COLONING AND SELECTIONPRESENTED BYDR. GHALIA QAYYUM

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

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.

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

First clone• Sanford was the first cloned cell strain, isolated by capillary

cloning from mouse L-cells [Sanford et al., 1948].

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.

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)

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

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

Multi well Plates, petri plates and flask

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.

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.

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.

ConfluenceAfter 24 hours

70% confluency

100% confluence

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

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.

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

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

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

FLOW CHART

Resected Tissue

Cell or tissue culture in vitro

Primary culture

Secondary culture

Sub-culture

Cell Line

Sub-culture

ImmortalizationSuccessive sub-cultureSingle cell isolation

Clonal cell line Senescence

Transformed cell line

Immortalised cell line

Loss of control of cell growth

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

Steps In Passaging Cells

General procedure cell cloning

Trypsinize the cells: a single cell suspension. trypsin used at 0.05 μg/mLUnder-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

General procedure 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.

FeedingFor 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 .

Use of Conditioned Medium

Conditioned MediumMedium 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

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.

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

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.

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.

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;

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.

IMPORTANT ADVISE FOR CELL CULTURE WORKERS

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

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

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.