Showing all principle of prepration tissue culture and perfect factors and media to cultaviate cells.

1. The principle of tissue
culture technique
Presented by
Dr. Hadeel Alboaklah
Day1 :

2. APPLICATIONS OF TISSUE CULTURE
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1. Studieson intracellularactivityeg. cell cycle and
differentiation, metabolism
2. Elucidation of intracellular flux eg. hormonalreceptors,
signal transduction
3. Studies related tocell tocell interaction eg. cell adhesion
and motility, metaboliccooperation
4. Evaluation of environmental interactions eg.cytotoxicity,
mutagenesis
5. Studies dealing with genetics eg. Geneticanalysis,
immortalization, senescence
6. Laboratory production of medical/ pharmaceutical
compounds forwide range of applicationseg. Vaccines,
interferon, hormones

3.  Animal cell culture are useful for the production of
many pharmaceutically/ medically importantproteins
which are asfollows:
1. Plasminogen
2. Interferon
3. Blood clotting factors
4. Hormones
5. Monoclonal antibodies
6. Erythropoietin
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4. Major development’s in cell culture
technology
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 Firstdevelopmentwas the useof antibioticswhich
inhibits the growth ofcontaminants.
 Second was the useof trypsin toremoveadherentcells
tosubculture further from theculturevessel
 Third was the useof chemicallydefined culture
medium.

5. Culture medium definition:
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A growth medium or culture
medium is a liquid or gel
designed to support thegrowth
of cells

6. Media Type Examples
Natural media
Biological Fluids
plasma, serum, lymph,
human placental cordserum,
amniotic fluid
Tissue Extracts
Extract of liver, spleen,
tumors, leucocytes andbone
marrow, extract of bovine
embryo and chickembryo
Clots coagulants or plasmaclots
Artificial media
Balanced salt
solutions
Eagle’s BS, Hank’sBS,
Basal media MEM DMEM
Complex media RPMI-1640, IMDM
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Types of Cell culture media

7. Natural Media
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 Very useful
 Lack of knowledgeof theexactcompositionof these
natural media

8. Artificial Media
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 Serum containing media
 Serum-free media (defined culturemedia)
 Chemically defined media
 Protein-free media

9. Basic Components of Culture
Media
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 Culture media (asa powderorasa liquid) contains:
 aminoacids
 Glucose
 Salts
 Vitamins
 Othernutrients
Therequirements forthesecomponents
vary among cell lines, and these
differencesarepartly responsibleforthe
extensive number of medium
formulations .

10.  Natural buffering system
 HEPES
 Phenol red as a pH indicator (yellow or purple)
 Inorganic salt
 Amino Acids (L-glutamine)
 Carbohydrates
 Proteins and Peptides (important in serum-free media.
Serum is a rich source of proteins and includes albumin,
transferrin, aprotinin, fetuin, and fibronectin
 Fatty Acids and Lipids
 Vitamins
 Trace Elements
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Decontaminate external surfaces of all vials, including the medium bottle, with ethanol or
isopropanol.
To formulate Endothelial Growth Medium (EGM™ Medium), transfer the contents of the
EGM™ SingleQuots™ Kit (Lonza Catalog No. CC-4133 containing Bovine Brain Extract [BBE];
Ascorbic Acid, Hydrocortisone, Epidermal Growth Factor [hEGF], Fetal Bovine Serum [FBS]
and Gentamicin/Amphotericin-B [GA]) to EBM™ Basal Medium with a pipette, and rinse
each vial with medium.
To formulate Microvascular Endothelial Growth Medium-2 (EGM™-2MV Medium), transfer
the contents of the EGM™-2MV SingleQuots™ Kit (Lonza Catalog No. CC-4147 containing
human Epidermal Growth Factor [hEGF], Vascular Endothelial Growth Factor [VEGF], R3-
Insulinlike Growth Factor-1 [R3-IGF-1], Ascorbic Acid, Hydrocortisone, human Fibroblast
Growth Factor-Beta [hFGF-β], Fetal Bovine Serum [FBS], and Gentamicin/Amphotericin-B
[GA]) to EBM™-2 Basal Medium with a pipette, and rinse each vial with medium.
NOTE: If there is concern that sterility was compromised during the supplementation process,
the entire newly prepared growth medium may be re-filtered with a 0.2 µm filter to assure
sterility. Routine re-filtration is not
Preparation of Culture Media

12. Thawing of Cells / Initiation of Culture
Process
NOTE: For
proliferation of these
cells, cells must be
cultured at 37°C±1°C,
5% CO 2, 90%±2%
humidity.
When initially plating
endothelial cells from
cryopreservation, the
recommended
seeding density is
provided in the table
below:

13. • To set up culture vessels, calculate the number of vessels needed based on the
recommended seeding density as well as the surface area of the vessels being used.
• Add the appropriate amount of medium to the vessels (1 ml/5 cm2) and allow the
vessels to equilibrate in a 37°C±1°C, 5% CO 2, 90%±2% humidity incubator for at least 30
minutes.
• Wipe cryovial with ethanol or isopropanol before opening. In a sterile field, briefly twist
the cap a quarter turn to relieve pressure and then retighten. Quickly thaw the cryovial in
a 37°C water bath being careful not to submerge the entire vial. Watch your cryovial
closely; when the last sliver of ice melts, remove it. Do not submerge it completely.
Thawing the cells for longer than 2 minutes results in less than optimal results.
• NOTE: Centrifugation should not be performed to remove cells from cryoprotectant
cocktail. This action is more damaging than the effects of DMSO residue in the culture.
• Carefully mix the cell suspension using a micropipette. Dispense cells into the culture
vessels set up in previous steps. Gently rock the culture vessel to evenly distribute the
cells and return to the 37°C±1°C, 5% CO 2, 90%±2% humidity incubator.
.

14. Maintenance
• Change the growth medium 16 to 24 hours after seeding and every other
day (every 48 hours) thereafter.
• When cell confluence is 25-45%, increase the media volume to 1.5 ml/5
cm2.
• When cell confluence is greater than 45%, increase the media volume to 2
ml/5 cm2.
• Warm an appropriate amount of medium to 37°C in a sterile container.
Remove the medium and replace it with the warmed, fresh medium and
return the flask to the incubator.
• Avoid repeated warming and cooling of the medium. If the entire contents
are not needed for a single procedure, transfer and warm only the required
volume to a sterile secondary container.

15. . Subculturing
The following instructions are for a 25 cm2 flask. Adjust all
volumes accordingly for other size flasks :
• Subculture the cells when they are 70%-85% confluent.
• For each 25 cm2 of cells to be subcultured:
• Thaw 2 ml of Trypsin/EDTA and allow to come to room
temperature
• Allow 7-10 ml of HEPES Buffered Saline Solution (HEPES-BSS)
to come to room temperature.
• Allow 5 ml of Trypsin Neutralizing Solution (TNS) to come to
room temperature.
• Remove growth medium from 4°C storage and allow
warming to room temperature.
• Prepare new culture vessels. 3. Subculture one flask at a
time

16. Common Cell Culture Media
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 Eagle’s Minimum Essential Medium (EMEM)
 Dulbecco’s Modified Eagle’s Medium (DMEM)
 Low glucose
 High glucose
 RPMI-1640
 Ham’s Nutrient Mixtures
 DMEM/F12
 Iscove’s Modified Dulbecco’s Medium (IMDM)

17. Criteria for Selecting Media
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18. Cell Line
Morpholog
y
Species Medium Applications
HeLa B Epithelial Human
MEM+ 2mM Glutamine+ 10% FBS +
1% Non Essential Amino Acids
(NEAA)
Tumourigenicity
and virusstudies
HL60 Lymphoblast Human
RPMI 1640 + 2mM Glutamine + 10-
20% FBS
Differentiation
studies
3T3 clone
A31
Fibroblast Mouse
DMEM + 2mM Glutamine +5% New
Born Calf Serum (NBCS) + 5% FBS
Tumourigenicity
and virusstudies
COS-7 Fibroblast Monkey DMEM+ 2mM Glutamine + 10% FBS
Gene expression
and virus
replication
studies
CHO Epithelial Hamster
Ham′s F12 + 2mM Glutamine + 10%
FBS
Nutritional and
gene expression
studies
HEK 293 Epithelial Human
EMEM (EBSS) + 2mM Glutamine +
1% Non Essential Amino Acids
(NEAA) + 10% FBS
Transformation
studies
HUVEC Endothelial Human
F-12 K + 10% FBS + 100 µg/ml
Heparin
Angiogenesis
studies
Jurkat Lymphoblast Human RPMI-1640 + 10% FBS Signaling studies
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19. Common media and their applications
Media Tissue or cellline
IMDM
Bone marrow, hematopoietic progenitorcells,human
lymphoblastoid leukemia cell lines
MEM
Chick embryofibroblast, CHO cells, embryonicnervecells,
alveolar type cells, endothelium, epidermis, fibroblast,
glia, glioma, human tumors, melanoma
DMEM
Mesenchymal stem cell, chondrocyte, fibroblast,
Endothelium, fetal alveolar epithelial type II cells, cervix
epithelium, gastrointestinal cells, mouseneuroblastoma,
porcine cells from thyroid glands, ovarian carcinoma cell
lines, skeleton muscle cells, sertoli cells, Syrian hamster
fibroblast
RPMI-1640
Tcells and thymocytes, hematopoietic stem cells, human
tumors, human myeloid leukemia cell lines, human
lymphoblastoid leukemia cell lines, mouse myeloma,
mouse leukemia, mouse erythroleukemia, mouse
hybridoma, rat livercells
Nutrient
mixtureF-10
and F-12
Chick embryo pigmented retina, bone, cartilage,adipose
tissue, embryonic lung cells, skeletal musclecells 15

20. Media Supplements
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 Serum in Media
 Basic nutrients
 Growth factors and hormones
 Binding proteins
 Promoteattachmentof cells to the substrate
 Protease inhibitors
 Provides minerals, like Na+, K+, Zn2+, Fe2+,etc
 Protectscells from mechanical damagesduring agitationof
suspensioncultures
 Acts a buffer
 Antibiotics

21. Aseptic conditions
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1. Switch on the laminarflow cabinet 20 mts priortostart
working
2. Swab all bottle tops & necks with 70%ethanol
3. If working on the bench usea Bunsen flame
4. Flameall bottle necks & pipette by passing veryquickly
through the hottest part of theflame
5. Avoiding placing caps & pipettesdown on the bench; practic
holding bottle tops with the littlefinger
6. Workeither left to rightorviceversa, so thatall material goe
to one side, oncefinished

22. 7. Clean up spills immediately & alwaysleave
the work place neat &tidy
8. Neveruse thesame media bottle fordifferent
cell lines.
9. If caps are dropped or bottles touched
unconditionally touched, replace themwith
newones
10. Necks of glass bottles prefer heat at least for
60 secs at a temperature of 200C
11. Never use stock of materials duringhandling
of cells.
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23. Contaminant’s of cell culture
Cell culture contaminants of twotypes
 Chemical-difficult to detect caused by
endotoxins, plasticizers, metal ions ortraces
of disinfectants that areinvisible
 Biological-cause visible effects on the culture
they are mycoplasma, yeast, bacteria orfungus
or also from cross-contamination of cells from
other cell lines
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24. Effects of Biological Contamination’s
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 They competes for nutrients with host cells
 Secreted acidic or alkaline by-products ceasesthe
growth of the hostcells
 Degraded arginine & purine inhibits the synthesis of
histone and nucleicacid
 They also produces H2O2 which is directly toxic to cells

25. Detection of contaminants
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 In general: turbid culture media, change in growth rates,
abnormally high pH, poor attachment, multi-nucleatedcells,
graining cellular appearance, vacuolization, inclusion bodies
and cell lysis
 Yeast, bacteria & fungi usuallyshows visibleeffecton theculture
(changes in medium turbidity orpH)
 Mycoplasma detected by direct DNA staining withintercalating
fluorescent substances e.g. Hoechst33258
 Mycoplasma alsodetected byenzyme immunoassay by specific
antisera or monoclonal abs or by PCR amplification of
mycoplasmal RNA
 The bestand theoldest way toeliminatecontamination is to
discard the infected cell linesdirectly

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