RU2033793C1 - Method of producing preparation having properties of bone marrow - Google Patents

Abstract

FIELD: biology. SUBSTANCE: coagulated blood is washed against plasma by physiologic salt solution, poured by 5 % solution of sodium citrate, allowed to stand within about 12 h and is then centrifuged. After complete hemolysis water is added into thus prepared precipitate and additional centrifugation is carried out. Hemolyzate is separated and poured by acetone. Thus prepared mixture is centrifuged, liquid is separated and precipitate being washed by acetone several times. Then centrifugation, filtration and drying are carried out. Thus prepared dry powder is cherry-colored. Modification of proposed preparation which has no antigen properties also may be prepared. Said process is carried out by adding concentrated hydrochloric acid into precipitate before its filtration and drying. Quantity of said acid is 0.1 of volume of acetone. Thus prepared mixture is allowed to stand within few minutes and then it is centrifuged. Thus prepared precipitate is washed several times by acetone, filtered and dried. Desired powder has slight grey color. EFFECT: improves efficiency of the method. 2 cl

Description

 The invention relates to biology, in particular to the processing and preservation of blood and bone marrow. The proposed method can be the basis for the production of drugs necessary for the treatment of people and animals affected by radiation sickness, and replacing the bone marrow, currently received from donors.

 In modern biology, methods of long-term preservation of bone marrow at moderately low and ultra-low temperatures in preserving glycerin-containing media have been tested and put into practice.

Known methods for processing and preservation of living cells, including bone marrow, deep freeze protected cryoprotectant freezing at a rate of 0.1-0.2 sec to a temperature ^of -196 C (SU, 1144673 N, N 1192828, cl. A 61 K 35/16 and others.). These are the most modern and advanced methods of preservation, allowing you to store bone marrow for a long time (up to 20 years).

 However, all known methods are complex and time-consuming, require expensive equipment and do not allow the storage of a sufficiently large amount of bone marrow. In addition, in bone marrow transfusion, in most cases, suppression of the recipient's immunological response is required.

Biocryogenic methods also have other disadvantages:
required the presence of liquid nitrogen, which greatly limits the regional application of these methods;
the survival rate of cryopreserved bone marrow is known to be very limited;
diglycerization of thawing bone marrow cells is also a complex and time-consuming process that inhibits the use of cryogenic canning.

 As a prototype, the patent PCT / US 87/00869, A 61 K 35/28, from 15.04.87, is considered.

 The principal feature of the proposed method is that to preserve the biological activity of the drug, coagulated blood is washed with saline, kept in a nuclease inhibitor and only after that further processing is used.

 In general terms, the procedure for obtaining a dry preparation is as follows.

 They take coagulated blood, wash it off from the plasma with saline, fill it with 5% sodium citrate (which is an inhibitor of nucleases) and incubate for about 12 hours; centrifuged and water was added to the resulting precipitate; after complete hemolysis, centrifuged again; the resulting hemolysate is poured with acetone and after shaking the mixture is centrifuged; the last operation is repeated several times, changing acetone; then, pure concentrated hydrochloric acid is added to the precipitate, again diluted with acetone, with continuous stirring in an amount of 0.1 of the volume of acetone and incubated for several minutes, centrifuged and the resulting precipitate is washed several times with acetone, then filtered and dried. As a result, a whitish-grayish powder is obtained. It can be stored for a long time at room temperature in a dark container.

 In addition, there is the possibility of preparing the specified drug individual for each person from his blood. In this case, treatment with hydrochloric acid is not provided. The resulting variety of the drug has a dark cherry color.

 If the drug obtained by the proposed method is mixed with Ringer-Locke solution, then after a few hours the growth of blood cells begins.

PRI me R. To 0.5 ml of twice-washed saline from plasma of erythrocyte mass obtained from clotted blood, 2.5-5 ml of 5% sodium citrate solution is added. Allow to stand for 12 hours at a temperature of + 3 + 5 ^C. centrifuged, the centrifugate is settled and to the residue was added 3 ml of water and left to stand until complete hemolysis. Centrifuged again, the clear hemolysate is sucked off. A triple volume of acetone is added to the hemolysate, shaken thoroughly and centrifuged again. The precipitate formed is washed twice with the same amount of acetone, followed by centrifugation. After the third wash, 5.4 ml of acetone was added to the precipitate, agitated and 0.6 ml of pure hydrochloric acid was added dropwise. Shake the contents of the tube after each drop. After 3-5 minutes, a saturated red precipitate falls to the bottom, the supernatant has the same color. It is centrifuged, the centrifugate is sucked off, the precipitate is washed several times with acetone until the supernatant ceases to stain. Filter through a paper filter. The filter cake was washed two or three times with acetone and dried in air. The dried preparation is stored in a dark glass dish with a ground lid.

 The ability of the drug to form cells is carried out in a micro camera under a microscope. For this purpose, 2-3 grains of the preparation are placed on a glass slide, onto which one or two drops of a Ringer-Locke solution or saline solution are applied and closed on top with a cover glass, the dimensions of which are slightly smaller than the glass slide. The glass slide is slightly pressed so that there are no air bubbles under it. To prevent drying out, the edges of the formed microcamera are lubricated with sterile liquid paraffin with a glass rod. With microscopy, after an hour or two, the formation of red blood cells, and even later bone marrow cells and even its pulp, as well as the embryos of blood vessels according to the embryonic type of hematopoiesis, is observed.

 The viability of the preparation obtained 10 years ago was periodically checked, and it did not lose its biological properties for the formation of blood cells in vitro.

 The possibility of obtaining from the blood of humans and animals, including a peripheral, dry preparation capable of long-term storage and possessing bone marrow properties when mixed with Ringer-Locke solution, provides medicine with great prospects. The drug can serve as the basis for the development of a new method of treatment of radiation sickness, some anemia and other diseases.

Claims (2)

 1. METHOD FOR PRODUCING A PRODUCT HAVING BONE MARROW PROPERTIES by treating peripheral blood of mammals, characterized in that the red blood cell mass is washed with physiological saline, sodium citrate is added, incubated, centrifuged, water is added to the precipitate, centrifuged, acetone is added to the supernatant, acetone is added to the supernatant. containing the desired product, washed with acetone, filtered and dried.  2. The method according to claim 1, characterized in that the precipitate after washing with acetone is diluted with acetone and concentrated hydrochloric acid is added.