1. Cell culture technique and its implication
341 Zoo
Dr. Gamal Badr
Associate professor

2. History of tissue culture
1885: Roux maintained embryonic chick cells in saline
1965:Ham introduced serum free culture medium that support living cells
1965: Harris & Watkins successfully fused human and mouse cells by virus
1975: Kohler & Milstein produced the first Hybridomas capable of secreting monoclonal antibodies.
1978: Sato developed a serum free medium with a cocktail of hormone and growth factors
!982: human insulin is produced
1985: human growth factors were produced
1986: lymphoblastoid gamaIFNlicences
1987:tissue type plasminogen activator(tPA) became commercially available
1989:Recombinant erythropoitin in trial
1990:Recombinent products in trial (factorVI.HBsAg, Il2, EGF, mAbs)

3. What is cell culture?
Cells, previously growing in a human or animal modified to grow in plastic or glass
In the body = in vivo
On plastic or glass = in vitro
Kept in an incubator to stay at body temperature
We use special media with nutrients so the cells can grow and divide

4. What can we do with cells?
Test pharmaceutical drugs
Watch disease mechanisms
Design potential treatments
Observe the regenerative process
How do cells and tissues repair themselves after damage from illness or injury?
Observe the developmental process

5. Cell Culture Protocols
The “Do’s” and “Don’ts” of cell culture
The Do’s
Use personal protective equipment (PPE)
Wear dedicated PPE for tissue culture facility and keep separate from PPE in the general lab environment.
Keep all work surfaces free of clutter.
Correctly label reagents including flasks, medium.
Only handle one cell line at a time.
Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol).
Wherever possible maintain separate bottles of media for each cell line.
Examine cultures and media daily for evidence of gross bacterial or fungal contamination.
Quality control all media and reagents prior to use.
Ensure that incubators, cabinet, centrifuges and microsocpes are cleaned.
Test cells for mycoplasma on a regular basis.

6. The Don’ts Do not continuously use antibiotics in culture medium.
Don’t allow waste to accumulate particularly within the incubators and culture area.
Don’t have too many ppl in the lab at any one time.
Don’t handle cells from unauthenticated sources in the main cell culture suite.
Avoid keeping cell line continually in culture without returning to frozen stock.
Avoid cell culture becoming fully confluent. Always sub-culture 70-80% confluency or as advised on ECACC’s cell culture data sheet.
Don’t allow media to go out of date.
Avoid water baths dirty.
Don’t allow essential equipment to become out of ccalibration.

7. Technique and instrument Laminar flow

8. Carbon dioxide incubator

9. Microscope

10. Tissue culture Ware

11. Culture Media Sterilization

12. Cell counting

13. Changing Medium

14. Passaging cells (subculturing cells)
Process of diluting cell number in order to keep cells actively growing
For adherent cells, when they cover the tissue culture dish, they need to be passaged
Otherwise, the cells will become unhealthy and stop growing

15. Other miscellaneous Equipment
Fridge Freezer for storing medium
Liquid nitrogen Container for cryopreservation of cells
Incubator for warming up of the medium
Bench centrifuges to separate out cell pellet.

16. Sterile technique
Procedures by which cultures are manipulated without infecting the worker or contaminating the cultures or the laboratory environment
Important for the cell culture
You want to be sure you are growing only the cells you want to grow a single unwanted cell can ruin an experiment or a multimillion $ production run
Important for the lab worker
Some cultured cells can pose health threats to workers if they are inhaled, ingested, or absorbed--sterile technique prevents exposure of the worker to cultured cells

17. Sterile technique: Bacteria
Media is sterilized in an autoclave
Very high pressure and temperature kills
Minimizing contamination through awareness
Inoculation loop, pipets, pipet tips, etc. should never touch contaminating surfaces
Containers holding media and other cell additives should be kept closed until needed, then opened briefly
Tubes and flasks are “flamed” whenever they are opened
The purpose of flaming is not to sterilize, but to warm the tube and create warm air convection currents up and away from the opening. This "umbrella" of warm, rising air will help to prevent the entrance of dust particles carrying contaminating microbes

18. Sterile technique: Tissue culture
Media is usually sterilized by filtration
Standard biological filters are 0.22 mm mm; these remove most microbes by trapping them in the filter
This does not remove all microbes (such as mycoplasm), and will not remove viruses
Unlike bacterial media, animal cell media cannot be autoclaved, because this would destroy many of the growth factors and other molecules needed for cell growth
Working with cells in a laminar flow hood
HEPA filter
Disinfect
70% Ethanol is sprayed in hood, onto bottles entering hood
Minimizing contamination through awareness
Inoculation loop, pipets, pipet tips, etc. should never touch contaminating surfaces
Containers holding media and other cell additives should be kept closed until needed, then opened briefly

19. Sterilization methods
Autoclave
Applies heat under high pressure; this increases the boiling point of water to 121ºC (normal boiling point of water is 100ºC)
15-20 min. is sufficient to kill most microbes
Filtration
Large volumes: suction filter
Small volumes: syringe filter
UV radiation
Causes mutations to form in the DNA of microbes, causing genetic damage and eventual death
Used to sterilize surfaces (such as the surface of laminar flow hoods)

20. What is in the media? Dulbecco’ Modified Eagle’s Media (DMEM)
Contains glucose, some proteins, and essential salts
Contains a pH indicator (phenol red) Media looks pink/red at pH 7.2
Acidic -yellow or orange (cell growth, bacterial growth)
Basic -purple (no cell growth, not enough CO2)

21. Media preparation
Process of combining and sterilizing ingredients of a particular medium
Proper media prep is essential for cell culture systems!
If the media is lacking any components, the cells would either die or be unhealthy
If the media is not properly sterilized, cells will be contaminated with microorganisms
If glassware, pipettes, etc. used to prepare media are not properly cleaned, cell cultures can be contaminated with chemical residues
As a result, the cells would not produce the desired product effectively

22. More media components Antibiotics (penicillin and streptomycin)
Prevent bacterial contamination
Salts and buffers
To simulate in vivo environment
Serum
Portion of blood after the cells and fibers have clotted
From cow, horse, sheep
added to media as a nutrient source for growing cells
Lipids, proteins

23. Phosphate Buffered Saline
Used to wash/remove excess serum that inhibits the function of TRED.
Must be warmed in the water bath before use so cells are not shocked by cold liquid.
Trypsin EDTA
An enzyme used to detach the cells from a culture dish.
EDTA binds calcium ions in the media that would normally inhibit trypsin.

24. Bleach
Used to destroy any remaining cells in dishes and tubes before they are tossed in the trash can.
Add enough to change media to clear,
wait 5 minutes,
rinse solution down sink
throw away the dish/flask/plate in the trash can.

25. Potential sources of contamination in cell culture
Equipment
Glassware, instruments, incubators.
Solutions
Media or reagents added into media
Room air
Work surfaces
Operators
Hands, hair, clothing, breath, etc.
Incoming cells

26. Types of contamination in animal cell culture systems
Biological
Bacteria
Fungi
Cross-contamination by other cell cultures
Chemical
Residues left from detergents or disinfectants on glassware, pipettes, instruments, etc.
Metal ions, other impurities in water
Endotoxin: highly bioreactive part of the cell wall of some types of bacteria (endotoxin molecules are shed from bacteria and are left behind even after bacteria die)

27. Characteristics of microbial contamination in cell culturespH
Sudden change in pH is often a strong indicator of contamination
Turbidity
Media looks cloudy
Microscopic evaluation
Can see individual microorganisms, often because their motion can be seen easily under the microscope

28. Further detection of contamination in cell cultures
Mycoplasma
Smallest free-living prokaryotes
Not killed easily by many antibiotics
Contamination can’t be seen by microscopic evaluation
Mycoplasma testing should be done routinely (several tests are available)
Long-term effects of mycoplasma contamination include reduced growth rate, changes in cell shape and metabolism, and chromosome abnormalities
Endotoxin
LAL test: an extract from the blood of horseshoe crabs is used to test for endotoxin (horseshoe crab blood contains a protein that binds endotoxin & can be detected)

29. Minimizing contamination
Contamination is a fact of life when dealing with cell cultures
Very difficult to prevent entirely, but good lab practices can keep contamination incidents to a minimum
Proper cleaning and sterilization of glassware, pipettes, and other lab instruments.
 Practicing sterile technique when working with cell cultures

30. Types of cell culture
Primary Cell culture
Continuous Cell lines

31. Adherent vs Suspension cells for tissue culture
Adherent cells: cells grow in a single layer (called a monolayer) attached to the tissue culture dish
Cell growth is limited by available surface area on which cells can grow
To passage adherent cells, the cells must be released from the dish (done either enzymatically, chemically, or mechanically)
Suspension cells : cells are suspended in liquid as single cells or as free-floating clumps of a few cells
To passage suspension cell cultures, a proportion of the cells in culture are diluted into a larger volume of medium

32. Secondary or continuous Cell Lines
Attached Cell Lines
Name
Species and tissue of origin
Morphology
MRC-5  (Prod. No )
Human lung
Fibroblast
HELA  (Prod. No )
Human cervix
Epithelial
VERO  (Prod. No )
African Green Monkey Kidney
NIH 3T3  (Prod. No )
Mouse embryo
L929  (Prod. No )
Mouse connective tissue
CHO  (Prod. No )
Chinese Hamster Ovary
BHK-21  (Prod. No )
Syrian Hamster Kidney
HEK 293  (Prod. No )
Human Kidney
HEPG2  (Prod. No )
Human Liver
BAE-1  (Prod. No )
Bovine aorta
Endothelial

33. SUSPENSION Cell lines Suspension Cell Lines Name
Name
Species and tissue of origin
Morphology
NSO  (Prod. No )
Mouse myeloma
Lymphoblastoid-like
U937  (Prod. No )
Human Hystiocytic Lymphoma
Lymphoblastoid
Namalwa  (Prod. No )
Human Lymphoma
HL60  (Prod. No )
Human Leukaemia
WEHI 231  (Prod. No )
Mouse B-cell Lymphoma
YAC 1  (Prod. No )
Mouse Lymphoma
U 266B1  (Prod. No )
Human Myeloma
SH-SY5Y  (Prod. No )
Human neuroblastoma
Neuroblast

34. Storing and maintaining Continuous Cell Lines

35. Advantages of Cell Culture
Cell types kept Constant and homogenous.
There is direct access to the cells so effect of toxicity of drug and chemicals studied without being lost to other tissues and excreted.
Replace/ reduce the number of animals. Legal, moral, ethical issues?(animal rights Group).
Control of the environment (pH, osmotic pressure, temperature, oxygen and Carbon dioxide tension.)

36. Disadvantages of Cell Culture
Contamination can be Chemical (culture medium) Or Biological (adding antibiotics).
Finding a Happy Environment.
De-differentiation
Origin of Cells
Major differences from in-vivo

37. Implication for Cell Culture
Model system (many specialized functions are restored)
Toxicity Test (viability of cells)
Cancer research
Virology
Cell Based manufacturing
Genetic councelling
Genetic engineering
Drug screening & development
Gene Therapy

38. Production of monoclonal antibodies

39. Tissue Engineering Carbon nanotube scaffolding Name of scaffoldig
Made up of
Nanofiber
Like carbon
Textile
Polyglycolide
Gas Foam
Foam like structure due to CO2 gas

40. Growing cells in 3D Forming Tissue in Bioreactor

41. Application of Monoclonnal Antibodies
IMMUNOLOCALIZATION
IMMUNOBlotting
Cancer Treatment

42. Immunolocalization

43. Immunoblotting

44. Cancer Treatment

45. Stem cell research Embryonic Cell Lines Adult Cell Line
Homeiopoitic Stem Cell

46. Different types of stem cells
Early embryonic stem cells
Totipotent: can become any kind of cell in the body
Blastocyst embryonic stem cells
Pluripotent: can become virtually any kind of cell in the body
Umbilical cord stem cells:
Multipotent: can differentiate into only a limited number of cell types
Adult stem cells:

47. Future of stem cell-mediated therapy {stem cells to replace damaged organs}
Stem cells are induced to differentiate into a cell type that is damaged or missing in the patient
Cells are grown on tissue-promoting matrix or scaffolding
Healthy tissue is transplanted into patient--unlike other organ transplants, tissue is an immune match to patient, so no immunosuppression drugs would be necessary

48. Medical applications of embryonic stem cell research
Diabetes
Caused by body’s destruction of insulin-producing cells in the pancreas.
Researchers in Spain (2001) used mouse embryonic stem cells that they differentiated into glucose-responsive, insulin-producing cells; the cells reversed diabetes symptoms when injected into the spleens of mice.

49. Animal cloning
The successful "cloning" of mammals has resulted in a flurry of research, Dolly July ‘96
Reproductive cloning
Remove nucleus from egg cell
Add somatic cell
from adult donor
Grow in culture to produce an
early embryo (blastocyst)
Implant blastocyst in surrogate mother
Remove embryonic stem cells from blastocyst and grow in culture
Induce stem cells to form specialized cells (therapeutic cloning)
Clone of donor is born
(reproductive cloning)
Donor cell
Nucleus from donor cell
Therapeutic cloning