WO2008034387A1

WO2008034387A1 - Method for preparing repairable piglet pancreatic island cells

Info

Publication number: WO2008034387A1
Application number: PCT/CN2007/070708
Authority: WO
Inventors: Junying Zhang; Bin Wang; Zhangcun Wang; Qingtang Fan
Original assignee: Zhengzhou Tongyuan Biotechnology Co., Ltd.
Priority date: 2006-09-18
Filing date: 2007-09-17
Publication date: 2008-03-27
Also published as: CN100554407C; CN1920009A

Abstract

Method for preparing repairable piglet Pancreatic Island cells, which comprises following steps: (1) choosing donator: choosing healthy piglet, getting pancreas by opening abdomen under aseptic condition, removing integument, vessels, connective tissues to Hank`s liquid at 4?; (2) separating and purifying pancreatic island cells: separating pancreatic island cells from external secretion tissues by mechanical grinding, and then obtaining pancreatic island cells by Dextran discontinuous density gradient centrifugation; (3) culturing pancreatic island cells: conducting CO2 culturing with microsphere carriers without serum, using RPMI-1640 culture solution supplemented with fibronectin, packaging the pancreatic island cells in the APA biological film to form microspheres during the course of culture, culturing in the CO2 incubator at 37 ?, changing culture solution every 48 hours, and getting the piglet pancreatic island cells in the forms of microspheres with good activity and high purity.

Description

Method for producing repairable piglet islet cells

Technical field

The invention relates to the field of biological cell technology, in particular to a method for repairing piglet islet cells, which can be clinically applied to islet transplantation of diabetic patients. Background technique

Diabetes is a global disease that threatens human health. The incidence is 3-5%; mortality is second only to cardiovascular disease and tumors. It is predicted that by 2025, the number of diabetic patients worldwide will reach 300 million. Among them, type I diabetes patients (IDDM) will account for 5-10%; these people will depend on exogenous insulin for life. Long-term injection of insulin can cause local skin irritation, itching, induration, infection, subcutaneous fat atrophy or fibrosis. Injecting insulin every day not only causes inconvenience and pain to the patient, but more importantly, the application of insulin can only control hyperglycemia and prevent the occurrence of chronic complications. A large number of experiments over the years have shown that heterologous insulin cell transplantation can not only reverse the symptoms of hyperglycemia in patients with type I diabetes (IDDM), but also prevent the occurrence and development of chronic complications. However, the application of islet cell transplantation to clinical needs to address several key technical issues. One is the source of islet cell donors. The second is the isolation and purification of islet cells. The third is islet cell culture, training, and induction. The fourth is the choice of transplant site. These key technologies must be solved by applying modern bio-technologies. Otherwise, the lack of that part will lead to the failure of islet cell transplantation for the treatment of type 1 diabetes, resulting in recurrence of diabetes.

The current status of islet cell transplantation in the treatment of type 1 diabetes: 1990s: Clinical application of islet transplantation has become a hot spot in transplantation research. According to relevant information, from 1989 to 2003, more than 70 patients with type I diabetes in three countries had received porcine islet transplantation. In the early stage, researchers at home and abroad used human embryonic islets for transplantation. The mechanism of type I diabetes is caused by autoimmune damage. Although allografts do not produce rejection, autoimmune damage to transplanted islets cannot be avoided. . Coupled with the lack of human embryo donors, ethics and other factors limit the promotion and development of this work. Later, it was found that pig insulin has been clinically applied for more than ten years. The safety and effectiveness have been fully proved. From the molecular biology and histology analysis, the insulin structure of human and pig is very similar, all have A chain, B chain, 51 Amino acid composition; A chain contains 21 amino acids, B chain 30 amino acids. The difference is that the 30th amino acid of human B chain is threonine; the 30th alanine of pig B chain. The difference in amino acids did not affect the specific stereostructure, so there was no change in biological functional activity. Such porcine islets as a donor have attracted great interest from researchers. In order to obtain purified islets, collagenase digestion was used in the separation of the pancreas. This method can be used to obtain purified islets in the laboratory, but neglects the irreversible side effects of islet and collagenase contact. Collagenase is prone to gel formation during digestion. This thing is not Easy to elute, easy to adhere to the biofilm affects the absorption of nutrients during the growth and regeneration of islet cells, and also affects the discharge of cellular metabolic products. In addition, collagenase is a strong chemotactic agent that adheres to the islet cell membrane and has strong chemotaxis to macrophages. This not only does not reduce the rejection, but also increases the rejection of the immune system. The digestion of collagenase is prone to produce a large number of clotting factors, which increases the risk of intravascular blood coagulation after transplantation, and is not conducive to long-term survival in the blood after islet transplantation. In terms of cell culture, each laboratory uses RPMI 1640 medium, plus calf serum or fetal bovine serum. However, the composition of the serum is complicated and it is easy to cause adverse culture effects.

Therefore, there is an urgent need in the art to provide an effective method of obtaining islet cells. Summary of the invention

The present invention aims to provide a method for producing repairable islet cells of piglets. The invention provides a method for producing repairable islet cells of piglets, which comprises the following steps: (1) isolating and purifying the islet cells: mechanically grinding the islets of the piglets and separating the exocrine tissue and then centrifuging through discrete density gradient to obtain islets Cell

(2) culturing islet cells: sodium alginate-polylysine-alginate microspheres coated with islet cells are cultured in RPMI-1640 culture medium containing fibronectin (FN), serum in the culture solution The content is 0.

In another preferred embodiment, the end time from separation to purification is 4 to 6 hours by Dextran discontinuous density gradient centrifugation in step (1).

In another preferred embodiment, the amount of fibronectin is 5-30 mg per liter of the medium volume. In another preferred embodiment, the step (1) further comprises the steps of: selecting an islet cell donor: the newly-born, non-breasted healthy piglets are aseptically opened to the abdomen to remove the capsule, blood vessels, and connective tissue. It was placed in a 4 °C Han s solution. The piglet is preferably born within 24 hours and is a progeny of the cross. In another preferred embodiment, the specific steps of the step (1) for isolating and purifying the islet cells are: 1 isolating islets

a. Using a VI 6 vibrating cell bead mill to separate the islets from other pancreas, grinding lOmin; b. Put the slurry into 4°CHank solution and over 500 μm metal stainless steel mesh; c. Re-grind the mesh without repeating the mesh. Repeat the mesh several times to make the slurry pass 500 μ ηι metal without embroidering. Steel mesh

d. The collected filtrate was centrifuged at 1500 r/min for 5 minutes at 4 ° C to collect sediment.

R, discard the supernatant;

2 Purification: Centrifugal with dextran discontinuous density gradient, operating as follows:

a, with 15ml, 27% Dextrna and precipitate R thoroughly mixed, based on this slowly added 4ml, 27% Dextran; 6ml, 23% Dextran; 4ml, 11% Dextran and 4ml Hank-s; b, centrifuge at 4 ° C 600r / min for 4min; c, then force the mouth velocity to 2500r / min centrifugation lOmin; d, collect 11% - 23% two layers of islet cells; e, wash twice with RPMI1640 solution, discard The supernatant, collect the cell pellet S; f, mix the cell pellet S with 10ml RPMI1640, take a microscopic examination, observe the islet morphology, activity, purity, quantity, survival rate;

g, from the separation to the end of purification time is controlled at 4 6 hours. A preferred method for producing repairable piglet islet cells of the present invention comprises the following steps: (i) selecting islet cell donors: donor selection of newly born, unbreasted healthy piglets, aseptically lowering the pancreas, removing the pack Membrane, blood vessels, and connective tissue were juxtaposed in 4 °C Hank solution;

(ϋ) Isolation and purification of islet cells: Islets are separated from exocrine tissue by mechanical grinding, and then islet cells are obtained by Dextran discontinuous density gradient centrifugation, and the morphology, activity purity, quantity, and survival rate of the sample are taken from the separation to the end of purification. Time control at 4 6 hours;

(iii) Culture of islet cells: cultured with serum-free microsphere carrier, using RPMI-1640 culture medium, bovine serum is not contained in the culture medium, and fibronectin (FN) is added, and the amount is added according to the culture. The base volume is 5-30mg per liter plus FN. During the culture process, the islet cells are encapsulated in the APA biofilm to form microspheres. The morphology, activity and count of the microcapsules are detected at appropriate time, and cultured in a constant temperature 37 ° C C0 ₂ incubator. Change the culture medium for 48 hours, and regularly take the culture solution to determine the insulin content. Observe and record the islet cells to differentiate, grow and secrete insulin in the microcapsules to determine the termination culture time, and finally obtain the microcapsules of the newborns with high activity and high purity. . Accordingly, the present invention provides an efficient method of obtaining islet cells. detailed description

SUMMARY OF THE INVENTION The object of the present invention is to develop a method for producing repairable piglet islet cells in response to the problems of the prior art described above.

The object of the present invention is achieved by the following scheme: The method for producing repairable piglet islet cells of the present invention comprises the following process steps:

(1) Selection of islet cell donors: The donors were selected from newly born, unbreasted healthy piglets, and the pancreas was taken aseptically under laparotomy, and the envelope, blood vessels and connective tissue were removed and placed in Hank liquid at 4 °C;

(2) Isolation and purification of islet cells: Islets are separated from exocrine tissue by mechanical grinding. Then, the islet cells were obtained by Dextran (dextran) discontinuous density gradient centrifugation, sampling microscopic morphology, active purity, quantity, and collection, and the separation from the separation to the end of purification should be controlled at 4 6 hours;

(3⁄4, islet cell culture: C02 culture was carried out using serum-free microsphere carrier, RPMI-1640 culture solution was used, and fibronectin was added. During the culture process, islet cells were encapsulated in APA biofilm to form microspheres. The morphology, activity and count of the microcapsules were detected, cultured in a CO2 incubator at 37 °C, and cultured for 48 hours. The culture medium was periodically taken to determine the insulin content. The islet cells were differentiated, grown and secreted in the microcapsules. The function is to determine the termination culture time, and finally a large number of islet cells of the microcapsules with active and high purity are obtained.

In the present invention, the piglet is born within 24 hours and is a hybrid progeny.

The specific steps for isolation and purification of islet cells are:

1 separation of islets

a. Using a VI 6 vibrating cell bead mill to separate the islets from other pancreas (grinding l Omin );

b. Put the slurry into the 4 °C Han s solution and pass the 500 μ m metal stainless steel mesh;

c. Re-grinding the mesh without passing through the mesh, repeating the mesh repeatedly, and passing the slurry through the 500 μ ηι metal stainless steel mesh; d. The collected filtrate was centrifuged at 1500 r/min for 5 minutes at 4 ° C, and the precipitate R was collected, and the supernatant was discarded;

a. Mix 15ml, 27% Dextrna (Glucan-Shanghai Reagent II) with the precipitate, and slowly add 4ml, 27% Dextran, 6ml, 23% Dextran;

4ml, 11% Dextran and 4ml Hank-s; b, centrifuged at 4 °C, 600r/mi n for 4min;

c, then the speed of the mouth to 2500r / min centrifugation l Omin;

d, collecting 1 1% - 23% two layers of islet cells;

e, wash twice with RPMI-1640 solution, discard the supernatant, collect cell pellets S

f. Mix the cell pellet S with 10ml RPMI-1640, take a microscopic examination, observe the islet morphology, activity, purity, quantity, and survival rate;

g, from separation to the end of purification should be controlled at 4 6 hours.

The innovations of the invention are as follows:

<1> Choice of donor

1. Just born no more than one day (not breastfeeding) piglets. Because he just left the mother, he was not in contact with the outside world. As long as the paternal paternal maternal system is carefully examined, the occurrence of infectious diseases can be eliminated, and the safety after transplantation can be guaranteed.

2. Taking advantage of the advantages of hybridization, the cells are easily mutagenized and easily adapt to the new environment.

3. Porcine islet cells are basically mature in the late embryo. The exocrine part of the pancreas develops behind the pancreas. This makes it easy to distinguish between islets and exocrine tissues. It facilitates the purification of islets.

4. Just one day piglets (not breastfeeding) and fetal pigs do not need any surgery; compared with young pigs, they are not exposed to external safety; compared with adult pigs, islets are just mature and have low antigenicity; they can be used for training and induction. The plasticity of hybrid and immature islets develops in the hope of human beings and is low in cost, which is suitable for industrial production.

<2> Isolation and purification of islet cells.

Due to the differentiation and development of porcine islets to the late stage of the embryo, the secretory tissues of the pancreas are still immature during childbirth. Therefore, it should not be separated by conventional collagenase digestion. The irreparable side effects of the enzyme on islet cells are the main reasons for the growth, renewability, activity, and function of islet transplantation after transplantation. Therefore, a VI6 vibrating cell bead mill is used. Islet by mechanical grinding Separated from exocrine tissue. Islet cells were then obtained by Dextran (dextran) discontinuous density gradient centrifugation.

<3> The culture is carried out using a serum-free microsphere carrier.

1. Add fibronectin (FN) to the medium and add F-30 5mg per liter of medium to promote adherent growth of the cells. Adhesion is a necessary condition for the growth of animal cells.

2. During the culture process, the islet cells are encapsulated in the A-P-A biofilm to form microspheres, which not only protects the islet cells from the rejection reaction, but also creates conditions for islet cell fusion and adherence.

A-P-A is an abbreviation for sodium alginate-polylysine-sodium alginate microcapsule technology. (references:

Foreign Medical Surgery Volume, Vol. 27, No. 2, 2000. P75— 77 )

Compared with the prior art, the cell separation, purification and culture techniques used in the present invention have the advantages that: due to the islet graft, the number of islets is directly lost during the usual purification process, and often due to forced oscillation, high-speed centrifugation, preparation It is easy to die if the time is extended. It has now been demonstrated that cell scaffolds are tightly linked to the extracellular matrix by transmembrane adhesin molecules, forming a large network of structures and functions. When the cells are isolated or transplanted, the cell scaffold, the extracellular matrix complex ruptures, and the adhesin molecule transmits a cell death signal. Experiments have shown that cell detachment from its extracellular matrix will cause apoptosis through a laminin molecule-mediated death signal. When the experts observed incomplete digestion during the separation process, the islets were embedded in a circle of extracellular matrix, and the apoptotic rate was very low (< 10%), while the highly purified islets had extremely high apoptosis. The rate (90%), and the former continues to have good insulin secretion. This indicates that the isolated islet grafts are highly inactive and functionally deficient. If fibronectin is added during the separation process, the apoptosis of free islets can be reduced, and the islet survival rate can reach more than 90%, the number is larger, and the volume is larger. These large islet cell clusters are not easily lost due to portal vein blood flow or pressure. Piglet paternity, maternal archives cattle safety report (accepted by statutory department) Pedigree file, epidemic report, medical report: Newborn pig "Newborn day (not breastfeeding) Health, cleansing" Anatomy of the pancreas (disinfection, under sterile) Open the abdomen to take the pancreas ^ pancreas (decapsulation, blood vessel, connective weaving 4 °C Hank's solution containing islet tissue blocks (VI6 vibrating cell ball mill grinding minutes, over 500 μπι metal mesh mixture ( Washed with Hank's solution at 4 °C three times, centrifuged the precipitates of the dextran discontinuous density gradient centrifugation (Dextran), collected ll % _23 % j | islet islet cells (sampled microscopic morphology, active purity, Quantity, collection ^" ~ ~ after the islet island ^! Cell "alginate-polylysine-alginate (APA) electrostatic high-pressure microcapsule coating ^ microcapsules (detection microcapsule diameter of 0. 25 - 0. 35 Uniform, no damage, no nakedness, form, activity Microcapsule islet culture

Change culture 3⁄4)

Insulin release

The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, may be arbitrarily combined.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in which the specific conditions are not indicated in the following examples are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. The present invention is further described below in conjunction with specific steps:

First, the choice of heterologous islets:

1. Characteristics of islets of newborn piglets:

(1) Both human and pig insulin molecules have an A chain and a B chain, all composed of 51 amino acids.

The A chain contains 21 amino acids and the B chain contains 30 amino acids. And there is a disulfide chain connection between the A and B chains. The structure is very similar, the difference is only one amino acid difference. The 30th amino acid of human insulin B chain is threonine. The 30th amino acid of pig insulin B chain is alanine. (2) Porcine insulin has been used in clinical treatment for patients with type 1 diabetes for more than a decade, which fully demonstrates the safety and efficacy of porcine insulin for human use. (3) The islet of the newborn pig is weakly immunogenic and produces little rejection. (4) Porcine islet cells survive and increase in culture with human fresh serum. It indicates that human serum has no cytotoxic effect on porcine islets, which is beneficial to the training, induction and growth after transplantation. (5) The newly born piglets, especially the pancreatic cells, have not yet differentiated. Cultivate in a different environment to give play to the plasticity of newborn pigs. (6) Newborn pigs have less contact with the outside world. As long as there is no infectious disease in the maternal, paternal, or zoonotic diseases, newborn pigs will not have infectious diseases, but the mother and father of newborn piglets should have pedigrees, medical records, physical examinations, and epidemic prevention files. It can only be applied after it has been approved by the relevant statutory authorities.

2. Biosafety measures:

(1) Animal cells must be completed in a short period of time from ex vivo to culture, but for human safety, newborn piglets are also tested for viral antibodies (hepatitis virus, immunodeficiency virus, cytomegalovirus) before taking the pancreas and dying. (2) Islet cells should be followed up during the culture process to observe abnormal conditions. (3) mechanical properties of the microcapsules prior to islet transplantation, biofilm control of macromolecules; Nutrients, the permeability of islet cells themselves metabolites; cell morphology, purity, quantity, insulin release, etc. must be carefully detected and recorded.

Second, the anatomy of newborn piglets:

1. Physical examination, numbering, weighing, source, gender, physical condition, organ, abnormality of limbs, body temperature, viral antibody examination.

2. Wash off surface contaminants with tap water.

3. Disinfect the body surface with alcohol.

4. Wrap with sterile gauze.

5. Enter the sterile dissection room through the transfer window.

Third, the newborn piglets dissected to take the pancreas:

1. Under sterile conditions, piglets are anesthetized with ketamine and injected intraperitoneally (17 mg per kilogram of body weight).

2. Fix the newborn piglets on the dissection table (width *deep *height) 1200 X 750 X860mm.

3. Caesarean section: First check the degree of anesthesia of the piglets, body temperature, heart rate, and breathing are normal, then disinfection and laparotomy according to the surgical routine. Find the pancreas along the duodenal mesentery. <1> Weighing and recording. <2> Remove the head of the pancreas (rough end) Collect the pancreas and the tail of the pancreas (stenosis), weigh it, and wash twice with 4 Hank solution.

4. In a sterile tray, remove the islet membrane, blood vessels, and connective tissue visible to the naked eye with a surgical scissors.

4. Separation of islets:

1. Use the VI6 vibrating cell bead mill to separate the islets from other pancreas (grinding lOmin).

2. Place the slurry in Hans solution over a 500 μm metal stainless steel mesh.

3. Re-grinding through the mesh structure, repeating the mesh repeatedly, and passing the slurry through the 500 μηι metal stainless steel mesh.

4. The collected filtrate was further centrifuged at 1500 r/min for 5 minutes at 4 ° C, and the precipitate R was collected, and the supernatant was discarded.

V. Purification:

Using a dextran discontinuous density gradient centrifugation, the procedure is as follows: 1. Mix well with 15ml, 27% Dextrna and precipitate R, and slowly add 4ml, 27% Dextran; 6ml, 23% Dextran; 4ml, 11% Dextran and 4ml Hank-s;

2. Centrifuge at 4 ° C, 600 r / min for 4 min;

3, then accelerate to 2500r / min centrifugation lOmin;

4. Collect 11% - 23% two-layer islet cells;

5. Wash twice with RPMI-1640 solution, discard the supernatant, and collect cell pellets.

6. Mix the cell pellet S with 10ml RPMI-1640, take a microscopic examination, observe the islet morphology, activity, purity, quantity, and survival rate;

7. From the separation to the end of purification should be controlled at 4 6 hours.

Sixth, islet count:

1. The islet cell pellet collected by gradient centrifugation was stained with dithizone (DTZ) solution [dithizone 10 mg; 99.5% dimethyl sulfoxide 3 ml; 25 % ammonia water 50 μ 1] and counted under a microscope (microscope) Islet cells are red or magenta, round in shape, full of orange-yellow cytoplasm, translucent, clear edges, other tissues and cells are not colored, contrast is very clear) islet cells are 50-300 μ ηι in diameter, varying in size, repeat Sample three times and average.

2. Trypan blue staining was used to determine the islet life and death, live cells were not stained, and dead cells were blue. (under the microscope).

3. A patient needs 36-400,000 islets per transplant. (About 20-25 newborn pigs are provided by the pig pancreas).

Seven, islet microcapsule technology:

The microcapsule islet cells were prepared by using the Α-Ρ-Α microcapsule technique with an electrostatic high-pressure microcapsule generator from 36 to 400,000 live islet cells (Reference: Foreign Medical Surgery Volume, Vol. 27, No. 2, 2000) Period 75-77).

Morphological identification of microcapsule islets:

(1) Light microscopy, the diameter of the microcapsules should be 0.25-0.35

(2) Microcapsule breakage rate was observed by shaking at 150 times/min for 24 hours in a 37 °C constant temperature water bath shaker

(3) Fluorescent staining: Preparation of pyridine orange (AO) with isotonic phosphate buffer [670 umol/L] And iodopropion (PI) [750 μ ηιοΐ / υ storage solution stored at 4 ° C, before the preparation of (AO) concentration of 0. 67 mol / L and (PI) concentration of 57 μ mol / mixture For staining, the living cells are green and see fluorescence, and the dead cells are red. The difference is obvious.

Formula for islet count:

Four times sampled islet ιο ³ χ suspension volume islet number = X

4 N

[N = Quantitatively absorb the microliters of the suspension using a micropipette]

(1) Islet recovery formula after purification:

Recovery rate = number of islets after microscopy / number of islets before purification X 100 %

(2) Islet purity estimation:

Purity was estimated by the ratio of the number of islets to the number of exocrine tissue cells under an inverted microscope.

Eight, microcapsule islet cell culture:

1. Incubate in a constant temperature 37 ° C, C0 ₂ incubator, change the culture solution for 48 hours, and periodically take the culture solution to determine the insulin content. It is observed that the islet cells differentiate, grow, and secrete insulin in the microcapsules to determine the termination culture time, and generally need to be cultured for 4 to 8 days.

2. RPMI-1640 medium was added to fibronectin for serum-free culture to induce cell adherent agglomeration growth and differentiation.

Nine, cultivated islets, it is best to transplant the same day, cold storage has a fix on cell growth and transplantation. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entirety in their entireties in the the the the the the the the In addition, it is to be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the present invention.

Claims

Rights request

A method of producing a repairable piglet islet cell, characterized in that it comprises the following process steps:

(1) isolating and purifying islet cells: mechanically grinding to separate the islets of the piglets from the exocrine tissue, and then islet cells are obtained by discontinuous density gradient centrifugation;

(2) culturing islet cells: sodium alginate-polylysine-alginate microspheres coated with islet cells were cultured in RPMI-1640 medium containing fibronectin, and the serum content of the culture solution was 0. .

The method according to claim 1, wherein the separation time from separation to purification is 4 to 6 hours by Dextran discontinuous density gradient centrifugation in the step (1).

The method according to claim 1, wherein the amount of fibronectin is 5-30 mg per liter of the medium volume.

The production method according to claim 1, further comprising the step of: (step):

Choosing islet cell donors: Fresh, unfed healthy piglets were randomly placed in the pancreas to remove the capsule, blood vessels, connective tissue and placed in Hank's solution at 4 °C.

The production method according to claim 4, wherein the piglet is born within 24 hours and is a hybrid progeny.

The method according to claim 1, wherein the step (1) of separating and purifying the islet cells is:

1 separation of islets

a, using VI6 vibrating cell ball bead mill to separate the islets from other pancreas, grinding lOmin;

b. Put the slurry into 4°CHank solution and pass the 500 μm metal stainless steel mesh; C. After re-grinding through the mesh structure, the mesh is repeatedly passed, and the slurry is passed through a 500 μΐΙ1 metal stainless steel mesh;

d. The collected filtrate is centrifuged at 1500 r/min for 5 minutes in an environment of 4 ° C, and the precipitate R is collected, and the supernatant is discarded;

a, with 15ml, 27% Dextrna and precipitate R thoroughly mixed, based on this slowly added 4ml, 27% Dextran; 6ml, 23% Dextran; 4ml, 11% Dextran and 4ml Hank-s;

b, centrifuged at 4 ° C 600r / min 4min;

c, then the speed of the mouth to 2500r / min centrifugation lOmin;

d, collecting 11% - 23% two layers of islet cells;

e, wash twice with RPMI1640 solution, discard the supernatant, collect cell pellet S;

f. Mix the cell pellet S with 10ml RPMI1640, take a microscopic examination, observe the islet morphology, activity, purity, quantity, and survival rate;

g, from the separation to the end of purification time is controlled at 4 6 hours.

7. A method of producing a repairable piglet islet cell, characterized in that it comprises the following process steps:

(i) Selection of islet cell donors: donor selection of newly born, unbreasted healthy piglets, pancreas was taken aseptically under laparotomy, and the envelope, blood vessels, and connective tissue were removed and placed in a 4 °C Hank solution;

(ii) Isolation and purification of islet cells: Islets are separated from exocrine tissue by mechanical grinding, and then islet cells are obtained by Dextran discontinuous density gradient centrifugation, and the morphology, activity purity, quantity, and survival rate of the sample are taken from the separation to the end of purification. Time control at 4 6 hours;

(iii) culturing islet cells: cultured with serum-free microsphere carrier, using RPMI-1640 culture medium, bovine serum is not contained in the culture medium, and fibronectin is added, and the amount is added according to the volume of the medium per liter plus FN 5-30 mg. During the culture process, the islet cells are encapsulated in the APA biofilm to form microspheres. Timely detection of microcapsule morphology, activity, and counting, culture in a constant temperature 37 ° C C0 ₂ incubator, change the culture solution 48 hours, and regularly take the culture solution to determine the insulin content; observe and record the islet cells in the microcapsule differentiation, growth, secretion Insulin function is used to determine the termination culture time, and finally the islet cells of the microcapsules with good activity and high purity are obtained.