JPS60500058A - Preservation method of living organic tissue by freezing - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Background of the invention 1. Field of invention The present invention relates to cryopreservation of fruits, vegetables, animal bodies, and organic tissues in general. In particular, fine Preserve or maintain the cell structure so that it can be completely restored to its original conditions by recomposition. Storage, transportation, long-term storage, or other conditions that may completely or partially abort biological activity. It concerns the freezing of cellular constructs for various desired uses.

2. Description of prior art From storing and transporting edible fruits and vegetables to facilitating surgical tissue treatment or medical and medical sciences. The study of fresh plants and animal organisms, up to the point where biological activities are no longer possible for scientific observation. Freezing cells has a wide range of utility. Unfortunately, most cellular structures If the cell wall network cannot withstand damage due to the formation of ice crystals The increasing volume and sharp edges of the ice crystals create a hole in the cell wall, causing the cell to collapse. Burst. This results in both a loss of tissue turgor pressure and a loss of natural fluid. fruits and In the case of fresh vegetables, the product can be eaten after thawing, but it is no longer tasty or attractive. not. In animal tissue systems, intense metabolic disruption occurs and often leads to death. bring about. Furthermore, air bubbles or frames formed by trapped gas during freezing It is released during deicing and enters the bloodstream. Severe pain and immune division occur, causing heart damage. Embolism in the viscera or brain spinal cord can lead to death.

Traditional methods of solving this problem typically allow for expansion with ice crystal formation. It involves partial dehydration of organic cells to obtain Yoharu's method is Ram (L amb) U.S. Patent No. 3.21.9,463 (November 26, 1965) Are listed. Ram's method uses a high level vacuum (absolute pressure 4.6 mm Hg i It is a two-step method that requires After thawing, the ingredients were boiled down to reconstitute them. Water must be replaced.

In the case of complex or dense cellular structures, a uniform distribution is obtained in this way. is difficult. Additionally, tissues with delicate cellular structures often lose their cellular structure upon dehydration and reorganization. Unable to withstand the stress associated with large amounts of material penetrating the wall.

Summary of the invention The removal of at least a portion of the dissolved gases from the intercellular secretions of the cellular composition composition without substantial damage to cell structures or embolization It has been found that freezing and recomposing the body is possible.

Therefore, the present invention achieves complete termination of life in a wide range of organic tissues and thaws the ice. After that, you can find a way to return to full life.

DESCRIPTION OF THE PREFERRED EMBODIMENT According to the method of the present invention, organic material in the form of cellular compositions is subjected to progressively reduced pressure. Cooled to freezing point or below freezing point. Reduced pressure is necessary to separate the gases dissolved in the fluid between the cells. considerably lowering the concentration of the fluid while avoiding substantial loss of the secreted fluid itself through evaporation. It is carried out to a certain extent. The term ``gas'' or ``gaseous substance'' is used in the text to refer to normal atmospheric Denotes a substance that is in a gaseous state under certain conditions of temperature and pressure.

The reduction in dissolved gas content reduces the specific volume that occurs when fluid secretions between cells freeze. The increase decreases as well.

The present invention shows that if this reduction in frozen volume is allowed to occur, cell wall damage accompanied by ice crystal formation can be prevented. It is based on the finding that it is sufficiently preventive.

The vacuum is applied at a rate that avoids evaporation of secretions. This can be reduced. Generally, about 0 per minute ．． 01 kl? /cr++2 (1.0 kilopascals) to 0.1 kg/cm2 (10,0 kilopascals) (7.1 mmHg to 7.7.3 mm H5 per minute) It is also advantageous to have a pressure reduction rate within the range of . A suitable decompression rate is approximately 0.031< 97 cm2 (3,0 kilopascals) to 0.07 kg/cm2 (7,0 kilopascals) Pascal) (23,0mm Hg to 55.6111111 Hg) Optimally is approximately 0.05 kL’an2 (5,0 kilopascals) to 0.06’j/am 2 (6,0 kilopascals) range (38,3mm Hg to 46.Orrrm Hg) Nomo Note 6 Le.

Sufficient amount of dissolved gas without causing evaporation of cell secretions during decompression The pressure is reduced to a temperature that causes the contents to escape from the solution and penetrate the cell wall. actual The level of vacuum is not critical and depends on the type of organic tissue, dissolved gas content, and cell It can vary widely depending on the structure and properties of the cell secretions. Preferably, At least about half of the dissolved gas content is released. For most organic materials, atmospheric pressure Approximately 0.9 kg/crn2 (90 kilopascals) to 0.6 kg/crI] 2 (60 kilopascals) (70 to 300ffIl'llHg absolute pressure). Best results are obtained by reducing pressure to level. Within this range, approximately 0 below atmospheric pressure ．． 8 kg7’crr+2 (80 kilopascals) to 0.65 kg/crn2 (65 kilopascals) range (150mmHg to 260nvnHg absolute pressure) preferable.

The optimal vacuum level depends not only on the type of organic matter, but also on the growth of the organic matter. Varies depending on altitude and climate. Especially at high altitudes or in relatively cold or warm climates. The same is true for plants such as vegetables and fruits that grow in borders. grows at high altitudes The relatively fragile cell walls that occur in plants make the cell structure more susceptible to rupture. , facilitates the diffusion of gases and vapors. In such cases, it is necessary to avoid cell dehydration at the same time. Special care must be taken to keep the cell structure intact. This is a decompression This can be achieved by reducing the degree and rate of depressurization. Similar adjustments can be made to In plants growing in warm climates, this is due to fluctuations in the amount of dissolved gases contained in cell secretions. is often necessary.

When cell walls are particularly thick or strong, preliminary decompression and recompression can be used to determine the decompression response of the cell. The shrinkage cycle can improve the flexibility of cell walls. Decompression and recompression Both parts of the cycle are performed at the above rates, although a smaller pressure drop is used. Ru. Generally, effective regulation is approximately 0.2 kL’cm” (20 kPa) below atmospheric pressure. Skull) to 0.4 kg/crn2 (40 kilopascals) (450 f This can be achieved by reducing the pressure from fIITIHg to 600 mHg (absolute pressure). Wear.

Depressurization is performed either before or during the cooling step.

Preferably, the depressurization and cooling are carried out simultaneously to allow the escape of gas, while reducing the loss of moisture. Gain optimal control to avoid When doing it at the same time, it spills out on the − side. phase of cooling and depressurization to avoid the formation of ice crystals before the gas can escape. The ratio must be determined.

Appropriate proportions are readily determined by routine experimentation. After thawing and recomposition of the frozen body, Those with ruptured cellular structures are easily determined by the lack of turgor pressure, while those that undergo dehydration The orders of magnitude indicate significant changes in density and partial loss of turgor pressure. ordinary cooling device Both cell rupture and dehydration can be avoided by reducing the pressure at the rate mentioned above. results can be easily obtained.

The final temperature is any temperature at which the intercellular fluid becomes a frozen solid. This is the property of solute and solute concentration thus vary with the type of organic tissue. In general, the freezing point of normal water Temperatures in the range from about -10°C are sufficient.

Cooling can be accomplished by any conventional technique.

When cooling and depressurizing are performed at the same time, as mentioned above, the dissolution occurs before sufficient crystal formation occurs. The cooling rate must be adjusted to allow gas to escape from the liquid. Therefore , rapid freezing is advantageously avoided.

In a preferred embodiment of the method of the invention, prior to the formation of large crystals inside the cell/ ” - Cooling and depressurization are applied to promote the formation of a type crystal mass first. is accompanied by agitation and/or vibration of the cell tissue. smaller cells are smaller It occupies the volume, deforming the cell shape becomes smaller, and it also breaks the cell membrane. Few sharp edges or points. The speed and degree of agitation or vibration are not critical; These allow the escape of dissolved gases without abrasive or other damage to cellular structures. can be varied as much as is possible.

Inducing molecular movement within a tissue sufficient to disrupt or reorganize crystal formation Any device capable of doing so may be used. This ranges from gentle stirring to audible range frequencies. It is within the range of vibrations of numbers. For best results, all secretions within the cell structure This can be achieved by gentle stirring that continues at a certain rate until it freezes into a solid. .

For most applications, it is approximately 2.5 cm (1.0 inch) to 25.0 cm (10,0 inch). lateral at a rate of approximately 25 to 100 cycles per minute with a displacement amplitude of Best results are obtained with anti-oscillation.

When processing dense or bulky materials by the method of the invention, Care must be taken to ensure that refrigeration takes place. Due to heat transfer limitations The interior of lettuce heads, etc., crystallizes more slowly than the outer cell layer or parts near it. Ru. Therefore, once the final temperature and vacuum levels are reached, complete cooling is achieved. In addition to continuing agitation during use for a sufficient period of time to ensure freezing, the above conditions must be met. It is often necessary to maintain

Additionally, optionally, humid or saturated air may be placed around the object being frozen. Other devices can be provided to provide circulation and prevent dehydration. Circulation device is installed as appropriate for cooling system. often incorporated into the structure of the installation. Under no circumstances should it be placed in an excessively dry environment. This must be avoided when cooling.

When a living animal undergoes life termination by freezing and decompression according to the method of the present invention, Minimize and suppress anxiety using carbon dioxide or other effective gaseous anesthetics can be promoted. The anesthetic was removed during decompression and the animal was allowed to fully recover after deicing. can. In addition, the organization's rapid Cooling is enhanced by the use of a helium-oxygen mixture that provides an outlet for the cooling chamber. ・Once the required amount of m-lysis gas is able to escape, the tissue is completely Once frozen, the organism is kept free at atmospheric pressure or in a partial vacuum with the release of molten gas. It can be stored frozen until the expiry date. Cell structure does not thaw upon refreezing Remain untouched as long as possible.

If you want to return the organic material to its original state, gradually warm the organic material to ambient temperature. This makes it easier to thaw the ice. Freezing within cellular structures as organic matter is warmed Crystals dissolve in cell secretions until atmospheric gases reach their equilibrium configuration.

Establishment of complete turgor pressure and return to initial conditions is achieved by subjecting organic matter to pressures slightly above atmospheric pressure. It is easily done by applying force. This is especially true for animals in which organic matter is aborting life. Preferable when consisting of cells or whole animals. In most preferred embodiments, excessively large Pressurization exceeding atmospheric pressure is performed before the ice thaws, and the intercellular pressure decreases before the inside of the cell returns to a fluid state. Reestablish power.

The amount of overpressure is not important unless it itself causes harm to the cell structure. stomach. Pressures in the range of approximately 5% to 10% above ambient pressure are sufficient for most applications. be. Similarly, the rate of repressurization is not critical and there are no necessary forces during depressurization. Therefore , a rate slightly faster than that used for decompression is possible, and is representative of such rates. is 9 78 table fig-500058 (4) partial [], 05 kg/cm” (, 5 kilopascals) to 0.1 to 97cm2 (10 kilopascals) ( (from 38 mm Hg to 77 plates Hg per minute). Once the ice has completely thawed The organic matter is then allowed to return to ambient temperature, where excess gas in the cell membrane is released. be done.

The method of the invention applies to fruits, vegetables, inedible plants, seeds, edible meat, living cells and All plant or animal products, including tissues, animal and human organs, whole animals, and humans. can be applied to the freezing and recovery of animal objects. Vegetables such as fruits and vegetables If the material is used, when such material is processed as soon as possible after harvest. Get the best results. The method of the present invention is useful for preserving botanicals in medical and scientific preservation. and improved storage and transportation of objects of animal origin and for surgical or general medical purposes. Medical abortion of damaged or decomposed tissue and organic strains and biological preservation measures. It has utility in the formation of suspended animation in living cells and complex lineages.

The following examples are provided for illustrative purposes and are not intended to limit or in any way limit the invention. It does not stipulate that.

Six standard laboratory mice and six lettuce lettuce were set at -4°C. Placed in an open 0.06 m” (2.2 cubic feet) portable freezer, This refrigerator was placed inside a high-altitude simulator with vibration capability. Set up a vent The pressure inside the simulator is maintained at a constant rate for approximately 20 minutes using a high-pressure high-vacuum pump. Pressure CI 6 at an altitude of 11,100 meters (67,000 feet) 4mmHg absolute pressure or -0,782ki9/cm2 (-78,2 kilopascals) ]. Approximately 7.6 cm (6 inches) of excursion and 60 m/min during decompression Lateral vibration was applied to the device system at a rate of 70 cycles. Once the specified pressure is reached Once reached, the refrigerator was closed and continued to vibrate for 1 hour and 45 minutes.

Next, stop this vibration, open the refrigerator again and gradually return the pressure to atmospheric pressure over 20 minutes. I was able to do that. Furthermore, 0.1 atm is induced into the simulator, the knot is released, and the door opens. made it easy. So I took out the mouse and the lettuce.

The mouse showed no signs of vitality and was clearly frozen to the touch. is. The mouse is placed in each cage along with the cloth used to line the bottom of the cage. I took it out. Next, the mouse and cloth were placed in a cardboard box for observation. From the decompression chamber About 45 minutes after I took it out, a mouse seemed to sigh. next 15 minutes The remaining four mice moved a little and showed recovery in their daily functions. The sixth-scented mouse is It took about 10 minutes to make a move.

After removal from the decompression chamber, water was drained for about 1 hour and food was given to all mice.2, for the next 1 hour. Over the years, the six mice appeared to be in excellent health and had normal behavior and eating patterns. Indicated.

The lettuce was also completely frozen solid when I took it out of the vacuum chamber at 9am. Paper towel for bulbs This made it possible to detect any leakage caused by cell rupture when the ice was placed on top.

During the 15 minutes, the lettuce thawed significantly and no leakage of cell contents was observed. times A small area that did not heal appeared near the 9th edge of some leaves. This is the leaves before freezing. This was caused by a scratch. However, most of the lettuce is rotated by complete turgor pressure. Recovered.

f! The apparatus used in Example B from l.2 has a transparent vacuum chamber placed in a chemical stirrer, All of this was placed inside a large refrigerator that people could walk inside. Decompression is small Achieved by pressure readings from a cage at the top of the vacuum chamber using a vacuum pump. .

Place the heads of salad bowl lettuce on the ○ha U S scale in the vacuum chamber, and The rule indicated that the amount of lettuce was 505 gr at the beginning of the experiment. chamber sample During cooling, the pressure was gradually reduced to -0.77 kg7'cm2 over 20 minutes, Meanwhile, the agitator was running at approximately 50 cycles per minute with a lateral displacement of approximately 7.6 cm (3 inches). It was moving. The frozen mold temperature was approximately -7°C. During decompression, the weight of lettuce is 10g. It decreased by r±2.

Once the required vacuum level was reached, it was maintained for an additional 4 hours. next, The recompression rate when the vacuum chamber was connected to a vent and the pressure was gradually increased to atmospheric pressure was 0.05 per minute. kj9/cm2 (5 kilopascals) to 0.06 kg/cm2 (6 kilopascals) ). Next, remove the lettuce from the vacuum chamber and freezer, and prepare it so that it can be thawed. It was placed on a paper towel in the laboratory. Laboratory temperature was 20°C (68°F).

For comparison purposes, two additional lettuces of the same variety were treated in the same way as a reference standard. was applied. However, one of them was simply placed in the freezer without depressurization or stirring. One was vacuumed and frozen without stirring. The same method was applied in other respects.

Within 10 minutes of removal from the freezer, collect the first reference standard sample (which may involve both vacuum and agitation). (frozen without) showed stains on the towel, indicating cell rupture and damage. second light The test standard sample (frozen under reduced pressure but without stirring) was a stain on the towel. Although it did not show a loss of turgor pressure, it became flexible and resembled seaweed or seaweed.

The remaining sample (frozen with both vacuum and agitation) maintained full turgor pressure after thawing. There was no evidence of leakage of secretions. This time too, the slight JS part of one leaf is clearly scratched. 6 salad bowls that did not recover due to small breaks in the capillaries acting in that area. Tass were selected for the experiment; one was collected from the garden on the day of the experiment and the others were harvested later. At least four days later, I got it from a produce dealer. These three heads are the reduction in Example 2. It was packed neatly into the pressure chamber.

The heads were gradually depressurized and stirred as described in Example 2. Partial vacuum stabilizing After stirring for 45 minutes under the level, it was observed with the naked eye that the heads were not completely frozen. Ta. Stirring was then continued at low pressure for an additional 2 hours and 15 minutes to achieve complete freezing. accomplished.

Gradual recompression could then be carried out over a period of 20 minutes. All 6 heads reduced It was removed from both the pressure chamber and the freezer and placed on paper towels in the laboratory as described above. Reta As the ice thawed, cell secretions were observed leaking from the leaves that were in contact with the sides of the chamber.

The inner leaves showed no leakage.

Heads taken on the day of the experiment returned to total turgor pressure. The head balls that were taken in from the vegetable garden after 4 days are The turgor pressure returned only partially.

A series of 10.2 cm (4 The pot was placed in a vacuum chamber and treated according to the method of Example 2. in one pot One chrysanthemum in the container was slightly higher than the height of the room, so it did not touch the top of the freezer compartment. was.

The final partial vacuum is maintained for 2 hours with agitation, at which time the plants are recompacted. This made it possible to remove the ice from both the room and the freezer and thaw it in the laboratory. within 1 hour All of the plants were completely thawed. The leaves show the same structure and turgor that they had before the experiment. However, the V color was slightly darker. Next, the plants were observed for four weeks in a room. All except the stem that was touching the top of the flower (flowers remain as they are, but buds bloom and grow) continued.

4 Hozuki tomatoes, 3 Beefsteak tomatoes, and 2 Italidents Freshly picked whole tomatoes were placed in the vacuum chamber of Example 2 and treated as described above. . Additionally, 2 Hozuki tomatoes and 2 Italian tomatoes, also freshly picked. was placed on a stirrer and outside the vacuum chamber for comparison purposes. Finally, 4 to 6 days The endive that had been taken in earlier in the day was similarly placed in a vacuum chamber.

The final partial vacuum was agitated for 4 hours due to the high moisture content of the tomatoes. maintained. The vegetables are then recompressed and removed from the room and freezer to thaw in the laboratory. Placed on a paper towel.

As a result of thawing, the control tomatoes (which were agitated but not depressurized) suffered from cell rupture. The skin showed signs of wrinkling and had a soft pulp-like consistency. remaining tomatoes retains its original shape and firmness and retains water droplets on its surface due to sudden exposure to warm air. there was. However, a few of the ripe tomatoes were darker than the first.

Endive showed mixed reactions after thawing. A5 Some leaves did not recover and shriveled, while other leaves returned to full turgor.

At altitudes between 760m and 1220m (2500 to 4000 feet) 4 pieces of Mekinkan lettuce, including 2 grown costicia and 2 red head heads. Processed as described in Example 2. Cell rupture or secretion leakage should not be observed after the ice cubes have thawed. It wasn't noticed. However, the turgor pressure has decreased, and the leaves have rubbery shark tissue and the heads are not visible for the first time. It had a shiny surface.

In addition, 4 pieces of lettuce of the same type were added at a vacuum of -0,77 kg/am2 (-77 kg). Lopascal) but only -0.70! ? /cm2 (0.70 kilopascua”) (217++m+Hg absolute pressure) Ta. These heads return to their total turgor pressure after thawing, and their texture and surface appearance remain the same as before freezing. It was the same as

The above description is for illustrative purposes only. The invention is limited to the features or embodiments described. It is not limited.

Numerous modifications and variations within the scope of this invention will be apparent to those skilled in the art. .

international search report In+s+nmanil＾ppHcallan Na,? CT/82/0 Continuation of 1888 page 1

Claims (9)

[Claims] (1) A method for freezing an organic tissue, comprising: (a) contacting said tissue; A gaseous substance that can be dissolved inside the tissue by lowering the pressure of the air and preventing moisture from evaporating from the tissue. releasing at least a small portion of the substance from the tissue; (b) an organic compound comprising the step of cooling the tissue to a temperature at or below the freezing point of the tissue; How to freeze tissues. (2) According to claim 1, steps (a) and (b) are performed simultaneously. Method. (3) Steps (a) and (b) above are performed simultaneously, and the tissue is stirred at the same time. or vibrated to induce molecular movement within the tissue sufficient to prevent crystal formation by freezing. A method according to claim 1 for inducing. (4) The steps (al and (bl) are performed simultaneously, and the tissue is heated at approximately 25 cycles per minute. The method according to claim 1, wherein the method is simultaneously stirred at a rate of 100 cycles to 100 cycles. . (5) The maximum pressure achieved in the above step (al) is approximately 0.90 kl below atmospheric pressure. lJ/cm” (90 kilopascals) to approximately 0.60 kg/cII]2 (60 kilopascals) 2. The method according to claim 1, wherein: (6) The minimum pressure achieved in the above step (al is approximately 0.80 kg/min below atmospheric pressure) crn2 (80 kilopascals) to approximately 0.65kg/cm2 (65 kilopascals) ). (7) The above step (al is approximately -0.03 kg/an2 (-3 kilopascal) per minute Approximately -0.07kl? Claims carried out at the rate of /era2 (-7 kilopascals) The method according to the first term of the range. (8) The step (a) is -0,05 kg/crrI2 (-5 kilopascals) per minute. Claims carried out at a rate of approximately -0,06 kg/am2 (-6 kilopascals) from The method according to paragraph 1 of the box. (9) At least about half of the gaseous substance dissolved in the tissue is released. The method according to paragraph 1 of the box. fil+ The organic tissue is a vegetable, and the method is performed within about 1 day of intake. A method according to claim 1. αυ　A method for freezing organic tissue, the atmospheric pressure in contact with the organic tissue Force from approximately -0,03 kg/am2 (-6 kilopascals) to approximately -0,07 kg per minute /aT12 (-7 kilopascals) below atmospheric pressure approximately 0.9 kg/an2 (9 Minimum pressure from 0.6 kg/am2 (60 kilopascals) to approximately 0.6 kg/am2 (60 kilopascals) a gaseous substance that dissolves into the tissue with little water evaporation from the tissue. At least about half of the material is released while the tissue is heated to a temperature of about -10°C to about 0°C. and agitating the tissue at a rate of about 25 to about 100 cycles per minute. A method for freezing organic tissue with stages. u21 A method for freezing vegetables, the atmospheric pressure in contact with the vegetables from about -0,05 kg/■2 (-5 kilopascals) to about -0,06 kg/a per minute Approximately 0.8 kg/c+n2 (80 kg) below atmospheric pressure at a rate of n2 (-6 kilobascal) Minimum pressure from approximately 0-65 kg/cm" (65 kio) escal) A gas that can be dissolved in the vegetables without evaporating moisture from the vegetables under low pressure. release at least about half of the material, while heating the vegetable material to about -10°C to about 0°C. The vegetable material is cooled to a temperature of about 25 to about 100 cycles per minute. cooling of the vegetable material, which comprises a step of stirring and is carried out within about one day of the intake of said vegetable material; Method for freezing.