실험 8. Tissue culture techniques

[colony]

I. Introduction

1. Cells

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Primary culture
When cells are isolated from a tissue, grown in vitro, and before subculture.
Primary culture present in the original tissue, which were unable to attach and survive in vitro. Furthermore, If the culture proliferates, then any dividing or slow growing cells will be diluted. Hence it may be necessary at this stage to select specific cell types by cloning, selective culture, or physical separation.
 
Subculture
A monolayer culture may be transfered to a second vessel and diluted by dissociating the monolayer in trypsin.
 

2. Role of serum in cell culture

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Role of serum
Hormonal factors simulating cell growth and functions : Insulin, Glucocorticoids
Attachment and spreading factors : collagen, fibronectin
Transport proteins carrying hormones, minerals, lipid : Albumin carries vitamins, lipid and hormones among others, Iron-saturated transferrin
 
Advantage of using Low-serum and serum free media
Improved reproducibility between cultures, avoidance of lot-to-lot variations of sera
Reduced risk of contamination (virus, fungi, Mycoplasma)
Improved economy
Easier purification of culture products
Less protein interference in bioassys
Avoidance of serum cytotoxicity
Prevention of fibroblast overgrowth in primary cultures.
Selective culture of differentiated and functional cell types from heterogeneous population of primary cultures without preferential stimulation of variant subpopulations
 
Disadvantage of serum free media
Althrough greater economy is possible with many cell lines, supplementing medium with hormones and growth factors can be as expensive as serum.
Serum free media are often highly specific to one cell types, so a different medium may be required for each cell line carried.

II. Materials

1. Baby Hamster Kidney cell(BHK-21)
2. PBS(phosphate buffered saline) (per liter)
         NaCl               10.00g
         KCl                   0.24g
         Na₂HPO₄         1.44g
         KH₂PO₄           0.25g  pH 7.0
         bottle in 400ml amounts. Autoclave to sterilize
3. DMEM - fetal calf serum(10%)
4. Tissue culture flasks and dishes
5. Trypsin/EDTA
6. Hemocytometer, CO₂ incubator

III. Experimental Procedures

1. Culturing BHK cells - General cell handling

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All solution and equipment that come in contact with the cells must be sterile
Always use proper sterile technique in a laminar flow hood
All incubations are performed in a humidified, 37℃, 5% CO₂ incubator
The medium BHK cells is DMEM
Use cells that are 80-90% confluent for all experiments
For general maintenance of cells, pass BHK cells when they are 80-90% confluent and split at 1:5 dilution
Cell may be passaged 60-70 times before restarting a culture from frozen stocks
 

2. Counting cells

The cell count can be expressed as number of cells per ml medium. Counting Chambers or hemocytometers are modified glass slides engraved with grids of known area(Fig 8-1). They are supplied with glass cover slips of precise thickness so that, when correctly applied, the volume of liquid under the grid is known. The chamber has a depth of 0.1 mm, and the area of grid is 1 mm², therefore the volume is 0.1 mm³.
 

3. Initiating cell culture from frozen stock

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Remove the vial of cells from the liquid nitrogen tank and thaw at 37℃ water bath.

 

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Just before the cells are completely thawed, decontaminate the outside of the vial with 70% EtOH, and transfer the cells to a 15 ml conical tube.
Add 9 ml of prewarmed (37℃) complete DMED medium dropwise to cells.
Centrifuge in a table top centrifuge at 1000 rpm for 5 min. Decant the medium. (The remove the DMSO from the cells).
Resuspend the cells in 10 ml of complete DMEM, transfer to a T-75 flask, and incubator at 37℃. Incubation for 1-2 days should be 80-90% confluent monolayer.
 

4. Passaging the BHK cells.

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When cells are 80-90% confluent, remove all medium from the flask.
Wash cells once with 10 ml PBS to remove serum. Serum contains inhibitors of trypsin.
Add 2 ml of trypsin/EDTA solution to the monolayer and incubate 1-2 min. at RT until cells  are detached.
Add 15 ml of complete DMEM to stop trypsinization and briefly pipet the solution to obtain a single cell suspension.
Decant proper amount(4/5-19/20) of cell suspension.
Add complet DMEM to the flask.
 

5. Store the BHK cells.

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Add 2 ml of trypsin/EDTA solution to the monolayer and incubate 1-2 min. at RT until cells  are detached.
Add 10 ml of PBS to stop trypsinization and briefly Centrifuge the solution.
Add 5-10% serum/DMSO solution 1 ml
let the solution to -70℃ one day.
Transfer this solution to nitrogen tank.

IV. Notes

1. DMEM has four times the concentration of amino acids and vitamins of BME as well as additional nonessential amino acids and added ferric nitrate.
2. CO₂ incubate has 5-10% CO₂ which this allows the cultures to be maintained at the normally optimal pH range of 6.9-7.4 and to act as a buffer system.
3. All cryopreservative should have high solubility in water and low toxicity to cells and it is used to prevent cell damage during freezing process. Cryoprotective has DMSO, Glycerol, Ethylene glycol Dextrans.
4. Cell can be stored indefinitely without significant loss of viability provided they are frozen in the correct media and are cooled and thawed in the correct manner.

V. References

1. R.I. Freshney Animal cell culture 1986 IRL Press
2. Butler, M. and D. Rickwood Cell culture Labfax 1992 Bios scientific

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