A device and method for isolating pancreatic islet tissue

Abstract

The invention provides a device and method for islet tissue separation, comprising a digestion tank for accommodating a pancreatic organ and performing a digestion operation; a water bath for heating a digestion fluid; antibody-carrying magnetic beads for binding to surface cells of islet tissue; a magnetic separator for separating positive tissue and negative tissue in pancreatic digested tissue; a first container for receiving negative tissue, digestion fluid and antibody-carrying magnetic beads separated by the magnetic separator after pancreatic digestion; a second container for receiving positive tissue, buffer and antibody-carrying magnetic beads separated by the magnetic separator; and a third container for receiving buffer and antibody-carrying magnetic beads. The invention has a simple principle, is easy to prepare, is convenient to use, has an ideal islet tissue separation result, can achieve large-scale batch production, and promotes the separation of islet tissue, and promotes the research and application of islet transplantation in clinical and scientific research fields.

Description

Technical Field

[0001] The invention relates to an immunomagnetic bead cell separation technology, and in particular to a device and a method for separating pancreatic islet tissue in the pancreas. Background Art

[0002] Immunomagnetic Beads Separation Techniques (IMBS) first appeared in the 1970s. The basic principle is to combine uniform superparamagnetic microspheres synthesized from magnetic materials with antibodies that have undergone affinity chromatography to capture and separate target substances from mixed suspensions. It is used for the detection of a variety of microorganisms and molecular biology. It is considered to be a sensitive and simple method for separating target analytes. Its detection limit can theoretically reach 1-10 bacteria per gram. After entering the 21st century, the application of IMBS has gradually increased both internationally and domestically. The Escherichia coli O157 immunomagnetic beads separated by Dynabeads were once included in the Chinese national standard. BeadRetriever TM Fully automatic immunomagnetic bead sorting system (BeadRetriever TM The automatic magnetic activated cell sorting system is an automated operating system developed from this. The entire solution is based on Dynabeads R Immunomagnetic bead sorting technology (IMS), combined with the Dynal 701 fully automatic magnetic bead sorter, can quickly purify and concentrate various biological samples, and complete the enrichment and separation of pathogenic bacteria. TM Dynabeads provided by the company R , mainly including Salmonella (Dynabeads R anti-Salmonella), Listeria (Dynabeads R anti-Listeria), O157H7 Escherichia coli (Dynabeads R anti-E.coli O157) etc.

[0003] Cell transplantation refers to the technology of transplanting an appropriate amount of free living cells, tissues or cell clusters with normal physiological functions from human organs and tissues into the blood vessels, body cavities or tissue organs of the recipient (human or animal) to achieve the purpose of treatment. The clinical application of cell transplantation is becoming more and more extensive, among which bone marrow and hematopoietic stem cell transplantation has attracted much attention. It can be used to treat hereditary combined immunodeficiency disease, hereditary diseases such as severe thalassemia, severe aplastic anemia, and blood system malignancies including various leukemias. In addition, there are pancreatic islet cell cluster transplantation for the treatment of type 1 diabetes, liver cell transplantation for the treatment of severe hepatitis hepatic coma, spleen cell transplantation for the treatment of severe hemophilia A, and testicular Leydig cell transplantation for the treatment of male sexual dysfunction (low testosterone).

[0004] Cell transplantation (such as pancreatic islet transplantation, bone marrow transplantation, stem cell transplantation, hepatocyte transplantation, etc.) has shown an increasingly high application value in the treatment of diseases such as diabetes, cancer, liver disease, genetic diseases and tissue damage. In some treatment areas, such as diabetes and leukemia, it has greatly surpassed the treatment methods of large organ transplantation and radiotherapy and chemotherapy. It has become an important direction for the development of surgical organ transplantation and one of the key funding directions of the National 863 Program. It belongs to the emerging biomedical industry. The research and application of tissues and cells is a new way and new hope for the treatment of cardiovascular diseases, diabetes, nervous system diseases, liver diseases, etc. It is a hot spot for international competition in the medical field. This emerging biomedical industry has the advantages of high technology, high added value, low cost and less pollution, and is a key support direction of the government.

[0005] Islet tissue or cell cluster transplantation is to transplant the separated islet tissue or cell cluster from donor or autologous body into the recipient's liver sinusoids, renal capsule, body cavity or other vascularized parts. The transplanted islets can secrete insulin, glucagon and C-peptide in real time according to the changes of blood sugar in the recipient, so as to achieve the purpose of accurately regulating the patient's blood sugar. Islet transplantation is a good way to cure clinical type 1 diabetes and end-stage type 2 diabetes. Islet transplantation has attracted much attention from clinicians due to its advantages such as low requirements for pancreatic donors, simple transplantation operation, almost no need for surgery, and few side effects. On the other hand, whole pancreas transplantation has high requirements for donors, complex exocrine gland processing, and many postoperative complications. In recent years, with the application of new immunosuppressive regimens, the quantity and quality of basic research on islets, islet isolation and purification have also been greatly improved. Islet transplantation has made great progress in the clinical treatment of diabetes. According to a report in 2012, the insulin-independence rate has reached 50% 5 years after islet transplantation, which is no different from whole pancreas transplantation (52%). The medical community has always promoted islet transplantation as the most ideal method for treating diabetes. The clinical application of islet transplantation has become an unstoppable trend. As Dr. Sutherland, the originator of pancreatic transplantation, predicted at the World Transplantation Conference, islet transplantation is gradually replacing pancreatic transplantation in the world and has become a boon for diabetic patients. In the next few decades, islet transplantation may become the best choice for treating patients with unstable blood sugar in type 1 diabetes and end-stage type 2 diabetes.

[0006] The research on islet transplantation for the treatment of patients with unstable blood sugar type 1 diabetes and end-stage type 2 diabetes has been going on for more than 40 years around the world, especially after the team led by Professor Sharpiro of Canada announced the "Edmonton Program" in 2000. It has been proved that islet transplantation can not only control the blood sugar of patients, but more importantly, it can inhibit and improve the occurrence of complications of patients. However, the Edmonton islet transplantation treatment plan is not perfect enough, and the results of patients being completely free from insulin dependence for a long time are not very ideal. The clinical application of islet transplantation has made rapid progress in recent years. As early as 2004, the US NIH invested 170 million US dollars to start the standardization of clinical islet isolation and transplantation procedures and the evaluation of 7 new immunotherapy programs. Among them, the immunotherapy program named CIT 07 was used to treat 48 clinical patients. After the operation, all patients were free from insulin treatment, and some patients have been completely free of insulin treatment for more than 10 years. The survival time of islets has reached the level of full pancreas transplantation. Countries such as Canada, Europe and Australia have approved islet transplantation to be included in medical insurance, and autologous islet transplantation has long been included in medical insurance in the United States. At present, with the aggravation of population aging, the incidence of diabetes, pancreatic diseases and other diseases in China is increasing year by year, and cell transplantation technology will inevitably develop gradually and vigorously in China.

[0007] The purpose of islet separation and purification is to extract intact islet tissue or cell clusters from pancreatic digested tissue. The separation method currently used is mainly density gradient centrifugation, the main principle of which is that islet cell clusters have a lower density than exocrine / ductal tissue. If these types of cells have been effectively separated during pancreatic digestion, we can purify the islet cell clusters by density gradient method. The most important progress in the large-scale purification of human and large animal islets is the use of COBE 2991 cell separator for islet purification. This centrifuge was originally designed to separate different cellular components in blood, but based on the density difference of different tissues after pancreatic digestion, the machine is also suitable for separating human islets.

[0008] Density gradient centrifugation is also called equilibrium density gradient centrifugation. A centrifugal force field is applied to a solution of small molecules for a long time to achieve sedimentation equilibrium, and a certain density gradient appears from the liquid surface to the bottom in the sedimentation tank. If a small amount of macromolecules or tissue or cell solution is added to the solution, the part of the solution with a higher density than the solvent will settle, and the part with a lower density than the solvent will float up, and finally gather to form a ribbon of sediment at the position where the centrifugal force and buoyancy are balanced. This phenomenon can be used to determine the floating density of nucleic acids or proteins, or to separate cells or tissues based on their differences. Since M. Meselson, F. W. Stahl, and J. Vinograd successfully separated [15N] DNA and [14N] DNA in 1958, this method has achieved many results. This method mostly uses concentrated cesium chloride solution, so the name cesium chloride concentration gradient centrifugation is sometimes also used. Rubidium chloride, cesium bromide and other solutions can also be used.

[0009] Centrifuges are also often used in the separation of cells or tissues for purification purposes, using the density difference of pre-prepared density gradient liquids such as sucrose for separation. Its working principle is: when there is a difference in sedimentation coefficient between different particles, under a certain centrifugal force, the particles each settle at a certain speed, forming zones in different density gradient areas. The density gradient medium should be pre-formed, and the maximum density of the medium should be less than the density of all sample particles.

[0010] If the density gradient is discontinuous, tissues will overlap at density interfaces with similar densities; the density gradient can also be continuous, so that cells can be effectively separated based on the precise density of the gradient fluid.

[0011] There are some related literatures on the preparation of density gradient solutions abroad, some of which have been used for islet purification, including sucrose, Ficoll, dialysed Ficoll, Ficoll Hypaque, Metrizamide, Percoll, Ficoll Conray, Dextran, bovine serum albumin solution, Ficoll Diatrizoic, Odixanol and Nycodenz, etc. In addition, there is an introduction to 50 density gradient media for isopycnic separation, which says that it will not change the fact that the density of islets and the density of exocrine tissue overlap. The two density gradient media commonly used for human islet separation and purification are Euro-Ficoll and high osmotic pressure bovine serum albumin (500 mOsm / kg / H2O). The advantages and disadvantages of Ficoll and bovine serum albumin for cell separation have been discussed in some literature. The advantages of Ficoll are that its solution is clear, autoclavable and non-animal derived. Its disadvantages are that it aggravates cell aggregation and significantly increases permeability by binding water at high concentrations. The advantages of bovine serum albumin are that it has a certain protective effect on cells and reduces cell aggregation, but its disadvantages are that it comes from animals and is easily contaminated.

[0012] Density gradient solutions for islet isolation are also constantly being improved. For example, density gradient solutions are prepared by adding additional hydroxyethyl starch solution or colloidal silica (Percoll) to UW solution; new density gradient solutions are composed of two solutions, Ficoll density gradient solution (1.100 g / mL) and UW solution. Although this modification reduces the range of the density gradient, it allows more digested tissue to be loaded (up to twice the original amount) and can be effectively separated in one round of purification. This speeds up the overall pancreas processing time (the original continuous density gradient technology often requires 2 rounds of purification). However, this adjustment may not be suitable for all islet isolation centers, because the optimal operating temperature of UW solution is 4°C, so in order to ensure islet activity, COBE 2991 must be modified and operated at this temperature, and is often placed in a cold room.

[0013] The conventional islet isolation process for humans or animals also includes the collection of islet tissue after digestion, centrifugal washing to remove collagenase, purification collection, centrifugal washing to remove purification solution, etc. Cell processing is time-consuming and complicated, and requires contact with a variety of different reagents and equipment, which affects the activity of the final cells. At the same time, due to the complexity of the source of donor organs, the density difference between islet tissue and exocrine tissue is not consistent; part of the exocrine gland tissue in the same pancreas may have a density close to that of islet tissue due to edema, which makes it difficult to obtain a large amount of high-purity islet tissue or cell clusters using conventional continuous density gradient separation methods, which often leads to the failure of islet separation, resulting in waste of organs and a large amount of reagents and labor.

[0014] Obtaining a large number of high-purity, active islet cell clusters plays an important role in their in vivo transplantation and improving the effect of diabetes treatment. Currently, density gradient centrifugation is a more commonly used method among these separation methods, but it still has insurmountable defects. In order to reduce the failure rate of islet separation, improve the purity of isolated islets and the amount of islet tissue, and reduce the operation time of islet separation, this patent designs a new continuous immunomagnetic bead islet separation system, which lays the foundation for the development and application of the system. Summary of the invention

[0015] In view of the deficiencies of the existing technology, the present invention provides a device and a method for separating islet tissue in the pancreas.

[0016] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present invention is: a device for isolating pancreatic islet tissue, comprising a digestion tank for accommodating pancreatic organs and performing digestion operations; a water bath for heating digestion fluid; antibody-carrying magnetic beads for binding to the surface cells of pancreatic islet tissue,

[0017] Also included is a magnetic separator for separating positive tissue and negative tissue in pancreatic digested tissue;

[0018] A first container for receiving negative tissue, digestion fluid and antibody-carrying magnetic beads separated by a magnetic separator;

[0019] A second container for receiving the positive tissue, buffer and antibody-carrying magnetic beads separated by a magnetic separator;

[0020] a third container for receiving a buffer and magnetic beads with antibodies;

[0021] The digestion tank, water bath, first container and magnetic separator are connected through a first conduit, the second container, third container and magnetic separator are connected through a second conduit, the third container and first container are connected through a third conduit, and peristaltic pumps are provided on the first conduit, the second conduit and the third conduit.

[0022] The magnetic separator is provided with a first channel, a second channel, a third channel and a fourth channel, wherein the first channel, the second channel, the third channel and the fourth channel are interconnected, and the multi-pole magnetic separator is provided with an electromagnet on one side of the interconnected channel close to the third channel and the fourth channel.

[0023] The first channel and the second channel are connected to the first conduit respectively, and the third channel and the fourth channel are connected to the second conduit respectively.

[0024] The inlet of the second conduit connected to the second container is located at the bottom of the second container. A screen is provided in the second container with an aperture of 1-50 microns. The outlet of the second conduit connected to the second container is located at the top of the second container.

[0025] The inlet of the second conduit connected to the third container is located at the bottom of the third container, an electromagnet is provided at the position of the bottom of the third container connected to the third conduit, the outlet of the second conduit connected to the third container is located at the top of the third container, and the outlet of the third conduit connected to the third container is located at the bottom of the third container.

[0026] The diameter of the antibody-carrying magnetic beads is 10 nanometers to 100 micrometers.

[0027] The cross-section of the interconnecting channel is circular, semicircular, elliptical, semi-elliptical, trapezoidal, triangular, rhombus, rectangular or square.

[0028] A method for separating pancreatic islet tissues comprises the following steps: placing the pancreas in a digestion tank for digestion, and then adding antibody-carrying magnetic beads to the exposed surface cells of the pancreatic islet tissues to form positive tissues, thereby the positive tissues, antibody-carrying magnetic beads and buffer solution are separated into a second container through a magnetic separator, the tissues not combined with the antibody-carrying magnetic beads after the pancreas digestion form negative tissues, the negative tissues, antibody-carrying magnetic beads and digestion solution are separated into a first container through a magnetic separator, the positive tissues, antibody-carrying magnetic beads and buffer solution are separated into a second container through a magnetic separator, the antibody-carrying magnetic beads and buffer solution not combined with the pancreatic islet tissues in the second container enter into a third container through a second conduit, the antibody-carrying magnetic beads enter into the first container through the third conduit under the pulling action of an electromagnet, and the buffer solution in the third container enters into the magnetic separator through the second conduit for recycling.

[0029] The negative tissue, antibody-carrying magnetic beads and digestion solution in the first container enter the digestion tank for re-digestion and combination, and the cycle is repeated until the positive tissue is completely separated.

[0030] Beneficial effects of the present invention: The present invention provides a device and method for islet tissue separation. The present invention has a simple principle, is easy to prepare, and is convenient to use. The islet tissue or cell cluster separation results are ideal and can be mass-produced on a large scale, which promotes the separation of islet tissue or cell clusters and promotes the research and application of cell transplantation in clinical and scientific research fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a structural schematic diagram of the present invention;

[0032] Figure 2 It is a schematic diagram of the structure of the magnetic separator of the present invention. DETAILED DESCRIPTION

[0033] like Figure 1-2 As shown, a device for islet tissue separation includes a digestion tank 100 for accommodating a pancreatic organ and performing a digestion operation;

[0034] A water bath 200 is used for heating the digestive fluid; the water bath heats the digestive fluid by hot water, so that the heating effect is stable and uniform.

[0035] Antibody-carrying magnetic beads for binding to cells on the surface of pancreatic islet tissue;

[0036] It also includes an electromagnetic separator 300 for separating positive tissue and negative tissue in the digested pancreatic tissue; the pancreatic islet tissue combined with the antibody-carrying magnetic beads is called positive tissue, which is the tissue that needs to be extracted in the experiment, and other substances in the pancreas and the antibody-carrying magnetic beads not combined with the pancreatic islet tissue are called negative tissue.

[0037] A first container 400 for receiving negative tissue, digestion fluid and antibody-carrying magnetic beads separated by a magnetic separator;

[0038] A second container 500 for receiving the positive tissue, buffer and antibody-carrying magnetic beads separated by a magnetic separator;

[0039] A third container 600 for receiving a buffer solution and magnetic beads with antibodies;

[0040] The digestion tank 100, the water bath 200, the first container 400 and the magnetic separator 300 are connected through a first conduit a, the second container 500, the third container 600 and the magnetic separator 300 are connected through a second conduit b, the third container 600 and the first container 400 are connected through a third conduit c, and a peristaltic pump 700 is provided on the first conduit a, the second conduit b and the third conduit c.

[0041] The magnetic separator 300 is provided with a first channel 310, a second channel 320, a third channel 330 and a fourth channel 340, and an interconnecting channel 350 is provided between the first channel 310, the second channel 320, the third channel 330 and the fourth channel 340. The magnetic separator 300 is provided with an electromagnet 360 on one side of the interconnecting channel 350 close to the third channel 330 and the fourth channel 340.

[0042] The first channel 310 and the second channel 320 are respectively connected to the first conduit a, and the third channel 330 and the fourth channel 340 are respectively connected to the second conduit b.

[0043] An electromagnet 610 is provided at a portion of the bottom of the third container 600 connected to the third conduit c.

[0044] The inlet of the second conduit b connected to the second container 500 is located at the bottom of the second container 500, and the outlet of the second conduit b connected to the second container 500 is located at the top of the second container 500. There is a screen 800 in the second container, and the screen aperture is 1-50 microns.

[0045] The inlet of the second conduit b connected to the third container 600 is located at the bottom of the third container 600, the outlet of the second conduit b connected to the third container 600 is located at the top of the third container 600, and the outlet of the third conduit c connected to the third container 600 is located at the bottom of the third container 600.

[0046] The diameter of the antibody-carrying magnetic beads is 10 nanometers to 100 micrometers. Preferably, the diameter of the antibody-carrying magnetic beads is 10 nanometers to 50 micrometers. In actual operation, antibody magnetic beads within this size range are generally selected for operation.

[0047] The cross-section of the interconnecting channel 350 is circular, semicircular, elliptical, semi-elliptical, trapezoidal, triangular, rhombus, rectangular or square. The cross-sectional shape of the interconnecting channel 350 is not limited thereto. The above shapes are only common regular shapes. Other regular shapes or irregular shapes may also be used, which will not be elaborated in detail.

[0048] A method for islet tissue separation comprises the following steps: placing the pancreas in a digestion tank for digestion, combining magnetic beads with islet tissue membrane protein antibodies with the surfaces of islet cell clusters exposed by digestion to form positive tissue, thereby separating the positive tissue, magnetic beads with antibodies and buffer solution into a second container through a magnetic separator, tissue not combined with magnetic beads with antibodies after pancreatic digestion to form negative tissue, and separating the negative tissue, magnetic beads with antibodies and digestion solution into a first container through a magnetic separator, magnetic beads with antibodies and buffer solution not combined with islet tissue in the second container entering a third container through a second conduit, and magnetic beads with antibodies entering the first container through a third conduit under the traction of an electromagnet, and the buffer solution in the third container entering the magnetic separator through the second conduit for recycling.

[0049] The negative tissue, antibody-carrying magnetic beads and digestion solution in the first container enter the digestion tank for re-digestion and combination, and the cycle is repeated until the positive tissue is completely separated.

[0050] Beneficial effects of the present invention: The present invention provides a device and method for islet tissue separation. The present invention has a simple principle, is easy to prepare, is convenient to use, has ideal tissue and cell separation results, can achieve large-scale batch production, and promotes islet tissue separation, and promotes islet transplantation research and application in clinical and scientific research fields.

[0051] Example 1

[0052] A device and method for isolating pancreatic islet tissue according to Example 1 of the present invention, the device design and the application results in isolating pancreatic islets from rabbits are as follows:

[0053] The mesh aperture of the second container of the device is 50 microns, the cross-section of the interconnecting channels between the first channel, the second channel, the third channel and the fourth channel of the magnetic separator is circular with a diameter of 0.5 cm, the surface magnetic field strength of the electromagnet of the multipolar magnetic separator is 680 mT, the magnetic field strength of the electromagnet of the third container is 800 mT, the buffer solution is Hank's solution (containing 10% fetal bovine serum), the magnetic beads are commercially available with a particle size of 100 nanometers and are coated with amino groups (-NH), the polyclonal antibody of rabbit islet membrane protein is homemade, and the antibody coating of the magnetic beads is completed before the islet separation experiment begins.

[0054] Collagenase perfusion and acquisition of pancreas: adult rabbits (2-3 kg) were taken, fasted for 12 hours, and anesthetized by injection of 3% sodium pentobarbital 60 mg / kg through the ear vein. The abdomen was shaved and disinfected in sequence. The abdominal cavity was opened along the linea alba, the intestinal tract was pushed to the right side of the abdominal cavity, and the intestine was pushed down along the duodenum to the small intestine. The common pancreatic duct was found and clamped with an artery clamp to the entrance of the small intestine. The rabbit was killed by injection of air needle into the ear vein. A 30G1 / 2 needle was used for retrograde pancreatic duct intubation and perfusion of collagenase V (1 mg / mL, the solvent is Hank's solution) about 15 mL. After the pancreas was fully expanded, ophthalmic scissors and forceps were used to quickly and completely remove the expanded pancreatic tissue along the small intestine wall, duodenal wall and stomach wall.

[0055] Pancreatic digestion and islet separation: Cut the expanded pancreatic tissue slightly, place it in a 100mL digestion tank containing 30mL of collagenase of the same concentration, connect pipelines a, b and c, add Hank's solution and magnetic beads coated with rabbit islet membrane protein antibody to the first container, turn on the peristaltic pump connected to the digestion tank in pipeline a, the speed is 100rpm, and exhaust the air in the digestion tank and pipeline; turn on the buffer peristaltic pump on tube b, exhaust the air in tube b and turn off the peristaltic pump; shake and digest the pancreas in a 37±0.5℃ constant temperature water bath until the pancreatic tissue is digested to chyle and mud (about 10min). Turn on the peristaltic pump on tube a connected to the interface of the magnetic separator 320 at a speed of 110rpm, turn on the peristaltic pump on tube b at a speed of 80rpm, intermittently turn on the electromagnet power supply (on 5s, off 5s), observe the changes in the tissue amount in the first and second containers, and observe the changes in the amount of magnetic beads in the third container. When the amount of magnetic beads in the third container is large, the C-tube peristaltic pump is turned on, the electromagnet of the third container is turned off, and the magnetic beads are transported to the first container.

[0056] When the amount of tissue in the first container gradually decreases and almost no islet tissue is found after staining with dithizone solution (DTZ), turn off the peristaltic pumps of tubes a, b, and c, take out the islet tissue in the second container, add 4°C Hanks solution, and wash the tissue twice by centrifugation at 2000r / min. After discarding the supernatant, the separated islet tissue is obtained and cultured with CMRL 1066 (containing 10% fetal bovine serum and penicillin-streptomycin) in a 37°C 5% CO2 incubator.

[0057] Experimental results: The isolated islets were of different sizes, but all were relatively complete with clear edges. Some islets were wrapped by a small amount of exocrine gland tissue. The purity of the islet tissue obtained after DTZ staining could reach 90%. The number of islets was 2839±248, and the islet equivalent was (1547.3±342.8)IEQ. After 6 hours of culture, the islet activity was 92.3±4.8% after staining with fluorescein diacetate-pyridinium iodide (FD-PI) staining solution.

[0058] Example 2

[0059] A device and method for isolating pancreatic islet tissue according to Example 2 of the present invention, the device design and application results in isolating pancreatic islets in rats are as follows:

[0060] The mesh aperture of the second container of the device is 10 microns, the cross-section of the interconnecting channels between the first channel, the second channel, the third channel and the fourth channel of the magnetic separator is elliptical, with a long diameter of 0.6 cm and a short diameter of 0.4 cm. The magnetic field strength of the electromagnet surface of the magnetic separator is 890 mT, and the magnetic field strength of the electromagnet of the third container is 800 mT. The buffer solution is RPMI 1640 solution (containing 10% fetal bovine serum). The magnetic beads are commercially available with a particle size of 200 nanometers and are coated with amino groups (-NH). The rat islet membrane protein polyclonal antibody is homemade, and the antibody coating of the magnetic beads is completed before the islet separation experiment begins.

[0061] Isolation of pancreatic islets: Adult rats (250-300 g) were taken, and the abdomen was opened after isoflurane inhalation anesthesia. The common bile duct was clamped to the entrance of the duodenum with an artery clamp. The common bile duct was retrogradely cannulated with a 24G indwelling needle, and 12-15 mL of type V collagenase solution (concentration 1.0 mg / mL) was perfused. The pancreatic tissues of three rats that were expanded after collagenase perfusion were removed, slightly chopped, and placed in a 100 mL digestion tank containing 20 mL of collagenase of the same concentration, and pipes a, b, and c were connected. Hank's solution and magnetic beads coated with rat islet membrane protein antibody were added to the first container, and the peristaltic pump connected to the digestion tank in pipe a was turned on at 80 rpm to exhaust the air in the digestion tank and the pipe; the peristaltic pump of the buffer on pipe b was turned on to exhaust the air in pipe b and turn off the peristaltic pump; the pancreas was shaken and digested in a constant temperature water bath at 37±0.5℃ until the pancreatic tissue was digested to chyle and mud (about 8 minutes). The peristaltic pump on the tube a connected to the interface of the magnetic separator 320 was turned on at a speed of 90 rpm, the peristaltic pump on the tube b was turned on at a speed of 70 rpm, and the electromagnet power was turned on intermittently (on for 5 s, off for 5 s), and the changes in the amount of tissue in the first and second containers were observed, and the changes in the amount of magnetic beads in the third container were observed. When the amount of magnetic beads in the third container was large, the peristaltic pump on the tube c was turned on, the electromagnet of the third container was turned off, and the magnetic beads were transported to the first container.

[0062] When the amount of tissue in the first container gradually decreases and almost no islet tissue is found after staining with dithizone solution (DTZ), turn off the peristaltic pumps of tubes a, b, and c, take out the islet tissue in the second container, add 4°C RPMI 1640 solution, and wash the tissue twice by centrifugation at 2000r / min. After discarding the supernatant, the isolated islets are obtained and cultured with CMRL 1066 (containing 10% fetal bovine serum and penicillin-streptomycin) in a 37°C 5% CO2 incubator.

[0063] Experimental results: The isolated islet tissue had complete appearance and clear edges, and most of the islets were not wrapped by exocrine gland tissue; the purity of the islets obtained by DTZ staining could reach 90%; the number of islets obtained from three rats was 1923±368, equivalent to (2147.5±574.6) IEQ; after 6 hours of culture, the islet activity was 91.4±3.7% after FD-PI staining.

[0064] Example 3

[0065] A device and method for isolating pancreatic islet tissue according to Example 3 of the present invention, the device design and application results in isolating pancreatic islets in adult Bama pigs are as follows:

[0066] The mesh aperture of the second container of the device is 5 microns, the cross section of the interconnecting channels between the first channel, the second channel, the third channel and the fourth channel of the magnetic separator is semicircular with a diameter of 1.0 cm, the magnetic field strength of the electromagnet surface of the magnetic separator is 560 mT, the magnetic field strength of the electromagnet of the third container is 800 mT, the buffer solution is CMRL 1066 liquid (containing 10% fetal bovine serum), the magnetic beads are commercially available with a particle size of 50 nanometers and are coated with amino groups (-NH), the polyclonal antibodies of porcine islet membrane proteins are homemade, and the antibody coating of the magnetic beads is completed before the islet separation experiment begins.

[0067] Pancreas acquisition process: Miniature pigs (male or female, weighing 30-60kg) were anesthetized by intramuscular injection of ketamine, and the hair in the abdominal surgical field was shaved, rinsed, re-fixed to the operating table, and continued to inhale isoflurane for anesthesia; the abdominal skin, muscle layer and peritoneum were cut along the linea alba with a scalpel, the pancreatic tissue was found along the duodenum, and the urine in the bladder was extracted with a 50mL syringe to expose the abdominal aorta under the bladder; a disposable infusion tube was inserted into the abdominal aorta for bleeding, which lasted about 3-5 minutes; when the bleeding was almost over, HBSS frozen into ice chips was poured around the pancreas to cool the pancreas; two surgical operators cooperated to obtain the pig pancreas, paying attention to careful operation and trying not to damage the pancreatic parenchyma, otherwise it would affect the subsequent collagenase perfusion; the pancreatic tissue was completely removed, rinsed with cold sterile HBSS to remove the blood on the surface, placed in a sterile organ preservation box with an ice bath, put in cold HBSS or UW liquid, and taken to the biosafety cabinet to prepare for the next step of iodine disinfection and trimming.

[0068] The method of collagenase perfusion of porcine pancreas based on peristaltic pump is as follows: a plastic basin with crushed ice is placed in the biosafety cabinet, and the trimming tray is placed on the ice; the newly obtained porcine pancreas is first disinfected with iodine tincture for 1-2 minutes, and then rinsed with HBSS+10U / mL sodium heparin, and then placed on the trimming tray, and HBSS+10U / mL sodium heparin is added; the porcine pancreas is simply trimmed to remove the exposed blood vessels and bloody connective tissue on the surface of the pancreas; a small piece of tissue is cut off from a branch of the pancreas to expose the pancreatic duct, and a 24G indwelling needle is used for cannulation. After success, it is ligated and fixed with 3-0 surgical sutures; a perfusion tray is replaced on the crushed ice, and the temperature monitor and perfusion pipeline are connected. Pour a small amount (about 30 mL) of HBSS containing 10 U / mL heparin sodium into the perfusion tray; turn on the peristaltic pump at a speed of 100 rpm, first use the HBSS solution containing 10 U / mL heparin sodium to remove the bubbles in the perfusion line (fill the perfusion line), and ensure that the perfusion system does not leak; then add the prepared collagenase solution to the perfusion tray (the preparation method of the collagenase solution: before pancreatic perfusion, weigh about 300 mg of collagenase V into 40 mL of HBSS solution containing 10 U / mL heparin sodium, slowly dissolve it at 2-8°C, and filter the collagenase V solution into a sterile 500 mL bottle with a 0.22 μm microporous filter membrane. Add 30 mL of 1 M HEPES to the 500 mL sterile bottle and mix to dissolve; add about 150 mL of HBSS containing 10 U / mL heparin sodium, and rotate and shake to mix evenly.) Connect the perfusion line to the indwelling needle interface.

[0069] Turn on the peristaltic pump at 100 rpm to circulate collagenase, clamp the pancreatic duct with leakage, and perfuse for about 5 minutes. Stop perfusion when the pancreas is fully expanded.

[0070] Cut the fully expanded porcine pancreas into small pieces of about 5 cm, place them in a 500 mL digestion tank, add the excess collagenase solution after pancreatic perfusion, and then add 2 mL of recombinant nuclease, connect pipelines a, b and c, add Hank's solution and magnetic beads coated with porcine islet membrane protein antibody to the first container, turn on the peristaltic pump connected to the digestion tank in pipeline a, the speed is 100 rpm, and the air in the digestion tank and pipeline is discharged; turn on the buffer peristaltic pump on tube b, discharge the air in tube b and turn off the peristaltic pump; shake and digest the pancreas in a 37±0.5℃ constant temperature water bath until the pancreatic tissue is digested to chyle and mud (about 12 minutes). Turn on the peristaltic pump on tube a connected to the interface of the magnetic separator 320 at a speed of 120 rpm, turn on the peristaltic pump on tube b at a speed of 90 rpm, intermittently turn on the power supply of the electromagnet (on 5s, off 5s), observe the changes in the tissue amount in the first and second containers, and observe the changes in the amount of magnetic beads in the third container. When the amount of magnetic beads in the third container is large, the C-tube peristaltic pump is turned on, the electromagnet of the third container is turned off, and the magnetic beads are transported to the first container.

[0071] When the amount of tissue in the first container gradually decreases and almost no islet tissue is found after staining with dithizone solution (DTZ), turn off the peristaltic pumps of tubes a, b, and c, take out the islet tissue in the second container, add 4°C CMRL 1066 solution, centrifuge at 2000r / min to wash the tissue twice, discard the supernatant to obtain the separated islets, and culture them with CMRL 1066 (containing 10% fetal bovine serum and penicillin-streptomycin) in a 37°C 5% CO2 incubator.

[0072] Experimental results: The isolated porcine islet tissue had a complete appearance with slightly rough edges, and some islets were wrapped by exocrine gland tissue; the purity obtained by DTZ staining was 85%; the number of islets obtained from a 30 g porcine pancreas was 315,623±33,285, equivalent to (208, 597.8±12,475.7) IEQ; the islet activity after 6 hours of culture was 89.6±6.5% after FD-PI staining.

Claims

1. A device for isolating pancreatic islet tissue, comprising a digestion tank for accommodating pancreatic organs and performing digestion operations; a water bath for heating digestion fluid; and magnetic beads with antibodies for binding to surface cells of pancreatic islet tissue, characterized in that: Also included is a magnetic separator for separating positive tissue and negative tissue in pancreatic digested tissue; A first container for receiving negative tissue, digestion fluid and antibody-carrying magnetic beads separated by a magnetic separator; A second container for receiving the positive tissue, buffer and antibody-carrying magnetic beads separated by a magnetic separator; a third container for receiving a buffer and magnetic beads with antibodies; The digestion tank, water bath, first container and magnetic separator are connected through a first conduit, the second container, third container and magnetic separator are connected through a second conduit, the third container and first container are connected through a third conduit, and peristaltic pumps are provided on the first conduit, the second conduit and the third conduit.

2. A device for isolating pancreatic islet tissue according to claim 1, characterized in that: The magnetic separator is provided with a first channel, a second channel, a third channel and a fourth channel, wherein there is an interconnecting channel between the first channel, the second channel, the third channel and the fourth channel, and the magnetic separator is provided with an electromagnet on one side of the interconnecting channel close to the third channel and the fourth channel.

3. A device for isolating pancreatic islet tissue according to claim 2, characterized in that: The first channel and the second channel are connected to the first conduit respectively, and the third channel and the fourth channel are connected to the second conduit respectively.

4. A device for isolating pancreatic islet tissue according to claim 1, characterized in that: The inlet of the second conduit connected to the second container is located at the bottom of the second container. A screen is provided in the second container with an aperture of 1-50 microns. The outlet of the second conduit connected to the second container is located at the top of the second container.

5. The device for isolating pancreatic islet tissue according to claim 1, characterized in that: The inlet of the second conduit connected to the third container is located at the bottom of the third container, an electromagnet is provided at the position of the bottom of the third container connected to the third conduit, the outlet of the second conduit connected to the third container is located at the top of the third container, and the outlet of the third conduit connected to the third container is located at the bottom of the third container.

6. The device for isolating pancreatic islet tissue according to claim 1, characterized in that: The diameter of the antibody-carrying magnetic beads is 10 nanometers to 100 micrometers.

7. A device for isolating pancreatic islet tissue according to claim 2, characterized in that: The cross-section of the interconnecting channel is circular, semicircular, elliptical, semi-elliptical, trapezoidal, triangular, rhombus, rectangular or square.

8. A method for islet tissue separation using the device according to claim 1, characterized in that: The method comprises the following steps: placing the pancreas in a digestion tank for digestion, and then adding antibody-carrying magnetic beads to the exposed surface cells of the pancreatic islet tissue to form positive tissue, thereby the positive tissue, the antibody-carrying magnetic beads and the buffer solution are separated into a second container through a magnetic separator, the tissue not combined with the antibody-carrying magnetic beads after the pancreas is digested forms negative tissue, the negative tissue, the antibody-carrying magnetic beads and the digestion solution are separated into a first container through a magnetic separator, the positive tissue, the antibody-carrying magnetic beads and the buffer solution are separated into a second container through a magnetic separator, the antibody-carrying magnetic beads and the buffer solution not combined with the pancreatic islet tissue in the second container enter into a third container through a second conduit, the antibody-carrying magnetic beads enter into the first container through the third conduit under the pulling action of an electromagnet, and the buffer solution in the third container enters into the magnetic separator through the second conduit for recycling.

9. A method for islet tissue isolation according to claim 8, characterized in that: The negative tissue, antibody-carrying magnetic beads and digestion solution in the first container enter the digestion tank for re-digestion and combination, and the cycle is repeated until the positive tissue is completely separated.

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