43 replies to this topic

[Kimer Med]

Is there any reason we couldn't begin running our own longevity studies?

 

There are many things that I think those of us who are on the leading edge would like to know that will *never* be investigated by conventional labs.

 

Which animals and strains would be best to use? (Mice, rats, guinea pigs, other?) How many animals do you need to get good statistical results? Are any special facilities needed, or could it be run from a basic warehouse or equivalent? How long would it take someone to feed and otherwise maintain the animals each day? How many animals per cage? How big do the cages need to be? How should the cages be configured / outfitted? Rough idea of cost?

 

Input from someone who has worked in these types of labs before would be most welcome.

 

[Mind]

There have been some crowd-funded efforts recently among some of our members and some people have run small "phase zero" trials with mice and pets at home. Here is one: https://www.longecit...periments-home/ discussion.

 

Good trials probably require dozens or maybe hundreds of mice. Quality control of the mouse habitat/environment is critical and hard to manage for novices.

 

Otherwise, people are now getting more biomarker testing and this should lead to better quantification of rejuvenation efforts. Here is a program that LongeCity has started: https://www.longecit...gingbiomarkers/

[Kimer Med]

Biomarkers will be great when they're truly proven.

 

One nice thing about longevity studies is that the lab animals wouldn't need any testing, and only minimal monitoring. You basically just want to know how long they live. That should dramatically simplify things compared to studies that are looking at blood chemistry, organ states, body function, and so on.

 

Regarding the best choice of lab animals, here are typical lifespans:

 

Mice: 1 to 3 yrs

Hamsters: 2 to 3 yrs

Gerbils: 2 to 3 yrs

Rats: 2 to 4 yrs

Guinea Pigs: 5 to 7 yrs

 

Although Guinea Pigs are appealing due to the fact that they share our lack of Vit C, the shorter lifespan of mice would seem to make them a better choice for a relatively low-budget program.

 

Perhaps this should be organized as a formal attempt to win the Methuselah Mouse Prize. I have to say I'm excited by the idea of working to develop a program that would make a mouse immortal.

 

I'm imagining a plan where we would have a control group plus four or five study groups, each with a different mix of interventions. Rather than studying a single compound or intervention, as nearly every academic lab does, the idea would be to mirror programs we could and would do as humans.

 

After reading a few papers, a typical setup seems to be:

 

C57Bl/6 mice (relatively inexpensive and readily available commercially)

2 to 5 mice per cage

Maintained at 22 +/-0.5°C

12-12 hour day-night cycle

Cages and bedding changed once/week (sterlized with an autoclave)

 

The number of mice per study group seems to vary between roughly 12 and 30. Some studies use all-male mice; others use mixed-sex.

 

Overall, this seems very doable.

 

Edited by AceNZ, 25 January 2019 - 11:19 PM.

[theone]

Recently, I have been considering how best to approach this same issue.

I have come to the conclusion that if you want something done a particular way it may be easier to do it yourself.


Here is what I came up with so far.

Primary focus of the trial:

- To determine if Fisetin + a NAD+ precursor (NMN/NR) increases
maximum late life span (old Mice).

- 30 Mice total (15 control)

Secondary focus:

- encouraging public engagement by live streaming the entire trial

- encourage contributions (for future trials)

I was wondering what your thoughts are on doing this in house?  
 

[Kimer Med]

I have come to the conclusion that if you want something done a particular way it may be easier to do it yourself.

Yes, exactly.
 

Here is what I came up with so far.

Primary focus of the trial:

- To determine if Fisetin + a NAD+ precursor (NMN/NR) increases
maximum late life span (old Mice).

- 30 Mice total (15 control)

Secondary focus:

- encouraging public engagement by live streaming the entire trial

- encourage contributions (for future trials)

I was wondering what your thoughts are on doing this in house?

That seems completely doable to me.

C57BL/6 mice are available from Jackson Labs:
https://www.jax.org/strain/000664

Price varies by age and sex. The oldest available are 24 weeks, at $86.80 each for males -- so, about $2,600 total for 30.

You would need 6 to 8 cages, plus food, water, controlled lighting, and a room with a fairly constant temperature, plus a small autoclave, to help avoid accidentally introducing contamination.

 

Live streaming and the like is easy these days, although I'm not sure that mice would be very engaging subjects for a live stream.

 

I'd say around $5K for a self-run study like that, not including someone's time to feed the mice, clean the cages, track results, and so on.

 

There are a number of other supplement combinations that would be interesting to look at.

 

For example, attacking all of the "conventional" aging targets at once: Insulin/IGF-1 (Acarabose), mTOR (Rapamycin), AMPK (Metformin), SIRT1 (Resveratrol), ROS (CoQ10?), Inflammation (Curcumin?), Autophagy (Spermidine) and Senescence (Fisetin).

 

A second group might use drugs and supplements that attack the SENS targets.

 

A third group might have a mix that's close to what forum members would actually consider taking.

 

[theone]

I appreciate your feedback.

I would prefer mice in the age range of 18-24 months. This age correlates with humans ranging from around 55-70 years of age. This would significantly reduce the projects time frame (cost).


I would also prefer to optimize feeding with some type of automation. Would you happen to have any suggestions?


Can  all of these substances to be incorporated into the feed?
 

Insulin/IGF-1 (Acarabose), mTOR (Rapamycin), AMPK (Metformin), SIRT1 (Resveratrol), ROS (CoQ10?), Inflammation (Curcumin?), Autophagy (Spermidine) and Senescence (Fisetin).

 

 

Are these supplements stable at room temperature (let's assume we are using an auto feeder)?
 

Edited by theone, 01 February 2019 - 05:13 PM.

[Kimer Med]

I appreciate your feedback.

I would prefer mice in the age range of 18-24 months. This age correlates with humans ranging from around 55-70 years of age. This would significantly reduce the projects time frame (cost).

I'm not aware of a commercial source for mice that old. If you want to start a study at 18 months, you may have to raise the mice to that age yourself.

My understanding is that mice live from 1 to 3 yrs (12 to 36 months), so if your youngest mice are 18 months, some will have already died.
 

I would also prefer to optimize feeding with some type of automation. Would you happen to have any suggestions?

Can  all of these substances to be incorporated into the feed?
 
Are these supplements stable at room temperature (let's assume we are using an auto feeder)?

I can't recommend a particular model, but I do know auto-feeders exist. One issue you would want to take into consideration is whether you want to have food available at all times, or if you want to limit availability to once per day or so, as a form of intermittent fasting.

Yes, I think all of the substances could be incorporated into the feed. They are stable at room temperature. You would just need to protect them from oxidation until shortly before consumption (as with food).

[Mind]

FYI, I tried to order research varieties of mice before. They required some proof that I was running a "certified/regulated" lab.

[Kimer Med]

FYI, I tried to order research varieties of mice before. They required some proof that I was running a "certified/regulated" lab.

 

Doesn't surprise me. Even lab equipment suppliers can be limiting in terms of who they will sell to.

 

For those of us located out of the US, there may also be issues on the export / import side.

 

I've had a lot of success in the past working with companies who wouldn't normally want to sell to me, though, so I'm not concerned too much, particularly since we wouldn't have to be especially picky in terms of which mouse strain we use.

[theone]

Let's do some shopping. 

Laboratory Equipment:

IVC cage system for mice:
https://www.alibaba.....49715e25A4lAd3
 

Autoclave
https://www.alibaba....44b036Wldc9&s=p

Medical latex gloves
https://www.alibaba....c1cb8VPSq0G&s=p

Laboratory Non Woven Face Mask
https://www.alibaba.....12001b155KmnEz

Disposable lab coats
https://www.alibaba....a3809vYnyiK&s=p

Disposable Scrub Pants
https://www.alibaba.....7e6d5f096nhQ4G

Waterproof Disposable CPE Shoe Cover For Lab And Cleanroom
https://www.alibaba....c2f4efDCiNN&s=p

Rubber-Tipped Forceps (mice handling)
https://www.alibaba.....661e11a7D0vd6B

 

Laboratory electronic analytical balance scale

https://www.alibaba....57b6fXucsmh&s=p

 

Biohazardous Disposal Bags
https://www.alibaba.....7ded298betqzLk

 

Animal  Pellet Maker

https://www.aliexpre...2918000213.html
 

 

I would prefer to buy everything new. I have an irrational fear of used lab equipment   Please feel free to add to the list.
 

 

PS:

The plan is to convert approximately 70 square feet of office space into a small HEPA filtered lab room .  I will take some pictures of the proposed space  on Monday. 
Last but not least, I have a maximum budget of $15K Canadian ($25K if we qualify for R&D tax credits).
 

 

Edited by theone, 02 February 2019 - 04:51 PM.

[Kimer Med]

Good list. I like the idea of using IVC cages. With the right control system, hopefully that will address temperature control, as well as air quality. I'll be interested in hearing what they cost.

 

FWIW, I've had mixed experiences with Alibaba. In particular, if something goes wrong, you have almost no recourse, even with so-called "buyer protection." Even though they're sister companies, Aliexpress is much better in that regard (of course product availability differs significantly between the two).

 

A few more items that come to mind:

• Biological waste storage (properly marked and sealed)
• Lights with a 12/12 hour timer (something like grow lights that simulate daylight would be great)
• Water filter (probably don't want to use tap water)
• Mouse Chow or equivalent (standardized food)

It looks like the IVC cages are empty, which means the interiors will need to be filled-out, including:

• Bedding (wood shavings)
• Food and water dispensers
• Some form of mental and physical stimulation. Pipes/tunnels, platforms, chains and exercise wheels would be a good start:

 

The following video is probably overkill for lab mice, but still has some good ideas:

 

 

May need to do some experimentation on the best way to mix the supplements with the food. I'm sure others have solved this problem; I'm just not personally aware of exactly the right way to do it. Maybe mix chow and nutrients in a blender (being careful not to overheat the mix), and then re-press somehow back into solid chunks? (Controls would have food processed the same way, just without the supplements).

 

I would be surprised if this type of work *wasn't* covered by R&D Tax Credits, at least in the US and New Zealand. Canada likes to be "different," though, so who knows.

 

[Kimer Med]

Here's a link to a manual food pelletizer that might work:

 

https://www.aliexpre...2918000213.html

 

Fortunately, mice don't eat that much.

 

[Kimer Med]

I'm not aware of a commercial source for mice that old. If you want to start a study at 18 months, you may have to raise the mice to that age yourself.

 

Update: JAX is now offering B6 mice with ages ranging from 25 to 78 weeks:

 

https://www.jax.org/...strains/aged-b6

 

Not cheap, though. 18 months = 77 weeks --> $303 each.

[theone]

FWIW, I've had mixed experiences with Alibaba. In particular, if something goes wrong, you have almost no recourse, even with so-called "buyer protection."

 

We need to do our due diligence, especially for the big ticket items.

 

I'll be interested in hearing what they cost.

 

Request for quote has been sent (with 10 mice cages to start). Comparative pricing https://www.bid-on-e...nimal-cages.htm

 

A few more items that come to mind

 

Give me a few days to put something together. Hopefully we can find something that everybody can agree with.

 

May need to do some experimentation on the best way to mix the supplements with the food.

 

This is one of my main concerns with the trial.  We have no relevant experience in this type of food preparation. Any feedback will be greatly appreciated. We are good at following instructions.

 

I would be surprised if this type of work *wasn't* covered by R&D Tax Credits, at least in the US and New Zealand. Canada likes to be "different," though, so who knows.

 

I believe your understanding is correct.
 

Not cheap, though. 18 months = 77 weeks --> $303 each.

 

These mice are more expensive than what I had anticipated. We need to  determine which has the lowest long-term cost. Maybe 40 weeks at $137.42 would make more sense.

Edited by theone, 02 February 2019 - 06:07 PM.

[Kimer Med]

This is one of my main concerns with the trial.  We have no relevant experience in this type of food preparation. Any feedback will be greatly appreciated. We are good at following instructions.

 

Here's a link to a video that shows how a small manual pelletizer works:

 

 

Might take a little work to get the process down in terms of moisture content, but it seems pretty straightforward.

[aribadabar]

..., ROS (CoQ10?C60oo?),...

 

What do you think? Or too big a confounder as it is purported to be a longevity/ healthspan (no detected tumors upon necropsy) extender on its own if Baati is to be believed?

[theone]

Nothing has been ruled out for consideration.
What is important is that we create simple daily routines. We need to make it nearly impossible to screw up.  This will mean avoiding  substances that require special handling.

[theone]

I would also like to incorporate live metric streaming into this trial. Specifically the overall physical activity between the control group vs experimental group. I am confident we could put together a video recognition API for object detection and object recognition. The raw videos will be available to download.

Edited by theone, 04 February 2019 - 05:46 PM.

[theone]

The plan is to turn this small room into tiny laboratory. Clearing out the clutter was already on our to-do list. 

 

Big things have small beginnings.

[Kimer Med]

That small room looks great. Does it have reasonable ventilation?

 

Love the idea of live metric streaming, too.

 

Regarding c60oo: depends how many study groups we have, but my thinking was that the first group should use compounds that have been well-studied in mice by longevity researchers. One of the Big Pieces that's missing, IMO, is how well they work *together*, rather than just independently. There's been some hints at potential big strides in that area with two compounds, but the full gamut has never been tried together, AFAIK.

 

In that context, c60oo would be on the fringe, since there haven't been very many studies yet, and since there's still some controversy about its effectiveness. If we end up having more than one study group, doing c60oo alone or with one or two other compounds might be an interesting option, though.

 

[theone]

Does it have reasonable ventilation?

 

 

The ventilation system will need to be upgraded. We should be able to create a positive pressure environment (HEPA Filtered).

[theone]

Hi Everyone, and welcome to the end of Week 1.

What changes do I have to report?

1. Tiny room has now been cleaned of it's clutter

 

http://www.canaca.com/images/pic1.jpeg

http://www.canaca.com/images/pic2.jpeg

2. Placed an order for two Quiet Inline Duct Booster Fans (air supply will be  HEPA filtered)

https://www.amazon.c...0?ie=UTF8&psc=1

3. Sent quote requests for one IVC system (multiple suppliers)

4. Placed an order for 1  Animal Food Maker

https://www.aliexpre...2918000213.html

Priorities for next week:

1. Install inline Duct Boosters

2. Run standby generator power to tiny room (off our existing server room)

3. Make the room air tight (positive air pressure)
 

[Kimer Med]

Excellent! Sounds like some good progress.

 

I would suggest submitting a request for quote to JAX for the mice, too. They may require some paperwork / documentation before they will sell or ship to you.

 

If there's anything I can do to help from here, please let me know.

 

[theone]

Your help would be greatly appreciated.

Specifically in the food and supplement preparation (finding the correct process).  Please let me know if you would be interested? If so I would be happy to ship you a pelletizer.

I have a few more questions.
 
Would it be safe to sterilize this Activity Kit in the Autoclave?

https://www.amazon.c...XB5WD54EBJ8NFTR

Should we only purchase Female mice (Male mice are territorial)?

 

[Kimer Med]

Some plastics can be safely autoclaved; others can't. Of those that can, some will wear out after a certain number of autoclave runs.

 

http://www.used-auto...m/autoclavable/

 

Summary: Polypropylene, Polymethylpentene, Polycarbonate, PTFE Resin and Polymethyl Methacrylate are all good choices.

 

Unfortunately, I didn't see the type of plastic that activity kit was made from in the Amazon listing. Probably best in that case to assume it's not safe.

 

In one of the videos I linked earlier, they used things like metal chains and plugs, and hard-plastic piping -- inexpensive, and readily autoclavable. Cardboard tubing (toilet paper tubes) and the like would be good, too, since it can just be thrown out after a while, and replaced.

 

I'll look into all female vs. all male vs. mixed-sex mice. When I was reviewing studies, I mainly saw all-male, but I'm not sure why.

 

I may be able to help with the supplement prep aspect. Let me think about it a bit more before you send me a pelletizer.

 

[theone]

It might make more sense just to go with stainless steel (considering the small amount of cages).

 

https://www.ottoenvi...rat-round-4x6-l

https://www.ottoenvi.../minie-jogger-3

https://www.ottoenvi...limbing-tower-3
https://www.ottoenvi...mouse-relaxer-3

https://www.ottoenvi...limbing-tower-3

https://www.ottoenvi...-rattle-3-and-5

 

 

Please take your time. No pressure. 

[Kimer Med]

Nice finds there. Sure, stainless would certainly be the easiest to keep clean / autoclave (just be sure to check the overall dimensions vs. the autoclave size).

 

You could probably hand-craft up a few things using items readily available from most hardware stores, although it's a trade-off of time vs. expense.

 

[theone]

I am adding a class I Bio-safety cabinet to the shopping list.

Call it overkill but I would rather error on the side of caution.

https://www.alibaba.....1e8c776bduSoLc
 

[Kimer Med]

I am adding a class I Bio-safety cabinet to the shopping list.

Call it overkill but I would rather error on the side of caution.

 

Nice.

 

Are you thinking of preparing the nutrient mixes in there, or do you have another use in mind?

[theone]

Yes, that's correct. Even though the risks in this study are very small,  I would still prefer not to be exposed to Rapamycin and Metformin dust (over long periods of time).