CELL CULTURE

Question 1

What is cell culture?

Answer 1

removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment

Question 2

Culture stage after isolation of cells
from tissue and proliferated under
appropriate conditions.

Answer 2

Primary Culture

Question 3

• derived from primary culture
• genotypic and phenotypic uniformity

Answer 3

cell line

Question 4

• Positively selected subpopulation of a cell line done by cloning or other methods.
• Acquires additional genetic changes.

Answer 4

cell strain

Question 5

divide only a limited number of
times before losing their ability to
proliferate (senescence).

Answer 5

Finite cell lines

Question 6

• some cell lines become immortal through a process called transformation.
• when a finite cell line undergoes
  transformation and acquires the ability to divide indefinitely

Answer 6

Continous cell lines

Question 7

process where some cells become immortal

Answer 7

transformation

Question 8

cell types based on morphology

Answer 8

• fibroblast
• epithelial
• lymphoblast
• endothelial
• neuronal

Question 8

adherent cell cultures

Answer 8

• Monolayer attached to an artificial
  substrate
• Appropriate for most cell types,
  including primary cultures- Requires periodic passaging
• Cells are dissociated enzymatically or mechanically
• Growth is limited by surface area- Requires tissue
  -cultured treated vessel
• Used for cytology, harvesting
  products continuously, etc.

Question 8

suspension cell cultures

Answer 8

• Free-floating in the culture medium
• Appropriate for cells adapted to
  suspension culture and a few other
  cell lines that are non-adhesive
• Easier to passage, requires daily cell counts and viability determination
• Does not require enzymatic or
  mechanical dissociation
• Growth is limited to concentration of cells in medium
• Can be maintained in vessels not
  tissue-culture treated
• Used for bulk protein production,
  batch harvesting, etc

Question 9

cell line selection

Answer 9

1. SPECIES
2. FINITE or CONTINUOUS CELL LINES
3. NORMAL or TRANSFORMED CELLS
4. AVAILABILITY
5. GROWTH CHARACTERISTICS
   * Population doubling time
   * Ability to grow in suspension
   * Saturation density (yield per flask)
   * Cloning efficiency
6. STABILITY
7. PHENOTYPIC EXPRESSION

Question 10

CELL CULTURE CONDITIONS

Answer 10

● A substrate or medium with essential nutrients (amino
acids, carbohydrates, vitamins, minerals).
● Growth factors
● Hormones
● Gases (O2, CO2)
● A regulated physico-chemical environment (pH, osmotic
pressure, temperature)

Question 11

Basic cell culture equipment

Answer 11

1.Cell culture hood/biosafetly cabinet
2. incubator
3. optical microscope
4. water bath
5. centrifuge
6. ultra low freezer
7. autoclave
8. cell counter
9. refrigirator

Question 12

basic cell culture suuplies

Answer 12

1. Cell culture flasks and well plates
2. Micropipettes
3. Electric pipette controller
4. Serological pipettes
5. Cell culture media and reagents
6. Centrifuge tubes
7. Centrifuge tube racks

Question 13

Lab safety for cell culture

Answer 13

1. Biosafety levels
2. Personal Protective equipment
3. Safety data sheet
4. safe laboratory practices

Question 14

biosafety level for human tissue and cell culture

Answer 14

biosafety level 2

Question 15

what is PPE for (Personal Protective Equipment)

Answer 15

Control and or minimize direct
contact or exposure from
reagents and samples.

Question 16

SAFETY DATA SHEET

Answer 16

Toxicity, health effects, storage,
disposal, protective equipment,
handling spills, etc

Question 17

SAFETY laboratory practices

Answer 17

Knowing the DOs and DON’Ts in
the laboratory, follow SOPs,
aseptic techniques, etc.

Question 18

Biological Contaminations

Answer 18

1. Bacteria
2. Yeast
3. Molds
4. Virus
5. Mycoplasma

Question 19

how is bacteria contamination detected

Answer 19

-Detected visually
-Cloudy culture
-pH change

Question 20

how is yeast contamination detected

Answer 20

-Detected visually
-Clear/turbid culture
-No pH change

Question 21

how is molds contamination detected

Answer 21

-Detected visually
-Clear/turbid culture
-No pH change

Question 22

how is virus contamination detected

Answer 22

-Detected by EM,
ELISA, PCR, and
immunostaining

Question 23

how is mycoplasma contamination detcted

Answer 23

-Behavior & metabolism change -Detected by PCR, ELISA, immunostaining, microbioassays, autoradiography, etc

Question 24

how to prevent cross contamination

Answer 24

1. obtain cell lines from reputable cell banks 2. Label cultures clearly and properly 3. check cell identity 4. practice aseptic techniques before and after

Question 25

example of reputable cell banks

Answer 25

ATCC or ECACC

Question 26

cell identity checker

Answer 26

karyotyping, DNA barcoding, isozyme analysis, STR analysis

Question 27

how to practice aspetic techniques

Answer 27

- Good personal hygiene, sterile work area, and proper handling of reagents/media.

Question 28

Applications of Cell Culture

Answer 28

1. Studying the physiology and biochemistry of cells and organs 2. Effects of drugs and toxic compounds 3. Mutagenesis and carcinogenesis 4. Drug screening and development 5. Manufacturing of biological compounds 6. Tissue engineering and stem cell research

Question 29

5 basics steps of cell cultures

Answer 29

1. preparation of cell culture media 2. thawing of cryopreserved cell line 3. splitting of cultured cell line 4. collection of cultured cell line 5. Cryopreservation of cultured cell line

Question 30

Preparation of cell culture media

Answer 30

1. Mix the following reagents in the cell culture media bottle. a. Culture media solution =435 mL b. Fetal Bovine Serum 10-20% = 50 mL c. L-glutamine 1% = 5 mL d. Penicillin/Streptomycin 1% = 5 mL e. Amphotericin 1% = 5 mL = 500 mL

Question 31

THAWING OF CRYOPRESERVED CELL LINE

Answer 31

1. Thaw the frozen cell line using a water bath (37˚C). Gently share the cryotube. 2. Pour the thawed cell line in a centrifuge tube containing 10 mL culture media. 3. Centrifuge at 1,500 rpm for 5 mins. 4. Decant the supernatant. 5. Resuspend the pellets (cells) in a culture flask with 20 mL culture media. 6. View under an optical microscope. 7. If crowded, split or transfer 10 mL of it in a separate culture flask. 8. Add 10 mL culture media on both culture flasks. *Each flask must contain 20 mL culture media

Question 31

SPLITTING OF CULTURED CELL LINE

Answer 31

*View the cell culture if they are growing properly and if the confluency reached more than 70%. 1. CLEANING a.Discard the old culture media. b. Add 5mL of phosphate buffer saline (PBS) in your culture flask. c.Gently mix the flask. d. Completely discard the PBS. 2. SPLITTING a. Add 2 mL of trypsin in your culture flask. Gently spread the reagent. b. Incubate for 3 mins at 37˚C. c.View under a microscope to check if the cells are completely detached. d. Add 40 mL culture media. e.Split or transfer 20 mL in a new culture flask.

Question 32

COLLECTION OF CULTURED CELL LINE

Answer 32

1. Discard the old culture media. 2. Perform CLEANING. 3. Add and spread 2 mL of trypsin to detach the cells. 4. Incubate for 3 mins at 37˚C. 5. Add 20 mL new culture media. 6. Collect and transfer the cell culture suspended in the culture media in a culture flask.

Question 33

CRYOPRESERVATION OF CULTURED CELL LINE

Answer 33

After COLLECTION 1. Transfer the collected culture line suspended in the culture media in a centrifuge tube. 2. Centrifuge at 1,500 rpm for 5 mins. 3. Discard the supernatant. 4. Add 5 mL freezing media. 5. Transfer the cell line in cryotubes. Label the tubes with name, date, and cell line. 6. Put the cryotubes in a cryobox, and store in -80˚C freezer. Cells should be frozen 1˚C/min. 7. *Transfer in a liquid nitrogen storage for longer cryopreservation