Frequently Asked Questions

Aggregate Freezing

We have a project that is focused in moving jPSC-derived cells that have been matured for a specific application toward human clinical trials. They are large mulit-cellular 3D structures as well as dissociated cells. ...

Question:
We have a project that is focused in moving jPSC-derived cells that have been matured for a specific application toward human clinical trials. They are large mulit-cellular 3D structures as well as dissociated cells. I was wondering if you have any experience/advice on methods for freezing large clumps of cells.

Answer:
A variety of cell types can be cultured in aggregates: hepatocytes, neuronal progenitor cells, etc. Freezing this system can have certain challenges. We will give you a brief overview of options.

General recommendations: In general, having wide variations in the size of the aggregate makes it difficult to achieve a uniform freezing response. If at all possible, keep the distribution of sizes as small as possible.

Option #1: Reduce the size of the aggregates to ~100-200 um in diameter. That results in a ~300 cells per aggregate. Using a controlled rate freezing protocol in which you carefully structure of the cooling protocol to reduce undercooling (e.g. the temperature at which ice forms in the extracellular solution).

Option #2: If for whatever reason you need to freeze the entire aggregate and it is larger than that listed in Option #1, you should use vitrification. There are several vitrification protocols that have been published for aggregates in the last few years. I would recommend that you start with those published protocols.

Alternatives to DMSO

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?

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. Clinical studies documenting the adverse reactions associated with infusion of DMSO indicate that nearly all patients experience mild adverse reactions-a small number experience more serious adverse reactions. Most physicians will just recommend giving the patient medication to ameliorate the adverse effects. Most patients however will tell you that the adverse reactions to DMSO were very unpleasant and in fact, some patients will tell you that the adverse reactions are the worst part of the process for a bone marrow transplant (that is saying a lot). Removing the DMSO is time consuming and labor intensive and results in a significant loss of cells.

The development of alternatives to DMSO will also be driven by development of new therapies. For example, there are cell therapies being developed that will require multiple infusions of cells. If the cells are frozen in DMSO, this may result in teh repeated infusion of DMSO into the patient. It is unlikely that regulatory bodies will find this acceptable. Therefore alternatives to DMSO will be needed.

In addition, DMSO is associated with epigenetic effects on cells. For embryonic stem cells preserved using DMSO, these effects result in larger fraction of cells differentiating. It will be important to establish alternative cryoprotective agents taht do not alter cell behavior and potentially biomarker expression.

ATE Blood Clot

My technician froze our blood samples after centrifuging but did not transfer the serum to a separate tube. ...

Question:
My technician froze our blood samples after centrifuging but did not transfer the serum to a separate tube. The frozen sample now has the clot at the bottom and the serum at the top. We want to perform Lipid profiles, Glucose and Creatinine assays. Can we thaw and use these samples? Or is there a way to remove the serum without disrupting the clot?

Answer:
I would recommend not using the samples. When you froze the clotted blood with the serum, the cells inside the clot will have lysed (red blood cells and unavoidable) and potentially disruption of the clot (it may or may not be visible). The products of cells lysis will be released into the sample and may bias measurements depending upon the method that you are using to analyze the sample when thawed. The most common methods of measuring creatinine will be influenced by the presence of lysed blood cells.

ATE oxygenation

When thawing cells, should you feed the cells oxygen? What is the ideal temperature versus time for thawing cells?

Question:
When thawing cells, should you feed the cells oxygen? What is the ideal temperature versus time for thawing cells?

Answer:
There is very limited scientific literature on oxygenating cells either pre-freeze or post thaw. As a result it is difficult for me to make recommendations in this matter. It would be difficult logistically to add oxygen during the thawing process unless you h ad an oxygenation unit that was sterile docked to a bag while in a warm water bath. Anything that is performed on the cells during thawing (1) reduce the warming rate; and (2) subject the cells to more physical stress. There have been a fair number of studies now that have looked at modifying culture conditions to improve post thaw recovery. Most of those studies have looked at adding apoptosis inhibitors to the culture medium to reduce post thaw apoptosis.

In terms of the thawing rate, for conventionally, slow cooled samples (not vitrified sample), rapid thawing is best. That translates to >60 C/min warming rate. You can estimate your overall warming rate by timing how long it takes to warm and have any visible ice crystals disappear from the bag/vial.

Biospecimen Protocols

I was just wondering if you have protocols to: 1. Preserve whole blood clot after removing the serum 2. Isolate and preserve PBMCs from buffy coat in peripheral blood as well as bone marrow 3. Preservation of RBCs after removing plasma and buffy coat.

Question:
I was just wondering if you have protocols to: 1. Preserve whole blood clot after removing the serum 2. Isolate and preserve PBMCs from buffy coat in peripheral blood as well as bone marrow 3. Preservation of RBCs after removing plasma and buffy coat.

Answer:
As a general policy, we do not distribute protocols for biospecimen collection. There is however an organization that is preparing and distributing standard protocols.

http://biospecimens.cancer.gov/resources/sops/default.asp

I would be checking this website regularly for protocols that will be distributed.

Design Rules/Heuristics on Cell Preservation

One of the main complexities in cryopreservation is that a lot of the scientific literature is specific to particular cell/process conditions. ...

Question:
One of the main complexities in cryopreservation is that a lot of the scientific literature is specific to particular cell/process conditions. I'm sure I don't have the comprehension of a real cryobiologist, but compared to other disciples, I find it difficult to bridge cryopreservation sciences into engineering practice. It would be very useful if there were a set of design rules or some simple heuristics to assist in development.

Answer:
I understand that it appears that we cannot seem to make protocol development more rational and systematic and perhaps less labor intensive. Part of the problem is that our current understanding of freezing damage is quite frankly severely limited. Until we have this understanding, we will not be able to improve preservation for cell types that are currently poor responders to freezing. We are hopeful that work that we have published spectroscopy of cells in the frozen state is a good start. We are continuing to work on this issue and it is an important one.

Secondly, there should be a method to compress and accelerate development of optimal protocols for cells that respond well but current protocols cannot be used or new and emerging applications. We are working on that right now. Stay tuned.

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

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

Question:
Do you know of any article showing analyte degradation in plasma or serum when being stored 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. 522-526.

Lee, D.H., et al., Proteomic Analysis of the Effect of Storage Temperature on Human Serum. aAnnals of Clinical and Laboratory Science, 2010. 40(1): p. 61-70.

Panesar, N.S. and L.C.W. Lit, Stability of serum thyroid hormones following 8-11 years of cold storage. Clinical Chemistry and Laboratory Medicine, 2010. 48(3): p. 409-412.

Freezing blood clots

We have an upcoming project, which involves long-term storage of blood clots in a -80 freezer. ...

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 is not the best container. I would use a bag. I will send you a link to a company that makes bags and can even customize the opening and volume of the bag to your needs. Cost may be an issue and these bags will be more expensive than a criovial. We will try to assemble more options for bag supplies for frozen storage and put them on the website.

The second issue is storing the samples at -80oC. Without knowing the biomarkers that you are interested in, it is hard to know if this is the best temperature for storage of blood clots. We will be looking into the issue and watch for updates on the website.

Freezing whole blood to use for isolation of CTCs

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?

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.

On the first issue (using a frozen and thawed sample to isolate CTCs), you will have to check manufacturers specifications for the specific device that you intend to use. The manufacturer should specify as to whether or not the technology can be used with frozen and thawed cells. Freezing alters in particular the cell membrane, which may in turn influence the ability of certain technologies to isolate the CTCs.

The second issue involves freezing whole blood. I am assuming that you will use a protocol designed to preserve the mononuclear cells in the sample. That being the case, the red blood cells (which will be the vast majority of the cells in the whole blood sample) will have lysed upon thawing. Granulocytes and platelets will also be damaged/activated. Basically, freezing whole blood will result in a sticky mess as the lysed cells will be releasing the intracellular contents (for example DNA is very sticky). Aggregation/clotting that results from freezing whole blood may entrap the target cell of interest (CTC) making it more challenging to isolate. The very different freezing response of the diverse cell types present in whole blood will make it a real challenge to isolate CTCs from frozen whole blood.

Freezing whole cord blood

There are private cord blood banks that offer cryopreservation of whole blood. This should minimize the risk of losing cells, especially since we do not know which cells will be valuable in the future. ...

Question:
There are private cord blood banks that offer cryopreservation of whole blood. This should minimize the risk of losing cells, especially since we do not know which cells will be valuable in the future. What is your opinion on this and what kind of protocol do you imagine they use? I read a posted question about freezing whole blood with the expert expressing his/her concern of the sticky mess that all the lysed red blood cells would cause...Please feel free to elaborate.

Answer:
Any hematopoietic product (blood from veins, bone marrow, cord blood) will contain large numbers of red blood cells and much smaller numbers of white blood cells. The biology and biophysics of these two cell types are very different. To date, methods of freezig both cell types successfully have not been developed (nor is it highly likely that it will).

To my knowledge, the only cells that have exhibited biological/therapeutic significance are nucleated (remember, mature red blood cells do not have a nucleus) and would typically be found in the white blood cell fraction (depending upon method of separation of cells.

That being said, freezing whole blood will require that you try to preserve the white blood cells and that will result in killing of the red blood cells. Also keep in mind that 93% of the cells are red blood cells and 7% (roughly) are white blood cells. You are killing off a lot of red blood cells. Dead cells are really sticky and will potentially cause aggregation. You want the nucleated cells but formation of aggregates may trap those high value cells and result in their losses. There you have the conundrum: red blood cells depletion techniques may result in loss of valuable cells but freezing whole blood will probably result in aggregation, which has the same outcome.

Information resources on cryopreservation

I want to understand cryopreservation.

Question:
I want to understand cryopreservation.

Answer:
Wonderful. Your request is rather broad so we will suggest some resources that will help you understand the scientific principles for the field or quite simply use cryopreservation more effectively.

1. Books that will give you background as well as communicate current research:

Advances in Biopreservation, ed John G. Baust, CRC Press, Boca Raton, FL, USA, 2005. This book covers a range of topics from preservation fundamentals to specific techniques. Systems studied include macromolecules, cells, and tissues (native and engineered).
Life in the Frozen State, Ed. Fuller, B.J., Lane, N, Benson, E. E, CRC Press, London, 2004. The preservation of cells, tissues, and gametes and supplemented by an extensive discussion of plant, bacteria, fungi preservation.

2. Journal articles that describe elements of a preservation protocol. These are more practical guides for individuals interested in developing new protocols or debugging problems with existing protocols:

Hubel, A. Cryopreservation of cells used therapeutically. In: K. Loper (ed). In: Cellular Therapty: Principles, Methods and Regulations, AABB Press, Bethesda, MD, 2010.
Aksan A, Hubel A. "Biopreservation of cells for use in biotechnology and cell therapy", Bioprocessing J, 2009.

3. Participate in our education program. Specifically, attend our short course, "Preservation of molecular, cellular and tissue biospecimens", offered at the University of Minnesota, May 18-20, 2010 (http://www.biocor.net/preservation-of-cells-tissues-and-gamets-overview). Hundreds of people from all over the world have found this to be an excellent method by which to understand cryopreservation.

4. Join the Society for Cryobiology or read its journal, Cryobiology.

Kitchen Freezer

What do you know about storage of cells at temperatures higher than liquid nitrogen? What about storage at -20oC?

Question:
What do you know about storage of cells at temperatures higher than liquid nitrogen? What about storage at -20oC?

Answer:
-20oC is roughly the temperature of the freezer in your kitchen. If you have observed changes in food stored in your freezer (i.e. freezer burn), you will know on some level that storage of frozen biological systems at that temperature is not appropriate. Most solutions are not completely frozen at this temperature and the high concentrations present are typically damaging to biological systems. In addition, biochemical activity of cells is diminished but still present at that temperature. That being said, there have been a limited number of studies of cells being stored at the temperature in the under cooled state. A description of this study can be found at Cryobiology. 22(6):537-46, 1985. It is an interesting article and will give you insight into issues associated with storage -20oC.

Liquid Storage

We are interested in short-term storage of hematopoietic cells. What infomraiton is out there about this topic?

Question:
We are interested in short-term storage of hematopoietic cells. What infomraiton is out there about this topic?

Answer:
Liquid storage is common for hematopoietic cells. Umbilical cord blood is one example. The unit is typically collected on a maternity ward and shipped in the liquid state to the central processing facility where it is red blood cell depleted and cryopreserved. It is also fairly common for certain types of bone marrow or peripheral blood stem cell trasnplants to be stored short term prior to transplantation. We have published work in this area and here are the references:

Burger S, Hubel A, and McCullough J. "Development of an infusible-grade solution for non-cryopreserved hematopoietic cell storage," Cytotherapy, 1: 123-133, 1999.

Schmid J, McCullough J, Burger S, and Hubel A. "Non-cryopreserved bone marrow storage in STM-Sav, an infusible-grade cell storage solution," Cell Preserv Tech, 1(1):45-51, 2002.

Hubel A, Carlquist D, Clay M, and McCullough J. "Cryopreservation of cord blood after liquid storage," Cytotherapy, 5(5):370-376, 2003.

Hubel A, Carlquist D, Clay ME, and McCullough J. "Liquid storage, shipment and cryopreservation of cord blood," Transfusion, 44:518-525, 2004.

Other authors have published on this area as well and I would encourage you to look up these additional references.

Liver Biospecimens

What are the best practices for collecting and preserving liver tissue for future DNA and RNA expression? ...

Question:
What are the best practices for collecting and preserving liver tissue for future DNA and RNA expression? We are finding that many of our previously stored samples do not have adequate amounts of RNA and need to make a shift in collection technique.

Answer:
It is not surprising that you are having difficulties. Liver or any other tissue that is highly metabolically active in vivo (e.g. heart) will be particularly challenging. Hepatocytes (the parenchymal cells in the liver) when stressed will rapdily progress toward apoptosis. These and a host of other factors will make collecting high quality liver biospecimens challenging. Unfortunately, this question is beyond the scope of what we can handle on "Ask the BioCoR Expert". You need to fill out a service request. Then, we can go over step by step your existing collection and processing protocols for weaknesses that may be causing your problems. We can also propose some other modifications that may improve quality further.

One common error with post thaw assesment

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

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 intact and viable; (2) cells that are intact but not viable; and (3) cells that are neiterh viable nor intact. Measurement of cell viability alone (typically defined as the number of viable cells post thaw divided by the total number of cells present) biases the measure of viability upward by failing to account for cells that have simply lysed and disappeared during the preservation process. Calculating both viability and yield helps to correct the bias and give you a more meaningful measure of post-thaw recovery of cells.

There are several other common erros that happen during post thaw assessment. We cover those common errors in our short course.

Pooling and Freezing Cells

Hi, I have been working on hepatocytes, and went through literature search. However, I am not clear on whether hepatocytes or any cell extracts (taken from different donors) can be pooled after freezing.

Question:
Hi, I have been working on hepatocytes, and went through literature search. However, I am not clear on whether hepatocytes or any cell extracts (taken from different donors) can be pooled after freezing.

Answer:
We get this question on a regular basis. If I understand what you are asking correctly, you are suggesting that cells can be thawed, pooled and then refrozen, the answer is, no. Well, perhaps a more accurate answer is: not without significant losses. What you are proposing to do is performed prepeated freeze/thaw cycles on the cells (in this case, hepatocytes). This technique is actually used in therapies such as cryosurgery for the purpose of destroying the cells. It is noteworthy that hepatocytes are, in particular, sensitive to freezing and post thaw recoveries for even a single freeze-thaw cycle are poor.

Post Thaw Recovery of CD34+ Cells > 100%

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. ...

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 the counts that you have observed reflect different factors. Secondly, what you have observed is not unusual and has been observed (and published) by a variety of other investigators. This issue has not been studied rigorously and should be. We believe that two factors may account for the measurements. Clearly additional studies must be performed to confim these hypotheses.

Improper gating: Flow cytometric analysis of fresh hematopoietic stem cell products has been extensively studied and there have been attempts to standardize sample preparation and analysis. Fresh cell and frozen cells are significantly different. Frozen cells have been subjected to high levels of dehydration and it is highly likely that forward and side scattering of the cells post thaw differs from that of non-frozen cells. Studies of changs in cell volume and morphology post thaw (compared to pre-freeze) have been published for other cell types (not HSCs) and it is highly likely that HSCs exhibit similar differences. Therefore, gating and analysis for frozen and thawed cells may need to be adjusted to get more accurate results.

Accounting erros: Please keep in mind that flow cytometric analyses express CD 34 as a function of intact cells. During freezing, there are three different populations: intact and viable cells, intact cells that are not viable and cells that are neither intact nor viable. That third population is frequently ignored. Cell lysis is one of the most common manifestations of cell damage during freezing. If you do not correct your counts to account for cell lysis, you will bias your post thaw assessment measures higher (and in some cases over 100%).

Quality of plasma samples

We have large number of serum and plasma specimens lying in freezers, collected over a long period and with no specific QC protocol. ...

Question:
We have large number of serum and plasma specimens lying in freezers, collected over a long period and with no specific QC protocol. What QC tests should I perform on these samples now, prior to issuing out, to ensure they meet at least some quality standards for downstream research?

Answer:
Currently, there is no single test or panel of tests capable of determining the quality of plasma samples for various downstream applications.

It is known that:

(1) Methods of collection will influence quality of the plasma sample (duration of time between collection from a patient and centrifugation to separate cells out from supernatant).

(2) Storage temperature/duration.

(3) Number of freeze-thaw cycles.

A couple of recent review articles will be helpful in understanding the influence of these factors on specific biomarkers:

Hubel A, Spindler R, Skubitz A, "Storage of human biospecimens: selection of the optimal storage temperature", Biopres & Biobanking, 12(3): 165-175, 2014.

Hubel A, Aksan A, Skubitz APN, Wendt C and Zhong X. "State of the art in preservation of fluid biospecimens", Biopreservation & Biobanking, 9: 237-244, 2011.

If you do not know the manner by which the samples have been processed, storage duration and temperature and number of freeze thaw cycles, it is unlikely that the samples can be used for a wide variety of downstream applications.

Reagents for short term preservation

Hello, I am trying to come up with a list of potential reagents that can be used to store mammalian cells at 4 degrees for up to 3 days that are commonly used. ...

Question:
Hello, I am trying to come up with a list of potential reagents that can be used to store mammalian cells at 4 degrees for up to 3 days that are commonly used. Could you please let me know some of the more common reagents used for this purpose? Any insite would be much appreciated since some of my literatures searches are giving me a lot of different formulations.

Answer:
Short-term storage of cells is very important but little research has been performed to develop proper solutions for storage of cells short term. This is an area that we at BioCoR are ver interested in and have worked on recently. Most short-term storage solutions are formulated for organs and the mechanism of damage for organs (ischemia-reperfusion) is different than cell suspensions (reactive oxidative species). I am sorry that I cannot give you a simple list of reagents. I can give you categories of molecules that are typically added: ions/osmolytes, antioxidants, buffering agents, with limitations on nutrients (typically simple sugars and amino acids). New, fit-for-purpose formulations for short-term storage solutions are sorely needed for cell therapy products.

Storing ascites

Hello, what is the best way to cryopreserve and store ascites fluid?

Question:
Hello, what is the best way to cryopreserve and store ascites fluid?

Answer:
Many biofluids (ascites included) are complex containing cells as well as fluid. In our laboratory, we process the cells and the supernatant separately so that we can perform functional assays on the cells later and the supernatant can be analyzed for the presence of biomarkers. The cells need to be frozen in specialized solutions to permit them to survive the stresses of freezing and thawing. The most common solution is a 10% solution of dimethylsulfoxide. In addition, cells need to be stored on liquid nitrogen or their viability decreases with time in storage.

Storing in a mechanical freezer at -150 C versus liquid nitrogen

Have you heard of anyone storing hematopoietic stem cell products in a -150 C mechanical freezer? I would be interested in knowing more about this. Is it safe? How does it affect the shelf life? I assume it would shorten it significantly.

Question:
Have you heard of anyone storing hematopoietic stem cell products in a -150 C mechanical freezer? I would be interested in knowing more about this. Is it safe? How does it affect the shelf life? I assume it would shorten it significantly.

Answer:
Storage in LN2 in the vapor phase is pretty close to -150 C so from a temperature perspecitve, storage at this temperature should be ok. There are two basic problems with mechanical freezers. (1) A -150 C mechanical freezer has a two stage compressor. The compressor is what fails on a mechanical freezer so three is a concern as to the duration that you can count on functioning of the freezer. A -80 C freezer may last a decade if you take very good care of it. If you use a -150 C mechanical freezer, your temperature has to be well monitored and you have to have a backup storage unit ready with short notice should the compressor fail. (2) Your shelf life of products may be shorter if you are using an upright mechanical freezer versus a chest. Cold air falls out of upright units when the door opens resulting in temperature fluctuations that can influence the shelf life of the project.

What is considered a successful viability range for a frozen cell line like EBV?

What is considered a successful viability range for a frozen cell line like EBV?

Question:
What is considered a successful viability range for a frozen cell line like EBV?

Answer:
Preservation of EBV transformed lymphocytes.

We typically achieve 85% viability (number of cells with intact membranes divided by total number of cells post thaw) and a yield of 78% (number of viable cells post thaw/number of viable cells pre-freeze) for lymphocytes transformed with SV40. These results are achieved with a 10% DMSO solution and a finely tuned controlled rate freezing protocol with an average cooling rate of 1 C/min. If you are getting considerably lower values, you may want to debug your protocol. We go through the debugging process in detail in our preservation short course.
We do not have direct experience with EBV transformed lymphocytes but we have a service project in this area scheduled for Spring 2010. Please check back in Summer 2010 for a published protocol.

Note #1: Other methods of immortalizing lymphocytes (transposons, etc) may influence freezing behavior.

Note: #2: Primary lymphocytes (lymphocytes that have not been immortalized) have a distinctly different freezing response. Furthermore, highly purified lymphocyte populations will respond differently to freezing than mixed lymphocyte populations.

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

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

Question:
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.

Sumida S, Matuski R, Sui W and Taira, S, 18 years Cryopreservation of Human Bone Marrow, Cryobiology, 28(6). p. 555, 1991.

What to freeze in: bags, vials, straws?

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

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 storage: straw, vial and bag. Straws are used typically to store small volumes (<1ml). Vials contain volumes ranging from ~1 to 5 ml. Bags can have the widest range of volumes but most commonly from 30150 ml (volume of liquid to be placed in the bag). Tissues may also be frozen in bags (albeit a very different construction from bags to contain cell suspensions or biofluids).

When storing a specimen from a single patient, you may actually need all three containers (straw, vial, bag) depending upon the assay for a specific biomarker. It is important to think about the volumes that you may need before you freeze and select the appropriate container. Each container has unique problems and limitations. We discuss in the short course advantages and disadvantages of the containers commonly used to store biospecimens.

It may also be helpful for you to know that some manufacturers hve flexible manufacturing capabilities and can custom manufacture a cryogenic biospecimen container to meet your individual needs (size, type of port, geometry, etc).

Would warming of serum to -15 to -10 C alter our anaysis of protein and small molecule metabolites?

One of the compressors on our -80 degree C freezer went out last night and the temperature climbed to -15 to -10 degrees C over a 12-14 hour period of time. ...

Question:
One of the compressors on our -80 degree C freezer went out last night and the temperature climbed to -15 to -10 degrees C over a 12-14 hour period of time. We had importatn serum samples stored in this freezer and we have now transerred them to another -80 C freezer. However, we are wondering if warming of serum to -15 to -10 C would alter our anaysis of protein and small molecule metabolites. Do you have any information that might shed some light on this question?

Answer:
We are sorry to hear about your equipment failure. There are two issues: (1) potential damage to your sample and (2) preventing future problems of this nature.

Warming the sample above the eutectic temperature (in this case ~-26 C), you will subject the proteins in your serum to high salt concentrations and interactions with the ice phase. Both of which can be damaging. It is highly likely that this warming has caused irreversible denaturation and aggregations of serum proteins. Small metabolites 'may' be ok.

Secondly, there are many, many investigator who store samples in their laboratory where the samples cannot be continuously monitored. In order to prevent future problems, we would strongly suggest that you install a monitoring system that permits someone to be notified in the case of an equipment failure. If you have access to a biorespository on your campus, you may also consider using this facility. Most repositories have monitoring systems and routine maintenance protocols to reduce/prevent failure of critical equipment.