JPH07500504A - Langerhans in its pure form - 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] Langerhans in its pure form The objective of the present invention is to achieve a high purity level of 98% or more compared to the amount of purified cell particles. Preparations of isolated islets of Langerhans having and methods of making such preparations, and diagnostic and/or therapeutic use of its preparations on animals or animals; .

In 1922, Banting and Be5t developed an insulin for the treatment of diabetes. Since the introduction of phosphorus, diabetic keto-acid coma, with its fatal consequences, has become completely rare. Ta. However, treatment with insulin is seen in a wide range of diabetic cases. As of 1966, the health Alleviating the limitations of commonly known insulin therapy through pancreatic transplantation Efforts have been made. However, the risks of surgical manipulation, especially that part of the recipient Providing long-term immunosuppressive treatment to prevent the body's rejection of In the opinion of diabetologists, pancreas transplantation as an option should not be justified. stomach.

Therefore, an intact organ obtained from the pancreas of a brain-dead organ donor (such as an accident victim) Langerhan's other effort, rather than transplanting organs, is to produce insulin. Efforts to separate the islet and transplant only it, introducing it between the veins (hepatic portal vein) Although this is the most prominent application, such efforts have been made for a long time. animal Numerous studies have shown that islets implanted within the portal vein have a invades and absorbs insulin production, which depends on demands like blood sugar levels, leading to diabetes Known to regulate sexual metabolism and prevent life-threatening diabetes complications . Furthermore, studies conducted on numerous animals have shown that low levels of immune In contrast to pancreas transplantation, islet transplantation can be used with immunosuppressive treatment in diabetic recipients. It is known that it is not needed or is only needed temporarily.

Similarly, there is evidence of islet transplantation in diabetic humans. The only female patient However, after islet transplantation, most patients survived without insulin use for only 2 years (Warnoc et al. k G, L, et al, Diabetologia 35: 89-9 5.1992) o Lack of success in more trials may be due to insufficient purity level. of non-island tissues such as exocrine fragments, ductal elements, lymph nodes and blood vessels. Illustrated by transplantation of poorly separated islet suspensions. Such insufficient purity level Bell's islet suspension is characterized by very high immunogenicity, and after transplantation, the body rejects it. Endangered by the frequent occurrence of reactions.

Immunosuppressive treatment protocols proven effective in heart, liver and kidney transplants Despite the use of coal, impure islet suspensions are rejected at an early stage. Dedicated The focus of the Monke's interest is on the use of donor organs, especially in their purest form. Each was a purification operation of a transplanted islet suspension or a preparation thereof.

In published PC=T application no. 1088109667, cell suspensions, e.g. For example, a suspension containing Langernon's islets separated from the connective tissue 4, By subjecting it to so-called density gradient centrifugation, it is possible to A method is described with the aim of obtaining a purified cell suspension. Numerous publications, especially , London N, J, M, et al, : l5let puri In a general discussion such as fication. Pancratic l5let C e1l Transplantation (Ricordi C,.

Editor), R, G, Landes, ＾ustin, Texas, In IIs^, pp 113-123.1992: cells of various densities and/or mainly for variable sedimentation (also called isopycnal centrifugation) of cell aggregates. A special method called density gradient centrifugation based on Its application as a purification method for suspensions with bodies is described and the method A purity level of over 90% was obtained compared to the amount of purified cell particles. . Therefore unwanted cells such as exocrine fragments, ductal elements, lymph nodes and blood vessels Contamination of useful islet suspensions by particles is easily observed under a light microscope at 100% magnification. can be distinguished.

The present invention provides a method for producing preparations of isolated islets of Langerhans with particularly high levels of purity; and to reduce apparent in vitro immunogenicity through the use of standard tests. purpose.

The problem lies in the isolation of purity levels greater than 98% compared to the amount of purified cellular material. Book containing islet of Langerhans preparations and methods of making such preparations Solved by invention.

The isolated Langer/N7 islet preparation according to the invention is characterized by: Uses a single separation means based primarily on variable sedimentation of cellular material of varying densities After purification, the purity level of Langernon's islets compared to islet suspensions is increased.

The isolated islets of Langerhans according to the present invention further features the following: As a sign High capacity loss is less than 3%; most purified cell material has a diameter greater than 100 μm E; Reduction in immunogenicity exceeds 75% on average: Potential microbial contamination is reduced; Miku (b) Fluorescence-based viability indicators remain unchanged; in vitro glucose stimulation Increases in insulin secretion indicators average more than 50%; and one of several immunomodulatory treatments Increased recovery of high volumes after one step.

These features make one separation based primarily on variable sedimentation of cellular material of different diameters. After applying the method, the following separation is still mainly based on variable sedimentation of cellular material of various diameters. This feature is obtained in comparison to pre-purified islet suspensions before applying the means. In detail, features of the inventor's method are described hereinafter.

Other characteristics are the The amylase content is less than 0.05% compared to the islet suspension before use of the separation method. Good too. The preparation also contains a physiologically acceptable carrier fluid.

The purity of the isolated islet of Langerhans preparation according to the invention is clearly confirmed under a light microscope. can.

The general definitions and terms in the description above and below are used within the framework of the description of the invention. In this context, the term “cell material” has the following meanings: )” also refers to cell fragments or particles, whole cells, and cell clusters. It includes complete cell clumps or fine clumps.

Isolated islets of Langerhans preparations are prepared from human and animal pancreas according to the following methods: preferably from a human donor organ for diagnostic and/or therapeutic treatment. However, it can be used on animals as well. Type 1 and II For therapeutics regarding the treatment of both diabetes, isolated Langerhans derived from human pancreas Islet preparations are preferred. There is limited human pancreas from available donors, and diabetes Due to the high proportion of patients, animal pancreas is also used. in such case porcine pancreas is preferred. For diagnostic purposes, isolated Langerha islet preparations are preferred. Isolated Langerhan from human pancreas for diagnostic use Isla preparations are another subject of the invention.

The preparation consists of a suspension of isolated islets of Langerhans in a physiologically acceptable introduction fluid. When used appropriately, the fluid is administered either to the ostium of the liver (portal vein of the liver) or to the splenic vein. , intramedullary, subgastric, intraperitoneal, intramuscular, subcutaneous, cerebral, intraperitoneal, intramuscular, subcutaneous, intracerebral, intraperitoneal, intramuscular, subcutaneous, intracerebral, Alternatively, it can be injected by intracanal injection.

The term "physiologically acceptable carrier fluid" The literature suggests that it is suitable for cleaning, diagnosis and transplantation procedures of islets of Ngerhans, respectively. means all aqueous media as described in . A suitable medium is typically Ha nk's balanced physiological saline solution (Hank's solution), minimum essential culture medium, Medium 199, RPM11640, CMRLI066, Rice Consin University liquid, etc. . The subject of the present invention also provides the above-mentioned cell material with a high purity level of more than 98%. This is a method for preparing an isolated islet of Langerhans suspension. This method is useful for operations such as: Characterized by aspects of the enemy, i.e. a) The pancreas may contain Golagenase and/or other proteases. by the use of a physiologically acceptable introduction fluid capable of The islets of Langerhans along with other cellular material are extracted from tissue connections through digestion. be; b) The extracted islets of Langerhans are mainly composed of variable sedimentation of cell aggregates of different diameters. separated from the additional cellular material using at least one separation means based on and if necessary C) Isolated islets of Langerhans reduce their potential immunogenicity/antigenicity and/or their respective islands are preserved.

Operation step a) This stage of operation is usually known and generally isolation and purification methods.

The term "distention" refers to expansion.

n). For the purpose of dilation, the pancreas, i.e. the output of the human or porcine pancreas, is oral tract, through which digestive enzymes produced in the exocrine part of the pancreas are released into the small intestine. A cannula is inserted into the conduit that exits; through this cannula, the , per gram of pancreatic tissue, approximately 0. Contains 1-1°0% (W/V) collagenase 2 ml of Hank's solution is retrogradely injected into the pancreatic duct.

This operational step is preferably carried out by “Lacy P, E, et al., Dia betes 16: 35-39 (1967) and Gray D, W, R, et al, Diabetes 33: 1055-1061 (1984 )”.

The term "digestion" 1 refers to the digestion operation. During the digestive process, the pancreas produces enzymatic and and under mechanical action, it is broken down into islets of Langerhans and cellular material. this The method is preferably carried out by “Ricordi C, et al.: Diabe tes 37: 413-420.1988” and PCT Application Publication No. WO It is carried out by the method described in pamphlet No. 88109667.

The term "protease" refers to neutral proteases, clostripain, trypsin, Enzymes known as elastase, dispase, etc. Refers to the known pure type of collagenase.

After the conclusion of operating step a), the isolated islets of Langerhans suspension in the medium indicated above can be obtained, but it is still not pure as it contains additional cellular material. do not have.

Operation step b) The term “separation means (measu "re of 5 separation)" refers to a generally known separation operation, Its application for isolation and purification of islets of Langerhans is still novel and its Therefore, it represents the subject matter of the present invention. This separation means is defined by the term "velocity sedimentation" (velo). city sedimentation)”, which was treated in international publication. It is based on the following operating variations: sedimentation at 1 gram, countercurrent centrifugation (tilt method), and re- Includes centrifugation in oriented band rotors and isokinetic gradient centrifugation. I like it here Preferred is the known operating variant commonly referred to as "isokinetic gradient centrifugation."

In one particularly preferred application of this operation, the isokinetic gradient centrifugation In combination with other separation means primarily based on sedimentation of variable density cell aggregates, bi et al. It will be done.

The term "separation means based primarily on sedimentation of cell aggregates of various degrees" is generally well known. It also refers to other separation methods of Langerhans isolation and purification. It has been applied for (London N, J”, supra, Page 5 ,reference). This separation means is referred to under the term “isopyknic centrifugation”. described in the international publication after ``ent centrifugation)'' has been done. A description of preferred applications is given in the Examples. Particularly suitable operation The applied form of the production is characterized by the following: b) Extraction of Langerhans is carried out by various A subsequent procedure based primarily on variable sedimentation of cell aggregates of varying density and diameter It is separated from the additional cellular material through the separation means.

In other applications of manipulation, cell aggregates of different diameters and different densities In a combination of separation means based primarily on variable sedimentation of Good too.

The isokinetic gradient centrifugation is preferably carried out by: “Pretlow T, G, ＾nal Biochea+ 41+248-255.1971 and Pretlow T , G, II and Pretlov T, P,, Ce1l 5epa ration:　Methods　and　5elected　Applica tion, Vol. 5. ^cadei+ic Pr■ 281-309.191117” physiologically acceptable linear It is carried out as described for the method in an aqueous medium with a density gradient. publication In the article, such a medium is given the term “gradient”. , in a so-called “gradient mixer” or during the process of centrifugation itself. It is formed. The gradient has the highest density level at the bottom of the centrifuge tube; The density decreases as you go above the surface. Suitable media for gradient formation are e.g. , Ficoll (trademark), Percoll (trademark) (phara+acia , Freiburg), polysucrose, polyvinino q, °←° Contains auxiliary substances such as lolidone, albumin or dextran You may. Concerning separation means based primarily on variable sedimentation of cellular material of different diameters As a result, the movement of cellular material sedimented through the gradient is such that its highest density reaches its lowest density. It is characterized by a lower movement of sedimentary substances. During the centrifugation process, sedimented cell material moves through the gradient at a very consistent speed. The separation means is such that the initial cell particles Centrifuge ends when the bottom of the tube is reached. This method essentially To a lesser extent, it is suitable for means of separating cellular material of different diameters.

Operation step C) Treatments that reduce potential immunogenicity and/or antigenicity, e.g. culturing, UV irradiation. radiation, gamma rays, incubation with antibodies, or encapsulation may be used for the aforementioned applications. It concerns the preparation method of ordinary cell material in culture for morphology, which It is also described in the documents mentioned in this application. Cryopreservation is preservation This is an appropriate next step.

The special purity of the islets obtained makes it possible for one element to have therapeutic and/or diagnostic potential. After applying all methods following separation measures representing an improvement over the above findings. better results can be achieved.

The following examples illustrate, but do not limit, the invention (temperatures (in degrees Celsius): regarding the location of the manufacturer or editor and publisher of the publication. Data is limited to locations in the Federal Republic of Germany.

Example: 1. Pancreatic tissue The pancreas was obtained from sows (German Federal Hybrid Breeding Program) by layer killing and exsanguination. Then, remove the intestinal tract while carefully keeping the membrane-encased organs from the surrounding adipose tissue. It is then removed, during which sterile precautions are taken. Warm ischemia for 10 minutes is included in the slaughter process. The organs thus obtained are cooled at 4°C/39°F. Euroco lins (trademark) solution (Fresenius, Ba d Homburg) and transferred to the island laboratory. Cooling depletion before starting island separation Blood time is 30 minutes.

2. Preparation and expansion of pancreas All other steps of high preparation are carried out under aseptic conditions. Section of the pancreas closest to the pancreas used for islet isolation. The weight of prepared organ pieces is 120 g . The outlet of the pancreas (pancreatic duct) is located at the cut between the right thigh and the proximal part of the pancreas. The cannula is inserted using a needle and tied by the surgical system.

The weight of the pancreatic section used from the proximal pancreas determines the volume of inflation fluid.

2 ml of distension fluid per pancreatic dam, i.e. a total of 240 ml, was added to the horizontally placed crab. It is injected retrogradely into the pancreatic duct through the urea. For expansion, use 0.4% collagen Serva, Heidelberg, catalog no. 17449) containing rice consin large solution (UW-solution) (DuPont, Bad n.

mburg) is used at a temperature of 22°C (72°F).

After the expansion is complete, the surrounding adipose tissue and connective tissue, organs covered by membranes, and non-distended parenchyma is removed (total 15 g excluding cannula i.e. The weight of the pancreas used is 105 g).

3. Digestion of the pancreas The islets of Langerhans are divided into exocrine fragments, ductal elements, blood vessel fragments, lymph nodes, etc. Ricordi for the isolation of human islets of Langerhans from connective tissue. C, et *r, : Diabetes 37:413-420.1988- and pcT application publication number? Described in pamphlet No. 088109667 isolated by the method.

For this purpose 1, an inflated pancreatic piece, the structure of which is described in PCT Application Publication No. WO 3810 7 pieces at the bottom of the 450m1 capacity digestion vessel described in pamphlet No. 9667. It is placed with a glass ball (marble). Then the lower part of the container, 400μ The size sieve, container lid, and piping system for collagen solution recirculation are As described in Figure 2 of the pamphlet of T application publication number WO88109667 be assembled.

The piping system is additionally filled with Hank's solution and a pump is used to recirculate that solution. and the flow rate is set to 200 ml/min. swollen pancreas and gala The digestion vessel containing the marbles was vibrated at a vibration rate of 300 times/min in the vertical axis direction. It is moved at an amplitude of 10 mm. The temperature inside the digester is controlled by the use of a heat exchanger. Rise to 32-34°C (90-94°F) at 3°C per minute. Recirculation phase (phase 1 ), the temperature is kept constant at this level.

Pancreatic digestion using microscopic observation of samples taken from the plumbing system at 1-2 minute intervals. The process continues carefully. After 14 minutes, there were many liberated islands in the sample. is checked first. Recirculation of Hank's solution containing collagenase is recommended at this point. The sample (phase I) is stopped and fresh Hank's solution without enzyme is added to the container. I) flowed at a flow rate of 250 m/min for 15 minutes until no islands were visible anymore. Ru. During phase II, the temperature drops from 32-34°C to 15°C in 15 minutes. play The detached islets are treated with collagenase and other proteins released by the liver. To protect from enzymatic degradation, the tissue suspension was placed in a chilled (8°C) cone mold. The bottom is placed in a 250 ml centrifuge cup containing 35 ml of Hank's solution.

These centrifuge cups filled with tissue suspension were heated at 4°C (39°F) and 120xg. Centrifuge for 2 minutes at 200 ml at 8°C (48'F), then resuspended and stored at 8°C (48'F). will be saved.

Representative samples were analyzed for islet number, islet volume, DNA content, insulin concentration and insulin concentration. Collected for measurement of mylase activity.

4. Isopycnic gradient centrifugation In addition, after centrifugation (2 minutes, 4°C, 120xg), the remaining liquid is discarded and the concentrated The condensate (pellet) was heated using heat-inactivated newborn calf serum (NC3゜Biochrom). m, Berlin) 15m I, resuspended in a volume of 32 ml and 6 Transferred to a 00ml glass cup and added Ficoll-sodium-diadrizoe. (Ficol 1-sodium-diatrl-5odiu solution 400ml filled with. This solution consists of Seromed™-ficoll 5epa r　a　　o　r　(1,090g/cc,　Biochros, Berl in) 500ml, IM HEPE S 12ml (Gibco, E ggenstein), penicillin-streptomycin (lO,0OOIU/ ml P,, 10. OOjg/ml S, Flow, Irvine, GB) 5 ml, I NN a OH 1 ml and Me d i um 199 (lx, Biochros, Berlin) by mixing 50ml It is produced by The contents of the glass cup (bottom layer) are 500ml of blood Transport bag (Biopack Model, Biotrans, Dreie ich), which is then transferred to Blood Cell Purifier 2991 (Cobe, Kir. chheim/Munich) (Blood cell Process4 P rocessing Set, Cobe, Catalog No. 9 12-647-819) combined according to The contents of the blood transport bag are then placed in its disposable bag. transferred into the file system. Blood Cell Processor Centrifuge 200OR per 2 minutes Accelerated by PM. 200ml of Medium199 centrifuges at 240ORPM onto the bottom layer using a hose pump at a flow rate of 5 Qml/min while accelerating to Layered. Subsequently, the disposable system is degassed and the supply line is Closed. After centrifugation for 10 min at 240 ORPM at room temperature, and same rotation using a gradient unloader. At speed, the following fractions are sequentially transferred from the “Bromo Sossing Set” to the centrifuge cup. Next transferred: Fraction 1: 180ml! Fraction IIa: 25 mj! Fraction 11b: 10mf Fraction IIc: 10 mj! Fraction IId: 10 mj! Both parts IIe: 25m1 Fraction III: 290 mjl' After stopping the centrifugation, the “processing sesoto” is removed and Fraction IV: 50ml is transferred to a glass cup.

Both components were suspended in Medium 199) containing 10% NC3 and 250% It was centrifuged for gS minutes, the A0 residual liquid was discarded, and the fraction was collected separately. 0% fetal bovine serum (ics, Biochrow, Berlin), 1% penile CiJ-streptomycin solution and 2mM glycyl glutamine (bioch rom, Berlin) containing Leibovitz Medium (Bioc hrom, Berlin): The suspension (lie, 1 sound) (Ial and evaluated for their islet purity (percentage of surface covered by islet tissue). It will be done. Both halves, which have a large number of islet clusters, have almost no exocrine contamination, followed by isokinetic gradients. suspended in a centrifuge.

Representative samples include islet number, islet volume, DNA content, insulin concentration, and Measurement of enzyme activity, in vitro glucose-dependent insulin secretion, and immunogenicity collected for.

5. Constant velocity gradient centrifugation 100ml polycarbonate round bottom centrifuge tube with a height of 16mm and a diameter of 31.5mm (Nalge, Braunschweig), Ficoll and Leib Bottom layer (cushion) made of ovitz Medium 4ml, height 15mm , 1.026 g/cc and a viscosity of 3.5 cP at 16°C.

Injected at

Using two gradient generators, Ficoll and lejbovitz Mediu 76 ml volume, height 120 mm, density rate 1.026 to 1. 017 g/c c and 16°C (60°F) viscosity range 3.50P to 2. OcP's A continuous gradient is added on top of the bottom layer (cushion).

On top of this continuous slope, a height of 8 mm and 50.000 island equivalents (1 island equivalent = volume compensation The corrected number of items and one island equivalent represent one island with a diameter of 150 μm (Ricordi C ．． etal. Acta Diabetol Lat 27: 185-19 5. 1990) and the remaining Leib Cell mass in Ovitz Medium is added. Bottom layer , a centrifuge tube with a gradient layer and a sample is placed in a centrifuge, whose state 1 Hettich Rotixa RP Centrifuge (Hettich, Tutt lingen) (contains a 5094 type pivotal rotor and a 100ml centrifuge tube) central force (with appropriate accessories and reduce tons) The distance between the cushion and the gradient is 22.2 cm, and the distance between the sample and the gradient is 10 cm.

It is 2cm.

The centrifuge tube is accelerated to 49QRPM in 30 seconds. In this RPM, the support A centrifugal force of 27.4×g acts on the layer between the sample and the gradient.

Centrifugation is performed at 16° for 60 seconds and decelerated to ORPM over 30 seconds.

A centrifuge tube is removed from the centrifuge; a first fraction consisting of 8Qml and Leibo A second fraction of 5 ml diluted with Vitz Medium is aspirated and further 1 Centrifuge at 120xg for 2 minutes at 6°. The residual liquid is discarded and the remaining cell suspension The solution is Leibovitz Medium or other culture medium and appropriate supplements. resuspended using additives.

Representative samples include islet number, islet volume, DNA content, insulin concentration, and Measurement of enzyme activity, in vitro glucose-dependent insulin secretion, and immunogenicity collected for.

The obtained Takano suspension can be used for subsequent applications, such as culture.

6. Exam islet number, islet volume, insulin concentration, microfluorescence survival test, in vitro glucose Gas-dependent insulin secretion is determined by the international standard method (Ricordi C. et al. , Acta Diabetol. Lad, supra) to each sump 11. L) and offshore is determined. Measurement of DNA content was performed according to “Labarca C. and Paigen K. , ^nal Biochem 102: 344-352 ．． amylase activity of individual samples. (Ma, α-amylase EPS (trademark) kit (Boehringer. nnheim. Mannheim) was used for L1 and Oki 1 was determined. Determine immunogenicity To do this, mixed lymphocyte-islet cultures are performed (Ulrichs K. et al. al. : Hort Metabol. Res. 25/S: 123- 127. 1990, see). 100 pig islets contain 105 human lymphocytes The cells were incubated at 37°C (99°F) for 3 days, followed by 10 μCi of 3H-thymidine. After addition, islets and xenolytic lymphocytes are mixed in culture for 20 hours. proliferation of lymphocytes , is correlated with 3H-thymidine incorporation, and is counted through a counter at 7 minutes. and compared with control values and expressed as stimulation index after transformation.

Table 1. Two sequential purification operations (isopycnic centrifugation and isokinetic gradient centrifugation) ) Results of preparation of islets of Langerhans from porcine pancreatic tissue.

Before IPGZ After IPGZ Before IKGZ Number of IKGZI islands 219.985 1 56.849 152.332 99.016 Island capacity (μf) 623 4 58 445 432 Amylase (U) 729, 644. 76 402, 773 165, 916 72. 748 Insulin (U) 379.6 236.3 212.6 151.5D N A (mg) 365.　 508 6, 643 2, 321 1. 820 Survival rate (%) 98. 7 98. 5 Perfusion - PI: 0.48 - PI: 4.44 Immunogenicity - 28. 5 2. 3 Island capacity 100% 73.5% 71.4% 69.3% Amylase 100 % 0. 055% 0. 023% 0.010% insulin 100% 62.2% 56.9% 39.9% D N A 100% 182% 0.6 3% 0. 5096 Table 2. Two consecutive purification operations (isopycnic centrifugation islets of Langerhans preparations from porcine pancreatic tissue using isokinetic gradient centrifugation). result.

Average value + SEM After IPGZ IKGZ posterior insula volume (μm) 321±44. 2 315.5 ± 39.4 Survival rate (%) 99.2 ± 0.3 99.1 + 0 ．． 4 Immunogenicity 100% * 24% ± 6.4% * (Irritation index after IPGZ = 10 0%) I PGZ: Isopycnal centrifugation IKGZ: isotonic centrifugation P■: Perfusion index * Transcript and translation of deviation correction sound in significantly significant promotion indicators) Submission form (patent (Article 184-7, Paragraph 1 of the Act) April 13, 1994

Claims (1)

[Claims] 1. Isolation laboratories with purity levels greater than 98% relative to the amount of purified cell material A preparation of Ngerhans Island, characterized in that: Uses a single separation means based primarily on variable sedimentation of cellular material of varying densities increased purity level of islets of Langerhans compared to islet suspension after Island capacity loss is less than 3%; Diameter of most purified cell material exceeds 100 μm: reduction in immunogenicity averages 75 μm %; Strong biological contamination is reduced: Viability indicators by microfluorescence remain unchanged; glucose stimulation in vitro increase in insulin secretion index of more than 50% on average; and After applying one separation means mainly based on variable sedimentation of cellular material of different densities, However, it is still possible to apply subsequent separation stages based primarily on variable sedimentation of cellular material of various diameters. One or several immunizations when compared to pre-purified islet suspensions, respectively. Increased islet volume recovery after conditioning treatment; after distension and release from the pancreatic tissue, but any subsequent separation measures performed. amylase content is less than 0.05% compared to the islet suspension before application; and a physiologically acceptable carrier fluid. 2. 2. A preparation according to claim 1, wherein the islets of Langerhans are obtained from humans. 3. 2. A preparation according to claim 1, wherein the islets of Langerhans are obtained from animal tissue. 4. 4. A preparation according to claim 3, wherein the islets of Langerhans are obtained from pig tissue. 5. 5. According to one of claims 1 to 4, in the form of a suspension to be injected parenterally. Preparation. 6. 6. A preparation according to claim 5 in the form of a suspension to be injected into the venous portal. 7. In the splenic vein, in the splenic pulp, under the capsule, in the omental flap, and in the splenic pulp (epiploi). cflap) by intraperitoneal, intramuscular, subcutaneous, intracerebral, or intratesticular injection. 6. A preparation according to claim 5 in the form of a suspension to be injected. 8. High purity level of >98% of cellular material compared to the amount of purified cellular particles 1. A method for producing an isolated islet of Langerhans preparation having a) collagenase and and/or other proteases. , inflate the pancreas and run it along with accompanying cellular material from its tissue conjunctures. islets of Gerhans and b) mainly based on variable sedimentation of cell conjugates of different diameters. Lange extracted from the accompanying cellular material by at least one separation means. Separate Luhans Island, c) reducing the obtained islets of Langerhans to reduce their high immunogenicity/antigenicity characterized by subjecting it to treatment and/or preserving the treated islets. 9. a) Use of a physiologically acceptable introduction fluid that the pancreas is capable of containing collagenase. 9. A method according to claim 8, characterized in that the expansion is performed by: 10. a) The islets of Langerhans and associated cell particles are enzymatically or characterized by being obtained from the connective tissue of the pancreas expanded by mechanical action The method according to claim 8 or 9. 11. b) Separation means based primarily on variable sedimentation of cell aggregates of different diameters, e.g. 11. The method according to one of claims 8 to 10, characterized in that it is a speed gradient centrifugation method. 12. b) Isokinetic gradient centrifugation is performed in a physiologically acceptable aqueous medium with a linear density gradient. 12. The method of claim 11, wherein the method is carried out in the body. 13. b) Isokinetic gradient centrifugation produces polysucrose with a molecular weight of approximately 400,000. 13. A method according to claim 12, characterized in that it is carried out in an aqueous medium containing. 14. separating the islets from additional cellular material comprises: b) placing the colloid in a suitable container; and a bottom layer consisting of a physiologically acceptable aqueous medium, followed by approximately 4°C. Density range 1.005-1.600 g/cc and viscosity range approximately at ~37°C Others have a continuous gradient with 1.1 to 15 cP in their built-in volume. Langerhan forming the medium layer and prepared according to step a) of claim 8. Claim 8 further comprising the step of adding a suspension of islets and additional cellular material. 13. 15. separating the islets from additional cellular material comprises: b) islets of varying density and diameter; The extracted Claims 8 to 9 include the step of isolating the isolated islets of Langerhans from additional cellular material. 14. 16. b) Separation means based primarily on variable sedimentation of cellular material of different densities are 16. The method according to claim 15, which is a gradient centrifugation method. 17. b) isopycnic gradient centrifugation is carried out in an aqueous medium with a discontinuous density gradient 17. The method according to claim 16, characterized in that: 18. b) Isopycnic gradient centrifugation yields polysucrose with a molecular weight of approximately 400.000. and sodium diatrizoate. 18. The method according to claim 17, wherein the method comprises: 19. The treatment step of step (c) The immunogenicity and/or antigenicity of antibodies can be determined by culturing, UV irradiation, gamma irradiation, Select from a group of commonly used operations consisting of incubation or encapsulation. 19. According to one of claims 8 to 18, the Method. 20. The preservation step of claim 8(c) comprises preserving the obtained islets of Langerhans in culture. Claims 8 to 1 include preservation in a state of The method described in one of 9. 21. An island of Langerhans obtainable by the method according to claim 8. 22. Claims for diagnostic and/or therapeutic use on the human or animal body A preparation according to claim 1. 23. 2. A preparation according to claim 1 for the treatment of diabetes in humans.