An update on my effort to isolate viable adipose derived stem cells for therapeutic reinjection and a call for advice

[Frank Garcia]

So I've scoured the internet for papers and guides to nonenzymatic procedures that can be done with simple inexpensive equipment and which yields an usable quantity of fat stem cells that are alive and well.

I've read a few and must say I'm not entirely sure if I understand the required steps.
Each of the following describes variations of the procedure. Almost all add some fancy element in one of the steps like a commercial kit, specialized syringe with filters built in , or custom made contraptions - get a load of this "Custom-made metallic disarmable pistons, concave cell-adhesive gaskets, closed cubic unit harnessing 3 different-sized sets of blade grids on each luer-lock port, rotating canal at the center of the cube to control the flow of the lipoaspirate, http://links.lww.com/PRSGO/B308).".

• https://www.nature.com/articles/s41598-017-10710-6#Tab3
• https://journals.lww.com/prsgo/fulltext/2020/02000/a_3_step_mechanical_digestion_method_to_harvest.27.aspx
• https://stemcells-aesthetica.com/isolation-process-of-stem-cells-from-fatty-tissue/
• http://www.irvinesci.com/protocol-for-mesenchymal-stem-cell-isolation

I'd like to use these to put together a protocol that uses no expensive or custom made equipment. I'd like to know if the plating and incubation is necessary and is filtering necessary? Also, is the VSF the end point or can ADSCs be isolated from VSF? any other feedback welcome.

[Dakota Hamill]

Pretty interesting pictures in some of the articles. Amazing you can
take out adipose tissue, diggest it, spin it, and harvest a plethora
of cell types.

As you mentioned, it looks like only 5% of that final fraction is
adipose derived stem cells. I imagine it will be difficult to select
only the ADSC's from that fraction with low-budget equipment. A flow
cytometry machine would be able to do it. A very careful hand and a
tiny pulled glass syringe might under a microscope.

I've done 0 human cell line culture in my life but even the lab next
to us who has an ozonator, secure room, hoods, everything, often
complains of contamination of fungal spores etc. It's not easy!

You may just take the cell mixture found in the VSF and culture that
and see what cells tend to dominate in it. It may be the stem cells
do well in culture and out-grow other types. It may be they do not,
and further purification to a monoculture is needed. How you'd do
that, I have no idea but it's interesting.

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[James Clement]

The subject line uses the term "therapeutic reinjection." Does this mean you're attempting to put the ASCs back into your body? If so, will you be testing the culture prior to injection for endotoxins? Have you planned on measuring the secretome of the cells, since ASCs have a tendency to produce proinflammatory cytokines (ILs 6, 7, 8 & 11, and TNF-alpha)? What is your intended biological endpoint for the therapy?

[Frank]

thanks for the feedback. very useful. So from your reading, these
processes end with the multicell SVF which is what they use as is in
their applications? i would consider using the filter at the end which
some of the protocols use. D you understand that to be for isolating he
ADSC's or for purifying all the live cells in the svf from surrounding
material?
Regarding contamination with fungi and the like, that's very important.
there are some protocols where the material remains in the same syringe
keeping it sterile.

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[Frank]

Thanks for the questions - which scare me. the answer is that yes i do intend to reinject  but only once I have a sound proven protocol and i have followed it successfully. I guess i have not thought of how i wold prove that I have done so.  

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[Dakota Hamill]

Maybe you mentioned this in your previous post but is there a specific
reason why you want to inject yourself with your own adipose stem
cells? Did you mention a rare disease which causes degradation of
adipose tissue? I saw in one of the articles a rare disease which
causes atrophy of one side of the face. I would assume all your care
would be covered under insurance by actual doctors if this is the
case. Wish you the best! I understand dealing with doctors can be
frustrating sometimes.

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[Frank]

I do not suffer from atrophied adipose tissue nor anything that is
simply a matter of having insurance. I am very interested in ADSC for
multiple conditions, the most invasive being periodontal bone
regeneration to reverse gum disease gum loss and the least being
subcutaneous applications to reverse fibroproliferative conditions and
rejuvenate aged skin. None of these would be covered by any insurance
that I know of.

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[Frank Garcia]

why didn't anyone tell me about dedifferentiated fat cells (DFAT)? Looking at the dates of many of the articles on DFAT cells I feel like I've been living under a rock.  Is this the answer to my question about teasing the 5% ADSCs out of the SVF? Seems too good to be true.