Complex as well as preparation method and application thereof

Abstract

The present application provides a complex for protecting a cell and / or tissue graft. The complex comprises a cell mixture and a cell / tissue transplant. The cell mixture is used to protect the cell / tissue graft. The transplantation body is at least one of hepatocytes, islet cells, hematopoietic stem cells, adrenal medullary cells, testicular interstitial cells, follicle cells, gland pituitary cells, thyroid tissue blocks, parathyroid gland tissue blocks and thymus tissue blocks. The cell mixture comprises at least one of mesenchymal stem cells, primary hepatocytes, primary fibroblasts, dendritic cells, primary hepatocytes and fibroblasts. The complex not only can solve the problems of oxygen deficit and lack of nutritional factors in the transplantation process of the transplantation body, but also can reduce the immunological rejection reaction of a receptor to an allosome or xenograft after the transplantation of the transplantation body. The invention further provides a preparation method and application of the complex. By utilizing the product, cells or tissues can be transplanted into the spleen, the abdominal cavity or the position below the greater omentum, so that the application range of the transplant is expanded.

Description

Technical Field

[0001] This application belongs to the technical field of cell or tissue transplantation preparation, and relates to the preparation and application of a complex for wrapping and protecting transplanted cells or tissues. Background Art

[0002] Cell or tissue transplantation follows the same immunological laws as solid organ transplantation. Taking islet transplantation as an example, allogeneic islets trigger immune-mediated rejection reactions and must be controlled with immunosuppressive drugs. However, doing so can lead to a decline in renal function and increase the risks of hyperlipidemia, infectious complications, and developing malignant tumors. At the same time, if immunosuppression is stopped, the recipient will develop immune sensitivity to islet donor tissue antigens.

[0003] In addition, since islets from multiple donors are usually required, it is difficult to find suitable donors for subsequent transplantation treatments. And due to the impact of the innate immune system on the survival of islet allografts, up to 5 - 6% of transplanted islets may be lost in the early stage after transplantation, so islets need to be transplanted from multiple donors to achieve insulin dependence.

[0004] In view of the fact that any existing method cannot effectively provide islet donor tissue, in Chinese patent application document CN1643133A, a new method of transplanting immature pancreatic tissue to mammalian recipients to increase the pancreatic mass of the recipients is proposed. This method harvests immature pancreatic tissue from mammalian donors and then forms blood vessels and matures the tissue in the pancreatic tissue. However, this method requires at least the generation of insulin - functioning tissue in the recipient, and this tissue needs to be chimeric with the endocrine gland, i.e., the pancreas, before it can be transplanted into the peritoneal cavity of mammalian recipients, so it has relatively large limitations. Summary of the Invention

[0005] Aiming at the problems that during the process of cell or tissue transplantation, the transplanted body is often hypoxic and lacks nutrient factors, and is vulnerable to the immune rejection reaction of the recipient to allogeneic or xenogeneic transplanted bodies, this application provides a complex to solve the above problems.

[0006] The first inventive point of this application lies in providing a complex.

[0007] Further, the complex includes a transplanted body and a cell mixture.

[0008] Further, the cell mixture wraps the transplanted body, thereby achieving the purpose of protecting the transplanted body.

[0009] Further, the cell mixture includes at least one of mesenchymal stem cells, primary hepatocytes, primary fibroblasts, dendritic cells, primary hepatocytes, and fibroblasts.

[0010] Further, the graft is at least one of hepatocytes, islets of Langerhans, hematopoietic stem cells, adrenal medullary cells, Leydig cells, follicular cells, adenohypophyseal cells, thyroid tissue, parathyroid tissue, and thymus tissue.

[0011] Further, 10%-100% of the surface area of the graft is wrapped by the cell mixture, and the size specification of the formed complex is 100 μm - 1000 μm.

[0012] Preferably, 50%-100% of the surface area of the graft is wrapped by the cell mixture.

[0013] The second inventive point of the present application lies in providing a preparation method of the above complex.

[0014] Further, the method includes the following steps:

[0015] S1. Extract the cells / tissues to be transplanted and place them in a buffer solution, and then place the cells / tissues to be transplanted in a primary culture medium for culture;

[0016] S2. Obtain the primary cultures and single-cell precipitates of each of mesenchymal stem cells, primary hepatocytes, primary fibroblasts, and dendritic cells in sequence, mix them to obtain a cell mixture, and dilute the cell mixture to a usable concentration range;

[0017] S3. Mix the cells in S2 with the cells / tissues to be transplanted to obtain a mixture, centrifuge the obtained mixture and then culture it to obtain the complex.

[0018] Further, in step S1, the cells / tissues to be transplanted include but are not limited to hepatocytes, islets of Langerhans, hematopoietic stem cells, adrenal medullary cells, Leydig cells, follicular cells, adenohypophyseal cells, thyroid tissue, parathyroid tissue, and thymus tissue.

[0019] Further, in step S1, the buffer solution is PBS buffer or Hanks buffer; the primary culture medium is DMEM medium, RPMI-1640 medium, or William's E medium, and 5%-25% of calf serum or fetal bovine serum, a double-antibody solution of 5%-1% penicillin and streptomycin, and 1%-2% of β-mercaptoethanol are added thereto.

[0020] Further, in step S2, prepare mesenchymal stem cells, primary hepatocytes, primary fibroblasts, and dendritic cells; then digest the primary cultures of each cell with trypsin; preferably, 50%-100% of the surface area of the cells or tissues to be transplanted is covered by the cell mixture.

[0021] Further, in step S2, the mesenchymal stem cells are derived from umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells of humans, monkeys, pigs, sheep, dogs, rabbits, rats or mice; the primary hepatocytes are extracted from the livers of humans, monkeys, pigs, sheep, dogs, rabbits, rats or mice; the primary fibroblasts are extracted from the skins of humans, monkeys, pigs, sheep, dogs, rabbits, rats or mice; and the dendritic cells are extracted from the bone marrows or spleens of humans, monkeys, pigs, sheep, dogs, rabbits, rats or mice.

[0022] Further, in step S2, the primary cultures of mesenchymal stem cells, primary hepatocytes, primary fibroblasts and dendritic cells are digested with trypsin until the cell density reaches the standard of cell dissociation. The standard of cell dissociation is that mesenchymal stem cells, primary hepatocytes, primary fibroblasts and dendritic cells are all digested into single cells, and the cell density is 1×10 2 -1×10 8 / mL.

[0023] Further, in step S2, after the digestion treatment, the primary cultures of each type of cell are treated with a neutralizing agent to neutralize the remaining trypsin, and then the cell suspension is collected and centrifuged at a speed of 500 - 2000 rpm to remove the supernatant. The obtained precipitate is resuspended in DMEM medium, 1640 medium or William's E medium, and after centrifugation, the supernatant is removed to obtain single cell precipitates of each type of cell. Then, the single cell precipitates of each type of cell are mixed and diluted to a usable concentration range. Preferably, the neutralizing agent is a medium containing 5% - 25% calf serum or fetal bovine serum, 1640 medium or William's E medium, or PBS buffer and / or Hanks buffer.

[0024] Further, in step S3, the cell mixture and the cell / tissue to be transplanted obtained in S1 are mixed in a well plate to obtain a complex precursor of the two, and then the well plate is placed in a centrifuge for centrifugation; preferably, the speed of the centrifugation treatment is 500 - 2000 rpm.

[0025] Further, in step S3, the well plate is a 6 - 384 well plate, and the 6 - 384 well plate carrying the complex precursor is centrifuged in a centrifuge for 0.5 - 6 minutes. Preferably, the acceleration and deceleration adjustment of the centrifuge is 0 - 9; preferably, the centrifugation temperature is 0 - 25°C, more preferably 15 - 25°C.

[0026] Further, in step S3, after centrifugation, the complex precursor is taken out of the well plate and placed in the above - mentioned primary cell medium, and cultured in a cell incubator for 1 - 14 days, preferably 1 - 7 days, more preferably 1 - 24 hours.

[0027] Further, during the above-mentioned culture process, the culture medium is changed every 12 - 96 hours; preferably, it is changed every 12 - 48 hours; more preferably, it is changed every 12 - 24 hours.

[0028] Further, in step S3, during the culture period, the cultured complex precursor is observed through a stereomicroscope every 12 - 24 hours to determine whether the complex precursor is formed into a standard spherical or ellipsoidal structure.

[0029] Further, if the surface of 10% - 100% of the cells / tissues to be transplanted is covered by mesenchymal stem cells, primary hepatocytes, primary fibroblasts, dendritic cells, and a mixture of primary hepatocytes and fibroblasts respectively after molding, then the complex precursor has been formed into a complex, that is, the culture of the complex is completed.

[0030] The third inventive point of this application is to provide the application of the above complex in organ transplantation.

[0031] Further, the complex can be used to promote organ / tissue regeneration for the transplantation project of endocrine organs / tissues.

[0032] Further, by using the complex, cells and / or tissues can be transplanted into the spleen, intraperitoneal cavity or under the greater omentum to expand the transplantation range of organs / tissues.

[0033] Compared with the prior art, this application has the following beneficial effects:

[0034] This application provides a mixture containing mesenchymal stem cells, primary hepatocytes, primary fibroblasts, dendritic cells, and a mixture of primary hepatocytes and fibroblasts for the current disease treatment, especially for the problems of hypoxia, starvation, immune rejection, etc. existing in cell or tissue transplantation. Various factors secreted by this mixture under the stimulation of hypoxia and starvation conditions can improve the hypoxia tolerance, anti-inflammatory, and anti-apoptotic factors of transplanted cells or tissues, protect and repair the transplant, and reduce the immune rejection of transplanted cells or tissues through the encapsulation and immune regulation functions of these cells, thereby protecting transplanted cells or tissues during and after transplantation.

[0035] Through the effective combination of the above cells, this application realizes the function of promoting organ / tissue regeneration, promotes the colonization and proliferation of the transplant to be, and thus is applied to the treatment of various diseases, and can also effectively replace the missing or damaged endocrine organs / tissues in the recipient.

[0036] By using tissue units instead of cells for transplantation, the present application can utilize the original vascular network of the tissue to rapidly integrate in the spleen, and growth factors are added to promote blood vessel regeneration, which can overcome the problem of insufficient vascularization. The secretions of endocrine organs do not require special structures or vascular networks, and can directly enter the bloodstream through the blood supply of the spleen to play a role, and the function will not be affected by ectopic transplantation. It can not only solve the problems of hypoxia and lack of nutrient factors during the transplantation of the graft, but also reduce the immune rejection reaction of the recipient to allogeneic or xenogeneic grafts after transplantation.

[0037] Using the complex provided by the present application, cells or tissues can be transplanted into the spleen, intraperitoneal cavity or under the greater omentum to expand the application range of the graft. Description of the Drawings:

[0038] Figure 1 It is a gross view of hepatopancreatic cell spheres.

[0039] Figure 2 It is for the detection of insulin secretion function of islets in vitro. The blue broken line represents hepatopancreatic cell spheres, and the red broken line represents single islets. Compared with single islets, the islet function of hepatopancreatic cell spheres is better, manifested in that the insulin secretion ability is more stable than that of single islets.

[0040] Figure 3 The left figure is the statistical result of the proportion of islet area after hepatopancreatic cell spheres are implanted into the spleen by immunofluorescence staining. The result shows that the retention rate of hepatopancreatic cell spheres implanted in the spleen is significantly increased compared with islet implantation; the right figure is a representative picture after hepatopancreatic cell spheres are implanted into the spleen. The green part is the islet part, and the red part is the hepatocyte part.

[0041] Figure 4 It is a representative picture after hepatopancreatic cell spheres are implanted into the spleen. The green part is the islet part, and the red part is the hepatocyte part.

[0042] Figure 5 It is the result of the blood glucose monitoring experiment for detecting the treatment of diabetic mice, and the related gene expression in the spleen after transplantation. The cell or tissue transplantation group, hepatopancreatic cell sphere transplantation group, normal group, and non-treatment group are set up, and blood glucose monitoring is carried out every two days. The results prove that after hepatopancreatic cells are transplanted into the spleen, they have better blood glucose maintenance ability than cell or tissue transplantation, and the islet function in them is close to that of the islets in the pancreas of normal mice.

[0043] Example 1

[0044] This example provides a cell preparation.

[0045] The cell preparation is composed of a cell mixture and a graft.

[0046] Further, the cell mixture includes at least one of mesenchymal stem cells, primary hepatocytes, primary fibroblasts, dendritic cells, primary hepatocytes, and fibroblasts.

[0047] Further, the graft is at least one of hepatocytes, primary islet cells, hematopoietic stem cells, adrenal medulla cells, Leydig cells, follicular cells, adenohypophysis cells, and thyroid.

[0048] Further, 10%-100% of the surface area of the graft is wrapped by the cell mixture, and the size specification of the formed complex is 100 μm - 1000 μm.

[0049] Preferably, 50%-100% of the surface area of the graft is wrapped by the cell mixture.

[0050] Example 2

[0051] This example provides a preparation method of the above cell preparation, and the method includes the following steps:

[0052] S1, Place the hepatocytes, islets, hematopoietic stem cells, adrenal medulla cells, Leydig cells, follicular cells, adenohypophysis cells, thyroid tissue blocks, parathyroid tissue blocks, and / or thymus tissue blocks to be transplanted in PBS or Hanks buffer and let them stand still on ice, and then place them in the primary cell culture medium for short-term culture;

[0053] In some embodiments, the cells / tissues to be transplanted above can be fresh or can be revived after cryopreservation.

[0054] In some embodiments, in some embodiments, the fresh hepatocytes, islets, hematopoietic stem cells, adrenal medulla cells, Leydig cells, follicular cells, adenohypophysis cells, thyroid tissue blocks, parathyroid tissue blocks, and thymus tissue blocks extracted are placed in PBS or Hanks solution and stand still on ice for 0.1 - 10 hours, and then cell counting is performed.

[0055] In some embodiments, after standing still on ice, the above hepatocytes, islets, hematopoietic stem cells, adrenal medulla cells, Leydig cells, follicular cells, adenohypophysis cells, thyroid tissue blocks, parathyroid tissue blocks, and / or thymus tissue blocks are put into the primary cell culture medium, and the primary cell culture medium is placed in a cell culture incubator and cultured at 37°C for 1 - 96 hours, preferably 24 - 72 hours, more preferably 48 - 72 hours.

[0056] In some embodiments, the primary cell culture medium is DMEM medium, 1640 medium or William's E medium, and 5%-25% fetal calf serum or calf serum, a double-antibody solution of 0.5%-10% penicillin and streptomycin, and 0.01%-2% β-mercaptoethanol are added thereto.

[0057] In some embodiments, the cell incubator is a 5% CO2 incubator, and its gas environment is 95% air and 5% CO2 by volume. Gas is one of the essential conditions for cell culture survival. The required gases are mainly oxygen and carbon dioxide. Oxygen participates in the tricarboxylic acid cycle, generating energy for cell growth and proliferation and synthesizing various components required for cell growth. When culturing cells under open conditions, the cells are generally placed in a mixed gas environment of 95% air and 5% carbon dioxide.

[0058] S2. Prepare mesenchymal stem cells, primary hepatocytes, primary fibroblasts, and dendritic cells independently, and obtain the primary cultures of each cell respectively.

[0059] In some embodiments, the mesenchymal stem cells are derived from umbilical cord mesenchymal stem cells or bone marrow mesenchymal stem cells of humans, monkeys, pigs, sheep, dogs, rabbits, rats or mice; the primary hepatocytes are extracted from the livers of humans, monkeys, pigs, sheep, dogs, rabbits, rats or mice; the primary fibroblasts are extracted from the skins of humans, monkeys, pigs, sheep, dogs, rabbits, rats or mice; the dendritic cells are extracted from the bone marrow or spleen of humans, monkeys, pigs, sheep, dogs, rabbits, rats or mice.

[0060] S3. Independently perform digestion treatment on the primary cultures of each cell until the standard of the target separation degree is reached, and obtain the primary cultures of each cell after digestion.

[0061] In some embodiments, trypsin is added to the medium to perform digestion treatment on the primary cultures of each cell, and adherent cells are released from the surface of the culture dish by digesting adhesion proteins, and at the same time, cells are also released from aggregates by digesting adhesion proteins; the digestion process stops until the standard of the target separation degree is reached, and finally the primary cultures of each cell after digestion are obtained.

[0062] In some embodiments, the standard of cell separation degree is that mesenchymal stem cells, primary hepatocytes, primary fibroblasts, and dendritic cells are all digested into single cells, and the cell density is 1×10 2 -1×10 8 / mL.

[0063] S4. After digestion, the primary cultures after cell digestion are independently treated with pre-cooled neutralizing agent to neutralize the remaining trypsin. The cell suspensions are collected, centrifuged, and the supernatant is removed. The obtained precipitate is resuspended in DMEM, 1640, or William's E medium, centrifuged for 0.5 - 6 minutes, and the supernatant is removed to obtain single-cell precipitates of each cell type.

[0064] In some embodiments, the neutralizing agent pre-cooled to 0 - 4°C is formulated as DMEM medium, 1640 medium, or William's E medium containing 5% - 25% calf serum or fetal bovine serum, or PBS buffer and / or Hanks buffer. The centrifugation speed is 500 - 2000 rpm, and the centrifugation time is 0.5 - 6 minutes.

[0065] S5. The single-cell precipitates of the above various cell types are independently resuspended in DMEM medium, 1640 medium, or William's E medium, and cell counting is performed.

[0066] S6. The cultured cells are placed in the primary cell medium and cultured for 3 - 12 hours, then taken out and aseptically selected using a stereomicroscope in a laminar flow hood or biosafety cabinet.

[0067] In some embodiments, after taking out the primary culture of each cell type from the primary cell medium, it is placed in a 2 - 10 cm culture dish and selected using a 20 - 200 μL pipette in a laminar flow hood. The circulating air speed of the laminar flow hood is 2 - 8 grades.

[0068] S7. Mesenchymal stem cells, primary hepatocytes, primary fibroblasts, dendritic cells, and the mixture of primary hepatocytes and primary fibroblasts are diluted to a usable concentration, and the cells are mixed with purified hepatocytes, islets, hematopoietic stem cells, adrenal medullary cells, Leydig cells, follicular cells, adenohypophysis cells, thyroid tissue blocks, parathyroid tissue blocks, and / or thymus tissue blocks in a well plate.

[0069] In some embodiments, mesenchymal stem cells, primary hepatocytes, primary fibroblasts, dendritic cells, and the mixture of primary hepatocytes and primary fibroblasts are mixed with the cells / tissues to be transplanted at a volume ratio of 1×10 2 -1×10 8 :1 to form a complex precursor.

[0070] S8. The well plate after adding the mixture is placed in a centrifuge for centrifugation.

[0071] In some embodiments, the complex precursor is placed in a 6 - 384 well plate.

[0072] In some embodiments, the 6 - 384 well plate carrying the complex precursor is centrifuged in a centrifuge for 0.5 - 6 minutes. The acceleration and deceleration of the centrifuge are adjusted to 0 - 9, and the centrifuge temperature is 0 - 25 °C.

[0073] S9. Place the well plate in a cell culture incubator for culturing;

[0074] In some embodiments, after centrifugation, the complex precursor is taken out of the well plate and placed in a mixed solution of primary cell culture media of different cell types contained in the complex. It is cultured in a cell culture incubator for 1 - 14 days, and the culture medium is changed every 12 - 96 hours during this period.

[0075] In some embodiments, the cell culture incubator is a 5% CO₂ incubator, and its gas environment is 95% air and 5% CO₂ by volume.

[0076] S10. Continue the culture. After observing under a microscope that the transplanted complex forms a uniform spherical structure, the complex for protecting the transplanted cells or tissues is completed.

[0077] In some embodiments, during the culture, the cultured complex precursor is observed through a stereomicroscope every 12 - 24 hours to determine whether the complex precursor is formed into a standard spherical or ellipsoidal structure; if it is formed into a standard spherical or ellipsoidal structure and 10% - 100% of the surfaces of the cells / tissues to be transplanted are respectively covered by mesenchymal stem cells, primary hepatocytes, primary fibroblasts, dendritic cells, and a mixture of primary hepatocytes and fibroblasts, then the complex precursor has been formed into a complex, that is, the complex culture is completed.

[0078] Example 3

[0079] This example provides a preparation process of a complex, which includes mouse islet cells and mouse hepatocytes, and the mouse hepatocytes wrap the mouse islet cells.

[0080] 1. The preparation process includes the following steps:

[0081] S1. The freshly extracted islets are placed in Hank's solution and left to stand on ice for 0.1 hour;

[0082] S2. Put the islets into the primary islet culture medium and culture them in a cell culture incubator at 37 °C in a 5% CO₂ cell culture incubator for 1 hour;

[0083] S3. Prepare primary hepatocytes and digest the mouse liver;

[0084] S4. Collect the hepatocyte suspension, centrifuge it at a speed of 500 rpm for 3 minutes, remove the supernatant, and resuspend the obtained precipitate in DMEM;

[0085] S5. Centrifuge again at a speed of 500 rpm for 3 minutes, remove the supernatant, and obtain a single-cell pellet of hepatocytes, with a cell density of 1×10 4 / mL;

[0086] S6. Resuspend the single-cell pellet of hepatocytes in DMEM medium and perform cell counting;

[0087] S7. After culturing, take out the islets and place them in a 10-cm culture dish. Use a 200-μL pipette to select islets. The selection environment is a laminar flow hood with a circulating air speed of level 2. Select islets with an islet equivalent between 1-3 IEQ for standby;

[0088] S8. Mix primary hepatocytes with islets. The mixing ratio of primary hepatocytes to primary islets is 100:1. Place the mixed islets and other cells in a 6- to 384-well plate;

[0089] S9. Place the 6- to 384-well plate with the mixture in a centrifuge and centrifuge for 1 min. Adjust the acceleration and deceleration to 0, and the centrifuge temperature is 0°C;

[0090] S10. Place the well plate in a cell culture incubator and culture the complex protecting the transplanted cells or tissues in the cell culture incubator for 1 day, changing the medium every 12 hours.

[0091] Observe once every 12 hours with a stereomicroscope to observe whether the complex precursor forms a standard spherical or ellipsoidal structure.

[0092] 2. Experimental results:

[0093] The complex is formed into a standard spherical or ellipsoidal shape, and 10% of the islet surface is covered by primary hepatocytes, indicating that the culture of protecting the complex is completed.

[0094] Example 4

[0095] This example provides a preparation process of a complex, and the complex includes rat islet cells and mouse hepatocytes, wherein the mouse hepatocytes wrap the rat islet cells.

[0096] 1. The preparation process includes the following steps:

[0097] S1. Place the freshly extracted islets in Hank's solution and let them stand on ice for 10 hours;

[0098] S2. Put the islets into the primary islet medium and culture them in a cell culture incubator at 37°C and 5% CO2 for 96 hours;

[0099] S3. Prepare primary hepatocytes and digest the mouse liver;

[0100] S4. Collect the hepatocyte suspension, centrifuge it at a speed of 2000 rpm for 6 minutes, remove the supernatant, and resuspend the obtained precipitate in DMEM.

[0101] S5. Centrifuge again at a speed of 2000 rpm for 6 minutes, remove the supernatant, and obtain a single-cell precipitate of hepatocytes with a cell density of 1×10 2 / mL.

[0102] S6. Resuspend the single-cell precipitate of hepatocytes in DMEM medium and perform cell counting.

[0103] S7. After culturing, take out the islets and place them in a 2-cm culture dish. Use a 20-μL pipette to select islets. The selection environment is a laminar flow hood with a circulating air speed of grade 8. Select islets with an islet equivalent between 1 - 3 IEQ for standby.

[0104] S8. Mix primary hepatocytes with islets. The mixing ratio of primary hepatocytes to primary islets is 1×10 6 :1. Place the mixed islets and other cells in a 6 - 384-well plate.

[0105] S9. Place the 6 - 384-well plate added with the mixture in a centrifuge and centrifuge for 3 min. Adjust the acceleration and deceleration to 5, and the centrifuge temperature is 15°C.

[0106] S10. Place the well plate in a cell culture incubator and culture the precursor complex in the cell culture incubator for 7 days, changing the medium every 48 hours during this period.

[0107] Observe once every 20 hours with a stereomicroscope to check whether the precursor complex protecting the transplanted cells or tissues forms a standard spherical or ellipsoidal structure.

[0108] 2. Experimental results:

[0109] The precursor complex is formed into a standard spherical or ellipsoidal shape, and 50% of the islet cell surface is covered by mouse hepatocytes, indicating that the culture of protecting the complex is completed.

[0110] Example 5

[0111] This example provides a preparation process of a complex, and the complex includes porcine hepatocytes and rat islet cells.

[0112] 1. The preparation process includes the following steps:

[0113] S1. Place the freshly extracted islets in Hanks buffer and let them stand on ice for 5 hours.

[0114] S2. Place the islets in S1 into the primary islet culture medium, and place the medium in a cell incubator with 5% CO2 and culture at 37 °C for 40 hours.

[0115] S3. Prepare primary hepatocytes, digest the pig liver, and collect the hepatocyte suspension.

[0116] S4. Centrifuge at 1000 rpm for 0.5 minutes, remove the supernatant, and resuspend the obtained precipitate in DMEM.

[0117] S5. Centrifuge again at 1000 rpm for 0.5 minutes, remove the supernatant, and obtain a single-cell precipitate of hepatocytes with a cell density of 1×10 6 / mL.

[0118] S6. Resuspend the single-cell precipitate of hepatocytes in DMEM medium and perform cell counting.

[0119] S7. After culturing, take out the islets and place them in a 5-cm culture dish. Use a 100-μL pipette to select islets. The selection environment is a laminar flow hood with a circulating air speed of level 4. Select islets with an islet equivalent between 100-300 IEQ for standby.

[0120] S8. Mix the primary hepatocytes with the islets. The mixing ratio of primary hepatocytes to primary islets is 1×10 8 :1. Place the mixed islets and other cells in a 6-384-well plate of a nucleic acid detector.

[0121] S9. Place the 6-384-well plate with the mixture added into a centrifuge and centrifuge for 6 minutes. Adjust the acceleration and deceleration to 9, and the centrifuge temperature is 25 °C.

[0122] S10. Place the well plate in a cell incubator and culture for 14 days. Change the medium every 96 hours during this period. Observe once every 24 hours using a stereomicroscope to observe whether the complex precursor forms a standard spherical or ellipsoidal structure.

[0123] 2. Experimental results:

[0124] The complex is formed into a standard spherical or ellipsoidal shape, and 100% of the islet surfaces are covered by pig hepatocytes, indicating that the culture of protecting the complex is completed.

[0125] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present application shall be included within the protection scope of the present application.

Claims

1. A complex, characterized in that, The complex is composed of a cell mixture and a graft. The cell mixture includes at least one of mesenchymal stem cells, primary hepatocytes, primary fibroblasts, dendritic cells, primary hepatocytes and fibroblasts. The graft is at least one of hepatocytes, primary islet cells, hematopoietic stem cells, adrenal medullary cells, Leydig cells, follicular cells, adenohypophysis cells, and thyroid glands.

2. The composite body according to claim 1, characterized in that, 10%-100% of the surface area of the graft is wrapped by the cell mixture, and the size specification of the formed complex is 100μm - 1000μm.

3. A method for preparing a composite body according to any one of claims 1-2, characterized in that, The method includes the following steps: S1. Extract the cells or tissues to be transplanted, place them in a buffer solution, and then place the cells or tissues to be transplanted in a primary culture medium for culture. S2. Obtain the primary culture and single-cell precipitate of any one of mesenchymal stem cells, primary hepatocytes, primary fibroblasts, and dendritic cells in sequence, mix them to obtain a cell mixture, and dilute the cell mixture to a usable concentration range. S3. Mix the above cell mixture with the cells / tissues to be transplanted to obtain a mixture, centrifuge the obtained mixture and then culture it to obtain the complex.

4. The preparation method according to claim 3, characterized in that, In step S1, the cells or tissues to be transplanted include, but are not limited to, hepatocytes, islets, hematopoietic stem cells, adrenal medullary cells, Leydig cells, follicular cells, adenohypophysis cells, thyroid tissues, parathyroid tissues, and thymus tissues. The buffer solution is PBS buffer solution or Hanks buffer solution. The primary culture medium is DMEM medium, RPMI-1640 medium, or William's E medium, and fetal calf serum or calf serum, double antibody solution, and β-mercaptoethanol are added to the above media.

5. The preparation method according to claim 3, characterized in that, In step S2, prepare mesenchymal stem cells, primary hepatocytes, primary fibroblasts, and dendritic cells; then digest the primary culture of each cell with trypsin.

6. The preparation method according to claim 3, characterized in that, In step S2, after the digestion treatment, treat the primary culture of each cell with a neutralizing agent to neutralize the remaining trypsin, then centrifuge the collected cell suspension, remove the supernatant, resuspend the obtained precipitate in DMEM medium, 1640 medium, or William's E medium, centrifuge again and then remove the supernatant to obtain the single-cell precipitate of each cell respectively. Then mix the single-cell precipitates of the above various cells and dilute them to a usable concentration range.

7. The preparation method according to claim 6, characterized in that, In step S2, the neutralizing agent is a culture medium containing 5%-25% by volume of calf serum or fetal bovine serum, 1640 medium or William's E medium, or PBS buffer and / or Hanks buffer; the available concentration range is 1×10 2 -1×10 8 cells / mL.

8. The preparation method according to claim 3, characterized in that, In steps S3 and S4, mix the cell mixture with the cells / tissues to be transplanted obtained in S1 in a well plate, then centrifuge the well plate in a centrifuge, and after centrifugation, continue to place the well plate in a cell culture incubator for culture.

9. The preparation method according to claim 9, wherein, The speed of the centrifugation treatment is 500 - 2000 rpm, and then continue to place the well plate in a cell culture incubator and culture it at 37°C for 1 - 96 hours.

10. Use of a complex as described in any one of claims 1 - 2, or a complex obtained by the preparation method as described in any one of claims 3 - 9, in cell or tissue transplantation.

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