CONSERVATION OF BODIES AND FABRICS (Latin conservare to store, keep) — methods of impact on the part (fabric, body, an extremity, a complex of bodies) isolated from a complete organism the physical, volumetric and biological factors allowing to keep its viability and full function during considerable life time out of an organism; it is used with the replaceable purpose at recovery operations.
To. lake and t. is an auxiliary action and a stage of organ and tissue transplantation (see. Transplantation ). For the first time conservation of the whole body (heart) A. Vyulpian in 1852 carried out, a podshivaniye of tinned body (a rat tail) — Bør (P. Bert, 1882), successful change of tinned blood vessels in an experiment — A. Karrel (1908). In clinic conservation of fabrics is the broadest (cornea) it was used by V. P. Filatov (1934). To. lake and t. it is eurysynusic in the surgical institutions which are engaged in organ and tissue transplantation.
The termination of delivery of oxygen and nutrients coming after a stop of blood circulation differently affects various bodies: one quickly perish, others — continue to remain viable. The bodies and fabrics keeping funkts, ability during certain time after their isolation from a complete organism are called perepaivayushchy bodies and fabrics.
E.g., after the termination of blood circulation the brain perishes in 5 — 6 min., a liver in 20 — 30 min., kidneys in 40 — 60 min., heart in 60 min., and the extremity can remain viable in these conditions within 4 — 6 hour. Cells of an organism possess a nek-eye a reserve of nutrients, but have no reserves of oxygen therefore in disturbance of their life activity a paramount role is played by an anoxia. For lack of oxygen energy demands of cells become covered due to oxygen-free splitting of glucose that leads to accumulation of nedookislenny products (milk to - you, etc.) and to acidulation of internal environment of fabric or body (see. Acidosis ). At acidosis cellular membranes are damaged, intracellular structures are broken, the enzymes causing self-digestion of cellular structures are released (see. Autolysis ), what leads to death of fabric or body.
Apply two types of influence to reduction of damages to the isolated fabrics and bodies: replaceable conservation (artificial maintenance of a metabolism by delivery of oxygen and nutrients) or the conservation suppressing a metabolism (artificial decrease in level of exchange processes, and consequently, and oxygen requirement, nutrients etc.). At To. lake and t. sometimes use a combination of both types of influence.
Bodies and fabrics depending on their role in an organism conditionally divide into two groups: gomovitalny and gomostatichesky. Gomovitalny transplants shall keep motor or secretory performance, and also ability of cellular elements to reproduction, i.e. shall be active. Gomostatichesky transplants perform basic or cover function, and preservation of life activity of their cells is not obligatory. Therefore requirements to conservation of various transplants different. It is natural that conservation of gomovitalny transplants shall be more physiologic, than gomostatichesky.
To. lake and t., as a rule, has no significant effect on easing immunol, incompatibility of the kept body or fabric as incompatibility is defined eventually by distinction of ancestral (genetic) features (see. Incompatibility immunological ). However it allows to have time from the moment of receiving a transplant before change, for a cut it is possible to make selection of the most compatible recipient that increases probability of a favorable result of transplantation. In some cases at conservation of gomostatichesky transplants (skin, heart valves) in solutions of aldehydes (formaldehyde, glutaraldegid) decrease in manifestations immunol, incompatibility due to reduction in transplants of amount of water-soluble proteins and the effect of «masking» which is caused chemical communication of aldehyde with proteinaceous components of fabrics is noted.
Departments To. lake and t. or the institutions having a stock of tinned transplants (bodies and fabrics) typed on immunol, to factors of compatibility, and making exchange of them with others to lay down. institutions as within the country, and beyond its limits, are called banks of fabrics and bodies.
Conservation allows to make bodies and fabrics, to create stocks of transplants in bank of fabrics for use them as required, to make exchange of transplants between various medical institutions, choosing the most suitable on immunol, compatibility for the recipient who is sometimes for many hundreds of kilometers from the place of receiving a transplant. Preserve such bodies as heart, blood vessels, lungs, a trachea, a liver, kidneys, endocrine glands, intestines, extremities, teeth, marrow, joints and fabrics — blood, skin, a bone, a cartilage, a fascia, a cornea, a sclera, a pleura, a pericardium, a peritoneum, heart valves, nerves, sinews, muscles, a wall of a bladder, an urethra, a firm meninx.
Preparation of transplants is carried out in court. - medical morgues or other medical institutions from the died people, and also from animals on slaughterhouses or meat-processing plants. Removal of organs is made the first 20 — 30 min. after ascertaining of death of the donor, and fabrics — in the first 2 — 24 hours. There are two ways of preparation of transplants: 1) in the conditions of strict asepsises (see); 2) without strict observance of rules of an asepsis. The way of preparation in the conditions of a strict asepsis is most widespread. At the same time all manipulations are carried out in the special operating room with use of sterile tools and linen. At preparation of basic and cover fabrics sometimes apply the second way. At the same time transplants after preparation will sterilize solutions of antiseptic agents, antibiotics or subject to gamma irradiation.
Ways of conservation of bodies and fabrics depend on a type of impact on a transplant.
1. Replaceable conservation: 1) biological perfusion; 2) normotermichesky hardware perfusion.
2. The conservation suppressing a metabolism: 1) cooling up to the temperature close to 0 degrees, in fluid salt mediums or in other liquids without nutrients: a) hypothermal perfusion, b) flowing method, c) besperfuzionny method; 2) cooling in firm environments up to the temperature close to 0 °; 3) freezing at a temperature below 0 °: a) at a temperature from 1 to — 50 °, b) at a temperature from — 51 to — 100 °, c) at a temperature from — 101 to — 273 °, d) freezing with drying (see. Lyophilizing ).
3. Combined (replaceable and suppressing a metabolism) conservation: 1) hypothermal perfusion by plasma substituting solutions; 2) a hypothermia in combination with hyperbaric oxygenation (see); 3) combination of a hypothermia, hyperbaric oxygenation and perfusion; 4) besperfuzionny cooling up to the temperature close to 0 °, in the fluid mediums containing blood, plasma, sugar, ATP, etc.
- 1 Replaceable conservation
- 2 The conservation suppressing a metabolism
- 3 The combined methods of conservation
- 4 Conservation of marrow
Biol perfusion — use in an experiment by V. P. Demikhov (1963, 1970) of the isolated heart-lung preparation or a cordial and visceral complex for preservation of the bodies entering them, and also extracorporal connection of bodies to vessels of the recipient or the intermediate donor — is a kind of this way K. lake and t. Biol, perfusion differs from usual blood supply of body only in the fact that the transplant is out of an organism; at the same time sterile thermostatic cameras are necessary for the kept bodies and the respiratory device for ventilation of the lungs in heart-lung preparation. During the use of heart-lung preparation without replacement of blood heart is reduced no more than 2 — 2,5 hours, and in case of periodic replacement to the circulating blood — 5 — 8 hours Lavender et al. (1966, 1968) at extracorporal connection of a kidney to the patient in some cases managed to support its viability up to 25 days. Use of heart-lung preparation for To. lake and t. did not find application in clinic in view of the difficulties of moral and ethical and legal character connected with withdrawal of a complex of bodies from a corpse and their «revival» yet. Besides, there are difficulties in management of breath, blood circulation and in maintenance of constancy of internal environment. Extracorporal connection of bodies also so far has limited use in view of organizational difficulties. Besides stay at the intermediate donor can strengthen immunol, incompatibility of tinned body at the expense of the antigens adsorbed by body at conservation.
Normotermichesky hardware perfusion differs from the previous type of conservation in the fact that blood circulation in the isolated body is carried out by means of the device artificial circulation (see). As an example it is possible to refer to widely known case of perfusion of the isolated head of a dog by means of an avtozhektor of a design of S. S. Bryukho-nenko (1930). At this way it is possible to keep viability of heart and liver during 3 — 6 hours, kidneys — to 3,5 days. Difficulties in maintenance of constant composition of perfused blood, gradual accumulation in blood of end products of exchange, destruction of erythrocytes in the cardiopulmonary bypass are shortcomings of this method of conservation.
The conservation suppressing a metabolism
the conservation Suppressing a metabolism includes the most extensive group of the methods based by hl. obr. on protective action of cooling or freezing. Options are ways K. lake and t. cooling in fluid salt mediums up to the temperature close to 0 °. Hypothermal perfusion by salt solutions by results concedes to hypothermal perfusion with belkovosoderzhashchy solutions because of approach of more expressed hypostasis of body a little, however allows to keep kidneys to the 24th hour. The flowing method differs from hypothermal perfusion in the fact that circulation of liquid is carried out not through a blood channel, and there is 'a washing of a transplant outside counting on diffusion processes through fabrics; this method of conservation is intermediate between perfused and besperfuzionny methods. The flowing method is applied for To. lake and t. and closed glands. Installation is mounted in the household refrigerator and adjusted on course of several drops of liquid in a minute. Terms of conservation by this method from 1 to 4 weeks.
Besperfuzionny cooling in fluid mediums up to the temperature close to 0 °, thanks to the simplicity and availability has quite broad use at To. lake and t. For increase in time of conservation of a kidney the metabolism in it is suppressed, except decrease in temperature, also other influences: use moderately giperosmotichny solutions causing braking of diffusion processes; apply the hyper potassium solutions which are artificially leveling transmembrane ionic gradients and saving the energy spent for operation of cellular «pumps»; add to composition of washout solutions of substance with antioxidant properties (gamma and hydroxy-butyric to - that, etc.). At conservation by a besperfuzionny method remote kidneys wash the cooled solution under pressure of 60 — 80 mm of mercury. through an artery before emergence from a vein of the pure flowing liquid. On washing of one kidney 600 — 1000 ml of perfusate, duration of washing of 2 — 5 min. are required. Washout solution represents or isotonic solution of sodium chloride with addition 0,5 — 1 ml of heparin and 5 — 10 ml of 10% of solution of novocaine for 1000 ml of liquid, or the special preserving solution (giperosmotichny, hyper potassium, with antioxidants). After washing of a kidney place in a dvukhstenny container (fig.) where temperature from 0 to 2 ° is maintained, and bring to clinic in the location of the recipient. Kidneys at the same time remain till 18 — 22 o'clock.
For To. lake and t. besperfuzionny cooling in fluid mediums use a large number of various liquids: simple chemical environments (isotonic and hypertensive solutions of sodium chloride, Tirod's liquid, Ringer's solution — Locke, water solutions of tetraethyl-diamino-triphenyl-carbohydride sulfate, weak solutions carbolic to - you, solutions of chloroamine, a mertiolat, tsialit, Diocidum, beta propiolactone, Furacilin, formaldehyde, a glutaraldegid, alcohol, the SKTN synthetic rubber); difficult chemical environments (Hanks's solution, L-6 solution with sodium citrate, triflaviny etc., buffer fiziol, solution with antibiotics, glyukozo - citrate and penicillinic solution, frigostable circles of Belyakov — the 4th, 31st, 31-zh, the 32nd, FS solution with lemon to - that and Rivanolum, TsOLIPK solution, solution with sodium lactate, Fedotenkov's solution, Beringer's circle with antihistaminic drugs, K G-2 LKG-1 and L solutions with gelatinous and heparin, Gelatinolum solution etc.). Conservation of fabrics besperfuzionny cooling in fluid mediums is carried out in the household refrigerator in usual glasswares. The transplants preserved on this way can be stored of 1 week to several months. Separate ways K. lake and t. (0.1 — 0,2% solutions of formaldehyde) fabrics up to 3 years allow to keep. So long terms of conservation during the use of solutions have a talk with formaldehyde chemical communication of the hydrated formaldehyde with enzymes, leads edges to blocking of their activity and thus stops process and satisfy for. To shortcomings To. lake and t. in fluid mediums refer need of periodic replacement of the preserving solutions, inconvenience of transportation in view of fragility of a container.
These shortcomings the way of conservation in firm environments is deprived. Sterile fabric transplants (preferential bone tissue) fill in with fusible fractions of paraffin (use paraffin with a chemical formula C 20 H 42 to C 23 H 48 , with t pl 42 °). Paraffin gidrofoben, does not get into fabrics of a transplant, does not exert on it an adverse effect. About a top the purpose apply also high-polymeric pitches. Duration of safety of fabrics in firm environments of 2 weeks up to 6 months.
Freezing found application generally for conservation of fabrics. Conditionally distinguish three modes of freezing: slow (from — 1 to — 50 °), bystry (from — 51 to — 100 °) and sverkhbystry (from — 101 to — 273 °). At slow freezing because of formation of a small number of crystallization centers in fabrics there are large crystals of ice which damage fabric structures, at the same time activity of some enzymes remains, so, process of an autolysis though slowly, but continues. At snap-freezing in liquefied gases (acetylene, ammonia, pro-tannins, hydrogen sulfide, ethylene, etc.) in fabrics small crystals are formed, enzymes and catalysts stop the activity. Supersnap-freezing in liquefied gases (oxygen, nitrogen etc.) leads to a vitrification (ostekleneniye) of fabric water and to the maximum safety biol, structures. Freezing to conservation of bodies (except for marrow) is not applied since crystals of ice damage internal structures of gomovitalny transplants, and it is not possible to carry out supersnap-freezing of the whole body in view of difficulty of receiving simultaneous uniform cooling of a massive transplant. At conservation of a bone tissue use preferential the mode from — 25 to — 35 ° as the most economic. Shelf-lifes at the same time — from several months to 1,5 years. To. lake and t. freezing carry out generally in large medical institutions since special refrigeration units, the service equipment and liquefied gases for this purpose are required.
A kind of conservation by freezing is lyophilizing (see). Process of drying is carried out from the frozen state at depression of 0,0015 atm during the 24th hour. The dried-up transplants (skin, bones, arteries) soldered in glass vessels can remain at the room temperature. Use of this method demands, except refrigeration units, existence of the equipment for evacuating and soldering of transplants; the method also does not allow to keep massive bone transplants since their drying and soldering presents great technical difficulties. Owing to this fact limited use for conservation of fabrics has lyophilizing.
The combined methods of conservation
the Combined methods of conservation were widely used more for preservation of bodies. They are based on a combination of the replaceable and suppressing a metabolism actions. Decrease in level of a metabolism at these methods is carried out by cooling or use pharmakol, means. Theoretical premises of use of cooling for preservation of bodies and fabrics in the isolated state is Vant Hoff's phenomenon, according to Krom decrease in temperature on 10 ° reduces the need of fabric for oxygen twice, and at t ° 2 — 4 ° oxygen requirement makes 4 — 5% of initial level. One of types of a combined method To. lake and t. — hypothermal perfusion; it is carried out by special devices with use of divorced blood, oxygenic plasma or serum, mix of plasma or serum with Hanks or Ringer's solutions (1: 4). This way allows to keep kidneys at t ° 10 ° to 48 hours, and in some cases to 5 days.
At a hypothermia in combination with hyperbaric oxygenation (supertension of oxygen to 4 — 5 atm) a replaceable role is played by excess of oxygen which gets into fabrics and causes oxidation of nedookislenny products of exchange; cooling in turn reduces the level of a metabolism. The special refrigerator capable to sustain supertension, cylinders with oxygen, regulators is necessary for this method: pressure and temperatures. Duration of conservation makes this way for a kidney of 24 — 48 hours, for heart 12 — 24 hours. Similar results are yielded by a combination of a hypothermia, hyperbaric oxygenation and perfusion. In view of complexity and lack of notable advantages before hypothermal perfusion the methods including hyperbaric oxygenation were not widely adopted.
The kind of a combined method can be counted up also besperfuzionny cooling in fluid mediums to temperature close to 0 °, in the presence of nutrients. For this purpose apply biol, Wednesdays (blood or plasma of the donor, blood or plasma of the recipient, ascitic liquid, embryonal extract), foodstuff (solutions of glucose, bee honey, boiled milk, fish oil, egg yolk or white), synthetic environments (the environment 199 — sugar, vitamins, purines, pyrimidines, ATP etc.; salt solutions with serum). Above-mentioned ways are applied generally to preservation of fabrics and have no essential advantages before To. lake and t. in solutions without addition of nutrients.
Sterilization. Ensuring sterility in process To. lake and t. it is reached by strict observance of rules of an asepsis at all stages of storage (sterile ware, the equipment, solutions etc.), and also processing in the course of storage and before transplantation by antiseptic agents (formaldehyde, glutaraldegid, beta propiolactone, ethylene oxide, alcohol, Furacilin, Rivanolum, etc.) or antibiotics (penicillin, streptomycin, tetracycline, Monomycinum, hlorotsid, etc.). Crops of pieces of transplants on a medium for bacterial, researches are made for control of sterility in the course of storage and before transplantation.
Control of the validity of tinned bodies and fabrics is carried out by various methods. Conditions To. lake and t. at first are fulfilled in an experiment on animals and only then are implemented into a wedge, practice. At both stages — experimental and clinical — various researches are conducted: morphological (usual and submicroscopy, vital and supravital coloring, luminescent microscopy, autoradiography, gistokhy, research of enzymes); Electrophysiologic (definition of conductivity, electric resistance); biochemical (studying of activity of tissue respiration by means of the device of Warburg or the polarograph, a condition of activity of enzymes of anaerobic glycolysis, inclusion in exchange of isotopes, studying of synthesis of DNA, definition of activity of a dehydrogenase by means of salts of tetrasol, etc.); researches of culture of fabrics in a medium; a research of specific function of bodies at recovery of blood circulation (for a kidney — release of urine, regulation of an electrolytic condition of blood, release of indigo carmine; for a liver — a bile production, synthesis of a glycogen and urea; for an extremity — safety of electro-excitability of muscles etc.); research funkts, abilities of body by control of terms of survival of animals recipients. Funkts, control methods of the validity are the most reliable and have a principal value for check of gomovitalny transplants. Morfol., elektrofiziol, and biochemical, researches have auxiliary character since the results received at their use reflect the private parties of manifestation of life activity.
The generalized data according to the characteristic of the main methods K. lake and t. are provided in the table.
Conservation of marrow
Ways of conservation
For conservation of marrow use two ways: storage in fluid mediums at positive temperatures (2 — 5 °), than is reached decrease in intensity of exchange processes in cells; freezing and storage of marrow at low and ultralow temperatures (— 70 — 196 °) that leads to considerable decrease or full suppression of exchange processes.
Conservation in fluid mediums at positive temperatures. It is known that morfol, full value and funkts, activity of cells of marrow remain in salt solutions at t ° 2 — 5 ° during 2 days, and in environments for culture of fabrics — 5 days. In the Soviet Union the greatest use in a wedge, practice was received by the preserving solutions developed in TsOLIPK by A. G. Fedotenkov and soavt. (1962) and in LIPKT. K. Mamysheva (1965) which basis is made by carbohydrates, proteinaceous components and anti-coagulating substances (trisubstituted sodium citrate, EDTA Na2), necessary for maintenance of exchange processes in cells, for creation of normal osmotic conditions and prevention the phenomenon of coagulation.
The preserving TsOLIPK solution No. 3 consists of two mixes: mixes No. 1 (sucrose — 4,3 g, glucose — 0,4 g, EDTA Na2 — 0.1 g, the water bidistilled — to 50 ml) and mixes No. 2 (trisubstituted sodium citrate — 1 g, 10% solution gelatin, prepared according to GF IX — 25 ml, the water bidistilled — to 50 ml). The mixes No. 1 and 2 poured in bottles for capture of blood will sterilize separately in the autoclave of 30 min. with a pressure of 1,2 atm. Before use of mix merge in the closed way with observance of rules of an asepsis. On 100 ml of marrow 100 ml of the preserving solution undertake (a ratio 1: 1).
The preserving LINDENS solution K contain 3 g of neutral citrate, 3,2 g of sucrose, 25 ml of 10% of solution gelatin, 0,89% of NaCl solution to 100 ml. Solution will be sterilized in the autoclave of 30 min. with a pressure of 1,2 atm. A ratio of marrow and the preserving solution 1:1. The marrow prepared on the above-stated solutions keeps morfol, full value and funkts, activity of myelocariocytes at t ° 2 — 5 ° during 5 days. Before use the marrow preserved in one of solutions is maintained one hour at the room temperature for fluidifying gelatin which is a part of solution. With lengthening of shelf-lifes of marrow the number of its yadrosoderzhashchy elements decreases and the number of dystrophic cells accrues. Megacaryocytes, segmentoyaderny neutrophils, then undifferentiated cells and unripe elements of a granulotsitarny row are the least steady. Lymphocytes and head elements of an erythrogenesis are steadiest. Stem hemopoietic cells are unstable in the course of conservation. The researches of viability of stem cells of marrow using a method of cloning of the hemopoietic fabric conducted by Till, Mack-Kallaky (J. E. Till, E. A. McCulloch, 1961), showed that best of all they remain (80 — 70%) within a day at t ° 2 — 5 ° in the environment of No. 199 and Hanks's solutions and TsOLIPK No. 3. Sterility of marrow is reached in the conditions of the most strict observance of an asepsis (the operating room, sterilization of air of the operating room, special clothes for the donor and personnel). Capture of marrow at donors is carried out by means of the syringe and Kassirsky's needle by multiple punctures of ileal bones and a breast (see. Sternal puncture , Trepanobiopsiya ). From corpses preparation of marrow is made by its aspiration in the closed system or washing away by means of perfusion of the preserving solutions. Preparation of marrow from the corpses which are at the room temperature shall be made in the terms which are not exceeding 5 — 6 hours from the moment of death. For preparation, storage, transportations and transplantations of marrow use standard glass bottles on 250 — 500 ml and plastic bags. Bottles with marrow cork a rubber bung and a metal cap which zavaltsovyvatsya around a neck. Plastic bags provide the best sealing since output tubes are soldered.
Conservation at negative temperatures
as a result of the researches directed to research of methods of long conservation of marrow the scientist of the different countries in the 50th 20 century were succeeded to find ways of protection of cells of a brain against destruction by means of the protecting substances, selection of the corresponding cooling rates and an otogrev and to reveal storage conditions. 2 groups of the substances having properties of protection of cells of marrow during the freezing are known. The first group includes the substances getting into a cell and possessing intracellular action (glycerin, a dimethyl sulfoxide, glucose, etc.), the second group — the substances which are not getting into a cell and possessing extracellular action (polyvinylpirrolidone, poly(ethylene oxide), a dextran, hydroxyethylstarch, gelatin, albumine, sucrose, etc.). During the freezing of marrow of the person the most valuable were 15% solution of glycerin and 8 — 10% solution of polyvinylpirrolidone. Protective action of glycerin is based on property to get into a cell, to create strong bonds with water thanks to what the percent of the freezing water and degree of salt content in a cell decreases. Its addition promotes overcooling of fabrics and exerts impact on inside - and extracellular crystallization, slowing down the crystal growth of ice. Polyvinylpirrolidone does not get into a cell. It envelops a cell, preventing her dehydration, forms bonds with the amino-acid remains of proteins, water molecules and electrolytes, lowering their concentration in the extracellular environment. Researches on studying of influence of the modes of freezing and cooling rates of marrow at separate stages showed that all process of freezing of marrow to t ° — 196 ° can be divided into 3 periods: the first period is time, during to-rogo there is a cooling of marrow from 15% with glycerin and 10% with serum from initial temperature prior to crystallization with a speed which is not exceeding 3 ° in 1 min.; the second period — crystallization of marrow within no more than 1,5 min.; the third (crystallization is finished) — comes final freezing of a bioproduct with a speed of 15 — 30 ° in 1 min. at a temperature from — 11 to — 40 °, with a speed of 5 ° in 1 min. at a temperature from — 40 to — 196 °. Slow freezing of marrow from 15% glycerin adversely affects erythrocytes. Hemolysis can be prevented by the maximum removal of erythrocytes from marrow that is reached by sedimentation them by means of present at TsOLIPK solution No. 3 gelatin. The bottle containing marrowy cells is centrifuged within 15 min. at 1200 rpm. The most part nadosadochny a layer is deleted, leaving 100 ml of a suspension in a bottle. The cells of marrow which settled at the bottom of a bottle carefully resuspend hashing. Further add the equal volume of the nadosadochny liquid containing 30% solution of glycerin and 20% of blood serum of the AB (IV) group to this volume of a marrowy suspension. Then the suspension of cells of marrow is spilled through system for hemotransfusion (with the filter) on 100 ml in standard aluminum containers with a capacity of 160 ml. Exposure of marrow from 15% glycerin before freezing shall be 30 min. Freezing of marrow is made according to the three-stage program: at the first stage (t ° from 10 to — 17 °) the camera of program freezing «the KV 1501I O» is cooled with a speed of 1 ° in 1 min., at the second stage (t ° from — 17 to — 120 °) within 3 min. the maximum supply of vapors of liquid nitrogen in the camera is made; at the third stage (t ° from — 120 to — 196 °) cooling rate of the camera makes 10 ° in 1 min. The three-stage program of freezing is reproduced by means of a special disk. It provides preservation of 90 — 85% of myelocariocytes and apprx. 70% of parent hemopoietic cells. The freeze-etch method of marrow from 15% glycerin and 10% serum ensures the best safety of stem hemopoietic cells, however washing of the protecting substance from marrow is a lack of this method.
During the freezing of marrow polyvinylpirrolidone uses the protecting solution Kiev in-that hemotransfusions, containing 18 g of polyvinylpirrolidone, 10 g of glucose, 4 g of lactose, 1500 units of heparin, 0,015 g of levomycetinum, to 100 ml of the bidistilled water; a ratio of marrow and the protecting solution 1:1. Freezing of marrow is made at the first stage (t ° from 0 to — 18 °) with a speed of 1 ° in 1 min., on the second (t ° from — 18 to — 80 °) with a speed of 10 ° in 1 min. then containers with marrow immerse in liquid nitrogen.
After defrosting there are morphologically safe 90 — 86% of myelocariocytes.
Long-term storage at ultralow temperatures. It is established that marrow of donors after 1 — 5 years of storage at t ° — 196 ° contains 85 — 80% of safe cells, i.e. almost as much, how many at storage in liquid nitrogen within 1 day, 1, 3, 6 months. Data of the electronic and microscopic analysis demonstrate that the cellular elements of marrow stored in liquid nitrogen within 1 day, 6 months and 2,5 years in the majority do not undergo any submicroscopic changes. For definition biol, full value of the frozen marrowy cells use in the vivo methods. So, the cloning of the hemopoietic fabric in a spleen of mice of the imbredny lines irradiated with lethal doses showed that the marrow frozen from 15% by glycerin and which remained within 5 years at t ° — 196 °, contains more than 50% of stem hemopoietic cells. Wedge, researches produce the convincing evidence of efficiency of transplantations of the autologous marrow stored at t ° — 196 °C glycerin, polyvinylpirrolidone or poly(ethylene oxide) for a long time.
Freezing by means of low temperatures
Is established that the best results of freezing of marrow from 15% and 10% serum are received by glycerin at a combination of raznotemperaturny electric refrigerators. For this purpose the container with marrow is placed in the refrigerator with t ° in the beginning — 20 ° for cooling to t ° — 9 ° (i.e. till the period of crystallization), then quickly transferred to the refrigerator with t ° — 70 ° and left in it on storage. At this mode of freezing in the first period marrow is cooled with a speed of 2 — 1V20 of 1 min., duration of a zone of crystallization makes 3 min., the speed of freezing of marrow in the third period (post-crystallizational) makes 5 — 3 — 1 ° in 1 min. Freezing of marrow using such mode provides preservation on average of 89% of yadrosoderzhashchy cells and 57% of stem hemopoietic cells. Preservation of stem cells in a viable state at t ° — 70 ° is provided within 1,5 months
Preparation of marrow for transplantation
Considerable influence on safety of marrowy cells render conditions of thawing of the frozen marrow. Bystry thawing of marrow from — 196 to 2 ° in the water bath is considered optimum at t ° 40 — 41 ° for 40 — 50 sec. Slow defrosting of marrow at the room temperature (30 min.) or in the refrigerator at t ° 4 — 5 ° (240 min.) gives low safety of marrowy cells, and in particular trunk. The thawed marrowy suspension before transplantation in the closed way (through system) is transferred from an aluminum container to glass bottles with a capacity of 500 ml. Add 50 ml of the glyukozosakharozny solution No. 1 cooled to t ° 2 — 5 ° to 100 ml of the thawed marrowy suspension (glucose — 24,3 g, EDTA Na2 — 0,05 g, 8% of solution of sucrose — to 50 ml). Contents of a bottle are mixed careful rocking. Then add 150 ml of the solution No. 2 cooled to 2 — 5 ° twice (sucrose — 24 g, EDTA Na2 — 0,3 g, the water bidistilled — to 300 ml) and mix a suspension. After that bottles centrifuge within 15 min. at 1200 rpm at t ° 5 °. The most part of nadosadochny liquid (300 — 350 ml) is deleted. The cells which settled on a bottom of a bottle resuspend hashing. The received suspension of 100 — 150 ml is filtered through system for hemotransfusion in a bottle with a capacity of 250 ml which is corked hermetically. The bottle with marrow is marked, and then transferred for transplantation. After thawing of marrow and decrease in concentration of glycerin to 3,3% by means of glyukozosakharozny solutions the quantity of safe myelocariocytes does not change in the first 4 hours of storage at t ° 2 — 5 °. In 20 — 24 hours the quantity of safe cells decreases by 1 — 17%.
the marrow Defrozen and prepared for transplantation can be transported during 8 hours by rail, car, airplane in the isothermal boxes for blood supporting t ° 2 — 5 °.
Storage of marrow is carried out in banks of two types. Large banks are created at some in-ta and on blood transfusion stations; in them there has to be stored autologous marrow of patients and marrow of donors typed but to histocompatibility antigens. Banks of this kind shall be based on the equipment using liquid nitrogen for freezing and long-term storage of marrow. For these purposes it is reasonable to use devices of program freezing of FTINT of AN of USSR and Ying-that cybernetics of AN of the Georgian SSR, and also vessels on 250 and 500 l of liquid nitrogen, each of which can hold 260 — 520 containers with marrow, with a capacity at 160 ml everyone. The second type — small banks at some to lay down. institutions (from< i in-you and clinics,-tsy) and to become about i! \hemotransfusions. They are intended for conservation for short terms (1 — 2 month) to hl. obr. marrow of patients. the Crimea after use of the raised doses of cytostatic means or radiation therapy will be required transplantation of autologous marrow. For this purpose it is reasonable to use a combination of electrorefrigerators to t ° — 20 and — 70 ° (— 80 — 90 °).
Methods of assessment of viability of tinned marrow
For assessment use methods of vital staining supravital dyes (1% water solution of eosine, 0,1% solution of tripanovy blue) with calculation of total number of yadrosoderzhashchy cells of marrow in Goryaev's camera. Apply to omnibus and multilateral estimate of viability of marrow morfol., funkts, and biochemical, the methods of a research characterizing biol, full value of myelocariocytes in the course of preservation: studying of cells in the painted smears, a submicroscopy, an autoradiography, tsitol. a method of identification of chromosomes — markers (Tbt6), use of distinctions of a sex chromatin of men's and female granulocytes, studying of phagocytal and respiratory activity of cells of marrow. Safety of stem hemopoietic cells is estimated by method of cloning of the hemopoietic fabric in a spleen of fatally irradiated mice with identification of splenic colonies gistol, researches and method of definition of the committed stem cells in culture on a semisolid agar.
Quality control of tinned marrow is exercised before its delivery for transplantation. At the same time each bottle with marrow is looked through by the specialist doctor. Main signs of unfitness of marrow: disturbance of tightness in packing of a bottle, existence of hemolysis of St. 100 mg of %, bacterial pollution, existence of considerable clots, low safety of myelocariocytes (less than 50%). Point such signs as opacification of a nadosadochny layer, emergence of a film, plaque, gas evolution to bacterial pollution of marrow. Assessment of the validity of posthumous marrow is made on the same signs, as assessment of marrow of donors. However also results of pathoanatomical necropsy and a blood analysis on syphilis and inf have crucial importance. hepatitis.
Table. Main methods of conservation of separate bodies and fabrics and their kliniko-experimental value
Designations: + the method is applied in an experiment and clinic; ++ the method is used in clinic in isolated cases (generally abroad); — the method is applied only in an experiment.
Bibliography: Urgent problems of organ transplantation, under the editorship of Yu. M. Lopukhin, M., 19 78, bibliogr.; B of ER P. Lektssh of zoology, the lane with fr., SPb., 1882; Preparation, conservation and sterilization of biological fabrics, under the editorship of M. V. Volkov, M., 1970; Maple of River. Preparation and conservation of fabrics, the lane from Czeches., Prague, 1962, bibliogr.; Conservation and transplantation of fabrics and bodies, under the editorship of G. Krystinov, the lane with bolg., t. 2, Sofia, 1975; Sh at m and to about in V. I., Sh t of e of N-gold of E. III. and About N and shch e of N to N. A. Preservation of bodies, M., 1975, bibliogr. To. marrow — L and in r and to S. S. Conservation of marrow, Kiev, 1975, bibliogr.; Low-temperature conservation of marrow, under the editorship of E. Ya. Pankov, Kiev, 1976, bibliogr.; Problems of hematology and transfusiology, under the editorship of O. K. Gavrilov, t. 1, page 88, M., 1976; Fedotenkov A. G., Danilova L. A. and Alekseeva L. P. Influence of various modes of freezing on proliferative activity and differentiation of parent cells of marrow, Probl, gematol, and modulation, blood, t. 16,> J\c 8, page 21, 1971, bibliogr.; Fedotenkov A. G., etc. A new method of preparation of the cadaveric marrow intended for transplantation, in the same place, to t. 8, No. 2, page 28, 1963, bibliogr.
G. M. Solovyov; A. G. Fedotenkov (gems).