Christopher

Thank you for your reply.  I am a Clinical Pathologist and I run the Stem Cell Laboratory at MDA here in Phoenix.  We freeze human peripheral stem cells for bone marrow transplant.  The cells we collect are CD34+ and are essentially large lymphocyte like cells. 

The controlled rate freezer runs at normal atmosphere, this is not a hyperbaric environment.  Nitrogen Gas is pulsed through a control valve  operated by a computer program that controls the rate of freezing.   DMSO is a cryoprotectant and we use a 5% solution and adjust the number of cells / ml of diluent to always freeze the same quantity per bag.  

DMSO is toxic to the cells (all human cells) when liquid.  The purpose of the DMSO is to have every part of the cell freeze at the same time rather than having the cytplasm freeze first, then the nucleoplasm, then the organelles and lastly the cell and nuclear membranes. Freezing sequentially forms ice crystals inside the cell that will poke throgh the cellular and nuclear membranes.The cells would die on thawing.  

The heat of fusion is a sharp increase in temperature that occurs when a liquid goes to solid form. The formation of a crystal latice (ice in this case). We want to see one peak of heat that indicates all cell elements froze at the same time. When this does not occur, the freezing curve shows a "script r" form which is formed by differnt pieaks indicating crystal has formed at different times. 

The question came up the other day, what happens after the heat of fusion (formation of ice) in a cell as the temperature drops to minus 200C?  I looked at the third law of thermodynamics which should address this, Still the math is there and we can predict what happens when. Now what I want to know is what happens to the ice crystal inside the cell as the temperature drops to near absolute zero?  In daily life we see that dry ice has differnt properties than regular ice, among other things, it tends to stick to organic tissues and fabric. It also affect electrical conductivity by the way (  we do not  run  voltage throgh the cells once frozen!) anyway, looking at quantum states, as Einstein Bose condensates form, the electrons are slowed down. Now, we are nowhere near Einstein Bose condesates,  so my question is: what happens to the crystal lattice as the temperature drops.  

Thanks

Sergio

Christopher.

If it wasn't for people like you that did all the research we wouldn't be doing what is common place today i.e..

Just a brief snapshot of the practice (FDA approved etc).

We mobilize the stem cells ideally after chemotherapy be it the last cycle of an appropriate combination of agents for the patient's diagnosis or just plain Cytoxan to hit the marrow and create a decrease in the stem cell niches.

After chemotherapy the BM will recover in 12 - 18 days.  We add G-CSF (Filgrastin)  at 10micrograms / Kg  sub cutaneous  once or twice a day.  This revs up the marrow to its full potential which can be up to 100x the daily production.

We follow the WBC counts (peripheral) until there is a significant increase (like doubling of counts). We only start collecting if the WBC count is over 3K. Usually the differential jumps up with first PMN's and lymphocytes shortly thereafter. The HSC is considered to be "a large lymphocyte". 

Once a peripheral CD34 count of 10 CD34/ul is present we use an apheresis machine to collect the patient (Terrumo Spectra or Optia).  This is just a fancy centrifuge with a hollow belt where the whole blood  separates.  The physics and optics of the centrifuge allows the collecting tube to be exactly in the MNC/Lymph layer, amazing that technology has come this far, knowing how thin a buffy coat is!.  Anyway,  we process 4 to 5 Total Blood Volumes (TBV) and collect within that layer trying to collect as few platelets and PMN's as possible.  Once that is done, the patient goes home and the cryo lab starts to work (we collected 2 patients today and are just started volume reduction, cell count and based on that dilution of the product to have a fixed number of cells/ bag).

Forgot to tell you that the anticoagulant of choice for apheresis is ACDA, because it has a short life and is readily cleared by the liver in 3 passes (that's the only part of Kreb's cycle I remember: Citrate goes to bicarb and is breathed out.). This also causes alkalosis and which drives K+ into the intracellular space, so there is a relative transient  hypokalemia that frequently has to be corrected. Citrate binds ionized calcium and Mg++.  Although the ACDA is given to the draw line, once mixed with  blood a lot of it is returned to the patient causing the above. The product sits ina collecton bag on an IV pole that is part of the apheresis machine. 

The heat of fusion does not affect the cell, as long as it is brief (1 min or less).  The only problem it causes is  hyperosmosis in the extracellular fluid that may not have frozen. This indeed if the diluent is not well calculated cause cell damage by osmotic shock when part of the cell is frozen and the extracellular is not.   Once that single peak is obtaines, then we decrease the temperature by about 1 degree per minute to minus 100C. The cassette containing the bag is then removed from the controlled rate freezer and stored in vapor phase of LN2 at about - 190 - 200C. There these cells can basically stay for a minimum of 10 years (NY Blood Center has products frozen for almost 30 years and they have shown to still be viable.

I would like to send you my presentation on Stem Cell Collection and another on cryopreservation along with examples of freezing curves that show what I described above.  

Again, thank you for taking the time to reply.

Sergio

[email protected]

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PS- we are looking for a cryopreservation technician for our lab (MT, MLT, or allied health with cryopreservation experience),  I am  pushing very hard to eventually get a PhD to be over the cryo lab like we had at the Mayo Clinic in Rochester where I trained.  Cryopreservation is a fascinating area and a lot remains to be done! 

Quite an accomplishment!

As to our lab tech position, it is advertised on www.bannerhealth.com. It is full-time with all benefits. I trained at Mayo and worked ther till Dec. 2013. There we had fully trained techs that were either med tech students that trained with us and stayed or  or hired people with at least 5 years experience in the cryo lab. No such luxury here. The program here was started in 2012. It takes about 5 months for someone with a BS to learn the technique and be able to operate independently.  The problem with that is that skill is very marketable.  This time we want to get someone who "can hit the road running". That is, someone who is familiar with the procedure, or has trained in the past to do cryopreservation.

On a totally different subject, I have some ideas for pilot projects using stem cells or other cell lines.

. One I would like to get rolling on is using Hibernation Factor on stem cells. This has never been done. While at the University of KY, I worked with a PhD chemist that identified and sequenced the factor.n He got as far as hibernating a primate and reviving it with Narcan.  The study was done  with grants from NASA and the Army. This is of public domain, and I have the pertinent literature in pdf format. The only study I did, was adding hibernating factor to red blood cells in storage  in a controlled study. The results were very interesting. This was published in an abstract format. To my knowledge no one has done anything with this since 1997. I am trying to find someone who wants to take this to the next level. . Sufices to say the hibernating factor is a Delta Opioid with 5 isomers. One  acts on the tyroid, another  has anticoagulant effect (really strange for an opioid!) and the other 3 never got worked-out.  Anyway, stay in touch.

 Have a good day

Sergio.