1. Cell Culture Mehrnaz kouhbanani nejad
Msc of hematology & blood banking
Kerman university of medical sciences

2. Introduction Cell Culture Organ Culture Ross Harrison in 1907
Cell culture widely available: the late 1940’s to early 1950’s
Cell culture has become one of the major tools used in the life sciences today
Tissue Culture
Organ Culture
Cell Culture

3. Cell Culture Systems
Monolayer Culture Systems: the ability of the cells to either grow attached to a glass or
treated plastic substrate
Suspension Culture Systems: floating free in the culture medium
Anchorage-Dependent: grow when attached to a suitable substrate
Anchorage-Independent: grow either attached to a substrate or floating free in suspension
always grow in suspension blood cells

4. What Are Cultured Cells Like?
Epithelial-like: cells that are attached to a substrate
and appear flattened and polygonal in shape.
Lymphoblast-like: cells that do not attach normally
to a substrate but remain in suspension with a spherical shape.
Fibroblast-like: cells that are attached to a substrat and
appear elongated and bipolar.

5. Functional Characteristics
origin (liver, heart, etc) how adapt to the culture conditions
Transformed Cells
Continuous cell lines
Finite cell lines

6. How Are Cell Cultures Obtained?
Cells are surgically removed from an organism and placed into a suitable culture environment, they will attach, divide and grow.
Explantation
Enzyme digestion
Combination of these methods
Primary Culture

7. Enzymes for Cell Detachment and Tissue Dissociation
Collagenase :
Collagenase cleaves the peptide bonds in native collagen.
widely used in isolation of cells from animal tissue
The most potent collagenase secreted by Clostridium histolyticum.
Enzymes for Cell Dissociation and Lysis / Issue 2, 2006

8. Enzymes for Cell Detachment and Tissue Dissociation
Hyaluronidase:
Hyaluronidase is typically used as a supplement to proteases for tissue dissociation.

9. Enzymes for Cell Detachment and Tissue Dissociation
Dnase:
DNAse is typically used to supplement proteases for tissue dissociation. DNase helps to reduce viscosity resulting from DNA released from damaged cells during harvesting.
Trypsin:
Trypsin is a serine protease commonly used for detachment of adherent cell lines and dissociation of tissues.
Enzymes for Cell Dissociation and Lysis / Issue 2, 2006

10. Isolation and culture of autologous goat DFs
Fresh skin specimens of the groin area were harvested from goat after anesthetization
The specimens were washed with sterile PBS intensively and cut into small fragments
The specimen fragments were then digested with 0.1% collagenase type I for 3 h.
Reconstructing spinal dura-like tissue using electrospun poly(lactide-co-glycolide) membranes and dermal fibroblasts to seamlessly repair spinal dural defects in goats Journal of Biomaterials Applications / The Author(s) 2015

11. Isolation and culture of autologous goat DFs
After that, the above mixture was filtered through a 100-mm
nylon mesh and centrifuged at 1200 g for 5 min.
Cells were collected from the bottom, washed with PBS,
re-suspended in the growth, and plated in 100mm culture plates.
The medium was changed twice a week.
The mesenchymal origin of DFs was confirmed by immunofluorescent staining with mouse-anti-goat vimentin and mouse-anti-goat fibronectin antibodies.
Reconstructing spinal dura-like tissue using electrospun poly(lactide-co-glycolide) membranes and dermal fibroblasts to seamlessly repair spinal dural defects in goats Journal of Biomaterials Applications / The Author(s) 2015

12. Isolation and Culture of BM-MSCs
Rats were euthanized, and femurs and tibias were removed aseptically.
A hole was then created in the knee joint end of each bone with a 26-gauge needle, and marrow was flushed.
Flushed bone marrow was centrifuged at 1200 ×g for 20 min. Cell pellets were resuspended in 5mL CCM and then transferred into T-25 flask and cultured.
The Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Media Topically Delivered in Fibrin Glue on Chronic Wound Healing in Rats BioMed Research International 2015

13. Isolation and Culture of BM-MSCs
Medium was changed, and nonadherent cells were removed twice a week. When adherent cells were subconfluent, they were detached with 0.25% trypsin-EDTA solution and reseeded at 2-3 T-25 flasks.
MSCs at passage 3 were used for the current experiments.
The Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Media Topically Delivered in Fibrin Glue on Chronic Wound Healing in Rats BioMed Research International 2015

14. Isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method
Five grams of human fat are incubated in 40 ml of warm sterile PBS containing 3% Pen/Strep for 30 min.
Then extra non-fat tissue (e.g., blood vessels and connective tissues...) were surgically removed under sterile conditions.
One gram of tissue is minced into pieces of 8–10 mm3 under sterile conditions.
Minced fat pieces were into two T70cm2 cell culture plates containing each 5 ml of complete growth medium [RPMI] and incubated for 3 days.
Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method
Data in Brief 6 (2016)

15. Isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method
Media is replaced with a new fresh growth media every 3 days.
On day six the explants cultures can be observed under light microscopy and cells appear to spread out of the fat pieces into the surroundings of the fat specimens.
At day 9, fat pieces are removed with a sterile pipet and the cell cultures were then washed twice with 5 ml of sterile warm PBS.
Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method Data in Brief 6 (2016)

16. What Are Some of the Problems Faced by Cultured Cells?
Avoiding Contamination
Biological:
fast growing yeast, bacteria and fungi
usually have visible
Chemical:
endotoxins, metal ions or traces of chemical disinfectants
invisible
Requirements to avoiding contamination:
proper training in and use of good aseptic technique on the part of the cell culturist.
properly designed, maintained and sterilized equipment, plasticware, glassware, and media.

17. What Are Some of the Problems Faced by Cultured Cells?
Finding A “Happy” Environment
Controlled temperature
Good substrate for cell attachment
Appropriate medium and incubator that maintains the correct pH and osmolality

18. How to Decide if Cultured Cells Are “Happy”
easiest to determine but is often the least useful
testing method , cells (20 to 200) are placed in a culture vessel and is measured the number of colonies
percentage of cells forming colonies is a measure of survival
colony size is a measure of growth rate
Morphology
which set of conditions (culture media, substrate, serum, plasticware) is better for the cells
measure cell survival or death , in vitro cytotoxicity assays.
Growth Rate
Plating Efficiency
Expression of Specialized Functions
the most difficult to observe and measure
biochemical or immunological assays and tests

19. What is Cell Culture Used For?
Toxicity Testing
Cancer Research
Virology
Genetic Engineering
Gene Therapy

20. Thanks for your attention