Animal cell and tissue Fatma Al-Qudsi ١
Chapter 7
Subculture and cell lines Chapter 7 Chapter 7
Subculture and cell lines
Subculture and cell lines
Animal cell and tissue Fatma Al-Qudsi ٢
Terminology
Subculture = Passage
• The transfer or subculture of cells from one culture vessel to another.
• Usually it involves the subdivision of a proliferation cell population,
enabling the propagation of a cell line or cell strain
Animal cell and tissue Fatma Al-Qudsi ٣
Terminology
Passage number
• The number of times a culture has been sub cultured
Generation number
• The number of doublings that the cell population has undergone
Animal cell and tissue Fatma Al-Qudsi ٤
Terminology
Cell line
• A propagated culture after the first subculture Cell strain
• A characterized cell line derived by selection or cloning Continuous cell line or cell strain
• Cell line or strain having the capacity of infinite survival.
Previously known as “established” and often referred to as
“immortal” Finite cell line
• A culture that has been propagated by subculture but is capable of only a limited number of cell generations in vivo before dying out
Animal cell and tissue Fatma Al-Qudsi ٥
Terminology
Recombinant
• An individual, meiotic genetic materials
originally present in two individuals end up in the same haploid complement of genes.
The reshuffling of genes can be either by independent segregation or by crossing
over between homologous chromosomes Recombinant DNA
• DNA generated in vitro from more than one source
Animal cell and tissue Fatma Al-Qudsi ٦
Cell line designation
New cell lines are given a code or designation eg.
NHB = Normal Human Brain
NHB1 = Normal Human Brain cell line no 1
NHB2-1= Normal Human Brain cell line no 2 clone no 1
Animal cell and tissue Fatma Al-Qudsi ٧
• A log book or computer database is kept for biopsies or specimens eg.
LT156 = Lung Tumor biopsy 156
• For finite cell lines the number of population doublings should be estimated and indicated after a forward slash eg. NHB2/2
• When dealing with a continuous cell line a “P”
number at the end is often used to indicate the number of passages since the last thaw from the freezer eg. HeLa-S3/P4
Cell line designation
Animal cell and tissue Fatma Al-Qudsi ٨
• When referenced in publications or reports it is helpful to prefix the cell line designation with a code indicating the laboratory in which it was derived eg.
WI= Wistar Institute
NCI = National cancer Institute SK = Sloan Kettering
• In publications or reports
– the cell line should be given its full designation the first time it is mentioned and in the materials and method
section
– the abbreviated version can then be used thereafter
• It is essential that the cell line designation is unique or confusion will arise
Cell line designation
Animal cell and tissue Fatma Al-Qudsi
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Contents of a biosafety cabinet during the subculture procedure. The only acceptable addition to the materials shown here are additional receptacles for additional cultures (i.e. more culture flasks, Petri dishes, or multi-well plates). The insert is a close-up of the culture flask showing the information that should be included on each new culture
J Insect Sci. 2002; 2: 9 Published online 2002 May 20 . .
Animal cell and tissue Fatma Al-Qudsi ١٠
Choosing a cell line
The following parameters should be considered when selecting a cell line
• Finite vs. Continuous
• Normal or Transformed
• Species
• Growth characteristic
• Availability
• Validation
• Phenotypic Expression
• Control Cell Line
• Stability
Animal cell and tissue Fatma Al-Qudsi ١١
Finite vs. Continuous
Continuous cell lines are
• Easier to maintain
• Grow faster
• Clones more easily
• Produces a higher cell yield per flask
• Adopt to serum-free medium more easily
• Therefore if there was a continuous cell line that expresses the right function it is advisable to use it.
Animal cell and tissue Fatma Al-Qudsi
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Properties of finite and Continuous cell lines
Properties Finite Continuous (transformed)
Ploidy Euploid, diploid Aneuploid, heteroploid
Transformation Normal Immortal, growth control
altered, and tumorigenic
Anchorage dependence Yes No
Contact inhibition Yes No
Density limitation of cell proliferation Yes Reduced or lost
Mode of growth Monolayer Monolayer or suspension
Maintenance Cyclic Steady state possible
Serum requirement High Low
Cloning efficiency Low High
Markers Tissue specific Chromosomal, enzymic,
antigenic Special functions (e.g., virus
susceptibility, differentiation) May be retained Often lost
Growth rate Slow (TD of 24–96 h) Rapid (TD of 12–24 h)
Yield Low High
Control parameters Generation no.; tissue-specific
markers Stain characteristics
•Freshney (2005), Culture of Animal Cells: A Manual of Basic Technique Published by John Wiley & Sons, Inc.)
Animal cell and tissue Fatma Al-Qudsi ١٣
Normal or Transformed
The researcher should decide whether the cell line should be malignantly
transformed or not.
Species
Nonhuman cell lines have fewer
biohazards restrictions and have advantage that the original tissue may be more accessible
Animal cell and tissue Fatma Al-Qudsi ١٤
Growth characteristics
• The researcher needs to consider
the following parameters in terms of growth rate
• Population doubling time
• Saturation density
• Plating efficiency
• Growth fraction
• Ability to grow in suspension
Animal cell and tissue Fatma Al-Qudsi ١٥
Availability
• If the researcher uses a finite cell line he or she should make sure that there is enough stocks available
• If the researcher uses a continuous cell line he or she should make sure that authenticated stocks are
available
Animal cell and tissue Fatma Al-Qudsi ١٦
Validation
• The researcher has to make sure that the selected cell line is not a result of cross-contaminations
Phenotypic expression
The researcher has to make sure that the selected cell line is made to
express the right characteristics
Animal cell and tissue Fatma Al-Qudsi
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Cell line Species Cell type Contaminant Species Cell type Source of Data 207 Human Pre-B leukemia REH Human pre-B leukemia DSMZ
2474/90 Human Gastric carcinoma HT-29 Human
Colorectal
carcinoma DSMZ 2957/90 Human Gastric carcinoma HT-29 Human
Colorectal
carcinoma DSMZ 3051/80 Human Gastric carcinoma HT-29 Human
Colorectal
carcinoma DSMZ ADLC-5 M2 Human Lung carcinoma HELA/-S3 Human
Cervical
adenocarcinoma DSMZ
AV3 Human Amnion HeLa Human
Cervical
adenocarcinoma ATCC BCC-1/KMC Human Basal cell carcinoma HELA/-S3 Human
Cervical
adenocarcinoma DSMZ BM-1604 Human Prostate carcinoma DU-145 Human
Prostate
carcinoma DSMZ
C16 Human
Fetal lung fibroblast
(MRC-5 clone) HeLa Human
Cervical
adenocarcinoma ECACC CHANG liver Human
Embryonic liver
epithelium HeLa Human
Cervical
adenocarcinoma ATCC; JCRB COLO-818 Human Melanoma COLO-800 Human Melanoma DSMZ
DAMI Human Megakaryocytic HEL Human Erythroleukemia DSMZ
ECV304 Human Endothelium T24 Human
Bladder
carcinoma ATCC ECV304 Human Normal endothelial T24 Human
Bladder
carcinoma DSMZ
EJ Human Bladder carcinoma T24 Human
Bladder
carcinoma ATCC; JCRB Some cross-Contaminatedd Cell Lines
•Freshney (2005), Culture of Animal Cells: A Manual of Basic Technique Published by John Wiley & Sons, Inc.)
Animal cell and tissue Fatma Al-Qudsi ١٨
Control cell line
The researcher has to make sure that if the selected cell line is mutant or abnormal that there is a normal
equivalent cell line Stability
The researcher has to make sure that the selected cell line is stable
Animal cell and tissue Fatma Al-Qudsi ١٩
Routine maintenance
• Once the culture is initiated it will need a periodic medium change followed by
subculture if the cells were proliferating
• It is important to examine the culture carefully to confirm the absence of
contamination or deterioration such as:
– Granularity around the nucleus – Cytoplasmic vacuolation
– Rounding up the cells with detachment from the substrate
Animal cell and tissue Fatma Al-Qudsi
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Unhealthy cells. Vacuolation and granulation in bronchial epithelial cells (BEAS-2B) due to medium inadequacy.
The cytoplasm of the cells becomes granular particularly around the nucleus and vacuolation occurs.
•Freshney (2005), Culture of Animal Cells: A Manual of Basic Technique Published by John
Wiley & Sons, Inc.)
Animal cell and tissue Fatma Al-Qudsi ٢١
Replacement of medium
Four factors indicate the need for medium replacement
• A drop in pH
• Cell concentration
• Cell type
• Morphological Deterioration
– Cells should be examined regularly to prevent apoptosis
Animal cell and tissue Fatma Al-Qudsi ٢٢
A drop in pH
• If the medium drops to pH6.5-6 ( goes from red to orange or yellow)
then the medium should be changed Cell concentration
• Cultures at high cell concentration use the medium faster than cultures at low cell concentration
Animal cell and tissue Fatma Al-Qudsi
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• Normal cells usually stop dividing at a high cell density because of
– cell crowding,
– growth factor depletion, – and other reasons.
• The cell blocks in the G1 phase of the cell cycle and deteriorate very little, even if left off for two or three weeks or longer.
• Transformed cells, continuous cell lines, some
embryonic cells, however, deteriorate rapidly at high cell densities unless the medium is changed daily or they are subcultured
Cell type
Animal cell and tissue Fatma Al-Qudsi
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Subculture of monolayer. Stages in the subculture and growth cycle of monolayer cells after trypsinization
•Freshney (2005), Culture of Animal Cells: A Manual of Basic Technique Published by John Wiley & Sons, Inc.)
Animal cell and tissue Fatma Al-Qudsi
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Procedure Pretreatment Dissociation agent Medium Applicable to
Shake-off None
Gentle mechanical shaking, rocking, or
vigorous pipetting
Culture medium Mitotic or other loosely adherent cells
Scraping None Cell scraper Culture medium
Cell lines for which proteases are to be avoided (e.g., receptor or
cell surface protein analysis); can damage
some cells and rarely gives a single-cell
suspension Trypsin* alone Remove medium completely 0.01–0.5% Crude
trypsin; usually 0.25%
D-PBSA, CMF, or
saline citrate Most continuous cell lines
Prewash + trypsin D-PBSA 0.25% Crude trypsin D-PBSA
Some strongly adherent continuous cell lines and many early-passage cells Prewash + trypsin 1 mM EDTA in D-PBSA 0.25% Crude trypsin D-PBSA Strongly adherent early-
passage cell lines Prewash + trypsin 1 mM EDTA in D-PBSA 0.25% Crude trypsin D-PBSA + 1 mM
EDTA
Many epithelial cells, but some can be sensitive to EDTA; EGTA can be used Trypsin +
collagenase 1 mM EDTA in D-PBSA
0.25% Crude trypsin;
200 U/mL crude collagenase
D-PBSA + 1 mM EDTA
Dense cultures and multilayers, particularly
with fibroblasts
Dispase None 0.1–1.0 mg/mL Dispase Culture medium
Removal of epithelium in sheets (does not dissociate
epithelium)
Pronase None 0.1–1.0 mg/mL Pronase Culture medium
Provision of good single- cell suspensions, but may
be harmful to some cells DNase D-PBSA or 1 mM EDTA in D-PBSA 2–10 µg/mL crystalline
DNase Culture medium
Use of other dissociation agents which damage
cells and release DNA
Cell dissociation procedures
•Freshney (2005), Culture of Animal Cells: A Manual of Basic Technique Published by John Wiley & Sons, Inc.)
Animal cell and tissue Fatma Al-Qudsi
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Stirrer culture. A small stirrer flask based on the technique design with a
capacity of 250-1000 ml. the cell suspension is stirred by a pendulum, which rotates in an annular depression in the base of the flask
•Freshney (2005), Culture of Animal Cells: A Manual of Basic Technique Published by John
Wiley & Sons, Inc.)
Animal cell and tissue Fatma Al-Qudsi ٢٧
Monolayer Suspension
Culture requirements
Cyclic maintenance Steady state
Trypsin passage Dilution
Limited by surface area Volume (gas exchange) Growth properties
Contact inhibition Homogeneous suspension Cell interaction
Diffusion boundary Useful for
Cytology Bulk production
Mitotic shake-off Batch harvesting In situ extractions
Continuous product harvesting Applicable to
Most cell types, including
primaries Only transformed cells
Monolayers VS. Suspension Culture
Animal cell and tissue Fatma Al-Qudsi ٢٨
