Talk:20.109(S13):Module 3

Protocols

Half the class at a time will work in the tissue culture room today. Today will be physically and mentally laborious, and you've all been working hard, so spend the rest of the afternoon however you see fit. (Whether that involves the FNT assignment, notebook prep, or a walk in the sunshine - are we still expecting sunshine?)

Chondrocyte or stem cell culture

Today you will work with primary cells that are directly isolated from bovine knee joints. Recently, your teaching faculty harvested cartilage fragments from two bovine knees, and sequentially digested them in pronase and collagenase enzymes to release the chondrocytes. Each joint typically yields > 50-100M cells. Stem cells were harvested from the bone marrow and grown up from a rare population by extended culture in bFGF (basic fibroblast growth factor). After cell isolation, aliquots of several million cells each were frozen and stored in liquid nitrogen.

Preparation

1. Begin by setting up your hoods. Prepare any standard equipment and solutions needed.
2. Note that the small beakers are for making a calcium chloride bath (not shared, one per person), and the large are for temporary waste in steps 10-12 below (shared, one per hood).
3. If you requested a special reagent or equipment, check with the teaching faculty.
4. If you are doing an alternative protocol (e.g., 2D culture or collagen gels), check with the teaching faculty.

Cell culture

1. When your hood is ready, thaw your aliquot(s) of frozen cells in the water bath. Avoid immersing the cap of the tube in the bath; just hold the body submerged. Agitate the vial slightly while you hold it. The cells should thaw in less than 5 minutes.
2. Spray the vial with 70% ethanol and take it into your hood. Using a P1000, add the cells drop-wise into the 15 mL conical containing 9 mL of pre-warmed medium. Spin at 800 g for 8 minutes.
3. Aspirate most of the medium off your cell pellet, then gently resuspend in 1 mL of medium using your P1000. Add 3 mL more of medium per vial, using a serological pipet for the addition and subsequent mixing of the medium and cells. Take 90 μL of cells into an eppendorf tube.
4. Add 10 μL of Trypan blue - this is a toxic material, so please be careful not to spill it! - to the eppendorf tube, and count your cells. Adjust your culture plan if you do not have as many cells as you expected.
   • No need to count all 4 corners today - perhaps count 2, especially if your cell count is high.
5. Separate the cells that will make up your two different cultures into two labeled 15 mL conical tubes. Note that the tubes may not all require the same amount of cells, depending on the cell densities you chose for the two cultures. Double-checking your calculations now may save you having to do an extra centrifugation step later!
   • Give any excess cells that you have to the teaching faculty, in case other groups want more cells.
6. Spin down your two conical tubes of cells at 800 g for 8 minutes.
7. Resuspend each sample of cells in the appropriate amount of the type and concentration of alginate that you chose.
8. Using the syringe that has been prepared for you, very carefully pull up the cells, then release them drop-by-drop into the beaker full of calcium chloride solution (20 mL). Recall that calcium effectively polymerizes the alginate, resulting in small gel beads filled with cells. Immediately discard the entire syringe into the RED sharps container (not the mayo jars) - do not try to remove or recap the needle.
   • Don't release too quickly or you will get a glob instead of distinct droplets, and try to match your release rate with your partner's.
   • Depending on the concentration of alginate that you chose, you may have between ~50-150 beads for 1 mL of alginate solution.
9. Allow the polymerization to proceed for 10 min. at room temperature. Then pour your beads into a 50mL conical tube.
10. Remove the calcium chloride solution from your beads using a large serological pipet (to better avoid aspirating the beads), and put this solution in the temporary waste beaker in your hood.
    • Ask the teaching faculty for tips on avoiding sucking up your beads. Basically, you want to keep the pipet close to the wall of the conical tube, so liquid can still be sucked up but the beads don't have room to be.
11. Now fill the conical tube with sodium chloride (20 mL), and gently invert it for 1-2 min. This is to remove excess calcium from the solution.
12. Remove the NaCl using a fresh pipet, then wash the beads again with fresh NaCl. Finally, wash the beads two times with DMEM culture medium (20 mL each time).
13. For each of your two samples, transfer the beads to the two leftmost wells of a 6-well plate, using a sterile spatula. Try to put approximately equal numbers of beads in the two wells.
14. Finally, add 6 mL of warm culture medium to each of your four sample wells, then put the two well-plates in the incubator.

The teaching faculty will exchange the culture medium as necessary (every other day).