Understanding your freezing curves

Understanding your freezing curves - Click here for document

December 2

Freezing blood clots

Question: We have an upcoming project, which involves long-term storage of blood clots in a -80 freezer.  In the past clots for this project were stored in 5 ml centrifuge tubes because of their wide opening.  This can't be good for the long term.  Are there any wide opening cryovials available?  What is the best container for clot storage?

Answer:  Your questions contains several issues.  The first is a container for storage of blood clots.  I agree that cryovial

Freezing whole blood to use for isolation of CTCs

Question:  We are interested in starting to bank whole samples with the future interest of isolating circulating tumor cells in the future.  Do you know of a protocol for the preservation of whole blood?

Answer:  You are basically describing two different issues.  The first issue involved preserving a blood specimen that can later be analyzed for the presence of circulating tumor cells (CTCs).  The second issue is a protocol for preserving whole blood.

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What is the 'shelf life' of hematopoietic stem cells stored on liquid nitrogen?

Answer:  There have been few studies of this issue.  One study examined the post thaw survival of HSC products frozen between 1973 and 1991 and then thawed at regular intervals.  This study observed that HSC products could be stored for between 35 and 156 years before complete loss of colony forming units.  Different types of colony forming units appeared to be more sensitive to storage than others.

Do you know of any article showing analyte degradation in plasma or serum when being srtored in -80?

Answer:  There have been several articles that have noted degradation of biomarkers for plasma and serum stored at -80°C. Here is a partial listing of the papers:

Hannisdal, R., et al., Analytical Recovery of Folate and Its Degradation Products in Human Serum Stored at -25 degrees C for up to 29 Years.  Journal of Nutrition, 2010. 140(3): p.

Post Thaw Recovery of CD34+ Cells > 100%

Question: As part of our stability program we have been thawing cryopreserved products to determine total nucleated cell counts (TNC) and CD34 recovery, as well as CD34 viability.  We have noticed that our recoveries for CD34 cells frequently exceeds 100%.  TNC recoveries are in the ange of 90%.  Our clinical labs cannot offer possible explanations for the unexpectedly high CD34 cell recovery.

Answer:    First of all, there is no evidence that stem cells proliferate during freezing so

Alternatives to DMSO

Question: I have heard you talk previously and you always mention the need to find alternatives to dimethylsulfoxide (DMSO), why?  Everybody uses it for preserving cells.  Why do we need the alternative?

Answer:    The full answer to this is rather long so I will try to summarize the issues as briefly as possible.  Many cells are used for therapeutic application.  Typically, cells frozen in DMSO are thawed at the patient's bedside and infused directly into the patient.  Clin

One common error with post thaw assesment

Question: We freeze hematopoietic stem cells and we getm uch higher viabilities than other centers that use the same protocol.  Why are our number different?

Answer:   In our experience post thaw assessment is performed incorrectly more than correctly.  When preserving cells, please remember to measure yield (defined as the total number of viable cells post thaw divided by the total number of viable cells pre-freeze).  During freezing, cells have three basic fates: (1) cells that are intac

Pooling and re-freezing cells

Question: In order to get enough cells, we freeze individual aliquots, thaw them, pool the cells and then refreeze them.  Will this harm the cells?

Answer:   The short answer is 'yes'.  All biospecimens (proteins, cells, tissues) suffer when they are repeatedly frozen and thawed.  Try to avoid at all cost repeatedly freezing and thawing any biospecimen.  For cell suspensions, freezing, thawig and then refreezing a cell will result in the majority of cells being dead.  In fact rep

What to freeze in: bags, vials, straws?

Question: We are freezing very small tissue fragments.  What container should we use for this specimen?

Answer: Good question.  Selection of a freezing container is very important.  Storage at low temperatures is adverse for most materials (materials get brittle at low temperatures).  Biospecimens stored at low temperatures should use containers designed to be used at low temperatures.

There are three different general categories of containers for low temperature