How to make human epidermal keratinocytes to form cell culture?(1)
1. How to develop human epidermal keratinocytes to form cell culture?
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Skin is not only a physical barrier to the body, but also plays an important role in the body's immunity and endocrine.
Horniness cells is the main part of epidermal cells, cutin forming cells in vitro can be therapeutic for the skin toxicology, burns, cosmetology and skin disease pathogenesis research provide a new way.
So how to do human epidermal keratinocytes?
(1) After achieving the fusion of 60% ~ 75%, remove the culture medium, and the single-layer cells were washed with 10ml 1:5000 ethylene diamine tetraacetic acid (Versene).
1 ~ 2 ml0. 05% trypsin - 0.53 m m EDTA in culture bottle in 36 ° C + 2 ° C incubation for 5 to 10 minutes.
When cells turned round, remove trypsin, incubation again in 36 ° C + 2 ° C.
(2) When approximately 90% of the cells were separated, the enzyme activity of trypsin was terminated using 10ml of soybean trypsin inhibitor.
The cell suspension was transferred to a 15ml sterile centrifuge tube and centrifuged at room temperature for 5 minutes.
The cell remakes the suspension and is centrifuged again as described above.
(3) The keratinocytes used 3 ~ 5ml full media to gently blow into suspension, and the cells were put into 75cm2 tissue culture bottles from about 1 ~ 3 x 106, and added 15ml complete media.
(4) Cell culture: 36 ° C + 2 ° C, 5% CO2 in the air.
The keratinocytes are replaced every 2 ~ 3 days, and can be passed on for the next generation after reaching 60% ~ 75%.
2. What should you pay attention to when you freeze the cells?
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Cell cryopreservation is one of the main methods for cell preservation.
At present, the most commonly used technique for cell cryopreservation is liquid nitrogen cryopreservation.
The method is puts cells - 196 ℃ in the liquid nitrogen cryopreservation, can temporarily out of the cell growth state and to save the cell characteristics, such as long as in need of recovery cells can be used in the experiment.
What should you notice when you freeze the cells?
(1) cells in the process of freezing in - 20 ℃ refrigerator placed time shall not exceed 1 hour, to prevent too much ice crystals and damage cells.
Also can skip - 20 ℃ this step directly into - 80 ℃ in the freezer, but doing so the cell survival rate lower.
(2) a large amount of heat is released when DMSO is diluted.
Therefore, DMSO cannot be added directly to the cell fluid and must be prepared in advance.
(3)DMSO must be a cell culture level.
New DMSO itself in aseptic condition, should immediately following the first open a bottle of a small amount of partial shipments in a sterile tube or in a bottle, 4 ℃.
Avoid repeated freeze-thaw to cause DMSO lysis to produce harmful substances.
And reduce pollution opportunities.
To filter DMSO, a DMSO must be selected for millipore.
The main components of cryopreservation: cryoprotectant, basic culture medium, serum or protein.
3. What are the characteristics of microbial contamination in cell culture such as bacteria and mycoplasma?
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The microbial contamination in cell culture can be divided into bacteria, mycoplasma, fungi and virus.
The main causes of pollution are the improper operation technique, poor operation room environment, the contaminated fetal bovine serum and the contaminated cells.
What are the characteristics of microbial contamination such as bacteria and mycoplasma in cell culture?
(1) bacteria: bacteria in ordinary black sand under inverted microscope, depending on the bacterial infection, had a different shape, culture generally turbid yellow, obvious effects on cell growth.
(2) mycoplasma: black, like the roar of multiple, nutrient solution culture generally turbidity commonly, chlamydial infection, the domestic many don't do mycoplasma negative serum detection, and mycoplasma is one of the most common microorganisms in bovine serum.
And it can't be removed by filtering.
After mycoplasma infects the cell, the cell disease is not obvious, but slowly.
(3) fungi: general culture clear, don't change color, the microscopic filaments, some fungus began to feel like pieces of dead cells, and is it lots and lots of small pieces of clear, like coral, unlike cell fragments cannot distinguish, slowly will grow very thin black filaments.
Fungi grow more slowly than bacteria, but once they are found, they are contaminated and difficult to revive.
(4) Virus: in the process of tissue culture, virus contamination can occur if the potential virus is not removed.
At present, no less than 20 serological viruses have been found in the cultivation of the original monkey kidney cells.
Although the cells of the virus do not affect the primary culture, it is not safe to produce vaccines.
Therefore, the potential virus is a difficult problem in the production and production of a large number of biological products such as vaccine and interferon.
After determining which microbial contamination of the cell, isolate the contaminated cell from the other cell lines, disinfect the culture vessel and the ultra-clean table with laboratory disinfectant, and check the HEPA filter.
High concentrations of antibiotics and antifungal agents may be toxic to some cell lines, so dose-response experiments determine the level of toxicity of antibiotics and antifungal agents.
This is especially important when using antibiotics such as amphotericin B and antimycin, such as tylenol.
4. When preparation of dry powder medium, what should you pay attention to?
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Now, most of the market is liquid medium, but there is still a small amount of dry culture medium.
Compared with the liquid medium, the advantage of dry powder medium is that the production cost is low and easy to transport, which can be kept for a long time.
But it can't be used directly, it needs to be made into liquid medium.
What should we pay attention to when making dry powder culture medium?
(1) The ingredients of dry powder are not included. The common ones include NaHCO3, glutamine, sodium pyruvate, HEPES, etc.
Some of these ingredients must be added, such as NaHCO3 and glutamine, and some are determined according to the experiment.
(2) The preparation is to ensure full dissolving, and the NaHCO3, glutamine and other substances will not be added until the medium is completely dissolved.
(3) The water used in the preparation shall be three steam water and the ion concentration is very low.
(4) The utensils used should be strictly disinfected.
(5) The prepared medium should be filtered immediately, and the sterilization is kept at 4 degrees.
(6) Liquid medium is mainly for the convenience of scientific research design medium, it is a guarantee of sterile solution after sterilization, when necessary, can be made without the endotoxin of solution, such as scientific research personnel's workload can be saved.
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