How to isolate amniotic stem cells from the placenta, at home!

/Thanks for spreading the idea of biotech diy: Make, Pharyngula, kottke.org, Scientific American Blog, BoingBoing, Digital Bio, Clock among others…/

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.

isolatestem

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

/Disclaimer: Attila Csordás and pimm.wordpress.com disclaims all responsibility for any resulting damage, injury, or expense./

Update: Smart comments from Make readers.

82 thoughts on “How to isolate amniotic stem cells from the placenta, at home!

  1. Pingback: Update from the Womb at

  2. Definately not for the squeamish, and I can imagine that a placenta can be difficult to get ahold of without some pretty tricky complications. I wonder if we’ll see human stem cells on the black market soon.

  3. I am soooooooo glad I kept the kid’s placentas in the freezer all this time. Now they have a practical use! Thanks for posting this.

  4. This is fabulous. I’m almost tempted- we have all those things in lab.

    My family did, at one point, have a placenta in the freezer. (Don’t ask.) But I expect -20 without cryprotectants would do the cells in. If it were blendered up in glycerol, now…

  5. Man, if only I hadn’t thrown out my home hemocytometer just last week! Drat!

    Very cool reading though, thanks! I was unaware of ‘DIY biotech” as a concept/movement…cool as a concept…but SO much room for abuse/injury by the untrained public. A little knowledge can be a dangerous thing…

    Then again, more public exposure to science is generally good…

    I’m also visiting via Pharyngula.

  6. This is public exposure to a lot of things. Interesting how we have such practical materials like placentas at our fingertips, but that religious morons have made stem cell research into an ISSUE. What you show here not only escapes that obstacle, but proves just how simple it is. Simple enough to do at home.

  7. Respect, man, total respect.

    I’d like to try this at home, so I need to get started on a placenta right away. Gentlemen, I need some help with that: any volunteers?

  8. To carry the placenta PBS is enough, no medium is needed.
    The freezing medium is regular Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2mM-L-glutamine.
    If you would like to grow the cells in vitro in Petri dishes (which is out of the range for the current project) you can use DMEM supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), epidermal growth factor (10 ng/ml) or in EBM-2 medium (Cambrex Bioscience, MD, USA).

  9. When I showed my poster on Euro Maker Faire in Brussels last year, one quite new dad said to me, that in an emotionally filled condition of a new dad it is hard and annoying to do the isolation of the placenta by himself so the option left is to rely on professionals or not to do anything. I appreciate that, but on the other hand there are the potential benefits of collecting the cells. Who knows, if the dad does the placenta work himself it could be interpreted as the hardworking paternal contribution to the birth, the other half of the exhausting job the mother does. Symmetrical thought, isn’t it?

  10. Dwillsworth, you are right, and I am sure you are a good father. :) I didn’t mean, that there must be some compelling and corresponding father job during the birth process, although I cannot exclude, that this could be a part of motivation for isolation for some people, later.

  11. Throw down your umbilical noose so I can climb right back……(Nirvana).

    Well it definitely beats getting the stem cells from bone marrow! However there is a slightly worrying property of placental tissue – viral gene expression.

    As I understand it, the placenta is mostly encoded from derived viral genes which enable, basically, parasitism of the mother. It’s called the germ line for a good reason.

    What I’m trying to say is that these cells might well be partly differentiated in a rather nasty way.
    I’m sure any side-effects will be played down by anyone fiddling around with them, though, so take reports of miracle cures with a pinch of salt.

    N

  12. You might want to explain sterile technique and how to put on sterile gloves, otherwise you’re going to definitely contaminate yourself and your specimen.

  13. RE: Attila’s advice: you’d need tissue-culture treated dishes, not petri dishes. Most mammalian cells will not stick to petri dishes. Also, the freezing medium must contain DMSO or ice crystals will form beneath the cell’s membrane killing all of them.

    Ummm…how exactly is this DIY? Where is a normal person supposed to buy trypan blue, trypsin, dmem, FCS, hemocytometers, 100uM strainers, a centrifuge, etc., etc. Moreover, most people are going to contaminate their cultures and 20 years from now doctors will wake up the cells only to find bacteria have eaten your child’s cells. Then you’d feel silly for having lugged that liquid nitrogen refrigerator from apartment to apartment for the last 20 years. And, I guess you’re going to have the liquid nitrogen guy come to your home every few weeks?

    Some things should be left to the pros. This is one of them. Plus, I can’t believe you’d save much money unless you already have a lab in the garage. I am currently building a lab and can tell you that a hemocytometer alone can set you back $200. I paid almost 8K for my centrifuge. The cheapest FBS will cost $200 for 500 ml. I can’t imagine that you’d save much of anything after buying what you’d need.

    Cute story, but detached from reality.

  14. Sterile, aseptic technique, sterile skills can be acquired easily, you don’t need a MD for that. One-two week class in a cell culture lab or in a hospital, see what lab technicans learn. Experts can organize this and so popularize it. Learning aseptic technique is not more different or harder than learning to use the basic tinkering tools, hammers, drills…
    The know-how is out there for all to learn.

    Every once a while a technology is moving from the pros to the non-pros, new institutions are needed to be set up. Think about m.i.t. fab labs and the whole personal fabrication movement.

    Concerning the costs: you can buy a cell centrifuge for a 1000-2000 bucks. See some fuge prices.
    Or you can rent it from a lab, as I think there is a possibility to loan a whole lab for some hours. It’s not impossible at all, but entrepreneurial spirit people have to calculate the prices well before starting anything.

    SciGuy says: RE: Attila’s advice: you’d need tissue-culture treated dishes, not petri dishes. Most mammalian cells will not stick to petri dishes. Also, the freezing medium must contain DMSO or ice crystals will form beneath the cell’s membrane killing all of them.

    Well, you can grow amniotic stem cells in normal Petri dishes, or flasks no problem with that, the cells will adhere, but for the isolation you don’t need to grow them at all.
    DMSO and freezing medium have been already mentioned in the main text. Look at this: DMSO Freeze Medium.

  15. Attila, much as I am in full suppport of extending healthy life, and bioDIY, I will share my green concerns here too (besides our morning discussion on it), as I wonder how the other readers see it.
    Undoubtedly, the most costly part of the placenta project is the span of 20 years’ storage. We not only need a refrigerator, but we need one that can keep the cells at about -190 Celcius centigrades.
    Hacking your home fridge could be an inexpensive solution (dunno), but even in that case, it would mean having to run a lot of family fridges, lots of electricity taken up etc. It seems to me that it might be unnecessary to have one separate fridge for every family unit. Especially, if you add that you need to provide some back-up in case of electricity failures.
    So it may sound strange but what I can envision as a long term solution is sth like the banking system. You keep your big money in a bank because it would be too dear to maintain home security. So we are back to cell banks again, but hopefully at a much lower price than present days’ costs. Or not?
    The snag is that if these cells can save your life and the cell-bank makes a fatal error, then it is a lot of risk for both. The higher the risk the higher the price – maybe hindering the introduction of lower prices.
    Question: are there any other advantages of keeping sth at approx. minus 200 degrees so that it comes down to a cost-efficient storage? Or should it be a community project (not unlike wifi umbrellas in SF) to have fridge hangars/ stem cell libraries for the locals?
    What do you think?

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  18. All I can say is wow. This is a very interesting blog with the huge technique that is required to do this, I’m my opinion, I would like to see a garage that you can actually do a sterile technique in. In the mist of a cluddered garage is the little placenta lab in the corner. :) All kidding aside, this is an interesting topic that I’m sure is going to become more real in the future with placenta research. Until then, a lot will be thrown away with no purpose.

  19. This is interesting, although stupid if you think you can find a garage clean enough to do clean technique let alone sterils procedure. This is a more beneficial approach to stem cell research to the taking of a human life for research purposes for maybe solutions.

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  23. I don’t know how you think this can be safely done at home. We do it in a lab setting with strict quality control and appropriate safety measures; even so, the cells are not certified for human therapeutics. While your procedure is largely accurate, I’d hesitate to add that most individuals would be hard pressed to acquire the reagents, much less use them safely and effectively. Interesting read but scary to think someone is doing it in a basement workshop.

  24. This is preferable to extracting stem cells from amniotic fluid, which I wrote about here.

    Not to mention the cool factor! Really amazing that this can really be done at home. What mos

    There are nutritional ways to make use of our own stem cells, as well. By supporting the body in releasing its own stem cells, more people may stay healthy, making planning for the worst unnecessary.

    Tracy Austin

    Organic Foods Mom

  25. Interesting. I prefer to leave it to the MD pros so I’ll be in Mexico next month getting the implant (to possibly help MS symptoms). Too bad the US is a third world country in their tolerance of individual choice for having this done without FDA approval.

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  30. I do have our placenta from our last child birth
    in the freezer will this method work??

    Also, I saved this child’s baby teeth in trehalose solution in the the freezer! I save these just for the purpose for stem cell salvaging and saving.

    Now will these procedures work is the question?

    AL

    AL@asu.edu

  31. Al,

    Although it may work to isolate a few viable cells, it is always preferred to recover the cells from fresh, not frozen, tissue. That said, I have never tried this procedure with frozen tissue, but suspect that 99% of the viable (meaning living and respiring) cells will be killed by the extreme conditions of freezing and the damage this is known to have on cell membranes still associated with tissues or organs.

    Especially important is how the tissue was frozen. If it was “snap” frozen in liquid nitrogen or “conditioned” in some manner prior to freezing, and the rate of freezing all all significant contributing factors to the ability to extract usable cells.

    Using a simple method like trehalose will help with the teeth. This is different too because the cells there, even though much more scarce, are in a thin layer and can be frozen without as much stress to the cells and in a much more rapid and uniform manner.

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  33. Thanks for this really interesting protocol! And really good blog.
    Bye from Italy
    Fabio

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  36. i am a dentist and i m searching about the project stem cells in dentistry..can this method be used without any changes in isolation of stem cells from dental pulp and periodontal tissues? or is there any change in the culture medium? and can u help me regarding the differentiation and the expansion of the isolated stem cells? plz

  37. Meenu,

    As a matter of fact, I attempted to use exactly this technique to isolate stem cells from my daughters recently extracted wisdom teeth. Unfortunately, my cultures became overwhelmed by contamination (yeast) and I was forced to abandon my experiment.

    I performed a simple enzymatic digestion of the whole tooth (all 4 teeth, actually), following essentially he same protocol. If I have access to more wisdom teeth (my wife still has hers available for “donation” and one more daughter who’s only 15, I would recommend extracting the dental pulp, rather than using the soft tissue extracted from the enamel.

    If you would like to collaborate on this, please contact me directly “hardy at gahaga dot com”
    I would like to try this again and am looking for someone like you (depending on where you are located)

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  41. Well Done. This is what is done in our lab ever single day for lots of money of course :). I wonder if there are blogs/articles like this on obtaining other stem cells? Particularly I am interested in dental pulp stem cells, liposcution, neural SC?

  42. From this wonderful blog. It shows what purity exist in the young. hopefully will not be destroyed by time and insanity.
    So, it appears that stem cells are not a DRUG, but refined meat. Why does the FDA with hold back use as a treatment, and health insurance not recognize applications of a food??

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