Antifreeze in stool/FMT capsules, glycerol vs maltodextrin, -80 (medical freezer) vs -20 (home freezer) Enhancing 

Enhancing the outcomes of FMT
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SFBayFMT5

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I was reading through the published information on how OpenBiome processes their stool samples into FMT preparations. They state that the stool is dissolved in a glycerol/saline solution, consisting of 12.5% glycerol in 0.9% NaCl, before storing in a -80C freezer (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082449/, see second paragraph under heading "Manufacturing").

I surmise that this solution plays the role of an antifreeze, similar to the antifreeze that HMorg offers to be added to their orders, although the HMorg antifreeze consists of maltodextrin and glycerol instead of glycerol and saline. Am I correct in inferring this?

If so, does adding antifreeze to capsules have any effect on their ability to stay preserved in a normal freezer? or are they still preserved just as well, only the bacteria do not sustain damage from ice formation the way they would if frozen without antifreeze?
 
SFBayFMT5 said:
although the HMorg antifreeze consists of maltodextrin and glycerol instead of glycerol and saline. Am I correct in inferring this?
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No. Openbiome and most other places use glycerol. But glycerol can be a laxative, so HMorg uses maltodextrin by default instead, but gives the option for recipients to choose glycerol.

-80 (medical freezer) generally preserves better than -20 (standard freezer). Glycerol & maltodextrin are very similar.

You can search the FMT page https://humanmicrobiome.info/fmt/ for "maltodextrin" for more info. And the "freezing" section https://humanmicrobiome.info/fmt/#freezing for general info on that.
 
Michael Harrop said:
No. Openbiome and most other places use glycerol. But glycerol can be a laxative, so HMorg uses maltodextrin by default instead, but gives the option for recipients to choose glycerol.
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What I meant was, does the antifreeze HMorg is using serve the same purpose as the glycerol other places are using? It sounds like the answer is yes.
Michael Harrop said:
-80 (medical freezer) generally preserves better than -20 (standard freezer). Glycerol & maltodextrin are very similar.

You can search the FMT page https://humanmicrobiome.info/fmt/ for "maltodextrin" for more info. And the "freezing" section https://humanmicrobiome.info/fmt/#freezing for general info on that.
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My question was, does having antifreeze added negatively affect (i.e. reduce) how long stool lasts in a standard freezer (either from an effectiveness/efficacy or a safety standpoint), by interfering with the preserving effect of cold? Or does it actually have a positive effect because the beneficial microbes don't get stressed as much?
 
Do you know which other providers use/did use antifreeze vs. which don't? Taymount says on their website that they do, I don't know about other sources.
 
I have looked at the papers that discuss using maltodextrin as a cryopreservative for FMT and/or other bacteria, including this one (https://www.nature.com/articles/s41598-019-45173-4), and ALL of them combine it with trehalose in various ratios, none with just maltodextrin alone. From what I can see, the trehalose is the component that actually replaces water in cells to keep them viable, the maltodextrin just dehydrates the bacteria. In one study involving a probiotic (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8732778/), skimmed milk was the best, but of all the carbohydrate preservatives (inulin, sucrose, and maltodextrin), maltodextrin performed in the middle overall, between the other two, and rather poor, at only 10% after 8 weeks (it was actually the worst at promoting survival during the initial freezing, but after storage at frozen temperatures, the initially best-performing inulin became the worst). Seeing this poor preservation of a single bacterium that is easy to isolate and grow doesn't seem to bode well for a complex mixture of potentially fragile bacteria like a FMT.

I don't know where you are getting your information that maltodextrin ALONE is comparable to glycerol in its effectiveness. Where did you read this?

Also, regarding glycerol--can the glycerol/saline mixture be encapsulated in the type of capsules you are using without dissolving/weakening them? It's clearly a liquid, albeit viscous.
 
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SFBayFMT5 said:
Also, regarding glycerol--can the glycerol/saline mixture be encapsulated in the type of capsules you are using without dissolving/weakening them?
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Yes, as long as you don't add too much saline.

I checked the citations of the links in the wiki and one is dead and another has no mention of "maltodextrin":
We selected the two well-known extracellular cryoprotectants maltodextrin and trehalose since they help in increasing the medium viscosity, thus limiting ice crystallization and osmotic imbalance that occurs during freezing 29,30. They differ from intracellular cryoprotectants such as dimethyl sulfoxide and glycerol, which can freely penetrate cell membranes and then compromise original biological and metabolic characteristics at revivification.
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That 29 and 30. That statement says both malto and trehalose are cryoprotectants, but the other paper indicates that it requires a mixture of both.

One of them contains this citation https://aiche.onlinelibrary.wiley.com/doi/abs/10.1021/bp049559j, but it's for drying, not freezing.

This one also indicates that the mixture is best https://link.springer.com/article/10.1007/s11694-023-01863-2.

This one's only for yeast https://www.sciencedirect.com/science/article/abs/pii/S0308814622026917.

Overall, I agree that it should be a maltodextrin-trehalose mixture. I changed the wiki page.

A few reasons I haven't bothered making donors do this is:

1. I've tried frozen, no antifreeze, vs fresh, and found no difference.
2. Other recipients have used frozen stool, no antifreeze, and experienced major improvements.
3. There is no apparent difference in recipient results between with/without antifreeze (may be partially due to MD-only), or glycerol vs maltodextrin.
4. It complicates things more for donors. Both with them having to create the mixture, and having to find and order more supplies for each new donor location.

It's doable for sure, but is it worth the extra effort? I would be particularly hesitant to have donors start adding it at this point where they're already unhappy about the lack of demand, and there isn't even enough demand to order blood & stool testing for new top donors.
 
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SFBayFMT5 said:
I was reading through the published information on how OpenBiome processes their stool samples into FMT preparations. They state that the stool is dissolved in a glycerol/saline solution, consisting of 12.5% glycerol in 0.9% NaCl, before storing in a -80C freezer (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082449/, see second paragraph under heading "Manufacturing").

I surmise that this solution plays the role of an antifreeze, similar to the antifreeze that HMorg offers to be added to their orders, although the HMorg antifreeze consists of maltodextrin and glycerol instead of glycerol and saline. Am I correct in inferring this?

If so, does adding antifreeze to capsules have any effect on their ability to stay preserved in a normal freezer? or are they still preserved just as well, only the bacteria do not sustain damage from ice formation the way they would if frozen without antifreeze?
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I have that question also. Does adding glycerin/maltodextrin stop the stool capsule from expanding? As it's liquid and physics? Help explain why maltodextrin or glycerin is even used when freezing?
 
Cryopreservation: An Overview of Principles and Cell-Specific Considerations

Abstract

The origins of low-temperature tissue storage research date back to the late 1800s. Over half a century later, osmotic stress was revealed to be a main contributor to cell death during cryopreservation. Consequently, the addition of cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), or propylene glycol (PG), although toxic to cells at high concentrations, was identified as a necessary step to protect against rampant cell death during cryopreservation. In addition to osmotic stress, cooling and thawing rates were also shown to have significant influence on cell survival during low temperature storage. In general, successful low-temperature cell preservation consists of the addition of a CPA (commonly 10% DMSO), alone or in combination with additional permeating or non-permeating agents, cooling rates of approximately 1ºC/min, and storage in either liquid or vapor phase nitrogen. In addition to general considerations, cell-specific recommendations for hepatocytes, pancreatic islets, sperm, oocytes, and stem cells should be observed to maximize yields. For example, rapid cooling is associated with better cryopreservation outcomes for oocytes, pancreatic islets, and embryonic stem cells while slow cooling is recommended for cryopreservation of hepatocytes, hematopoietic stem cells, and mesenchymal stem cells. Yields can be further maximized by implementing additional pre-cryo steps such as: pre-incubation with glucose and anti-oxidants, alginate encapsulation, and selecting cells within an optimal age range and functional ability. Finally, viability and functional assays are critical steps in determining the quality of the cells post-thaw and improving the efficiency of the current cryopreservation methods.

Keywords: cryopreservation, cryoprotectants, low temperature banking, freezing
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https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-024-04166-w#Sec8
The use of cryoprotectants such as glycerol help preserve organisms and tissues by lowering the freezing point of water to prevent crystallization and mechanical damage
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Just came across this:
Glycerol, which forms hydrogen bonds with water molecules and can penetrate both the cell wall and cell membrane, is widely used for preservation in microbiome research, and is recommended for the preservation of fecal microbiota transplant material in human medicine [55, 56]. The results in individual studies are somewhat mixed, with some finding improved preservation without any buffer [57, 58].
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The two citations:

Functional amplification and preservation of human gut microbiota (2017) https://www.tandfonline.com/doi/full/10.1080/16512235.2017.1308070

The CPAs used in this study were two cell-permeating, intracellular agents [glycerol (G) and DMSO (D), each at 10% (v/v)] and one extracellular, non-permeating,

We tested three different CPAs, either alone or mixed, and two different preservation durations, up to 6 months.
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It has better penetrating power than glycerol, this last also being known to cause a molecular reorganization of plasma membranes during freezing [Citation33]. It has therefore been suggested that glycerol should not be added more than few minutes before freezing [Citation34], a technically difficult requirement when working in an anaerobic chamber, as is needed for fecal microbiota processing. This may explain why glycerol had worse preservation reliability in this study on some checked parameters, even when used in association with other CPAs.
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Comparative methods for fecal sample storage to preserve gut microbial structure and function in an in vitro model of the human colon (2020) https://link.springer.com/article/10.1007/s00253-020-10959-4

This study aimed to assess the impact of three commonly used preserving methods, compared with fresh fecal samples used as a control, on the kinetics of lumen and mucus-associated microbiota colonization in the M-ARCOL model. Feces from two healthy donors were frozen 48 h at − 80 °C with or without cryoprotectant (10% glycerol) or lyophilized with maltodextrin and trehalose prior to inoculation of four parallel bioreactors

All the preservative treatments allowed the maintaining inside the M-ARCOL of a complex and functional microbiota, but considering stabilization time of microbial profiles and activities (and not technical constraints associated with the supply of frozen material), our results highlighted 48 h freezing at − 80 °C without cryoprotectant as the most efficient method.
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I added them to the wiki.
 
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