This document discusses various techniques for cell cloning and selection, including: - Primary cell cultures are sub-cultured at a 1:2 ratio while continuous cell lines replicate at higher rates. - Cloning involves isolating and growing cells with similar properties through dilution cloning or cloning on feeder layers. - Conditions like growth medium, serum, hormones, and carbon dioxide levels can improve cloning efficiency. - Colonies are selected through isolation with cloning rings or drug/growth factor resistance and further propagated. - Proper examination, good laboratory practices, and new automated systems help optimize the cloning process.

1. COLONING AND
SELECTION
PRESENTED BY
DR. GHALIA QAYYUM

2. CELL CULTURE OR TISSUE
CULTURE
• The term cell culture refers to the cultivation of dispersed cells
taken from an
• Original tissue
• A primary culture
• Or a cell line

3. CELL CULTURE OR TISSUE
CULTURE
• Primary cultures are generally sub cultured at a 1:2 ratio
(they are split in half with each passage).
• Continuous cell lines replicate at higher rates and are
sub cultured at a much higher split ratio.
• Passage number is generally the number of times the
cells have been sub cultured into a new vessel.

4. CLONE
• Its a group of identical cells that share a common ancestry,
meaning they are derived from a single cell.
• Poly clonal—derived from many clones
• Oligo clonal- derived from a few clones
• Mono clonal—derived from one clone
CLONING
• To isolate and grow cells with specialized similar properties

5. First clone
• Sanford was the first cloned cell strain, isolated by capillary
cloning from mouse L-cells [Sanford et al., 1948].

6. Why Do We Need To Clone?
• Model systems for
Studying basic cell biology, interactions between disease causing
agents and cells, effects of drugs on cells, process and triggering of
aging & nutritional studies
• Toxicity testing
Study the effects of new drugs
• Cancer research
Study the function of various chemicals, virus & radiation to
convert normal cultured cells to cancerous cells
Virology
Cultivation of virus for vaccine production, also used to study there
infectious cycle.

7. Why Do We Need To Clone?
• Genetic Engineering
Production of commercial proteins, large scale production of
viruses for use in vaccine production e.g. polio, rabies, chicken pox,
hepatitis B & measles
• Gene therapy
Cells having a functional gene can be replaced to cells which are
having non-functional gene
• To produce artificial tissues (skin, heart, cartilage)
• Use single cell (impossible in vivo)

8. Disadvantages
• Cell characteristics can be changed
• Cell adapts to different nutrients
• If mixed cells cultivated some types will disappeared.
• Activity of enzymes may altered by environment.
• Necessary expertise
• Quantity and cost

9. Cloning
• Cloning is relatively easy for continuous cell lines
• Difficult in primary cultures
• Cloning of cells carried out in
• Petri dishes
• Multi well Plates
• Flasks

10. Multi well Plates, petri plates and flask

11. Anchorage dependent or independent
cloning
• Cell colones derived from normal tissues are considered as
anchorage-dependent grow only on a suitable substrate OR
attached to a surface, seeding cells into a gel, such as agar or
agarose,
Cloning In Suspension
• Suspension cells are anchorage-independent e.g. blood cells
coloned in a a viscous solution, such as Methocel, with an agar or
agarose underlay.

12. characteristic growth pattern of
cells
Four phases
• Lag phase — Immediately after seeding of the
culture vessel, the cells grow slowly for recovering
from the stress of sub-culturing.
• Log or exponential phase — The cells enter a
period of exponential growth that lasts until the
entire growth surface is occupied or
• the cell concentration exceeds the capacity of the
medium.

13. characteristic growth pattern of cells
• Stationary phase — Cell proliferation slows and stops.
• Decline phase — If the culture medium is not replaced and
the cell number is not reduced, the cells lose viability and
their number decreases.
• Viability, genetic stability, and phenotypic stability,
• Cell lines need to be maintained in the exponential phase.
• This means that they need to be sub cultured before a
monolayer becomes 100% confluent.
Confluence
• Available substrate surface is covered by cells and growth
slows & ceases.
• Cells to be kept in healthy & in growing state have to be sub-
cultured or passaged, when they reach 70-90% confluence.

14. Confluence
After 24 hours

15. 70% confluency

16. 100% confluence

17. Dilution Cloning
• Dilution cloning was first introduced by puck and marcus, 1955
• It is the most widely used technique
• Based on observation that cells diluted below certain density form
discrete colonies

18. Plating Efficiency
• Reasons for low plating efficiency
• loss by leakage due to dilution
• cell derived diffusible factors too dilute
• improved media in conjunction with feeder cells increase
plating efficiency
Continuous cell lines generally have a high plating
efficiency in monolayer and in suspension because of
their transformed status.

19. Conditions That Improve Clonal
Growth
Basal Medium(nutrients)
A rich medium, such as Ham’s F12, or a medium that has been
optimized for the cell type
Serum
fetal bovine is better than calf or horse.
Hormones and drugs like
Insulin 1 × 10-10 IU/mL, has been found to increase the plating
efficiency
Dexamethasone (Hydrocortisone)
(10 μg/mL) improves the plating efficiency

20. Conditions That Improve
Clonal Growth
supplement media metabolites.
keto-acids
pyruvate
Polylysine
Fibronectin
Add 1 mg/mL
Prior included in rich medium such as Ham’s F12, Dulbecco’s
modification of Eagle’s MEM

21. Carbon dioxide
CO2 is essential for obtaining maximum cloning efficiency for
most cells, 5%- 2% used.

22. FLOWCHART
Resected Tissue
Cell or tissue culture in vitro
Primary culture
Secondary culture
Sub-culture
Cell Line
Sub-culture
Immortalization
Successive sub-cultureSingle cell isolation
Clonal cell line Senescence
Transformed cell line
Immortalised cell line
Loss of control
of cell growth

23. 1)Check confluency of cells
2) Remove spent medium
3) Wash with PBS
5) Resuspend in media with serum
4) Incubate with
trypsin/EDTA
6)Transfer to culture flask
StepsInPassagingCells

24. Generalprocedurecellcloning
Trypsinize the cells: a single cell suspension. trypsin used at 0.05
μg/mL
Under-trypsinizing = produce clumps
Over-trypsinizing = reduce the viability of the cells
cells round up and start to detach
disperse the monolayer to a single-cell suspension in 5 mL of
medium containing serum or trypsin inhibitor.
Count the cells, and dilute the cell suspension
1 × 105 cells/mL, concentration will give roughly 50 colonies per
Petri dish

25. Generalprocedure cell cloning
Seed petri dishes
Chemically modified plastic or coated with ECM proteins seeded with suspension culture
Humid CO2 incubator (2–10%).
Incubation
1st week for the colonies to form: if negative
2nd week continue to culture.
3rd week Feed the dishes, medium replaced, at least half of it and culture them.
If no colonies appear by 3 weeks, then it is unlikely that they will appear at all.
Feeding
For increasing density of cells If any growth factors, hormones, or other supplements are
to be added to the dishes, they should be added to the 0.6% agar underlay.
• Feeding mainly counteracts the loss of nutrients (e.g., glutamine),
• Replaces growth factors that have degraded or been depleted,
and compensates for evaporation.
Draw back
Increases the risk of contamination, so it is reasonable to leave dishes for two weeks
without feeding.

26. UseofConditionedMedium
Conditioned Medium
Medium that has been used for the growth of other cells acquires
metabolites, growth factors, and matrix products from these cells and is
known as conditioned medium.
• Conditioned medium can improve the plating efficiency by adding
into the regular growth medium.
PREPARATION OF CONDITIONED MEDIUM
• Harvest medium from homologous cells, or a different cell line, from
the late log phase. Filter, and dilute with fresh medium.
• Cells for conditioning: same cell line, another cell line like 3T3 cells
or mouse embryo fibroblasts
• Cloning medium: Ham’s F12, with 10% FBS
• Sterilizing filter: 0.45 m or 0.22 μm, filter

27. Procedure
• 1. Grow conditioning cells to 50% of confluence.
• 2. Change the medium, and incubate for a further 48 h.
• 3. Collect the medium.
• 4. Centrifuge the medium at 1,000g for 10 min.
• 5. Filter the medium through a 0.45-μm sterilizing filter.
• 6. Store the medium frozen at −20◦C.
• 7. Thaw the medium before use, and add it to cloning medium
• 1 part conditioned medium to 2 parts cloning medium.
Centrifugation, freezing and thawing, and filtration steps all help avoid
the risk of carrying any cells over in the conditioning medium
• If the same cells are used for conditioning as for cloning, then this
problem is less important,
• But better cloning may be obtained by using a different cell line or
primary mouse fibroblasts.

28. COLONING ON FEEDER LAYER
• The reason that some cells do not clone well may be related
to their inability to survive at low cell densities.
• to maintain cells at clonogenic densities but, at the same time,
mimic high cell densities is to clone the cells onto a growth
arrested feeder layer
• The feeder cells provide nutrients, growth factors, and matrix
constituents for the cloned cells to survive

29. COLONING ON FEEDER LAYER
• Procedure
1. Trypsinize embryo fibroblasts, from the primary culture and
reseed the cells at 1 × 105 cells/mL.
2. After 3 to 5 days (when cells are sub confluent and still
dividing) block further
(a) By irradiation: 60 Gy of radiation from X-ray machine
(b) By mitomycin C at 2 μg/1 × 106 cells.
It is important that the cells enter the cell cycle after the
mitomycin C or irradiation treatment or the DNA damage may be
repaired.
• the feeder cells will remain viable for up to three weeks, but
will eventually die out and are not carried over if the colonies
are isolated.

30. Examination of Cultures
• Observe morphology and viability of cultures
• Examine the medium in the vessel
• unusual pH shifts (yellow or purple color from the phenol red),
• turbidity, or particles.
• fungal colonies that float at the medium-air interface.
• With an inverted microscope at low power (40×), microbial
contamination
• Bacterial contamination will appear as small, shimmering black dots
within the spaces between the cells.
• Yeast contamination will appear as rounded or budding particles,
• while fungi will have thin filamentous mycelia
• Dead cells often round up and detach from the monolayer and
appear smaller and darker (not refractile) than healthy cells.

31. selection
• For selection of specific cell strains, the colonies need to be
isolated for further propagation.
• If monolayer cells are cloned directly into multi well plates,
then colonies may be isolated by trypsinizing individual wells.
• If cloning is performed in Petri dishes, there is no physical
barrier between colonies. This barrier must be created by
removing the medium and placing a stainless steel or ceramic
ring around the colony to be isolated.
• ISOLATION OF CLONES WITH CLONING RINGS
• Cloning rings
• Silicone grease;

32. selection
• Selection by resistance to a number of drugs such as
neomycin, genetacin
• Selection by different responses to growth factors possible to
stimulate one cell type with the appropriate growth factor
• advantage increased sensitivity of the more rapidly growing
cells or
• to kill the cells selectively with irradiation or cytosine., if an
inhibitor is known or a growth factor is removed, take one
population out of cycle.

33. IMPORTANTADVISEFOR CELLCULTURE
WORKERS
• Keep your cells happy OR tell when they’re not BY
• following common sense
• WITH
• GOOD LABORATORY PRACTICES (GLPs)

34. Robotic Cell Factory can churn out 500
cell cultures a month.

35. Robotic Cell Factory
• robot is designed to move around the first-generation cell
multititer plates, among various spots.
• Then an automated microscope checks the cells to assess their
growth,
• adjusting the light and focus as needed,
• the images are fed into a computer system.
• Special software determines how many cell colonies are
present on the plates,
• if there are enough, another robot is tasked with picking
them up.
• Using a hollow needle, it chooses cells measuring between
100 and 200 micrometers and transfers them to a new
container for continued growth.