Lucas Cranach: Fountain of Youth (1546)
Opening a can of worms - Life extension: bogus or real?.
Is aging a process that can be stopped, slowed down or even reversed? These were some of the questions that I wanted to see if I could answer by recreating life extension/longevity studies that I had read about in science publications where the life span of animal models was increased by 10-20% or more. These studies indicated that aging was a controllable process that could be hacked, by inhibiting mTor pathways. (http://en.wikipedia.org/wiki/Discovery_and_development_of_mTOR_inhibitors). It is speculated that this pathways has evolved in animals to allow them to survive when they come under stress by lack of food, and helps them survive until food is more abundant. This process can be hacked by CR caloric restriction or the use of Rapamycin, both mTor inhibitors. Once I got comfortable in the lab I would use the skills learned at the BioHack Academy to see if the experiments could be duplicated and if the results would be convincing (below - worm action-hero)
I read all the life extension/longevity documentation I could find, along with Youtube videos and the first decision I needed to make was a choice of animal model to use. Mice were too long lived my purposes, fruit flies could be hard to wrangle and prospect of watching yeast age didn't seem too exciting so I settled on c.elegans (microscopic earth worms). The use of these beautiful creatures was first pioneered by Nobel Prize winner Sydney Brenner (2002 Medicine) as an animal model. The life cycle is short 3-5 days, they are prolific reproducers and I could maintain them with a diet of e.coli. Conceptually I was ready to rock & roll.
C.elegans -Youtube https://www.youtube.com/watch?v=zc1P7lGSzdU
Wormbook - Maintanence http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
Getting worms - Go ahead and try this at home (take away - Scientists are really generous).
This email below was sent to (7) 'googled' researchers (3) replied positively: Jeroen van Zon of AMOLF research Amsterdam answered my request http://www.amolf.nl/.
The following is a request for assistance with a developmental biology experiment being conducted at the Waag Society of Amsterdam - institute for art, science and technology (www.waag.org). I understand you are using c. elegans at your lab and my request for a donation of c. elegans, NGM medium or any other cultivation material for the project. The organisms will be used to study the reproducibility of longevity experiments (details below) recently publish. Lab supervisors Pieter van Boheemen (Pieter@waag.org) or María Boto Ordóñez (mboto@ub.edu) may be contacted or Waag Society (lucas@waag.org).
Please forward this to any colleagues who may be able to help in this request. The organizations will be credited in the publically available results. Details of the project are below and I can be contacted for any further information. Thanks in advance for your cooperation, Tony Garcia.
The life extension/longevity experiment (conceptual 1st draft which is still evolving)
Developmental Biology - Reproduction of aging/longevity studies This project will explore the ability to reproduce aging/longevity studies results using mTOR inhibitors – Rapamycin/Sirolimus a biosynthesized molecule, Streptomyces hygroscopicus (biohazard 1) the original target bacteria which produces Rapamycin and Caloric Restriction CR. All three have been attributed to extending life in various animal and insect studies. The results of this project will be publically posted.
Rapamycin (also called Sirolimus): This chemical discovered by Suren Sehgal and is a product of bacteria found on Easter Island (also known as Rapa Nui). Biosynthesized it was approved by the US Food and Drug Administration in September 1999 and is marketed under the trade name Rapamune by Pfizer.
mTor inhibitors refers to (metabolic target of Rapamycin) this molecule inhibits IL-2 and other cytokines receptor-dependent signal transduction mechanisms, via action on mTOR, and thereby blocks activation of T and B cells. Since Rapamycin or mTOR inhibitors have been biosynthesized they have been used for a number of years for organ transplants to inhibit transplant rejection. (below - worm food OP50)
Bacteria - Streptomyces hygroscopicus (biohazard 1): Sirolimus (also known as Rapamycin) is a product of bacteria Streptomyces hygroscopicus. Rapamycin/Sirolimus was originally developed as an antifungal agent. However, this use was abandoned once it was discovered to have potent immunosuppressive and anti-proliferative properties. It has since been shown to prolong the life of mice and might also be useful in the treatment of certain cancers.
Calorie Restriction CR: Caloric Restriction without malnutrition has been shown to extend life in a variety of species, among them yeast, fish, rodents and dogs. It decelerates the biological aging process, resulting in longer maintenance of youthful health and an increase in both median and maximum lifespan. (below - worm family feeding on OP50 e.coli)
Test organism – c. elegans Cultivation - The life cycle of the worm is about 3 days. Feeding - In the lab, Escherichia coli (E. coli) strain OP50.
Longevity testing protocol Once c. elegans has be incubated, then synchronized and transferred to a colony plate. The target c. elegans colony will be divided into 4 control plus a control group and fed and monitored over a period of one life cycle.
This same protocol will be repeated over a number of colony life cycles with varying solutions of biologics, bacteria or food restriction. The longevity/health will be measured in days/hours/minutes of survival. The results will be calculated by averaging the totals of each group with specific dilution mixture. Part of the results will identify the optimal dilution regime that correlates to longevity and health.
Worm wrangling (the countdown to day Zer0)
The worms gifted to me from AMOLF researchers in Amsterdam were in two 6cm petri dishes. Along with these were two dishes of OP50 (e.coli worm food). I incubated them for a week and both colonies flourished. To make this experiment a success one has to be able to transfer the worms from one prepared petri dish filled with NGM (nemetode growth medium) to another. To feed the worm with a standardized amount of food OP50 (e.coli strain). To distinguish between long lived animals and younger ones (using markers of size, mobility etc). One has to be able manipulate the worms with confidence moving them from one petri dish to another to eventually synchronize them for a day zero. Only then can the experiment begin. All of the above it takes time, patience and a steady hand. _(moving worms with an injection needle) _
The heartbreak of worm wrangling (continued - the move to Zer0 hour)
My worm transfer to 3.5cm petri dishes was not totally successful. Out of 20 petri dishes transferred from the original colony only 3 were successful. I also realized that to synchronize the worms (creating a day zero for the start of the experiment) I needed to make a much larger batch of worms and then let the colony starve out leaving only their eggs. I would then separate the eggs into different dishes then introduce different nutrients to see whether the colony benefits from it. With time running out for the BioHack academy I am not sure what will be the next steps of the experiment. (Try this at home boys & girls!! - Reciepes for NGM mix, worm feed and care are included at the end of the blog)
Open worm – c.elegans in Silico
https://www.youtube.com/watch?v=r00FkkGFfrg
Why this experiment might not work (and why being ignorant can be a good thing)
Ten years ago John Ioannidies published a paper called 'Why most research findings are false'. The following is an part of the abstract: "There is increasing concern that most current published research findings are false. The probability that a research claim is true may depend on study power and bias, the number of other studies on the same question, and, importantly, the ratio of true to no relationships among the relationships probed in each scientific field." (full article - http://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.0020124)
I also emailed googled Longevity researchers like Matt Kaeberlein of Kaeberlein Lab for advice http://kaeberleinlab.org/ (Again scientist are super generous) Matt wrote: 'Best of luck with your C. elegans studies. If your experience is like ours, you will find that about half of what’s in the C. elegans aging literature is not consistently reproducible. There are a variety of factors that contribute to this, including often incomplete or inaccurate methods descriptions as well as insufficient replication in the initial reports. Relatively modest temperature and food density fluctuations can often obscure smaller lifespan effects as well. You will also want to consider how to handle censoring of animals for things like bagging (assuming you don’t use FUDR), rupture, burrowing, contamination, and crawling of the plates. It all makes a difference. That’s why I’ve adopted the approach that we stick to large magnitude effects in my lab that work every time we do the experiment'.
Let the race for long life begin! (delusions of a would-be King in the land of worms)
I was running out of time - but even with the uncertainty of getting any results I was committed to lead my worms on the path to long-life. I transferred my surviving c.elegans into three new 6cm petri dishes but I was not able to properly synchronize them. I learned that in lab work as in war - no plan survives contact with the enemy. This would not be the first compromise I would have to make on the road to longevity.
They say victory favors the bold - In week 8 of the 10 week Biohack course I had finally got my hands on a batch of Rapamycin, the mTor inhibitor. I had built up high expectations for my worms so I pushed ahead with my master plan hoping to snatch victory from the jaws of defeat by accelerating the experiment's timetable - But to do this I had to further compromise its scientific integrity.
I introduced Rapamycin into the three new petri dishes in dilutions of 1:200, 1:2000 & 1:20000 and prayed for long life and glory. I further adjusted the experiment eliminating CR caloric restriction from their menu. My worms had been through enough and I didn't want to inflict anymore hardship on them.
The results life extension/longevity experiment - if any (in the amount of time I have left) - will be challenging to interpret because of all the compromises I made with the experiment's protocols. How will I recognize the changes in the worms if mTor has been inhibited? Will I be able to differentiate between the long lived and newly hatched? Will I see changes in the older worms, like increased mobility? Questions, questions, questions - with few answers
Winding down the experiment (the importance of a good exit)
With the lab skills I've learned at the Biohack Academy I want to continue working with c.elegans until I can get convincing results regarding life extension. Also I have many more ideas for the use of c.elegans in other projects. When its all over I want to be able to retire my worms with a dignity fitting such an important, useful and beautiful creatures. Perhaps I can release them in the wilds of the Vondelpark so I can thank them for their contribution to science and humanity every time I bike through. This would be the way I'd like to see this experiment to end.
Trying out the longevity juice - (Rapamycin) - Philosophical questions
Is aging inevitable or is it a controlled process that can be slowed?
What will it take for this experiment to produce convincing results? Scaling up - How can human tests be designed for longevity?
What is compelling proof? If results of longevity experiment are convincing - who gets the juice?
Do we follow the Pharma-patent business model or should longevity be available for all?
What implications does longevity have for society? - Optimistic story (tweeking the societal order) and pessimistic story (limited resources)
What are the risk/benefit calculations involved?
Support documentation
NGM agar plates For 1 liter/333ml:
- 3g/1g NaCl
- 17g/5.7g Bacto-agar
- 2.5g/0.83g Bacto-peptone
- 1ml/0.33ml cholesterol (5mg/ml stock in 100% ethanol)
- dH20 to ~975ml/325ml
- autoclave, let cool to 55C (until you can touch it with your hand without discomfort), then add:
- 1ml/0.33ml 1M CaCl2 (stock)
- 1ml/0.33ml 1M MgSO4 (stock)
- 25ml/8.3ml 1M KPO4 pH 6 (stock)
- swirl thoroughly after each addition.
- When all added, pour plates. (~25 ml for 10cm plate, ~10ml for 5 cm plate) 10ml for 10cm plates, 5 ml for 6cm plates, 2ml for 3.5cm plates
- To remove air bubbles in agar, flame agar surface
- Dry plates upside-down with lids slightly ajar for 1-2 hours at 37C, or upside-up with lids closed for 2-3 days at room temperature.
- Store upside-down in sealed plastic box at 4C Because the autoclaving step takes 1.5-2hr, it is often more convenient to let the NGM agar solidy after autoclaving and then melt it in the microwave at a later point. In this case, make ~333ml in a 500ml bottle. If using multiple bottles, make each bottle separately because the agar doesn’t fully dissolve and is hard to divide up later. Melt agar by microwaving for 4min. Do not microwave too long or the agar will overboil. Small chunks of solid agar will melt while the agar is cooling down. The molten agar will be ready to be poured after 20-25 mins at RT. If you worry about the agar being too cold, microwave it for an additional 30s.
Seeding NGM agar plates with OP50
- Seed 50 ml B broth with single OP50 colony. Incubate at 37C overnight
- Add ~1 ml of OP50 in B broth to 10cm NGM plate.
- Swirl plates to distribute OP50, incubate overnight at 37C
- Store upside-down in sealed plastic bag at room temperature.
LB Amp plates (for RNAi bacteria)
- 10 g tryptone
- 5 g yeast extract
- 10 g NaCl
- (The above can be replaced by 25 g DIFCO LB broth)
- 15 g agar
- Add dH20 to 1 liter
- Autoclave, let cool to 55C (until you can touch it with your hand with discomfort), then:
- Add Ampicilin to 50 ug/ml, i.e. 1 ml 1000X Ampicilin stock
- Pour plates Freezing worms
- Wash worms from plate with 3x 5ml M9 solutions into 15 ml Falcon tube. Preferably use a plate that has just starved and contains many L1 larvae
- Spin worms down 1 min 2000 rpm
- Aspirate using clean tip to 1.5 ml
- Add 1.5 ml freezing solution. Work sterile to prevent contamination of freezing solution stock.
- Aliquot 1 ml into liquid nitrogen cryotubes (with a screw cap) 3 tubes fot each strain
- Freeze slowly to -80C. This is done by putting the cryotubes in the worm freezing box: two styrofoam racks (the ones that 15 ml Falcon tubes come in) on top of one another, fastened together by a rubber band.
- The next day, move two of the tubes to a permanent location in the freezer.
- Use the third tube for a test thaw: thaw the contents of the tube quickly by holding in your hand. Dump the contents onto a large seeded plate
- Check for live worms after a day or so.
- Note date of freezing and succesful testthaw in strain database (Gmail account)
Freezing bacteria
- Grow bacteria overnight in LB
- Add an equal volume of 60% glycerol (autoclaved)
- Aliquot 1ml into at least two cryovials
- Freeze at -80C
Synchronization by washing
- Grow plate with many eggs
- Wash plate with dH2O, aspirate to make sure all liquid is gone. Repeat if necessary.
- L1 larvae can be transferred to a new plate, if necessary, by washing larvae off with 2x ~5ml M9
Synchronization by bleaching
- Grow plate with many gravid hermafrodites
- Wash hermafrodites into 15 ml Falcon tube with dH2O
- Spin down, aspirate and add 6-8 ml worm bleach, depending on size of pellet
- Shake/vortex until adult hermafrodites have disappeared (about 5 mins), but not longer to avoid damage to eggs.
- Spin down, aspirate bleach and wash with dH20.
- Transfer eggs to new plate. To avoid overcrowding, use ~2000 eggs per plate
LB Medium
- 10 g tryptone
- 5 g yeast extract
- 10 g NaCl
- Add dH20 to 1 liter
- Autoclave Keep in fridge until further use
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