II BSCs require all biologically contaminated ducts and plenums to be under negative pressure.
▪ Pressure in the hood is lower than the external air pressure. Outside air to enter the hood but inside air cannot escape out.
▪ The exhaust air is treated with either double HEPA filtration or single HEPA filtration followed by air incineration and then exhausted outside.

Question 36

A Class II cabinet

Answer 36

is defined as a ventilated cabinet for personnel, product and environmental protection, often used for microbiological work to maintain sterile conditions.
▪ Cell culture or tissue culture hoods.
▪ BSL 2 and BSL 3 in conjunction with other protective measures required for these biosafety levels.

▪ Class II BSCs are designed with an open front with inward airflow (personnel protection), downward HEPA-filtered laminar airflow (product protection) and HEPA-filtered exhaust air (environmental protection).
▪ All Class II BSCs require all biologically contaminated ducts and plenums to be under negative pressure.
▪ Pressure in the hood is lower than the external air pressure. Outside air to enter the hood but inside air cannot escape out.
▪ The exhaust air is treated with either double HEPA filtration or single HEPA filtration followed by air incineration and then exhausted outside.

Question 37

The Class III biological safety cabinet

Answer 37

was designed for work with biosafety level 4 (BSL-4) microbiological agents and provides maximum protection to the environment and the worker.
It is a gas-tight enclosure with a non-opening, completely sealed, viewing window.

Question 38

Growth curve of cells in culture

Answer 38

➢ Lag phase: Latent phase before growths begins
➢ Log phase: Exponential growth phase.
➢ Stationary phase: increase in cell numbers gradually slows.
➢ Death phase: Cells die because of lack of nutrients, accumulation of toxic byproducts

▪ Lactic acid: by-product of cellular metabolism is toxic to the cells in higher concentration.
▪ Decreased pH
▪ Ever-increasing number of cells in a confined cell culture dish, consumption of nutrients eventually results in cell death.

Cells should be passaged before they reach the stationary phase.

Question 39

Subculturing or Passaging cells

Answer 39

Cells from a given culture are divided and split in
new cultures with lower cell density than the original culture.
▪ Includes:
cell dissociation,
counting cells,
determining optimal seeding density and
preparing new culture vessels with fresh media for further expansion
for passaged cells.
▪ New cell culture dish (new matrix/surface), addition of fresh growth medium and removal toxic metabolites allow
long-term maintenance of the culture.

Question 40

Cryopreservation

Answer 40

Long term cryogenic cell storage (Freezing in liquid nitrogen)
▪ Temperature of liquid nitrogen: -196°C
▪ Cells can be stored at this temperature for decades.
Long term cryogenic cell storage: Method
▪ Pellet cells by centrifugation
▪ Re-suspend in Freezing media is 90% serum (fetal bovine serum)/
10% DMSO (composition can vary)
DMSO: Dimethyl sulfoxide: Cryoprotective agents: prevent cell damage by intracellular ice crystals/
Reduce the freezing point of the medium and allow a slower cooling rate, Reduce the risk of ice crystal formation, which can damage cells and cause
cell death. Slow regulated freezing minimises cell damage

Question 41

Cell culture contamination

Answer 41

▪ Cultures can be infected through poor handling, from contaminated media, reagents, and equipment (e.g., pipets).
▪ Cell culture contamination: slow cellular growth, change in morphology, fast change of pH in media, and elevated quantities of death or floating cells in the culture are the consequence.
Bacterial contamination: rod-shaped or cocci
Macroscopic detection
• Increased turbidity of culture medium, medium appears cloudy.
• If medium contains phenol red as pH indicator, a rapid color change to yellow indicates a sudden decrease in pH

Question 42

Cell culture contamination

Answer 42

Macroscopic detection
• Increased turbidity of culture medium, medium appears cloudy.
• If medium contains phenol red as pH indicator, a rapid color change to yellow indicates a sudden decrease in pH

Fungi (molds)
• Thin filamentous mycelia and sometimes as denser clumps of spores.
• Fungi are usually very slow growing but in advanced stages of contamination often overtake a culture as a fuzzy growth (either whiteish or black)
Mycoplasma
• Cannot be detected by the naked eye or even by typical light microscopy
• Inhibit cell metabolism and growth, interference with nucleic acid synthesis.
• Detection by ELISA, PCR
• MycoFluor
nuclei as well as the mycoplasma, if the culture is contaminated.

Question 43

Good cell culture practice

Answer 43

▪ Dealing with contaminations in cell culture
Dispose safely of contaminated cells
Review aseptic technique, media preparation etc. Culture media/solutions used filtered or discarded Autoclave equipment where appropriate
Clean thoroughly work area
Decontaminate the CO2 incubators
▪ Maintain a clean cell culture environment,
Changing pipette tips between aspirating different cultures; Using autoclavable instruments;
Having dedicated media bottles for each cell line.

Question 44

Disinfection

Answer 44

▪ Alcohol (ethanol, iso-propanol)Effective concentrations-70% ethanol, 60% i-propanol Dehydrate and fixate
Effective against bacteria. Ethanol -against most viruses but not non-enveloped viruses
▪ Hypochlorites (e.g.Chloros, Presept)general purpose anti-viral
corrosive
Readily inactivated by organic matter (made fresh every day)
▪ Phenolics(Sudol, Hycolin) Not active against viruses Remain active in the presence of organic matter

Question 45

Advantages of cell culture

Answer 45

➢Cost effective as compared to animal studies or clinical trials
➢Easy to use
➢Immortalised cell lines provide an unlimited supply of material.
➢Use of cell culture bypasses ethical concerns associated with the use of animal and human tissue
➢Immortalised cell lines provide a pure population of cells which provide consistent and reproducible results

Question 46

Limitations of cell culture

Answer 46

➢ Not representative of physiological processes and interactions taking place in the whole organism.
➢ The genetic manipulation of cell lines may alter their phenotype, native functions and their responsiveness to stimuli.
➢ Serial passage of cell lines can cause genotypic and phenotypic variation over an extended period of time
➢ Contamination of cell cultures with other cell lines and microbes.