The rest of the image(s) and information after the jump….
Link to full sized image – here.
1. Googlise human amniotic stem cells and the potential of stem cell therapy. It is not fact but a promising possibility.
2. Consult with some expert about the whole field (you can skip this step, if you are self-assured).
3. Talk to the pregnant member of the family (or acquaintances of yours) months before birth, and convince her to store the amniotic cells at home.
4. Consult with her gynaecologist months before birth and convince him to put the placenta under the birth into a sterile flask full with ice. You can put it into Phosphate buffered saline solution (PBS, preparation here) with some antibiotics (Penicillin-Streptomycin) for the sake of sterility.
5. Technical preparations (I did not calculate the exact amount of money, which is needed fro the adventure, but it’s around some thousands of dollars, and that could cheaper than collect the cells via a commercial way): Set up a sterile hood at your garage. You can make one out of a household air purifier, for that see Home Mycology Lab by Philip Ross, in Make Backyard Biology issue, page 102. Rent or buy a normal light microscope (10x resolution will be enough), a centrifuge (1000rpm), and buy a liquid nitrogen refrigerator.
6. When the placenta is in your hand, process it within 4 hours. Use sterile gloves.
7. Put the flask with the placenta under the sterile hood. Take a sterile scissor and carefully cut the outside epithelial layer off. The more you cut, the more stem cells you get. The amnion layer is mechanically peeled off the chorion.
8. Wash the amnion in Phosphate buffered saline solution (PBS, preparation here) in several times (8-10X) to remove blood.
9. Mince the tissue thoroughly with sterile scissor.
10. To release amniotic epithelial cells, incubate the minced amnion membrane with trypsin (0.05%) for 10 minutes at 37°C. Take out the digested tissue from trypsin after 10 minutes, and discard the cells from this digestion to exclude debris. There are different kinds of trypsinization protocol, I follow here Miki et al.
11. Treat the remaining tissue in another tube of trypsin (0,05%) for 20 minutes at 37 °C (do this step once more if necessary to collect more cells).
12. Pool the cells from second (and third 20 minutes) digests.
13. Pass cell suspension through a 100 μm cell strainer.
14. Fuge the filtered cell suspension for 8 minutes at 1200 RPM (150-200g), room temperature.
15. Wash the cell pellet with PBS and fuge again.
16. Count the cells with a hemocytometer and it is advisable to determine the viability of the cells by exclusion of trypan blue dye, which is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, whereas dead cells do not. From one epithelium you can get as many as 10-60 million stem cells.
17. Prepare freezing medium. The freezing protocol is from the best lab manual, At the Bench: A Laboratory Navigator by Kathy Barker: 1ml/aliquot plus 10%. Freezing medium typically contain regular culture media, 10-20% serum, and 5-10% glycerol or DMSO.
18. Each ampoule will take 1×10(7) cells (or between 4×10(6) and 2×10(7) cells) in 1ml of medium.
19. Resuspend the pellet in freezing medium by pipetting gently.
20. In order to freeze the cells gradually and safe place the ampoules in -60°C or less and leave them there for 16-24 hours.
21. Put the aliquots with the cells in liquid nitrogen and store them. As Charles Platt says in Life and Death at Low Temperature (page 56): “liquid nitrogen is available in most urban areas (search for “liquid gases”), and it is generally inexpensive. It is nontoxic, but must be handled with caution, since its temperature of -196°C can cause serious injury to any exposed human tissue. Always wear heavy gloves and eye protection.”
22. Give the freezed cells to doctors, when it is needed for repair after X years.