A new controlled-rate cooling apparatus for freezing hematopoietic cells for storage at -196 degrees C

An apparatus has been constructed to cool biological material at a controlled rate. The material to be frozen is placed in glass ampuls which are immersed in an aluminum bath containing ethyl alcohol and the bath is placed inside a freezer cabinet. Liquid nitrogen is pumped intermittently into the cabinet by means of a single-speed electric pump. The rate of cooling is controlled by a device that varies the interval between successive pumping cycles. The temperature fall is monitored by thermocouples placed inside selected glass ampuls and recorded as a plot on moving graph paper.This simple instrument is capable of cooling at an accurately controlled rate over the range of 0 to 7 °C/min. We chose for our studies a cooling rate of 1 °C/min which we could maintain with an accuracy of ±0.1 °C. Temperature fluctuations were, however, observed at the freezing plateau and varied considerably in magnitude and temperature at onset even for the same material cooled under the same conditions. Mouse bone marrow cells frozen by our technique and stored for various periods of time may, on reconstitution, form colonies in vivo and in vitro identical in morphology and number to those from unfrozen control cells. Our results suggest that expensive and intricate devices may not be necessary to obtain optimal recovery of viable cells after storage in liquid nitrogen. The apparatus is now in regular use for the storage of human bone marrow cells intended for use in treatment of patients with leukemia refractory to conventional measures.     

Controlling cooling rates with a variable vacuum in a Dewar flask

A simple, inexpensive system to manipulate and accurately reproduce cooling rates was developed by modifying the system designed by Leibo and Mazur (5) and altered by Zeilmaker and Verhamme (12). Changing the volume of ethanol, the cooling bath medium, served to coarsely adjust the rate. Fine adjustments were made by changing the vacuum level drawn from the flask. Reducing vacuum level during phase transition increased heat flux and may have reduced cell destruction from latent heat of fusion.     

Influence of cooling rate on Saccharomyces cerevisiae destruction during freezing: unexpected viability at ultra-rapid cooling rates

The purpose of this work was to study cell viability as a function of cooling rate during freezing. Cooling rate strongly influences the viability of cells during cold thermal stress. One of the particularities of this study was to investigate a large range of cooling rates and particularly very rapid cooling rates (i.e., faster than 20000 degrees C min (-1)). Four distinct ranges of cooling rates were identified. The first range (A(')) corresponds to very slow cooling rates (less than 5 degrees C min (-1)), and results in high cell mortality. The second range (A) corresponds to low cooling rates (5-100 degrees C min (-1)), at which cell water outflow occurs slowly and does not damage the cells. The third range (B) corresponds to rapid cooling rates (100-2000 degrees C min (-1)), at which there is competition between heat flow and water flow. In this case, massive water outflow, which is related to the increase in extracellular osmotic pressure and the membrane-lipid phase transition, can cause cell death. The fourth range (C) corresponds to very high cooling rates (more than 5000 degrees C min (-1)), at which the heat flow is very rapid and partially prevents water exit, which seems to preserve cell viability.     

Cell size and water permeability as determining factors for cell viability after freezing at different cooling rates

This work studied the viabilities of five types of cells (two yeast cells, Saccharomyces cerevisiae CBS 1171 and Candida utilis; two bacterial strains, Escherichia coli and Lactobacillus plantarum; and one human leukemia K562 cell) as a function of cooling rate during freezing. The range of investigated cooling rates extended from 5 to 30,000 degrees C/min. Cell viability was classified into three ranges: (i) high viability for low cooling rates (5 to 180 degrees C/min), which allow cell water outflow to occur completely and do not allow any intracellular crystallization; (ii) low viability for rapid cooling rates (180 to 5,000 degrees C/min), which allow the heat flow to prevail over water outflow (in this case, cell water crystallization would occur as water was flowing out of the cell); (iii) high viability for very high cooling rates (>5,000 degrees C/min), which allow the heat flow to be very rapid and induce intracellular crystallization and/or vitrification before any water outflow from the cell. Finally, an assumption relating cell death to the cell water crystallization as water is flowing out of the cell is made. In addition, this general cell behavior is different for each type of cell and seems to be moderated by the cell size, the water permeability properties, and the presence of a cell wall.     

Prediction of local cooling rates and cell survival during the freezing of a cylindrical specimen

A finite element numerical model was implemented to simulate the freezing process of an aqueous salt solution in a cylindrical container. Local cooling rates within the container were computed for several defined cooling protocols applied at the boundary. Characteristic cell survival signatures were used to predict the associated local survival rates throughout the system. These calculations show that there are two definite time domains during a typical freezing process: (1) while the surface temperature is changing and (2) after the surface temperature reaches a constant storage value. The calculations also show significant spatial variations in the local cooling rates within the container and considerable local deviation from the volumetric average survival for various simulated freezing protocols.     

The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10 to 12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4 °C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P < 0.05). Sperm motility increased as cooling rate was increased for both strains (P < 0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70 °C/min and 100 °C/min cooling rate improved post-thaw motility of rat sperm.     

An analytical method to evaluate cooling rates during cryopreservation protocols for organs

A simple, graphical procedure has been established to estimate the cooling rates at a specified location and temperature in a biological medium frozen by imposing a constant cooling rate at the outer surface. The graphical procedure also facilitates the determination of the outer surface temperature at the instant a given temperature has been attained in a specific location. The results obtained through a numerical study using finite elements can be applied to biological media with various chemical compositions including different concentrations of cryoprotective agents.     

A simple sandwich-cryogen-jet procedure with high cooling rates for cryofixation of biological materials in the native state

Summary We evaluate a method for improved, ice-crystal-free cryofixation of various biological materials (macromolecules, subcellular fractions, whole cells in suspensions or as monolayers) without any chemical pretreatments. The principle of the method (as introduced byPscheid, Schudt, andPlattner 1981 for cell monolayer cultures) is as follows. Firstly, a thin sample layer is sandwiched between a thin copper object holder and a thermally insulating plastic sheet (Thermanox, which is also used as a cell culture substratum); secondly, a jet of melting propane is shot from a simple, inexpensive brass vessel onto the copper object holder. We now ascertained that this simple and inexpensive method allows cooling rates up to > 18,000 °C/sec and, thus, gives better results than comparable but more sophisticated and expensive procedures. The method also avoids all the disadvantages of some other rapid freezing methods. Specifically, the sample mass required is minimal, the sample remains fully hydrated and no changes of ionic or other conditions can occur. There is no hazard of passing a cold gas phase (cooling curves being very steep right at the beginning) or of impairment by mechanical impact (as it could be with cold metal block freezing). The expedient of using a thermal insulator as part of a sandwich sample allows cooling rates which are superior or at least equivalent to those obtained with a commercial double propane-jet device (and double copper sandwiches). The reason for this is that in practice a two-side propane jet onto a copper-copper sandwich can not reach both sides precisely at the very same time (as implied from our cooling rate measurements). The tentative transformation of our mono-jet into a double-jet variation (with a branched nozzle piece) did also not improve the results, when compared with a single propane-jet onto a copper-Thermanox sandwich.This work is taken in part from a Ph.D. thesis by G.Knoll.