X-Message-Number: 15560
Date: Mon, 05 Feb 2001 00:39:19 -0500
From: Paul Antonik Wakfer <>
Subject: Re: Mr Ettinger's - #15541, #15543, #15546
References: <>

>Message #15541
>Date: Fri, 2 Feb 2001 23:10:40 EST
>Subject: infamous last words
>In the past I have said that, time permitting, I will respond point by point 
>to criticisms and  arguments. But of course that does not mean that I will 
>answer every critical missive, especially when it is just the same old same 
>old, as is essentially the case with Grimes and Wakfer. Getting in the last 
>word is not that important.

This is, of course, a good practical ploy when one runs out of ways to
contrive answers to support one's position, ways to further confuse the
situation for the reader, and is not able to or does not wish to address
direct questions, such as those asked by Mr Grimes, or myself.

Here are some of mine:

1. How was the 26% glycerol tissue concentration measured?

2. Does CI use a refractometer for measuring glycerol concentrations?

3. Does each mortician who perfuses with cryoprotectants have a
refractometer, and, if not, then how do they measure the effect of their

4. In general, exactly what patient data is being collected and how is
this done?

>No, I won't pull a Wakfer and unsubscribe and resubscribe to Cryonet every 
>other week. But I'll acknowledge the law of diminishing returns and try to 
>allocate my time rationally.

There is no need to castigate my chosen method of accomplishing the same
goal. My unsubscribing is so that I am not bothered by the myriad of
irrelevant (to me at this time) postings to CryoNet. I can always
quickly search the CryoNet archives for pertinent past posts.

>Message #15543
>Date: Fri, 2 Feb 2001 23:31:01 EST
>Subject: Asymptote
>One of our people referred me to the web site of a company called Asymptote, 
>which makes equipment for freezing specimens for in-vitro fertilization.There 
>is some exposition of both freezing and vitrification. 
>The brief section on vitrification says the approach is promising but still 
>experimental (even for embryos, apparently, although some laboratories have 
>reported good survival), and the high levels of additives are potentially 
>cytotoxic. It also says practical problems include that of devitrification 
>during thawing *or storage.* It did not say at what temperature 
>devitrification (ice crystal formation) might occur during storage.
>Their own technology apparently involves freezing, not vitrification, and 
>leads to good results. 

In contrast here is a non-commercial article about pig embryo
cryopreservation which emphasizes that freezing does not work and
vitrification is necessary for success.


If you read the article and you understand biology, then you will
understand that they are cryopreserving pig embryos at a more advanced
stage of development than human embryos. At the more advanced stage
where some cell differentiation has already begun, the inter-tissue
connectivity is of essential importance just as it is for larger tissue
masses of organs including brains. Whereas at the earlier stage only the
survival of the individual cells is essential since even if only one of
the 8 or 16 cells survives, a fully competent fetus can still develop
from this totipotent cell (a cell which is not yet differentiated -
masked some of its DNA - so that it can still produce any kind of
successor cell).

>An interesting point, not seen or even contradicted in some discussions, is 
>that (for their particular application at least) it is BETTER if ice forms 
>sooner. If supercooling occurs with glycerol protection, for example--ice 
>forming only at lower temperatures--then there may be more damage than if ice 
>forms sooner at higher temperatures. In fact, they deliberately seed the 
>solutions to make ice form sooner. I won't go into the reasons for this just 
>now, since it's getting late.

This point is covered in Cryobiology 101. The reason for it, and the
difference with tissue preservation, is that for best cellular
preservation we need to get most of the water to leave the cell and form
ice in the inter-cellular space where it does not damage the cell
interior. Seeding the solution will cause this to happen early and, in
fact, to draw even more water out of the cell, leaving a highly
concentrated cytosolic solution (inside the cell) which then proceeds to
vitrify. In this manner, the best solution for the individual cell is
obtained, which is what is optimal if each cell is totipotent.
> Message #15546
> From: 
> Date: Sat, 3 Feb 2001 16:26:30 EST
> Subject: Joe Blow
> Wakfer challenged me about my statement that, while he said the 66%
> "viability" of rat brain slices by the K/Na criterion was an average over all
> the cells in the sample, his main man had said it was for each individual
> cell.

Actually, that is not at all what he meant, although I do admit that his
choice of words was not meant for understanding by someone with less
than college level understanding of biology. For details see below.
> I told him the information came from himself (Wakfer), and reminded him that
> the man didn't want to be named in public. Wakfer said to call him Joe Blow,
> but he insisted I find the document.
> O.K., I have found it. I think Wakfer published it also on Cryonet, but the
> document I have in hand at the moment was sent to a long list of people,
> including the editor of The Immortalist, and was dated 1/11/01. The subject
> line was "Hippocampal Slice Cryopreservation Project Status." The passage in
> question reads:

The full text was also posted in CryoNet #15324 by Kitty for me when I
was unsubscribed. I have highlighted below, the portions which apply to
points which have been made repeatedly in this latest round of dialogue.

	    By the time of the Asilomar meeting, the HSCP had
	managed to achieve a more than 10 times improvement in the
	state of the art of cryopreservation of large, integrated
	neural systems, the cryopreservation of which had not
	previously been reported in the formal scientific literature
	to our knowledge.
			 ** Hippocampal brain slices frozen in a
	variety of ways using the standard glycerol originally found
	by Suda to be optimal for cryopreservation of brains were
	found to yield only about 5% plus or minus 5% viability after
	thawing from dry ice temperature.**

This shows that Suda's result (so often referred to by Mr Ettinger) is
quite inapplicable to cryopreservation at lower temperatures. This also
relates to the physiological fact (stated in medical textbooks) that
nerve fibers and synapses do not need to be attached to live functioning
cells in order to be producing a lot of electrical activity.

					  **Hippocampal slices
	vitrified with the simplest and most elementary exemplar 
	of the new 21CM vitrification solutions (essentially Veg) 
	recovered with 53% of the potassium and sodium transport
	capacity of fresh, untreated slices.**

Nothing was stated about individual cells since the potassium-sodium
transport testing method is a bulk tissue assay. Such statements are
written for people with some advanced schooling.

						This accomplishment
	greatly understates the magnitude of the advance over the
	prior state of the art in that it is safe to assume that frozen-
	thawed slices sustain major structural damage as a result of
	the formation and dissolution of ice, whereas such damage
	is presumably absent after vitrification and warming. Therefore,
	the new methods are believed to achieve both immensely
	greater functional recovery and immensely greater structural
	preservation than has been possible with more conventional

	     Since the Asilomar meeting, an attempt has been made to
	further improve upon these results.  

					**The bottom line is that,
	at the time of this report (January 9, 2001), the viability of
	vitrified/rewarmed rat hippocampal brain slices has been
	increased to about 66%.**

Again, it should be patently clear to someone with 2 masters degrees and
high apparent intelligence that this means the measuring method was a
bulk assay for the slice as a whole. 

				  Progress was slower than hoped for
	because the thrust of the experiments was to exploit more
	sophisticated vitrification solutions, and this required a 
	re-optimization of the method for adding and removing the

			** The damage sustained by the slices is very
	sensitive to factors such as osmotic stress, temperature, and
	time, and the previous method of addition and washout was no
	longer the best.**

Thus, it has been shown by much experimentation that damage is very
sensitive to all those factors for which CI appears to give little
attention within their processing of human patients.

			**Also, due to the presence of non-penetrating
	cryoprotectants that require time to diffuse into the slices,
	we found that it was necessary to expose the slices to the
	highest concentrations of cryoprotectant for longer times in
	order to allow vitrification to occur.**

But at the low temperatures involved this still gave less toxic damage
overall as shown by the higher viability obtained.

	**When the previous equilibration time of 10 min was used, slices
	that were cooled to below -130oC and rewarmed gave variable and
	usually suboptimal results, but when equilibration time was
	extended to 20 min, then vitrification yielded the 66% recovery
	just noted.**

Showing just how important it is to reach complete equilibration,
something which is not achieved by CI patient methods.

		   **It is possible this can be improved slightly by
	even longer exposure to the cryoprotectant prior to
	vitrification, or by modifications of the solution that will
	allow for faster equilibration.**

Ie. to get faster penetration, something at which viscous glycerol is
extremely poor at doing.

	     **At the moment, 66% recovery of brain slice function is 
	equivalent to the recovery of K/Na ratio typically achieved for
	vitrified/rewarmed rabbit kidney slices by 21CM, a remarkable

Again, the term "slice function" clearly signifies, to any biologically
competent reader, the use of a bulk tissue assay.

			** There is indirect evidence that this may be
	sufficient for long term cellular survival after transplantation
	and in vivo recovery.**

This is just as I have been stressing in these discussions, since the
conditions above have already been shown to be sufficient for
transplanted rabbit kidneys. And before Mr Ettinger (masquerading as a
simpleton instead of the father of cryonics with two masters degrees,
and apparently highly knowledgeable about biology) objects that rabbit
kidneys were never vitrified let me explain. Yes, the successful
transplantation of rabbit kidneys with 66% cellular viability did not
occur after fully reduced temperature because sufficiently fast
rewarming methods were not available at that time. Such fast rewarming
methods would have been required for full vitrification because the
solution used was not concentrated enough for slower rewarming to
prevent recrystallization, and it could not be more concentrated because
it that would have made it too toxic. Now we are at the happy stage of
having less toxicity at a concentration which vitrifies even better than
with the old rabbit kidney experiments, so that this solution may not
need as fast rewarming as was needed before. At the same time somewhat
faster rewarming techniques are now available and these should prove
sufficient to prevent recrystallization with current or slightly
advanced solutions.
If this sounds complex, it is! This is not simple stuff, there are many,
many variables and conditions involved. However, once again for an
"expert" like Mr Ettinger, it should be easy enough to comprehend. If he
can't do so then perhaps he should politely ask rather than attack and
attempt to confuse the even less informed. (If Mr Ettinger considers the
above explanation an example of me being pretentious, then he doesn't
know the meaning of the word; this is *education*, not pretension.)

			**  We attempted to improve the K/Na ratio of
	rat hippocampal slices by culturing them for longer times after
	exposure to cryoprotectant to see if self-repair would be
	apparent, but at incubation times at least as long as 8 hours,
	there was no apparent improvement.**

This again has been alluded to as a negative finding, but coming right
after the above statement about transplantation success implies that
culturing of slices ("wounded" and without perfusion support) is *not*
as helpful to self-repair as is transplantation of a whole organ, and
perhaps not even as helpful as grafting of the slice to a whole body
healthy environment.

					**On the other hand, we believe
	that 66% recovery implies 66% recovery of the function of each 
	individual cell, rather than outright loss of 34% of the cells
	with full recovery of the remaining 66%.**

Now it becomes clear that by quoting the last sentence only, Mr Ettinger
loses its full meaning.
The previous sentence once again spoke of K/Na ratios of slices, not of
individual cells. The purpose of the (almost parenthetical) insertion of
this second sentence, which once more should have been clear to a man
with 2 masters degrees (Mr Ettinger), was to emphasis that the 66% did
not imply an outright loss of 34% of the cells. Unfortunately, the
choice of phrasing was poor enough that someone a) illiterate in
biology, b) with little understanding of the fact that such individual
cellular measurements would be exceedingly difficult and time consuming
(if even possible), or c) ignorant of the almost logical impossibility
of the K/Na ratio being the same for every cell, might very well get

						Several brain slices
	have been fixed using an electron microscopy grade fixative and
	will be examined structurally to verify general integrity of all
	cells and the preservation of brain slice structure after
	vitrification and rewarming.  Unfortunately, as of the time of
	this writing, the samples have not yet been processed and the
	structural results are not available.  If the project continues,
	this gap will be closed by processing these samples as well as
	additional samples that will further probe the issue of
	structural preservation after vitrification.

The project is continuing sufficient at least to get this accomplished
(currently until June 30, 2000) and I will be posting a more detailed
announcement about that shortly.

	     In January, we plan to use a vitrification solution that
	we believe is an improvement over the one adopted following the
	Asilomar meeting.  In February, we expect to have a further
	improvement ready for trial on brain slices.  The 3rd
	vitrification solution has already been shipped to Dr. Pichugin
	and has been received, along with more of the 2nd solution for
	direct head-to-head comparison.  At present, we have no reason
	to believe that we cannot exceed the 66% functional recovery
	level achieved so far.

Again, no additional progress reports on this are yet available, but
they will be posted here as soon as they are.

> "On the other hand, we believe that 66% recovery implies 66% recovery of the
> function of each individual cell, rather than the loss of 34% of the cells
> with full recovery of the remaining 66%." (He didn't mention the third
> possibility, which is almost certainly the correct one, viz., that the 66% is
> an average over all the cells in the sample, with variance unknown or
> unspecified, which I believe Wakfer also thinks.)

I don't just *think* so! I am in contact with the involved scientist at
least weekly and he has explained to me the exact method by which the
K/Na ratio is measured. It involves sealing the cell membranes, washing
out the intercellular space, homogenizing the cells, and the measuring
the ratio of K/Na in the resulting cellular soup. No individual cellular
measurements are made.
Furthermore, since this is a bulk assay which measures *nothing* about
the individual cells, it is extremely "pretentious" of Mr Ettinger to
continue to raise the notion of a variance in the measure between cells.
The individual bulk measurements from several slices could be supplied
and the variance of those could be computed, but that is all.

> It's not a huge deal. Joe Blow was just a trifle careless at the moment.

With that I agree. But any person who understands biology would not have
thought for a moment that the measurement was made on individual cells.

> I wouldn't rub his nose in it, or Wakfer's, except for Wakfer's non-stop
> pretentiousness and double standards.

I have no pretentiousness or double standards, this is like the pot
calling the kettle black.
Please tell me precisely what I have said which is ostentatious,
assumption of false dignity or authority, pretending or alleging
falsely, exaggerating for effect. This sounds much more like others to
Mr Ettinger has shown himself to be the knave by his constant use of
such abusive words and other phrases such as "rub his nose in it" which
is normally only used for dogs!

-- Paul --

The Institute for Neural Cryobiology - http://neurocryo.org
A California charitable corporation funding research to
perfect cryopreservation of central nervous system tissue
for neuroscience research & medical repair of the brain.
Voice-mail: 416-968-6291  Fax: 559-663-5511

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