JPS6354681B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for preserving fresh tissue that avoids immune reactions and enables long-term storage and injection transplantation. Since P. Niehans proposed the transplantation therapy of internal glands (P.Niehans: Die endokrinen Dru¨senund).
Surgical transplantation of not only glands but also organs, organs, and tissues has been widely introduced into therapeutic medicine, and is now used to treat the placenta for aging phenomena, leukemia, and cachex levels. The effect of bone graft transplantation has been attracting attention, but the transplantation technique is limited to surgical means of embedding tissue sections within the fascia, and therefore the transplantation effect depends on the freshness and biological activity of the tissue, and the transplantation technique is limited to the surgical method of embedding tissue sections in the fascia. The raw material is limited to fresh tissue immediately after extraction. However, even if the tissue is fresh, it self-dissolves over time, causing tissue protein denaturation and loss of biological activity. Even if heating or culturing methods are used to prevent this, local abscesses often occur and the effectiveness of the transplant is hampered.Furthermore, the antigenic nature of tissue proteins in the mature body is unavoidable, and antibody reactions on the recipient side are unavoidable. Even if pretreatment with immunosuppressants is used to prevent this, it cannot be said that it is always perfect. The expected effects of transplantation can be summarized as maintaining the biological activity of the transplanted tissue and avoiding immune reactions. When using humans as raw materials for transplantation, blood of the same type as the donor body is required to avoid immune reactions. In transplantation of allogeneic isotype, allogeneic homotype, and xenogeneic heterogeneous tissue from other animals, the tissue protein itself is foreign to humans, and the antigenicity of the mature tissue protein is the same as that of the donor. A reaction is inevitable, and even worse, with aggravated sensitization, death from shock is inevitable. Current transplantation techniques mostly rely on cows as transplant sources, except for the heart and kidneys, but it is inconvenient to obtain them at the time of use and there are restrictions on maintaining freshness, so although there is hope for the effects of transplants, it is not carried out. There is. Transplantation method using survivors who were on the verge of death from the nuclear bombing that occurred at the Yugoslav Institute for Nuclear Research (G.
Mathe; Reu France, Etudes Clin, et Biol,
(1959) used Hank's solution (heparin 15
mg/100ml and AB type serum inactivated by heating.
This is a complicated extraction method in which a quantitative amount of bone parasitic cells that have been centrifuged repeatedly in a 15% mixture (15% mixture) is transplanted into a blood transfusion set. The development of transplant therapy is not easy, as a facility such as a blood bank is required to constantly store transplant tissue, and immunosuppressants are required if tissue from a mature body is used as the transplant source. The reason why the transplant material of the present invention was obtained from cow, pig, and sheep fetuses or children less than 4 months old is that the internal and external embryonic mesenchymal tissues and internal glands are morphologically This is because they are immature in terms of function and function, and the antigenicity of tissue proteins lacks the ability to cause antigen-antibody reactions, but on the other hand, they are highly active in cells. In general, immunity is established four months after birth, and while the ability to produce antibodies is lacking during the in utero period or immediately after birth, self-acquired resistance is conferred (PBMedawar: Biological Problem
Grafting:ANN-NYAcad.Sci, 73:539,
1958). The application of fetal and immature animal tissues as transplant sources to humans therefore makes sense in order to avoid immunological reactions. Tissue culture is generally used as a method to preserve the biological activity of tissues, but even so, tissues that undergo autolysis over time, such as the liver, umbilicus, adrenal glands, kidneys, etc., which are particularly rich in enzymes, easily degenerate. In addition, freezing treatment at temperatures below -50°C causes large ice crystals that cluster inside and outside the tissue to damage cells, reduce tissue mass, and reduce biological activity; However, it causes denaturation of tissue proteins, and heat treatment deactivates tissue cells. In the present invention, as a means of preserving the biological activity of fresh tissue, fresh tissue sections are stored at -30°C in a freezing room.
Exposure to freezing and then reduce the pressure to below 30 while reducing the pressure in the freezer compartment.
The condition is that the water in the frozen tissue is gradually sublimated through stepwise low-temperature treatment from -40°C to 40°C, and that the water content is kept at less than 1% of the mass at the end. This technique is different from the freeze-drying method for food vegetables and R. Altmann's method, which dehydrates with sulfuric acid under vacuum and freeze-dries at -20°C.
Elementarorganism und ihre Bejiehangevzu
dev Zellen, 1890), said method results in loss of biological activity of the tissue. In the present invention, the tissue is cut into sections of a certain shape of about 20 x 20 mm in advance, and the tissue is frozen at a temperature of -30°C to maintain homogenization during freezing. The frozen water is sublimated while being exposed to ℃ to prevent tissue cell damage. The resulting dried tissue is easily penetrated by buffer solutions such as Ringer's solution and Tyrode's solution, and becomes a cell suspension, maintaining biological activity at the same level as fresh tissue. Therefore, if a fixed amount of this dried tissue is sealed in an ampoule and the air phase is replaced with an inert gas such as argon gas, it can be stored for a long time, and when used, it can be injected intramuscularly as a suspension in a buffer solution. It has the convenience of being able to easily perform percutaneous intracutaneous transplantation of tissue. Human tissues, including those of animals, contain 60-90% basic water. This salt affects the homogenization of the tissue depending on the freezing temperature and time. If the freezing time is prolonged, the salt concentration will increase, and the ice crystal particles generated inside and outside the tissue will become larger, damaging cells and preventing tissue homogenization.In addition, in order to shorten the freezing time, rapid freezing is performed at a temperature below -45℃. As expected, large ice crystals grow in clusters, leading to tissue degeneration. In this invention, based on repeated experiments, the optimum temperature for preventing the clustering of large ice crystals and obtaining a homogenized freeze-dried product is below zero.
The temperature must be 30℃, the frozen moisture must be sublimated to less than 1% of the mass, and the optimum temperature that does not cause tissue degeneration or cell damage is -40 to -40℃ in a vacuum.
It turned out to be 45°C. In other words, it is possible to maintain tissue cell activity even when 99% of the water contained in fresh tissue is sublimated, and the yield rate is 1/15 of that of raw tissue.
Based on this experimental knowledge, the object of the present invention could be obtained by the following method. Example Strong disease resistance of sheep and its tissue. Taking advantage of the fact that allergic reactions are negative during human transplantation, lambs around one month old are decapitated to death under anesthesia, the brain cortex is removed from the head, and the heart, liver, kidneys, placenta, etc. are separated by abdominal surgery. Each removed tissue was thoroughly washed with steam, and adhesion and blood sludge were removed with 3% sodium citrate solution. Sterilized with formalin aerosol, cut into 2mm x 20mm thick sections using a microtome, and separated into flasks for each tissue. After each flask was immersed in a dry ice or ethyl alcohol bath for 5 to 10 minutes to rapidly freeze it, it was placed on the shelf of a small electric freezer directly connected to a two-stage rotary vacuum pump and heated to -30°C. Operate the vacuum pump inside the frozen freezer.
Depressurize at 10 ^-2 mmHg pressure for 30 minutes and then reduce the pressure to -40°C to 45°C.
The pressure was reduced at ℃ for 30 minutes until the moisture content of the frozen tissue was 1% or less. The dried tissue thus obtained was transferred to a stoppered bottle or glass ampoule, and the empty phase was replaced with argon gas and sealed.
A day later, the package was opened and 100 mg of the solution was mixed with 1 ml of Ringer's solution to form a cell suspension within a few seconds, and when this was aspirated with an intramuscular injection needle, the entire amount could be aspirated. 100 mg of this dried tissue is equivalent to 1.5 g of fresh tissue, and its physicochemical properties are generally as follows. Physicochemical properties It is a yellowish-gray, brittle, porous dry tissue with a certain degree of humidity.It becomes soft in air in a few minutes, and permeates into a buffer solution in a few seconds, exhibiting a cell-suspending state. No denaturation of tissue proteins is observed, and it is heat resistant, and ultraviolet spectrometry shows high absorbance in a specific region of the ultraviolet region, indicating the presence of nucleic acid salts, which are cellular constituents (PMWalker;
General Cytochemical Methods 1, p-166,
Relationship between cell constituent substances and ultraviolet absorption values, 1958). A microscopic view of the tissue fixed with liquid paraffin and 96% ethanol and stained with hematoxin showed a cell composition comparable to that of fresh tissue, with ice crystal particles scattered in the interstices of the tissue, but no cell damage was observed. The total protein in any dry tissue is 60-85% of the tissue equivalent;
Free nitrogen shows a range of 40-60%. Even if the total protein contained in 100 mg of this dry tissue is 80 mg, it is a much smaller amount compared to the 200 to 1000 mg of serum protein in the tetan vaccine, and it is not antigenic enough to cause an antigen-antibody reaction like serum protein. . This dried tissue maintains the same level of biological activity as fresh tissue before freezing treatment, as evidenced by cholinesterase activity and peptidase activity, and the amino acid group leutin, phenylalanine, methionine, glutamic acid, arginine, Alanine, tyrosine, threonine, glycine, aspartic acid,
It can also be identified by detecting cystine, etc. Ketosteroid hormones are detected in dried tissues of the adrenal glands and placenta, but the amount ranges from 5 mg/100 to 8 mg/150, which is far lower than the pharmacodynamic potency of 17-ketosteroids. , glycogen, and lipidoids are detected, indicating a cellular composition that is generally identified as fresh tissue. Chemical analysis value of dried lamb organ tissue according to the present invention: value equivalent to 100 mg of tissue

【table】

[Table] Composition ratio (%) of protein albumin and globulin in the same as above

[Table] The above example is a laboratory example, but for industrial use, as shown in the figure, stainless steel is used with several flask shelves 2 inside and a heat pipe 3 installed under the shelf to facilitate heat exchange. A series of freeze-drying equipment is used in which a freezing chamber 1 and a smaller ventilation chamber 4 are directly connected, and a pipe 7 for a cooling gas generator 6 (F22 Freon refrigerant) and a decompression pipe 8 are placed inside the ventilation chamber. Paste the tissue sections on the inner wall of the flask and place them on a shelf in a stainless steel freezer, avoiding stacking them on the bottom.
A commercially available electric refrigeration gas generator with the ability to generate gas at -60°C using F22 as a refrigerant is driven to first fill the freezing compartment with cooling gas at -30°C for about 60 minutes through the ventilation chamber. Freeze the flask on the shelf and drive the vacuum pump 5 to achieve a temperature of 10 ^-2 mmHg.
The internal pressure of the ventilation chamber and freezing chamber is brought to a vacuum state by pressure,
Next, the cooling temperature is set to -40°C and the same treatment is performed for 90 minutes to sublimate the water content of the frozen tissue while preventing the outflow of cell constituents of the tissue section on the inner wall of the flask. When the dry tissue obtained at the end of this process is measured according to the moisture measurement method of the Japanese Pharmacopoeia General Tests, the humidity is 1%.
The following is shown. Its physicochemical properties are identical to those obtained with laboratory treatments.

[Brief explanation of the drawing]

The figure is a sectional view of the frozen tissue dehydration device according to the present invention. 1 is a stainless steel freezer compartment 2 is a flask shelf 3 for freezing and dehydrating fresh tissues; a heat pipe 4 is a ventilation chamber;

Claims (1)

[Claims] 1 Tissues of organs, organs, glands, reproductive organs, placenta, and arterial ducts of sheep, pigs, and cow fetuses or children under four months of age, washed with sodium citrate solution and sterilized with formalin aerosol. Freeze the tissue, then lower the temperature stepwise under reduced pressure to lyophilize it, sublimate it until the water content is less than 1% of the dried tissue, transfer a certain amount of it to a light-shielding container, and fill the empty phase of the container with an inert gas. A method of preserving fresh tissue that allows for intramuscular injection and transplantation as a suspension in the buffer solution at the time of use.