How to isolate amniotic stem cells from the placenta, at home!
Posted by attilacsordas on January 23, 2007
The placenta is a very valuable and scarce human tissue, although the proper recycling of it is not placentophagy, but the isolation of stem cells from its amnion layer, and storing them for later regenerative purposes for the whole family. Human amniotic epithelial cells (HAECs) from the placenta are alternative replacements of human embryonic stem cells, and have the potential to differentiate to all three germ layers in vitro. These cells are very close to those earlier and broadly multipotent amniotic fluid-derived stem cells, which made the big buzz lately on the web, published by De Coppi, Atala et al. in Nature Biotechnology. Here I would like to show, although I do not provide any warranty and can not give any guarantee, that isolating stem cells from the placenta is not more difficult than making a steak, and with proper preparation, investment and timing you can do it even at home or in a rent lab. The process is ethically non-controversial since the placenta is usually discarded after birth. Today, stem cell therapy is just a promising possibility, but in the not so distant future, self-aware citizens may manage their own stem cells, grow them in the garage, and store them in the fridge. If so, it could be a form of autonomous medical self-insurance. We are at the dawn of the bioDIY movement backed by open source science for anybody. I used Make magazine’s Backyard Biology issue as a reference, because it invented the basic language of bioDIY or home or garage biotech. Here is the algorithm at the cartoon and below are the detailed, although not self-including textual protocol. More details will come later, if asked.
1. Googlise human amniotic stem cells and the potential of stem cell therapy. It is not a 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 expectant mother+the would-be father months before birth, and convince her to store the amniotic cells at home.
4. Consult with her gynaecologist months before birth and convince him/her to put the placenta 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 for the adventure, but it’s around some thousands of dollars, and that could be cheaper than collecting the cells via a commercial way): Set up a sterile hood at your garage. You can make one out of a household air purifier, see Home Mycology Lab by Philip Ross, in Make magazine’s 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 pair of sterile scissors 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 a pair of another sterile scissors.
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 the 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 contains 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 frozen cells to doctors, when it is needed for repair after X years.
References in peer-review journals:
In ‘t Anker et al: Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004, 22:1338-1345
Bailo et al: Engraftment potential of human amnion and chorion cells derived from term placenta. Transplantation 2004; 78: 1439-1448
Miki et al: Stem cell characteristics of amniotic epithelial cells. Stem Cells. 2005 Nov-Dec;23(10):1549-59
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Update: Smart comments from Make readers.
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