Differences in HE Versions - Liquid Nitrogen Freezers

There has been some confusion about the different HE versions, these differences are outlined below.

• HE – Operates at -150°C, stainless steel turntable with pie shaped divisions
  
• HE+ – Same as HE except has more usable height and aluminum turntable
• HE Gen 2 – Same as HE except the turntable has rectangular divisions
• HE+ Gen 2– Same as HE Gen2 except has more usable height
• ETERNE – Operates at -190°C, aluminum turntable with pie shaped divisions

Increasing Sample Storage Temperature Above -132°C (Glass Transition Temperature of Water [GTTW]) - Part Two

Given these striking temperature effects observed with mock samples, we cycled viably cryopreserved PBMCs through the GTTW to observe the effects oh apoptosis. Using a programmable rate-controlled liquid nitrogen freezer, we exposed PBMC samples to one entire cycle of temperature change (-150°C to -114°C) for 10 min and then back to -150°C. Next, we exposed aliquots to multiple temperature cycles (10, 20 and 40). All samples were then assessed for the incidence of apoptosis using Hoechst staining. There was no considerable difference in the number of positively stained cells (% apoptotic nuclei) from PBMCs stored at -150°C compared to those that underwent 1, 10, 20 and 40 multiple temperature cycles.

Accordingly, we determined to examine this issue by incubating frozen PBMC samples at -114°C for a longer period (e.g., 48 hours) and then returning them to -150°C. Apoptosis values were considerably higher from PBMC samples held at -114°C than in PBMCs stored for the extent of the experiment at -150°C.

Our information illustrates that, when exposed to a warmer temperature for 48 hours, the quality of cryopreserved samples was negatively affected. We conclude that apoptosis occurred due to temperature fluctuations around the GTTW (-132°C). Similar findings were discovered when PBMCs where stored at -80°C for 48 hours. 

Increasing Sample Storage Temperature Above -132°C (Glass Transition Temperature of Water [GTTW]) - Part One

The vapor phase of liquid nitrogen LN2 (-150°C)is typically used to store PBMC’s. It is quite possible that Cryopreserved samples could possibly be frequently exposed to GTTW during retrieval of specimens, particularly from large active cryogenic repository collections. We have determined the rate and the degree that frozen samples will warm when removed from a liquid nitrogen freezer. A freezer rack enclosing mock samples was removed and laid on the top of an open liquid nitrogen freezer. With our computerized freezer monitoring system, we demonstrated that the mock samples' temperature had increased by between 14°C to 55°C, after just 5 minutes in this position, which equaled an average temperature of -114°C. This rise was well above the GTTW (-132°C).

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The Dangers of Overfilling your Cryogenic Dewar - Part Two

Overfilling problems occur When LN2 liquid nitrogen contacts with the pumpout plug or body for an extensive period of time. Extreme cold temperatures cause the o-rings to contract and harden, which can cause the o-rings to temporarily lose their sealing properties. Since all manufacturers utilize the same o-ring design, this problematic scenario can occur on any liquid nitrogen dewar. The plastic cap will not stop this from happening but will lengthen the time it takes for failure to initiate. It is vital that the pumpout remains covered. Liquid nitrogen may crack the plastic cap if it is spilled on it, (a sign of improper filling), and therefore a replacement cap should be applied to the pumpout without delay. These caps are inexpensive and MVE will supply replacement pumpout caps when they are needed.

 Years ago, MVE made use of a metal cap that held on to the pumpout body. Even though this kept dirt particles from distressing the o-rings, it did not alleviate pressure sufficiently and became a liability matter. For a short while, we used a hard plastic cover that relieved pressure but did not guard the plug from dirt. The design we presently use appears to be the safest and easiest method of shielding the o-rings without adding to the cost of the liquid nitrogen dewar. Although there are different styles, depending on the manufacturer, this appears to be the common design practiced throughout the industry. This filling safeguard is particularly significant on liquid nitrogen vapor shippers where the margin for failure recovery is even less. Liquid nitrogen vapor shippers are usually warm when LN2 liquid nitrogen charging is required. Because of the smaller inner volume and the additional liner to be cooled down, the initial flash off is far greater then if the liquid nitrogen dewar was cold. It is due to this fact that the liquid nitrogen vapor shippers usually require additional filling during the charging cycle. Proper filling directions are listed in the manuals provided with each MVE liquid nitrogen dewar, both liquid and vapor.

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The Dangers of Overfilling your Cryogenic Dewar - Part One

Customers occasionally express concern due to a sudden vacuum loss to a liquid nitrogen dewar. Such liquid nitrogen dewars were returned and immediately checked, then revacuumed and LN2 liquid nitrogen tested. Incorrect filling procedures, such as overfilling, were the main causes of the vacuum losses. This article explains why it is very important to follow careful filling protocol. I am addressing the issue in this month's Tech Tip to help explain why overfilling liquid nitrogen dewars during the charging process is damaging to the vacuum integrity of the aluminum liquid nitrogen dewar.

The pumpout plug has two purposes. The main and most obvious purpose is to preserve the vacuum in the liquid nitrogen dewar’s annular space. Provided that a vacuum is present, the pumpout plug will remain inserted into the pumpout body. The second purpose is to perform as a pressure relief plug in the occurrence of a ruinous inner leak. A leak developing to the inner section of the liquid nitrogen dewar would allow liquid nitrogen to enter the annular space, causing the plug to fall out and relieve any pressure build up. When the liquid nitrogen evaporates into a gas and begins to expand, a pressure increase in the annular space is created. Two o-rings seal the plug to the pumpout body, and are coated with a vacuum grease film to guarantee longevity. The pumpouts are then covered with a flexible composite cap which keeps dirt and contaminates from disturbing the o-rings.

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