Culture of colorectal tumor cells
Culture of colorectal tumor cells
The adenoma is usually digested with enzymes, and the cancerous tissue is minced with a scalpel blade. If well-differentiated colorectal cancer specimens are not easily separated from the cells after cutting, they can be separated by enzymatic digestion.
Operation method
Scheme 24.3 Culture of colorectal tumor cells
Principle
The adenoma is usually digested with enzymes, and the cancerous tissue is minced with a scalpel blade. If well-differentiated colorectal cancer specimens are not easily separated from the cells after cutting, they can be separated by enzymatic digestion.
Materials and Instruments
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Growth medium Wash solution Digestive solution
Culture Flasks
1. with wash solution (Wash solution: add 5% FBS, 200 U/ml penicillin, 200 ug/ml streptomycin and 50 ug/ml gentamicin. In primary culture, the effect of gentamicin is maintained for at least one week, and then removed). Wash the tumor specimen 4 times.
2. Place the tissue block in a small volume of wash solution that just submerges the block (avoid drying the tissue). Dissect the tissue block into approximately 1 mm3 pieces with a crossed scalpel blade or sharp surgical scissors.
3. Wash the tissue block 4 times by centrifugation (300 g, 3 min) in a bench-top centrifuge. The number of washes was determined empirically and based on cell contamination.
4. The tissue blocks were placed in a digestive solution (Digestive solution: DMEM, with antimicrobials added as in the wash solution, and 1.5 mg/ml collagenase (type IV, Worthington), 0.25 mg/ml hyaluronidase (type I, Sigma), and 2.5%-5% FBS. Although commonly used, collagenase from Worthington is not used in this way. Although Worthington collagenase is commonly used, culture grade collagenase from Sigma can also be used.) The digestion is usually done by shaking at 37°C, usually overnight (about 12-16 h). Since digestion is a slow process, it does not matter how long the tissue mass is in the digestive broth, but it is important that the digestive broth does not become acidic during the process. A low pH indicates that the tissue mass has been in the digestive solution for too long or that too much tissue has been placed in the digestive solution. Place about 1 cm3 of tumor tissue into 20-40 ml of digestive solution.
Non-enzymatic digestion
Adenomas always require enzymatic digestion. However, it is common to find that small clusters of cells are dislodged from the tumor tissue into the wash solution when the cancerous tissue is cut into 1 mm3 pieces with a scalpel blade. In this case, the following steps can be followed.
1. Collect the wash solution containing the dislodged cell clusters.
2. Leave the centrifuge tube for a few minutes to separate the tissue mass from the small clusters by gravity settling the clusters.
3. Aspirate out the supernatant.
4. Incubate the rest of the tissue mass directly, or place the mass of tissue into the wash solution and gently shake for 30-60 min to dislodge more small clusters, and collect the clusters and plant in a culture dish. Cell clusters are then collected and planted in a petri dish and cultured separately from the remaining tissue mass.
5. Wash all specimens 3 times before planting in a petri dish.
Standard conditions for primary culture
1. Cultivate epithelial tubules and cell clusters isolated from tumor tissues in 25 cm2 culture flasks pre-coated with collagen and planted with feeder cells in 4 ml culture medium.
2. Cultivate in an incubator containing 5% CO2 and 37 ℃.
3. Change the culture medium twice a week.
Tubules and cells begin to attach to substrate and epithelial cells migrate out within 1~2 d. Most tubules and small epithelial cells are not attached to the substrate. Most tubules and small epithelial masses attach within 7 d, but large organoids take up to 6 weeks to attach, although the attachment time varies.
If cells were inoculated in culture flasks precoated with collagen (Biocoat, B-D Biosciences), the epithelium adhered easily during primary culture. Cells grew very well when inoculated on the 3T3 feeder layer compared to no 3T3 word feeder layer. When epithelial cell clones are expanded to several hundred cells per clone, they become independent of the 3T3 feeder layer and it is not necessary to add leptosphere cells.
Succession Culture and Expansion
Most colorectal adenoma progenitor cells and adenoma cell lines cannot be passaged by conventional trypsin and EDTA mixture treatment [ Paraskeva et al., 1984, 1985 ]. Since dispersion of adenoma cells into single cells with 0.1 % trypsin (prepared with 0.25 mmol/L EDTA) resulted in poor cell growth, Dispase was used instead.
1. Dispase was injected onto the cell monolayer, just enough to submerge the cells (about 2.5 ml per 25 cm2 culture flask). Treat for 40-60 min for primary cells and 20-40 min for cell lines.
2. Once the epithelial cell layer begins to de-attach (it is the cell sheets that de-attach, not the single cells), blow with a pipette to promote de-attachment and disperse the cell sheets into cell clusters.
3. Wash and grow the cells under standard culture conditions. After a few days the cell clusters de-attach, so the solution should be changed gently.