- Email: [email protected]
Freezing under pressure: A new method for cryopreservation
YCRYO 3562
No. of Pages 5, Model 5G
24 December 2014 Cryobiology xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo 4 5 3
Q1
Freezing under pressure: A new method for cryopreservation
6
Q2
Nickolas Greer ⇑
7
Q3
Rissali LLC, USA
8 1 1 0 2 11 12 13 14 15 16 17 18 19 20
a r t i c l e Q4
Q5
i n f o
Article history: Received 3 September 2014 Accepted 15 December 2014 Available online xxxx Keywords: Freezing under pressure Cryopreservation under hydraulic pressure Red blood cells Cryoprotectant toxicity
a b s t r a c t The ability to preserve cells, tissues, and organs with minimal damage for an extended period of time is essential for advancements in medicine and research. Current methods for cryopreservation are based on using high concentrations (up to 60% v/v) of cryoprotective agents (CPAs) which are toxic to living cells. The effect of pressure and a low concentration of dimethyl sulfoxide (Me2SO) or glycerol, on hemolysis of human red blood cells (RBCs) after freezing and thawing were investigated. Pressure was applied during cooling and freezing the RBCs and minimum in hemolysis was reached at approximately 120 MPa. Either 5% v/v Me2SO or 8% v/v glycerol concentration in combination with 120 MPa pressure was sufficient to obtain 8% or less hemolysis of RBCs after cooling at a 35 °C/min or a 160 °C/min rate. The preliminary results suggest that the method may help to solve the CPAs toxicity problem. Ó 2014 Published by Elsevier Inc.
22 23 24 25 26 27 28 29 30 31 32 33
34 35
Introduction
36
Water is an essential component of life. The human body is 65% water. Upon freezing at ambient pressure, water transforms into relatively large, sharp crystals of ice, causing mechanical damage to cells and tissues. The ice formation also causes overconcentration of salts, dehydration, and osmotic balance disruption, often leading to cell death.
37 38 39 40 41
42
Cryoprotectant toxicity
43
Cryoprotective agents (CPAs) were developed to substitute a portion of water inside and around cells, reducing the size of ice crystals and limiting over-concentration of salts during freezing. Unfortunately, CPAs are toxic to live cells, tissues, and organs (bio-samples) and CPA toxicity increases with concentration and contact time in liquid state [9,1,8,10]. CPAs bind to proteins and other molecules, disrupt multiple bio-chemical pathways inside the cells, and cause osmotic imbalance [9]. See Tables 5 and 6 at [9] for more details. In addition, many tissues and organs have limited permeability even to the most penetrating CPAs, such as Me2SO, which makes loading high CPA concentrations difficult and time consuming. Therefore, alternative methods for cryopreservation with lower CPA concentration or CPA-free need to be
44 45 46 47 48 49 50 51 52 53 54 55
⇑ Address: 271 White Tree Ln, Ballwin, MO 63011, USA. E-mail address: [email protected]
developed to address the growing demand for cryopreservation of quality bio-samples.
56
Freezing under pressure for electron microscopy sample preparation
58
Freezing under pressure is not a new concept. Since the 1970s a method of freezing under pressure around 210 MPa (HPFe method) is widely used for sample preparation for electron microscopy [3,5]. The HPFe method works well for preserving ultrastructure of cells with the following limitations: developers of the HPFe method claim that the method works well for freezing up to 0.5 mm thick samples with relatively fast cooling rates (>60,000 °C/min) [3]. With slower cooling rates ice crystals become visible with an electron microscope and the vitrified portion of water becomes progressively smaller. Several polymorphs (forms) of ice were detected inside of the samples prepared using the HPFe method [14,5,18]. They include vitreous (non-crystalline, amorphous) ice, as well as crystalline ice forms: Ih (hexagonal ice), Ic, and III. The fast cooling rate (>60,000 °C/min) and the presence of ice inhibitors (bio-molecules and CPAs) inside the bio-samples account for the vitreous component, whereas the crystalline ice is more likely to form where cooling rate was slower (deeper into the bio-sample) or where spontaneous ice nucleation took place. The HPFe method helps to preserve samples without visible ice crystals when observing with an electron microscope [5,3]. Since the resolution limit of modern electron microscopes used for biological research is <10 nm, then the ice crystals detected by X-ray diffraction [5,14,18] have to be smaller than 10 nm.
59
http://dx.doi.org/10.1016/j.cryobiol.2014.12.005 0011-2240/Ó 2014 Published by Elsevier Inc.
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
57
60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82
YCRYO 3562
No. of Pages 5, Model 5G
24 December 2014 2
N. Greer / Cryobiology xxx (2014) xxx–xxx
Freezing under pressure
139 140
106
Some CPAs possess an unexpected property. They can protect bio-samples from negative impact of high pressure [7]. See Table 4 at [7] for details. The Fahy group found that high CPA concentrations can protect rabbit renal cortex from prolonged (30–60 min) exposure to high pressure without freezing [6,7]. See Table 5 at [7] for details. Since the negative impact of high pressure accumulates with time spent under pressure [12,13], low concentrations of CPAs (for example, 5% Me2SO) may also be sufficient for protecting bio-samples from short-term (2–3 min) exposure to high pressure.
For generating pressure a manual high pressure generator (pump) was used. The pump was made by High Pressure Equipment Co, Erie, PA, USA (HiP) using a manual hydraulic pump HiP 37-5.75-60 as a main component. Pressure was measured using a 140 MPa gauge (Pressure Products Industries, Warminster, PA, USA). For pressures above 140 MPa a 350 MPa gauge (Astra Products, Ivyland, PA, USA) was used. A high pressure tube HiP 60-HM4-10 (tube) was used for containing and freezing cell suspensions under pressure (Fig. 1). The tube (stainless steel 316, 1.6 mm I.D., 6.35 mm O.D., 20 cm long) was attached to a 1 mL syringe using a plastic tubing adapter. About 300 lL of cell suspension was drawn into the tube (Fig. 1). A rubber stopper (silicone rubber, 1.78 mm diameter, 6.5 mm long) was covered with white petrolatum USP and inserted 35–45 mm deep into the open end of the tube. The opposite end of the tube was closed with a cap (Butech #60CA4, Haskel, Burbank, CA, USA). Accuracy of pressure control was within 2–3% or +/-3 MPa at 120 MPa level. For freezing at ambient pressure (0.1 MPa) no pressurization of the cell suspension was performed. For all other pressures, the tube was connected to the pump and pressurized to the testing pressure. The tube was oriented vertically and submerged into a container filled with 2L of 75 °C isopropyl alcohol coolant (Fig. 1). After 3 min in the coolant, pressure at the pump was dropped. The tube was disconnected from the pump, and was plunged into a +32 °C water bath. The cell suspension was warmed at ambient pressure.
107
Materials and methods
Cooling rates
165
108
To test the above hypothesis on human red blood cells (RBCs) a method and an apparatus were developed and are presented below.
To obtain 160 °C/min cooling rate of the cell suspension, the tube was submerged into 75 °C alcohol with 2 mm/s velocity.
166
83 84
Effect of hydraulic pressure on viability of bio-samples without freezing
96
High hydraulic pressure can be harmful to bio-samples and pressure-induced damage increases with time spent under pressure [12,13,20,15]. Armand Karow’s team did research on pressure tolerance of mammalian organs and found that rat hearts can tolerate high hydraulic pressure up to 100 MPa (15,000 psi) for up to 10 min without apparent changes [13]. They also found that dog kidneys can tolerate 100 MPa for up to 2 min and the animals survived for at least 4 weeks after re-implantation of the kidneys [12]. Another research group demonstrated that dog hearts can survive 100 MPa for up to 30 min [19]. This data implies that pressure up to 100 MPa applied for a short period of time (up to 2–30 min) is relatively harmless to some organs.
97
Baroprotective properties of CPAs
85 86 87 88 89 90 91 92 93 94 95
98 99 100 101 102 103 104 105
109 110
Pump 111
Biological model
112
123
RBCs were chosen as a model for developing and preliminary testing the method. Unlike complex tissues and organs, it is relatively easy to analyze and quantify damages to RBCs, which is usually done by measuring free hemoglobin and looking at cell morphology. RBCs have no nucleus, DNA, or RNA, and therefore lack membrane repair capabilities, which make their membranes more sensitive to damages when compared to nucleated cells types with advanced membrane repair capabilities. Since damage to cell membranes is a direct result of ice crystal formation, repairing the membranes by the cells can be undesirable for testing cryopreservation methods. If the method works on RBCs, then there is a good chance it may also work on more complex bio-samples.
124
RBCs suspension preparation
125
Two human donors provided blood for this study. Whole venous blood was drawn in standard fashion after a 12 h fast and stored at +4 °C in BD Vacutainer™ REF 367841 containers lined with 3.6 mg of K2 EDTA to prevent coagulation (2 mL of whole blood per container). Hematocrit (Hct) was within 40–45%. The blood was used within 4 days of the draw. Three types of bio-samples were tested: (1) whole blood; (2) whole blood with 5% v/v Me2SO; (3) whole blood with 8% v/v glycerol. The cell suspensions were obtained by centrifuging whole blood at 3200 RPM for 3 min allowing separation of the plasma (supernatant) and RBCs (precipitate). Me2SO or glycerol was added to plasma. RBCs were resuspended in plasma to make 5% v/v Me2SO or 8% glycerol v/v concentration in whole blood. The Me2SO and glycerol were purchased from Sigma (St. Louis, MO).
113 114 115 116 117 118 119 120 121 122
126 127 128 129 130 131 132 133 134 135 136 137 138
water under pressure rubber stopper – moves up when pressure inside of the bio-sample rises due to water expansion upon freezing
tube
liquid bio-sample under pressure
liquid-solid boundary solid (frozen) bio-sample
cap
container is elevated with 1 or 2 mm/sec velocity -75°C alcohol
Fig. 1. Apparatus for freezing under pressure. The figure shows a cross-section of the tube loaded with whole blood, connected to the pump, and partially submerged into coolant. The apparatus is designed for freezing cell suspensions at a nearconstant pressure. When freezing of the cell suspension begins, pressure inside of the cell suspension rises due to ice expansion upon freezing. The rubber stopper will move upward, thereby keeping pressure inside of the cell suspension in a relatively narrow range (for example, 120 ± 3 MPa).
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164
167
YCRYO 3562
No. of Pages 5, Model 5G
24 December 2014 3
N. Greer / Cryobiology xxx (2014) xxx–xxx
169 170 171 172 173 174 175 176 177
To obtain 35 °C/min cooling rate of the cell suspension, the tube was wrapped with a 31 cm long 15 cm wide, 0.015 mm thick polyethylene film (15 layers), and was submerged into 75 °C alcohol with 1 mm/s velocity. Warming rate for all cell suspensions was 950 °C/min. The above cooling and warming rates are average rates of temperature change on the interval from 0 to 50 °C. As there is no easy way for measuring cooling rates inside of the tube under pressure, the rates of temperature change were measured at ambient pressure in the middle and inside of the tube filled with saline using an Omega TT-T-36-36 thermocouple.
100.0 90.0 80.0 Hemomisys, %
168
70.0 60.0 50.0 40.0 30.0
178
RBCs suspension analysis
179
186
In a healthy individual RBCs occupy 40–45% of total blood volume. When RBCs are damaged they release hemoglobin. By measuring the free hemoglobin, one can determine a degree of damage to RBCs (hemolysis). The free hemoglobin concentration is often used to estimate hemolysis of RBCs during cryopreservation [16]. Hemolysis was calculated as a percentage of free hemoglobin in RBCs suspensions after cryopreservation using the following equation:
187 189
% hemolysis ¼ ½ð100 HctÞ supernatant Hgb = total Hgb
180 181 182 183 184 185
190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205
The cell suspension was extruded from the tube by pushing on the rubber stopper with a stainless steel wire (1.37 mm diameter, 21 cm long). About 90lL of cell suspension, obtained from the middle portion of the tube, was centrifuged at 3200 RPM for 3 min. Hemoglobin concentration in the supernatant was measured using HemoCue Hb 201+ analyzer (HemoCue AB, Sweden). The analyzer was calibrated and its accuracy was confirmed with standard trilevel hemoglobin controls (Stanbio, Boerne, TX) and the following linearity test. A whole blood sample with 13.2 g/dL (within normal limits for human blood) concentration of hemoglobin was diluted 1:1, 1:4, 1:9, and 1:19 in an isotonic saline to obtain the following concentrations of hemoglobin: 50%, 25%, 10%, and 5%. Hemoglobin content in the resulting solutions was measured with the analyzer and plotted on a graph. The results of the test were linear with an insignificant deviation from a straight line.
206
Results
207
The effect of pressure on hemolysis of RBCs
208
Fig. 2 shows the effect of hydraulic pressure on hemolysis of RBCs after cooling/freezing whole blood samples at five pressure levels: 0.1, 70, 120, 160, 200 MPa. Minimum in hemolysis (28.2 ± 2.6%) was reached at 120 MPa. Minor to no morphological difference were noticed between the control RBCs (Fig. 3A) and RBCs, frozen under 120 MPa and thawed at ambient pressure (Fig. 3B).
209 210 211 212 213
214
The effect of pressure, cooling rate, and low CPA concentration
215
Another set of experiments was conducted to compare effect of pressure, cooling rate, and low CPA concentration on hemolysis of RBCs. RBCs were cooled/frozen at two cooling rates: 35 °C/min and 160 °C/min while being compressed at two pressure levels: 0.1 MPa (ambient pressure) and 120 MPa (Figs. 4 and 5). Three kinds of cell suspensions were tested: (1) whole blood; (2) whole blood with 5% v/v Me2SO; (3) whole blood with 8% v/v glycerol. Fig. 4 shows the effect of 120 MPa on hemolysis of RBCs. Blood samples were cooled at the 160 °C/min rate. RBCs, frozen at 0.1 MPa (ambient pressure) were mostly hemolyzed after the cryopreservation cycle. This was an expected result for cryopreser-
216 217 218 219 220 221 222 223 224 225
20.0 10.0 0.0 0.0
50.0
100.0
150.0
200.0
Pressure, MPa Fig. 2. The effect of pressure on hemolysis of RBCs after cooling/freezing whole blood at 160 °C/min rate at five different pressure levels (0.1; 70; 120; 160; 200 MPa) and thawing at ambient pressure. Mean ± SD (n = 4).
vation of RBCs at ambient pressure. However, 5% Me2SO in combination with 120 MPa decreased hemolysis of RBCs to 5 ± 1.2%. Fig. 5 shows the effect of 120 MPa on hemolysis of RBCs in whole blood with 5% Me2SO or 8% glycerol. Blood samples were cooled at the 35 °C/min rate. RBCs, frozen at 0.1 MPa, were mostly hemolyzed after the cryopreservation cycle. A low CPA concentration in combination with 120 MPa decreased hemolysis of RBCs down to 7.6 ± 1.4% for Me2SO, and 4.2 ± 1.2% for glycerol.
226
Discussion
234
The effect of hydraulic pressure, applied during cooling and freezing, on hemolysis of RBCs after thawing was investigated. Minimum in hemolysis (28.2 ± 2.6%) was reached at 120 MPa. The increase in hemolysis after freezing at 160 MPa and 200 MPa may be explained by the negative impact of elevated pressure.
235
The effect of pressure on ice crystal nucleation
240
Water content of whole blood is 83% v/v. According to the Bridgman’s diagram for phase transitions of water under pressure [2], at 120 MPa ice I nucleation begins at 12 °C or so. Ice nucleation temperature inside the tube (Fig. 1) was measured with the thermocouple at ambient pressure and was found to be around 0 °C for whole blood. Super-cooling of water is improbable in slowly cooled whole blood samples inside the tube due to multiple nucleation sites (RBCs). Thus, ice nucleation temperature in whole blood at 120 MPa is likely to be close to pure water (12 °C). To understand the process it helps to visualize simultaneous nucleation of multiple ice crystals on RBCs membranes and the inner walls of the tube at the liquid–solid boundary (Fig. 1) during freezing.
241
The effect of pressure on ice crystal growth
254
Ice crystal growth rate depends on the following three factors: pressure level, cooling rate, and composition of bio-samples. The effect of hydraulic pressure on ice crystal growth inside biosamples is not entirely understood. The effect of pressure may be explained by looking into the changes of density and viscosity of liquid water under pressure. For example, under 120 MPa pressure at 0 °C water density is increased by about 5.4% relative to ambient pressure. The viscosity of water at the liquid–solid transition point
255
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
227 228 229 230 231 232 233
236 237 238 239
242 243 244 245 246 247 248 249 250 251 252 253
256 257 258 259 260 261 262
YCRYO 3562
No. of Pages 5, Model 5G
24 December 2014 4
N. Greer / Cryobiology xxx (2014) xxx–xxx
(A)
(B)
Hemolysis, %
Fig. 3. The effect of 120 MPa pressure on morphology of RBCs. Blood cells before freezing (A) vs. after cooling/freezing whole blood under 120 MPa pressure at 160 °C/min rate and thawing at ambient pressure. (B) Whole blood was diluted 1:200 in lactated Ringer’s and observed on a hemocytometer at 400. No CPAs were used.
0.1MPa 120MPa
Whole blood (WB)
WB + 5% DMSO
Hemolysis, %
Fig. 4. The effect of 120 MPa pressure on hemolysis of RBCs after cooling/freezing whole blood (WB) and WB + 5% Me2SO at 160 °C/min rate and thawing at ambient pressure. Mean ± SD (n = 4).
120MPa
WB + 8% glycerol
Fig. 5. The effect of 120 MPa pressure on hemolysis of RBCs after cooling/freezing WB + 5% Me2SO and WB + 8% glycerol at 35 °C/min rate and thawing at ambient pressure. Mean ± SD (n = 4).
264 265 266 267 268 269 270 271 272
273
Cooling rates
296
Intracellular ice is one of the major damaging factors to RBCs membranes at ambient pressure. The 160 °C/min cooling rate is fast enough so RBCs have no time to lose liquid water during cooling and liquid water becomes intracellular ice. Thus, intracellular ice crystals formed under pressure inside RBCs when cooled at 160 °C/min rate have to be significantly smaller and/or have different shape when compared to intracellular ice crystals formed at ambient pressure. Cooling whole blood with 5% Me2SO at 160 °C/min rate resulted in a lower hemolysis (5 ± 1.2%) when compared to cooling at 35 °C/ min rate (7.6 ± 1.4%) (Figs. 4 and 5). Higher cooling rates may result in smaller crystals of ice and may account for the lower hemolysis of RBCs. Furthermore, there are two other reasons why the cooling rate of bio-samples should be maximized when freezing with CPAs under elevated pressure. First, CPAs are toxic to living cells and the toxicity increases with time spent in contact with liquid CPA. Second, pressure above 100 MPa can be lethal to mammalian organs if applied for longer than 2 min and the negative impact of pressure rises with increase of pressure and time spent under pressure [12,13]. Utilizing fast cooling techniques, it is possible to freeze
297
274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
0.1MPa
WB + 5% DMSO
263
the other hand, under high hydraulic pressure water molecules are compressed into (interlocked with) each other and their movement becomes restricted, reducing the probability of a water molecule becoming a part of the ice crystal and the growth rate of the crystal is inhibited. The high hemolysis after freezing at ambient pressure (Fig. 2) is primarily due to mechanical and osmotic stresses created by ice crystals in close proximity to RBCs membranes. Ice crystals formed at 120 MPa may be smaller, may trap solutes more efficiently, and may have smoother surface when compare to ice crystals formed at ambient pressure, thereby reducing osmotic and mechanical stresses to cell membranes. In addition, CPAs combined with pressure may further reduce the size of ice crystals and even convert a portion of water into a vitreous (non-crystalline) form during rapid freezing. Electron microscopy studies of frozen bio-samples are needed to determine the exact fate of the water molecules and ice crystals. Indirect evidences of ice crystal growth inhibition by pressure can be found in HPFe studies [3,5,14,18]. The size of individual ice crystals formed under 200 MPa with >60,000 °C/min cooling rate is often <10 nm (the resolution limit of electron microscopes) [5,3] and the size of ice crystals appear to increase with a reduction of cooling rate of the samples. More definitive studies on ice crystal growth rate under pressure are needed.
at 120 MPa and 12 °C is significantly higher (2.5 mPas), when compared to viscosity of water at the liquid–solid transition point at ambient pressure and 0 °C (1.7 mPas) [4,11]. For details see Fig. 2 at [4] and Figs. 6 and 7 at [11]. Adding cryoprotectants such as sucrose to water in combination with pressure further increases the viscosity of the solution [11]. For details see Fig. 4b at [11]. Ice crystals are ordered structures of water molecules. At ambient pressure movement of water molecules in liquids is relatively free. Any water molecule in close proximity to a growing crystal of ice has a high probability of becoming a part of the crystal. On
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316
YCRYO 3562
No. of Pages 5, Model 5G
24 December 2014 N. Greer / Cryobiology xxx (2014) xxx–xxx
335
large (20+ mm thick) bio-samples within 1–2 min, thereby limiting or eliminating impact of high hydraulic pressure and CPA toxicity prior freezing. When the bio-sample is in the frozen (solid) state both hydraulic pressure and CPAs have little to no effect on biosamples. Thus, the cooling rate of bio-samples should be maximized to reduce time spent under pressure, exposure to liquid CPAs, and size of ice crystals. The 35 °C/min and 160 °C/min cooling rates were investigated because they approximate the fastest cooling rates which can be achieved on the surface (160 °C/min) and in the center (35 °C/ min) of many large bio-samples, such as units of blood (400 mL) and organs (kidney) due to limited thermal conductivity of the bio-samples. The above cooling rates can be obtained by plunging the bio-samples into liquid nitrogen or by contact with cold metal plates. Low hemolysis after cooling with the two very different cooling rates makes it possible to keep hemolysis under control in the entire volume of a unit of blood, and may open up the possibility for successful cryopreservation of organs, such as heart and kidney.
336
The effect of low CPA concentration
337
344
The above results indicate that the cryoprotective effect of pressure can be amplified by 5% Me2SO or 8% glycerol (Figs. 4 and 5). The low concentrations of CPAs combined with 120 MPa pressure were sufficient for protecting cell membranes from mechanical and osmotic damages during ice formation. It appears that the low CPA concentration further inhibits ice crystal growth, resulting in even smaller ice crystals and a larger vitrified component of water when compared to freezing using pressure alone.
345
Prospective applications
346
367
Even though the FDA-approved method for cryopreservation of RBCs [16,21] often results in <15% post-thaw hemolysis prior processing, it requires 40% glycerol combined with lengthy and expensive processing using specialized equipment [21]. On the other hand, 8% glycerol is far less toxic than 40% [9] and adding and removing 8% glycerol can be done relatively fast in 1–2 simple steps. Thus, reduction in CPA concentration not only reduces toxicity, but also simplifies processing the bio-samples. Despite multiple attempts, scientists have not been able to cryopreserve and restore normal functions of complex bio-samples, such as mammalian tissues and organs. Cryopreservation protocols are based on using: (1) optimized cooling and warming protocols, which are impossible to achieve in most tissues and organs due to low thermal conductivity of biological tissues; and/ or (2) high concentration of CPAs, which are toxic to cells and are difficult to distribute inside tissues and organs. Inevitably, there will be too much CPA in some places (too toxic) and not enough in others (too many ice crystals). On the other hand, hydraulic pressure, applied to the bio-sample, can be rapidly and equally distributed throughout the entire volume of the bio-sample, creating favorable conditions for cryopreservation, which cannot be realized at ambient pressure.
368
Summary
369
Although it is not known how pressure prevents damage from ice crystals, the above results on hemolysis of RBCs indicate that
317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334
338 339 340 341 342 343
347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366
370
5
size, shape, and distribution of ice crystals, formed at 120 MPa in combination with a relatively low Me2SO or glycerol concentration, have little to no effect on integrity of RBCs membranes when compared to ice crystals formed at ambient pressure (Figs. 4 and 5). Pressure appears to be an amplifier of the cryoprotective properties of CPAs and may reduce concentration of CPAs needed for preservation and recovery of viable bio-samples. Since tissues and organs are highly susceptible to CPA toxicity [10,9], development of the low-CPA method may be another step towards successful cryopreservation of tissues and organs. More research is needed to optimize the method and determine if it is clinically applicable.
371
Uncited reference
383
[17].
373 374 375 376 377 378 379 380 381 382
Q6
384
References
385
[1] S.J. Baxter, G.H. Lathe, Biochemical effects on kidney of exposure to high concentrations of dimethyl sulphoxide, Biochem. Pharmacol. 20 (1971) 1079– 1091. [2] P.W. Bridgman, Water, in the liquid and five solid forms, under pressure, Proc. Am. Acad. Arts Sci. 47 (1911) 441–458. [3] R. Dahl, L.A. Staehelin, High pressure freezing for the preservation of biological structure: theory and practice, J. Electron Microsc. Tech. 13 (1989) 165–174. [4] T. DeFries, J. Jonas, Pressure dependence of NMR proton spin–lattice relaxation times and shear viscosity in liquid water in the temperature range 15 to 10 °C, J. Chem. Phys. 66 (1977) 896–901. [5] I. Erk, G. Nicolas, A. Caroff, J. Lepault, Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution, J. Microsc. 189 (1998) 236–248. [6] G.M. Fahy, A. Hirch, Prospects for organ preservation by vitrification, in: D.E. Pegg, I.A. Jacobsen, N.A. Halasz (Eds.), Organ Preservation: Basic and Applied Aspects, MTP Press, Lancaster, 1982, pp. 399–404. [7] G.M. Fahy, D.R. MacFarlane, C.A. Angell, H.T. Meryman, Vitrification as an approach to cryopreservation, Cryobiology 21 (1984) 407–426. [8] G.M. Fahy, The relevance of cryoprotectant ‘‘toxicity’’ to cryobiology, Cryobiology 23 (1986) 1–13. [9] G.M. Fahy, T.H. Lilley, H. Linsdell, M.S. Douglas, H.T. Meryman, Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms, Cryobiology 27 (1990) 247–268. [10] G.M. Fahy, Cryoprotectant toxicity neutralization, Cryobiology 60 (2010) S45– S53. [11] P. Först, F. Werner, A. Delgado, The viscosity of water at high pressures – especially at subzero degrees centigrade, Rheol. Acta 39 (2000) 566–573. [12] A.M. Karow Jr., W.P. Liu, A.L. Humphries Jr., Survival of dog kidneys subjected to high pressures: necrosis of kidneys after freezing, Cryobiology 7 (1970) 122–128. [13] A.M. Karow Jr., M. Shlafer, R. Sims, Contractile and ultrastructural effects of hypothermia and of high pressure on rat hearts during ischemia, J. Surg. Res. 11 (1971) 571–583. [14] J. Lepault, D. Bigot, D. Studer, I. Erk, Freezing of aqueous specimens: an X-ray diffraction study, J. Microsc. 187 (1997) 158–166. [15] A.G. Macdonald (Ed.), Effect of high pressure on biological systems, SpringerVerlag, Berlin, 1993. [16] H.T. Meryman, M. Hornblower, A method for freezing and washing red blood cells using a high glycerol concentration, Transfusion 12 (1972) 145–156. [17] G. Rapatz, J.J. Sullivan, B. Luyet, Preservation of erythrocytes in blood containing various cryoprotective agents, frozen at various rates and brought to a given final temperature, Cryobiology 5 (1968) 18–25. [18] K. Richter, High-density morphologies of ice in high-pressure frozen biological specimens, Ultramicroscopy 53 (1994) 237–249. [19] R.D. Robertson, P. Deshpande, L. Slegel, S.W. Jacob, Studies on the function of the canine heart exposed to sub-zero temperatures, JAMA 187 (1964) 574– 578. [20] P. Sebert (Ed.), Comparative high pressure biology, Science Publishers, Enfield, NH, 2010. [21] C.R. Valeri, Standard operating procedure. Glycerolization and deglycerolization of red blood cells in a closed system using the haemonetics ACP215. Available at: (retrieved 10.11.14).
386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
372
439
No. of Pages 5, Model 5G
24 December 2014 Cryobiology xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo 4 5 3
Q1
Freezing under pressure: A new method for cryopreservation
6
Q2
Nickolas Greer ⇑
7
Q3
Rissali LLC, USA
8 1 1 0 2 11 12 13 14 15 16 17 18 19 20
a r t i c l e Q4
Q5
i n f o
Article history: Received 3 September 2014 Accepted 15 December 2014 Available online xxxx Keywords: Freezing under pressure Cryopreservation under hydraulic pressure Red blood cells Cryoprotectant toxicity
a b s t r a c t The ability to preserve cells, tissues, and organs with minimal damage for an extended period of time is essential for advancements in medicine and research. Current methods for cryopreservation are based on using high concentrations (up to 60% v/v) of cryoprotective agents (CPAs) which are toxic to living cells. The effect of pressure and a low concentration of dimethyl sulfoxide (Me2SO) or glycerol, on hemolysis of human red blood cells (RBCs) after freezing and thawing were investigated. Pressure was applied during cooling and freezing the RBCs and minimum in hemolysis was reached at approximately 120 MPa. Either 5% v/v Me2SO or 8% v/v glycerol concentration in combination with 120 MPa pressure was sufficient to obtain 8% or less hemolysis of RBCs after cooling at a 35 °C/min or a 160 °C/min rate. The preliminary results suggest that the method may help to solve the CPAs toxicity problem. Ó 2014 Published by Elsevier Inc.
22 23 24 25 26 27 28 29 30 31 32 33
34 35
Introduction
36
Water is an essential component of life. The human body is 65% water. Upon freezing at ambient pressure, water transforms into relatively large, sharp crystals of ice, causing mechanical damage to cells and tissues. The ice formation also causes overconcentration of salts, dehydration, and osmotic balance disruption, often leading to cell death.
37 38 39 40 41
42
Cryoprotectant toxicity
43
Cryoprotective agents (CPAs) were developed to substitute a portion of water inside and around cells, reducing the size of ice crystals and limiting over-concentration of salts during freezing. Unfortunately, CPAs are toxic to live cells, tissues, and organs (bio-samples) and CPA toxicity increases with concentration and contact time in liquid state [9,1,8,10]. CPAs bind to proteins and other molecules, disrupt multiple bio-chemical pathways inside the cells, and cause osmotic imbalance [9]. See Tables 5 and 6 at [9] for more details. In addition, many tissues and organs have limited permeability even to the most penetrating CPAs, such as Me2SO, which makes loading high CPA concentrations difficult and time consuming. Therefore, alternative methods for cryopreservation with lower CPA concentration or CPA-free need to be
44 45 46 47 48 49 50 51 52 53 54 55
⇑ Address: 271 White Tree Ln, Ballwin, MO 63011, USA. E-mail address: [email protected]
developed to address the growing demand for cryopreservation of quality bio-samples.
56
Freezing under pressure for electron microscopy sample preparation
58
Freezing under pressure is not a new concept. Since the 1970s a method of freezing under pressure around 210 MPa (HPFe method) is widely used for sample preparation for electron microscopy [3,5]. The HPFe method works well for preserving ultrastructure of cells with the following limitations: developers of the HPFe method claim that the method works well for freezing up to 0.5 mm thick samples with relatively fast cooling rates (>60,000 °C/min) [3]. With slower cooling rates ice crystals become visible with an electron microscope and the vitrified portion of water becomes progressively smaller. Several polymorphs (forms) of ice were detected inside of the samples prepared using the HPFe method [14,5,18]. They include vitreous (non-crystalline, amorphous) ice, as well as crystalline ice forms: Ih (hexagonal ice), Ic, and III. The fast cooling rate (>60,000 °C/min) and the presence of ice inhibitors (bio-molecules and CPAs) inside the bio-samples account for the vitreous component, whereas the crystalline ice is more likely to form where cooling rate was slower (deeper into the bio-sample) or where spontaneous ice nucleation took place. The HPFe method helps to preserve samples without visible ice crystals when observing with an electron microscope [5,3]. Since the resolution limit of modern electron microscopes used for biological research is <10 nm, then the ice crystals detected by X-ray diffraction [5,14,18] have to be smaller than 10 nm.
59
http://dx.doi.org/10.1016/j.cryobiol.2014.12.005 0011-2240/Ó 2014 Published by Elsevier Inc.
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
57
60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82
YCRYO 3562
No. of Pages 5, Model 5G
24 December 2014 2
N. Greer / Cryobiology xxx (2014) xxx–xxx
Freezing under pressure
139 140
106
Some CPAs possess an unexpected property. They can protect bio-samples from negative impact of high pressure [7]. See Table 4 at [7] for details. The Fahy group found that high CPA concentrations can protect rabbit renal cortex from prolonged (30–60 min) exposure to high pressure without freezing [6,7]. See Table 5 at [7] for details. Since the negative impact of high pressure accumulates with time spent under pressure [12,13], low concentrations of CPAs (for example, 5% Me2SO) may also be sufficient for protecting bio-samples from short-term (2–3 min) exposure to high pressure.
For generating pressure a manual high pressure generator (pump) was used. The pump was made by High Pressure Equipment Co, Erie, PA, USA (HiP) using a manual hydraulic pump HiP 37-5.75-60 as a main component. Pressure was measured using a 140 MPa gauge (Pressure Products Industries, Warminster, PA, USA). For pressures above 140 MPa a 350 MPa gauge (Astra Products, Ivyland, PA, USA) was used. A high pressure tube HiP 60-HM4-10 (tube) was used for containing and freezing cell suspensions under pressure (Fig. 1). The tube (stainless steel 316, 1.6 mm I.D., 6.35 mm O.D., 20 cm long) was attached to a 1 mL syringe using a plastic tubing adapter. About 300 lL of cell suspension was drawn into the tube (Fig. 1). A rubber stopper (silicone rubber, 1.78 mm diameter, 6.5 mm long) was covered with white petrolatum USP and inserted 35–45 mm deep into the open end of the tube. The opposite end of the tube was closed with a cap (Butech #60CA4, Haskel, Burbank, CA, USA). Accuracy of pressure control was within 2–3% or +/-3 MPa at 120 MPa level. For freezing at ambient pressure (0.1 MPa) no pressurization of the cell suspension was performed. For all other pressures, the tube was connected to the pump and pressurized to the testing pressure. The tube was oriented vertically and submerged into a container filled with 2L of 75 °C isopropyl alcohol coolant (Fig. 1). After 3 min in the coolant, pressure at the pump was dropped. The tube was disconnected from the pump, and was plunged into a +32 °C water bath. The cell suspension was warmed at ambient pressure.
107
Materials and methods
Cooling rates
165
108
To test the above hypothesis on human red blood cells (RBCs) a method and an apparatus were developed and are presented below.
To obtain 160 °C/min cooling rate of the cell suspension, the tube was submerged into 75 °C alcohol with 2 mm/s velocity.
166
83 84
Effect of hydraulic pressure on viability of bio-samples without freezing
96
High hydraulic pressure can be harmful to bio-samples and pressure-induced damage increases with time spent under pressure [12,13,20,15]. Armand Karow’s team did research on pressure tolerance of mammalian organs and found that rat hearts can tolerate high hydraulic pressure up to 100 MPa (15,000 psi) for up to 10 min without apparent changes [13]. They also found that dog kidneys can tolerate 100 MPa for up to 2 min and the animals survived for at least 4 weeks after re-implantation of the kidneys [12]. Another research group demonstrated that dog hearts can survive 100 MPa for up to 30 min [19]. This data implies that pressure up to 100 MPa applied for a short period of time (up to 2–30 min) is relatively harmless to some organs.
97
Baroprotective properties of CPAs
85 86 87 88 89 90 91 92 93 94 95
98 99 100 101 102 103 104 105
109 110
Pump 111
Biological model
112
123
RBCs were chosen as a model for developing and preliminary testing the method. Unlike complex tissues and organs, it is relatively easy to analyze and quantify damages to RBCs, which is usually done by measuring free hemoglobin and looking at cell morphology. RBCs have no nucleus, DNA, or RNA, and therefore lack membrane repair capabilities, which make their membranes more sensitive to damages when compared to nucleated cells types with advanced membrane repair capabilities. Since damage to cell membranes is a direct result of ice crystal formation, repairing the membranes by the cells can be undesirable for testing cryopreservation methods. If the method works on RBCs, then there is a good chance it may also work on more complex bio-samples.
124
RBCs suspension preparation
125
Two human donors provided blood for this study. Whole venous blood was drawn in standard fashion after a 12 h fast and stored at +4 °C in BD Vacutainer™ REF 367841 containers lined with 3.6 mg of K2 EDTA to prevent coagulation (2 mL of whole blood per container). Hematocrit (Hct) was within 40–45%. The blood was used within 4 days of the draw. Three types of bio-samples were tested: (1) whole blood; (2) whole blood with 5% v/v Me2SO; (3) whole blood with 8% v/v glycerol. The cell suspensions were obtained by centrifuging whole blood at 3200 RPM for 3 min allowing separation of the plasma (supernatant) and RBCs (precipitate). Me2SO or glycerol was added to plasma. RBCs were resuspended in plasma to make 5% v/v Me2SO or 8% glycerol v/v concentration in whole blood. The Me2SO and glycerol were purchased from Sigma (St. Louis, MO).
113 114 115 116 117 118 119 120 121 122
126 127 128 129 130 131 132 133 134 135 136 137 138
water under pressure rubber stopper – moves up when pressure inside of the bio-sample rises due to water expansion upon freezing
tube
liquid bio-sample under pressure
liquid-solid boundary solid (frozen) bio-sample
cap
container is elevated with 1 or 2 mm/sec velocity -75°C alcohol
Fig. 1. Apparatus for freezing under pressure. The figure shows a cross-section of the tube loaded with whole blood, connected to the pump, and partially submerged into coolant. The apparatus is designed for freezing cell suspensions at a nearconstant pressure. When freezing of the cell suspension begins, pressure inside of the cell suspension rises due to ice expansion upon freezing. The rubber stopper will move upward, thereby keeping pressure inside of the cell suspension in a relatively narrow range (for example, 120 ± 3 MPa).
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164
167
YCRYO 3562
No. of Pages 5, Model 5G
24 December 2014 3
N. Greer / Cryobiology xxx (2014) xxx–xxx
169 170 171 172 173 174 175 176 177
To obtain 35 °C/min cooling rate of the cell suspension, the tube was wrapped with a 31 cm long 15 cm wide, 0.015 mm thick polyethylene film (15 layers), and was submerged into 75 °C alcohol with 1 mm/s velocity. Warming rate for all cell suspensions was 950 °C/min. The above cooling and warming rates are average rates of temperature change on the interval from 0 to 50 °C. As there is no easy way for measuring cooling rates inside of the tube under pressure, the rates of temperature change were measured at ambient pressure in the middle and inside of the tube filled with saline using an Omega TT-T-36-36 thermocouple.
100.0 90.0 80.0 Hemomisys, %
168
70.0 60.0 50.0 40.0 30.0
178
RBCs suspension analysis
179
186
In a healthy individual RBCs occupy 40–45% of total blood volume. When RBCs are damaged they release hemoglobin. By measuring the free hemoglobin, one can determine a degree of damage to RBCs (hemolysis). The free hemoglobin concentration is often used to estimate hemolysis of RBCs during cryopreservation [16]. Hemolysis was calculated as a percentage of free hemoglobin in RBCs suspensions after cryopreservation using the following equation:
187 189
% hemolysis ¼ ½ð100 HctÞ supernatant Hgb = total Hgb
180 181 182 183 184 185
190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205
The cell suspension was extruded from the tube by pushing on the rubber stopper with a stainless steel wire (1.37 mm diameter, 21 cm long). About 90lL of cell suspension, obtained from the middle portion of the tube, was centrifuged at 3200 RPM for 3 min. Hemoglobin concentration in the supernatant was measured using HemoCue Hb 201+ analyzer (HemoCue AB, Sweden). The analyzer was calibrated and its accuracy was confirmed with standard trilevel hemoglobin controls (Stanbio, Boerne, TX) and the following linearity test. A whole blood sample with 13.2 g/dL (within normal limits for human blood) concentration of hemoglobin was diluted 1:1, 1:4, 1:9, and 1:19 in an isotonic saline to obtain the following concentrations of hemoglobin: 50%, 25%, 10%, and 5%. Hemoglobin content in the resulting solutions was measured with the analyzer and plotted on a graph. The results of the test were linear with an insignificant deviation from a straight line.
206
Results
207
The effect of pressure on hemolysis of RBCs
208
Fig. 2 shows the effect of hydraulic pressure on hemolysis of RBCs after cooling/freezing whole blood samples at five pressure levels: 0.1, 70, 120, 160, 200 MPa. Minimum in hemolysis (28.2 ± 2.6%) was reached at 120 MPa. Minor to no morphological difference were noticed between the control RBCs (Fig. 3A) and RBCs, frozen under 120 MPa and thawed at ambient pressure (Fig. 3B).
209 210 211 212 213
214
The effect of pressure, cooling rate, and low CPA concentration
215
Another set of experiments was conducted to compare effect of pressure, cooling rate, and low CPA concentration on hemolysis of RBCs. RBCs were cooled/frozen at two cooling rates: 35 °C/min and 160 °C/min while being compressed at two pressure levels: 0.1 MPa (ambient pressure) and 120 MPa (Figs. 4 and 5). Three kinds of cell suspensions were tested: (1) whole blood; (2) whole blood with 5% v/v Me2SO; (3) whole blood with 8% v/v glycerol. Fig. 4 shows the effect of 120 MPa on hemolysis of RBCs. Blood samples were cooled at the 160 °C/min rate. RBCs, frozen at 0.1 MPa (ambient pressure) were mostly hemolyzed after the cryopreservation cycle. This was an expected result for cryopreser-
216 217 218 219 220 221 222 223 224 225
20.0 10.0 0.0 0.0
50.0
100.0
150.0
200.0
Pressure, MPa Fig. 2. The effect of pressure on hemolysis of RBCs after cooling/freezing whole blood at 160 °C/min rate at five different pressure levels (0.1; 70; 120; 160; 200 MPa) and thawing at ambient pressure. Mean ± SD (n = 4).
vation of RBCs at ambient pressure. However, 5% Me2SO in combination with 120 MPa decreased hemolysis of RBCs to 5 ± 1.2%. Fig. 5 shows the effect of 120 MPa on hemolysis of RBCs in whole blood with 5% Me2SO or 8% glycerol. Blood samples were cooled at the 35 °C/min rate. RBCs, frozen at 0.1 MPa, were mostly hemolyzed after the cryopreservation cycle. A low CPA concentration in combination with 120 MPa decreased hemolysis of RBCs down to 7.6 ± 1.4% for Me2SO, and 4.2 ± 1.2% for glycerol.
226
Discussion
234
The effect of hydraulic pressure, applied during cooling and freezing, on hemolysis of RBCs after thawing was investigated. Minimum in hemolysis (28.2 ± 2.6%) was reached at 120 MPa. The increase in hemolysis after freezing at 160 MPa and 200 MPa may be explained by the negative impact of elevated pressure.
235
The effect of pressure on ice crystal nucleation
240
Water content of whole blood is 83% v/v. According to the Bridgman’s diagram for phase transitions of water under pressure [2], at 120 MPa ice I nucleation begins at 12 °C or so. Ice nucleation temperature inside the tube (Fig. 1) was measured with the thermocouple at ambient pressure and was found to be around 0 °C for whole blood. Super-cooling of water is improbable in slowly cooled whole blood samples inside the tube due to multiple nucleation sites (RBCs). Thus, ice nucleation temperature in whole blood at 120 MPa is likely to be close to pure water (12 °C). To understand the process it helps to visualize simultaneous nucleation of multiple ice crystals on RBCs membranes and the inner walls of the tube at the liquid–solid boundary (Fig. 1) during freezing.
241
The effect of pressure on ice crystal growth
254
Ice crystal growth rate depends on the following three factors: pressure level, cooling rate, and composition of bio-samples. The effect of hydraulic pressure on ice crystal growth inside biosamples is not entirely understood. The effect of pressure may be explained by looking into the changes of density and viscosity of liquid water under pressure. For example, under 120 MPa pressure at 0 °C water density is increased by about 5.4% relative to ambient pressure. The viscosity of water at the liquid–solid transition point
255
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
227 228 229 230 231 232 233
236 237 238 239
242 243 244 245 246 247 248 249 250 251 252 253
256 257 258 259 260 261 262
YCRYO 3562
No. of Pages 5, Model 5G
24 December 2014 4
N. Greer / Cryobiology xxx (2014) xxx–xxx
(A)
(B)
Hemolysis, %
Fig. 3. The effect of 120 MPa pressure on morphology of RBCs. Blood cells before freezing (A) vs. after cooling/freezing whole blood under 120 MPa pressure at 160 °C/min rate and thawing at ambient pressure. (B) Whole blood was diluted 1:200 in lactated Ringer’s and observed on a hemocytometer at 400. No CPAs were used.
0.1MPa 120MPa
Whole blood (WB)
WB + 5% DMSO
Hemolysis, %
Fig. 4. The effect of 120 MPa pressure on hemolysis of RBCs after cooling/freezing whole blood (WB) and WB + 5% Me2SO at 160 °C/min rate and thawing at ambient pressure. Mean ± SD (n = 4).
120MPa
WB + 8% glycerol
Fig. 5. The effect of 120 MPa pressure on hemolysis of RBCs after cooling/freezing WB + 5% Me2SO and WB + 8% glycerol at 35 °C/min rate and thawing at ambient pressure. Mean ± SD (n = 4).
264 265 266 267 268 269 270 271 272
273
Cooling rates
296
Intracellular ice is one of the major damaging factors to RBCs membranes at ambient pressure. The 160 °C/min cooling rate is fast enough so RBCs have no time to lose liquid water during cooling and liquid water becomes intracellular ice. Thus, intracellular ice crystals formed under pressure inside RBCs when cooled at 160 °C/min rate have to be significantly smaller and/or have different shape when compared to intracellular ice crystals formed at ambient pressure. Cooling whole blood with 5% Me2SO at 160 °C/min rate resulted in a lower hemolysis (5 ± 1.2%) when compared to cooling at 35 °C/ min rate (7.6 ± 1.4%) (Figs. 4 and 5). Higher cooling rates may result in smaller crystals of ice and may account for the lower hemolysis of RBCs. Furthermore, there are two other reasons why the cooling rate of bio-samples should be maximized when freezing with CPAs under elevated pressure. First, CPAs are toxic to living cells and the toxicity increases with time spent in contact with liquid CPA. Second, pressure above 100 MPa can be lethal to mammalian organs if applied for longer than 2 min and the negative impact of pressure rises with increase of pressure and time spent under pressure [12,13]. Utilizing fast cooling techniques, it is possible to freeze
297
274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
0.1MPa
WB + 5% DMSO
263
the other hand, under high hydraulic pressure water molecules are compressed into (interlocked with) each other and their movement becomes restricted, reducing the probability of a water molecule becoming a part of the ice crystal and the growth rate of the crystal is inhibited. The high hemolysis after freezing at ambient pressure (Fig. 2) is primarily due to mechanical and osmotic stresses created by ice crystals in close proximity to RBCs membranes. Ice crystals formed at 120 MPa may be smaller, may trap solutes more efficiently, and may have smoother surface when compare to ice crystals formed at ambient pressure, thereby reducing osmotic and mechanical stresses to cell membranes. In addition, CPAs combined with pressure may further reduce the size of ice crystals and even convert a portion of water into a vitreous (non-crystalline) form during rapid freezing. Electron microscopy studies of frozen bio-samples are needed to determine the exact fate of the water molecules and ice crystals. Indirect evidences of ice crystal growth inhibition by pressure can be found in HPFe studies [3,5,14,18]. The size of individual ice crystals formed under 200 MPa with >60,000 °C/min cooling rate is often <10 nm (the resolution limit of electron microscopes) [5,3] and the size of ice crystals appear to increase with a reduction of cooling rate of the samples. More definitive studies on ice crystal growth rate under pressure are needed.
at 120 MPa and 12 °C is significantly higher (2.5 mPas), when compared to viscosity of water at the liquid–solid transition point at ambient pressure and 0 °C (1.7 mPas) [4,11]. For details see Fig. 2 at [4] and Figs. 6 and 7 at [11]. Adding cryoprotectants such as sucrose to water in combination with pressure further increases the viscosity of the solution [11]. For details see Fig. 4b at [11]. Ice crystals are ordered structures of water molecules. At ambient pressure movement of water molecules in liquids is relatively free. Any water molecule in close proximity to a growing crystal of ice has a high probability of becoming a part of the crystal. On
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316
YCRYO 3562
No. of Pages 5, Model 5G
24 December 2014 N. Greer / Cryobiology xxx (2014) xxx–xxx
335
large (20+ mm thick) bio-samples within 1–2 min, thereby limiting or eliminating impact of high hydraulic pressure and CPA toxicity prior freezing. When the bio-sample is in the frozen (solid) state both hydraulic pressure and CPAs have little to no effect on biosamples. Thus, the cooling rate of bio-samples should be maximized to reduce time spent under pressure, exposure to liquid CPAs, and size of ice crystals. The 35 °C/min and 160 °C/min cooling rates were investigated because they approximate the fastest cooling rates which can be achieved on the surface (160 °C/min) and in the center (35 °C/ min) of many large bio-samples, such as units of blood (400 mL) and organs (kidney) due to limited thermal conductivity of the bio-samples. The above cooling rates can be obtained by plunging the bio-samples into liquid nitrogen or by contact with cold metal plates. Low hemolysis after cooling with the two very different cooling rates makes it possible to keep hemolysis under control in the entire volume of a unit of blood, and may open up the possibility for successful cryopreservation of organs, such as heart and kidney.
336
The effect of low CPA concentration
337
344
The above results indicate that the cryoprotective effect of pressure can be amplified by 5% Me2SO or 8% glycerol (Figs. 4 and 5). The low concentrations of CPAs combined with 120 MPa pressure were sufficient for protecting cell membranes from mechanical and osmotic damages during ice formation. It appears that the low CPA concentration further inhibits ice crystal growth, resulting in even smaller ice crystals and a larger vitrified component of water when compared to freezing using pressure alone.
345
Prospective applications
346
367
Even though the FDA-approved method for cryopreservation of RBCs [16,21] often results in <15% post-thaw hemolysis prior processing, it requires 40% glycerol combined with lengthy and expensive processing using specialized equipment [21]. On the other hand, 8% glycerol is far less toxic than 40% [9] and adding and removing 8% glycerol can be done relatively fast in 1–2 simple steps. Thus, reduction in CPA concentration not only reduces toxicity, but also simplifies processing the bio-samples. Despite multiple attempts, scientists have not been able to cryopreserve and restore normal functions of complex bio-samples, such as mammalian tissues and organs. Cryopreservation protocols are based on using: (1) optimized cooling and warming protocols, which are impossible to achieve in most tissues and organs due to low thermal conductivity of biological tissues; and/ or (2) high concentration of CPAs, which are toxic to cells and are difficult to distribute inside tissues and organs. Inevitably, there will be too much CPA in some places (too toxic) and not enough in others (too many ice crystals). On the other hand, hydraulic pressure, applied to the bio-sample, can be rapidly and equally distributed throughout the entire volume of the bio-sample, creating favorable conditions for cryopreservation, which cannot be realized at ambient pressure.
368
Summary
369
Although it is not known how pressure prevents damage from ice crystals, the above results on hemolysis of RBCs indicate that
317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334
338 339 340 341 342 343
347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366
370
5
size, shape, and distribution of ice crystals, formed at 120 MPa in combination with a relatively low Me2SO or glycerol concentration, have little to no effect on integrity of RBCs membranes when compared to ice crystals formed at ambient pressure (Figs. 4 and 5). Pressure appears to be an amplifier of the cryoprotective properties of CPAs and may reduce concentration of CPAs needed for preservation and recovery of viable bio-samples. Since tissues and organs are highly susceptible to CPA toxicity [10,9], development of the low-CPA method may be another step towards successful cryopreservation of tissues and organs. More research is needed to optimize the method and determine if it is clinically applicable.
371
Uncited reference
383
[17].
373 374 375 376 377 378 379 380 381 382
Q6
384
References
385
[1] S.J. Baxter, G.H. Lathe, Biochemical effects on kidney of exposure to high concentrations of dimethyl sulphoxide, Biochem. Pharmacol. 20 (1971) 1079– 1091. [2] P.W. Bridgman, Water, in the liquid and five solid forms, under pressure, Proc. Am. Acad. Arts Sci. 47 (1911) 441–458. [3] R. Dahl, L.A. Staehelin, High pressure freezing for the preservation of biological structure: theory and practice, J. Electron Microsc. Tech. 13 (1989) 165–174. [4] T. DeFries, J. Jonas, Pressure dependence of NMR proton spin–lattice relaxation times and shear viscosity in liquid water in the temperature range 15 to 10 °C, J. Chem. Phys. 66 (1977) 896–901. [5] I. Erk, G. Nicolas, A. Caroff, J. Lepault, Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution, J. Microsc. 189 (1998) 236–248. [6] G.M. Fahy, A. Hirch, Prospects for organ preservation by vitrification, in: D.E. Pegg, I.A. Jacobsen, N.A. Halasz (Eds.), Organ Preservation: Basic and Applied Aspects, MTP Press, Lancaster, 1982, pp. 399–404. [7] G.M. Fahy, D.R. MacFarlane, C.A. Angell, H.T. Meryman, Vitrification as an approach to cryopreservation, Cryobiology 21 (1984) 407–426. [8] G.M. Fahy, The relevance of cryoprotectant ‘‘toxicity’’ to cryobiology, Cryobiology 23 (1986) 1–13. [9] G.M. Fahy, T.H. Lilley, H. Linsdell, M.S. Douglas, H.T. Meryman, Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms, Cryobiology 27 (1990) 247–268. [10] G.M. Fahy, Cryoprotectant toxicity neutralization, Cryobiology 60 (2010) S45– S53. [11] P. Först, F. Werner, A. Delgado, The viscosity of water at high pressures – especially at subzero degrees centigrade, Rheol. Acta 39 (2000) 566–573. [12] A.M. Karow Jr., W.P. Liu, A.L. Humphries Jr., Survival of dog kidneys subjected to high pressures: necrosis of kidneys after freezing, Cryobiology 7 (1970) 122–128. [13] A.M. Karow Jr., M. Shlafer, R. Sims, Contractile and ultrastructural effects of hypothermia and of high pressure on rat hearts during ischemia, J. Surg. Res. 11 (1971) 571–583. [14] J. Lepault, D. Bigot, D. Studer, I. Erk, Freezing of aqueous specimens: an X-ray diffraction study, J. Microsc. 187 (1997) 158–166. [15] A.G. Macdonald (Ed.), Effect of high pressure on biological systems, SpringerVerlag, Berlin, 1993. [16] H.T. Meryman, M. Hornblower, A method for freezing and washing red blood cells using a high glycerol concentration, Transfusion 12 (1972) 145–156. [17] G. Rapatz, J.J. Sullivan, B. Luyet, Preservation of erythrocytes in blood containing various cryoprotective agents, frozen at various rates and brought to a given final temperature, Cryobiology 5 (1968) 18–25. [18] K. Richter, High-density morphologies of ice in high-pressure frozen biological specimens, Ultramicroscopy 53 (1994) 237–249. [19] R.D. Robertson, P. Deshpande, L. Slegel, S.W. Jacob, Studies on the function of the canine heart exposed to sub-zero temperatures, JAMA 187 (1964) 574– 578. [20] P. Sebert (Ed.), Comparative high pressure biology, Science Publishers, Enfield, NH, 2010. [21] C.R. Valeri, Standard operating procedure. Glycerolization and deglycerolization of red blood cells in a closed system using the haemonetics ACP215. Available at:
386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438
Q1 Please cite this article in press as: N. Greer, Freezing under pressure: A new method for cryopreservation, Cryobiology (2014), http://dx.doi.org/10.1016/ j.cryobiol.2014.12.005
372
439