CN101139573A - Method for inducing islet cells differentiated from stem cells to form islet-like structure in vitro - Google Patents
Method for inducing islet cells differentiated from stem cells to form islet-like structure in vitro Download PDFInfo
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Abstract
体外诱导干细胞分化的胰岛细胞形成胰岛样结构的方法。将干细胞分化的胰岛细胞在经过硅烷处理的培养器皿中，用含有20％胎牛血清、细胞外基质、一定浓度钙离子和ATP的细胞培养基，通过悬浮培养的方式诱导细胞形成胰岛样结构。本发明具有以下有益效果：1.方法简便，诱导时间短，胰岛样结构形成率高；2.形成的胰岛样结构与天然胰岛非常相似，经过门脉移植后能稳定定位于肝窦中；3.诱导形成的胰岛样结构具有明显包膜，并具有与天然胰岛相似的基质成分。应用此方法诱导形成的胰岛样结构形态完整，可以达到胰岛移植的质量要求。
A method for inducing islet cells differentiated from stem cells to form islet-like structures in vitro. The islet cells differentiated from the stem cells were cultured in a silane-treated culture vessel with a cell culture medium containing 20% fetal bovine serum, extracellular matrix, a certain concentration of calcium ions and ATP, and the cells were induced to form islet-like structures by means of suspension culture. The invention has the following beneficial effects: 1. The method is simple, the induction time is short, and the formation rate of the islet-like structure is high; 2. The formed islet-like structure is very similar to the natural islet, and can be stably positioned in the hepatic sinusoid after portal vein transplantation; 3. .The induced islet-like structure has a clear envelope and has a matrix composition similar to that of natural islets. The shape of the islet-like structure induced by this method is complete, which can meet the quality requirements of islet transplantation.
Description
Technical field
The present invention relates to a kind of technological method that forms pancreatic islet-like structures of inducing, the islet cells that especially relates to a kind of external evoked differentiation of stem cells forms the method for pancreatic islet-like structures.
Background technology
Diabetes are one of diseases of serious harm human health, and the common method for the treatment of diabetes at present is to adopt insulin injection.Adopt insulinize can improve patient's carbohydrate metabolism disturbance to a certain extent, however it can not prevent effectively or reverting diabetes causes microangiopathies and complication thereof.Therefore, seek new treating diabetes means is the advanced subject of medical research always.Pancreatic islets transplantation is the effective ways for the treatment of insulin-dependent diabetes mellitus at present, and has experimental data to show, pancreatic islets transplantation not only can be corrected glycometabolic disorder, and can prevent or the microangiopathies of reverting diabetes.But, extremely lack owing to can be provided for the pancreas donor of pancreatic islets transplantation at present, limited pancreatic islets transplantation in clinical widespread use.Stem cell has self duplication and differentiation becomes various histiocytic potential, therefore stem cell is induced to differentiate into islet cells and might solves the needed cell of pancreatic islets transplantation source, has the great value that is applied to the diabetes cell therapy.
There has been many researchs report to induce differentiation to become insulin secretory cell the stem cell in multiple source recently, these insulin secretory cells has been transplanted in the animal body of diabetes and can be obviously improved carbohydrate metabolism disturbance.For example: the patent application 200510064431.5 of the disclosed application of State Intellectual Property Office of the People's Republic of China artificial " Peking University "; The main contents of patent application 02824367.6 innovation and creation such as grade of application artificial " JieLong Co.,Ltd " all are that stem cell is induced the method that is divided into insulin secretory cell.
Usually, the position of pancreatic islets transplantation is a sinus hepaticus, and this is because 1) sinus hepaticus can provide abundant blood confession for islet cells and islet, helps the survival of islet transplantation; 2) survival pancreas islet herein can be experienced changes of blood glucose fast and produce Regular Insulin release; 3) liver is the target organ of insulin action maximum, and excretory Regular Insulin can act on liver cell and promote the synthetic of glycogen, lowering blood glucose; 4) hepatic secretion panimmunity supressor helps to alleviate rejection.But if induce the single islet cells after the differentiation to be grafted directly to sinus hepaticus stem cell, cell may be moved to other tissue or organ with blood flow, can not guarantee its stable survival and function performance in sinus hepaticus.Make transplanted cells can be positioned sinus hepaticus, it must be induced becomes location and the function performance that pancreatic islet-like structures just helps cell, thereby treats diabetes effectively.
In former studies, there have the investigator to use in the Matrigel gel cultured method to promote insulin secretory cell to assemble to be agglomerating, but there are some deficiencies in this method: the rate of formation that at first is pancreatic islet-like structures is low, and the pancreatic islet-like structures that forms is not of uniform size; Secondly, formed pancreatic islet-like structures coating is not obvious or imperfect.Therefore the interaction of compositions such as there is coating outward in natural pancreas islet, helps preventing antibody in the blood, complement and islet cells can alleviate immunological rejection to a certain extent.In addition, its extracellular matrix of formed pancreatic islet-like structures is obviously different with natural pancreas islet.(Extracellular matrix ECM) is the important component part of pancreas islet microenvironment to extracellular matrix, by multiple different molecular composition, is the support that islet cells is rely and depended on, and helps the function performance of cell, and regulates the differentiation and the survival of cell.ECM has two kinds of forms, promptly between matter and extracellular basement membrane (BM).There are some researches show except that the capillary blood vessel basement membrane, seldom have cell to ask the distribution of matter composition between the pancreatic islet endocrine in the pancreas islet structure; The outer coating of pancreas islet structure then mainly is by ln (Laminin), and IV Collagen Type VI and fibronectin (Fibronectin) are formed.In carrying out the pancreas islet separating process, the digestion of enzyme might cause the destruction of pancreas islet basement membrane, but the destruction that concerns between this cell-matrix and the disappearance of basement membrane be the apoptosis of inducing cell often, so that brings into play in intravital survival of acceptor and function after influencing pancreatic islets transplantation.Nearest research shows that also in plain secretory cell differentiation of stem cell to pancreatic islet and pancreas islet growth course, extracellular matrix components also plays an important role.In the gel of collagen and ln formation, the survival ability of pancreas islet is stronger, can also make pancreatic islet endocrine keep the expression of Regular Insulin more enduringly.But the present report that does not also use extracellular matrix in the method for external evoked insulin secretory cell formation pancreatic islet-like structures does not have the report that the pancreatic islet-like structures of inducing forms the complete packet film outward yet.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method of islet cells formation pancreatic islet-like structures of external evoked differentiation of stem cells.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: the islet cells of differentiation of stem cells through in the cultivation vessel of silane treatment, use cell culture medium, by the mode inducing cell formation insulin-like cell structure of suspension culture.
Described cell culture medium is any substratum that is suitable for cell cultures, and adds 20% foetal calf serum, the glucose of 4.5g/L, the Ca of 1～2mmol/L
2+, 0.01～2mmol/L Triphosaden, 2mmol/L glutamine, 100U/ml penicillin and 100U/ml Streptomycin sulphate.
In the described cell culture medium, contain the extracellular matrix components similar with islet cells.
Described and the similar extracellular matrix components of islet cells comprise ln 0.1～20 μ g/ml, IV Collagen Type VI 0.1～20 μ g/ml and fibronectin 0.1～20 μ g/ml.
Described and the similar extracellular matrix components of islet cells comprise ln 0.5～10 μ g/ml, IV Collagen Type VI 0.5～10 μ g/ml and fibronectin 0.5～10 μ g/ml.
Described and the similar extracellular matrix components of islet cells comprise ln 1～5 μ g/ml, IV Collagen Type VI 2～8 μ g/ml and fibronectin 1～5 μ g/ml.
The optimum weight ratio of described ln, IV Collagen Type VI and fibronectin is 2: 5: 3.
Described suspension culture is at 37 ℃, carries out 24～72 hours cultivation in the incubator of 5%CO2 and 95% air, and in culturing process per 10～14 hours gently rotational oscillation once, each 1～3 minute.
This method of the present invention is compared with reported method in the past, has following beneficial effect:
1. simple to operate, pancreatic islet-like structures rate of formation height.Adopt suspension culture, promptly do not cultivate in the culture vessel of adherent growth, operate identical with the ordinary cells cultural method, so simple to operate at the treated cell that makes.But this method can make adding cell 80% all form pancreatic islet-like structures.
2. the pancreatic islet-like structures size that forms evenly, and is similar to natural pancreas islet, and the energy stable position was in sinus hepaticus after portal vein was transplanted.
3. the periphery of pancreatic islet-like structures has formed coating.By in substratum, adding and similar extracellular matrix material of islet tissue matrix such as fibronectin, ln and IV Collagen Type VI, can induce the pancreatic islet-like structures of new formation to form the extracellular matrix coating on every side, not only be beneficial to and transplant the complete of back pancreas islet 26S Proteasome Structure and Function, and avoid pancreatic islet-like structures to be subjected to the immune destruction of acceptor.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail
The pancreatic islet-like structures that Fig. 1 forms by method of the present invention;
The comparison of the pancreatic islet-like structures that Fig. 2 forms by method of the present invention and the extracellular matrix immunofluorescence dyeing of natural pancreas islet;
A: the ln of natural pancreas islet; B: the IV Collagen Type VI of natural pancreas islet; C: the fibronectin of natural pancreas islet; D: the ln of pancreatic islet-like structures of the present invention; E: the IV Collagen Type VI of pancreatic islet-like structures of the present invention; F: the fibronectin of wooden invention pancreatic islet-like structures.
Embodiment
Embodiment 1
A kind of islet cells of external evoked differentiation of stem cells forms the method for pancreatic islet-like structures, after comprising the steps: the 2nd generation people embryonic pancreas expansion of stem cells converged substantially to the T75 bottle, with 0.2% trysinization, counting is cultivated people's embryonic pancreas stem cell in the sterile culture pipe of crossing with silane treatment with the density of 3ml culture medium inoculated 120,000 cells then; Behind the silane treatment culture tube, cell can't attach to the cultivation tube wall and be suspended in the substratum, and the substratum of this suspension culture uses and contains the M199 substratum of 20% heat-inactivated fetal bovine serum, and contains the glucose of 4.5g/L, the Ca of 1mmol/L
2+, 0.01mmol/L Triphosaden, 2mmol/L glutamine, 100U/ml penicillin and 100U/ml Streptomycin sulphate; At 95% air, 5%CO
2, in 37 ℃ the wet environment, the embryonic pancreas stem cell was cultivated 6 hours in this condition of suspension culture; Form pancreatic islet-like structures.
Embodiment 2
A kind of islet cells of external evoked differentiation of stem cells forms the method for pancreatic islet-like structures, after comprising the steps: the 3rd generation people embryonic pancreas expansion of stem cells converged substantially to the T75 bottle, with 0.2% trysinization, counting is cultivated people's embryonic pancreas stem cell in the sterile culture pipe of crossing with silane treatment with the density of 3ml culture medium inoculated 1,000,000 cells then; Behind the silane treatment culture tube, cell can't attach to the cultivation tube wall and be suspended in the substratum, and the substratum of this suspension culture uses and contains the M199 substratum of 20% heat-inactivated fetal bovine serum, and contains the glucose of 4.5g/L, the Ca of 2mmol/L
2+, 2mmol/L Triphosaden, 2mmol/L glutamine, 100U/ml penicillin and 100U/ml Streptomycin sulphate; At 95% air, 5%CO
2, in 37 ℃ the wet environment, the embryonic pancreas stem cell was cultivated 10 days in this condition of suspension culture; Form pancreatic islet-like structures.
Embodiment 3
After the 3rd generation people embryonic pancreas expansion of stem cells converged substantially to the T75 bottle, with 0.2% trysinization, counting was cultivated people's embryonic pancreas stem cell in the sterile culture pipe of crossing with silane treatment with the density of 3ml culture medium inoculated 500,000 cells then; Behind the silane treatment culture tube, cell can't attach to the cultivation tube wall and be suspended in the substratum, and the substratum of this suspension culture uses and contains the M199 substratum of 20% heat-inactivated fetal bovine serum, and contains the glucose of 4.5g/L, the Ca of 1.5mmol/L
2+, 1mmol/L Triphosaden, 2mmol/L glutamine, 100U/ml penicillin and 100U/ml Streptomycin sulphate; At 95% air, 5%CO
2, in 37 ℃ the wet environment, the embryonic pancreas stem cell was cultivated 30 hours in this condition of suspension culture, form pancreatic islet-like structures.
The spontaneous island spline structure that is gathered into of method cultured human embryo tire pancreatic stem cells meeting by embodiment 1,2 and 3, the size of this pancreatic islet-like structures is more even, diameter is at 50～350 μ m, mean diameter 150 μ m, inverted microscope observe down and are very similar to natural pancreas islet (mean diameter 150 to 200 μ m); It is very high to use the efficient that the method for this suspension culture of the present invention induces pancreatic islet-like structures to form, add 80% of cell and all formed pancreatic islet-like structures.
Embodiment 4
Repeat embodiment 1, difference is: added a certain amount of extracellular matrix components in the suspension culture base, these extracellular matrix components are main extracellular matrix moietys of selecting natural pancreas islet coating for use, as fibronectin, ln, IV Collagen Type VI etc.; The time that adds extracellular matrix can be that suspension culture begins promptly to add various extracellular matrix materials, also can be the certain hour after the suspension culture, and for example suspension culture is 6 hours; Continue to cultivate certain hour, for example 24 hours behind the extracellular matrix that adds; The amount that adds extracellular matrix is 0.1 μ g/ml as the amount of fibronectin; The amount of ln is 0.1 μ g/ml; The amount of IV Collagen Type VI is 0.1 μ g/ml.
Embodiment 5
Repeat embodiment 2, difference is: added a certain amount of extracellular matrix components in the suspension culture base, these extracellular matrix components are main extracellular matrix moietys of selecting natural pancreas islet coating for use, as fibronectin, ln, IV Collagen Type VI etc.; The time that adds extracellular matrix can be that suspension culture begins promptly to add various extracellular matrix materials, also can be the certain hour after the suspension culture, and for example suspension culture is 72 hours; The amount of the extracellular matrix that adds is 20 μ g/ml as the amount of fibronectin; The amount of ln is 20 μ g/ml; The amount of IV Collagen Type VI is 20 μ g/ml.
Embodiment 6
Repeat embodiment 3, difference is: added a certain amount of extracellular matrix components in the suspension culture base, these extracellular matrix components are main extracellular matrix moietys of selecting natural pancreas islet coating for use, as fibronectin, ln, IV Collagen Type VI etc.; The time that adds extracellular matrix can be that suspension culture begins promptly to add various extracellular matrix materials, also can be the certain hour after the suspension culture, and for example suspension culture is 26 hours; The amount of the extracellular matrix that adds is 5 μ g/ml as the amount of fibronectin; The amount of ln is 5 μ g/ml; The amount of IV Collagen Type VI can be 8 μ g/ml.
Embodiment 7
Repeat embodiment 3, difference is: added a certain amount of extracellular matrix components in the suspension culture base, these extracellular matrix components are main extracellular matrix moietys of selecting natural pancreas islet coating for use, as fibronectin, ln, IV Collagen Type VI etc.; The time that adds extracellular matrix can be that suspension culture begins promptly to add various extracellular matrix materials, also can be the certain hour after the suspension culture, and for example suspension culture is 24 hours; The amount of the extracellular matrix that adds is 2 μ g/ml as the amount of fibronectin; The amount of ln is 5 μ g/ml; The amount of IV Collagen Type VI is 3 μ g/ml.
Embodiment 4,5,6 and 7 washes the pancreatic islet-like structures that forms 2 times with phosphate buffered saline buffer (PBS) after suspension culture finishes, and the preparation frozen tissue section, also prepares the frozen tissue section of the natural pancreas islet of isolating people simultaneously; Use the antibody staining of anti-fibronectin, ln and IV Collagen Type VI to find after fixing respectively, three kinds of extracellular matrix components are the obvious positive at the skin of pancreatic islet-like structures of the present invention, dyeing on the structural integrity parcel that is similar to the coating sample that whole pancreatic islet-like structures is formed by these three kinds of extracellular matrixs, this and natural pancreas islet is closely similar.
Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be qualification to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here can't give exhaustive to all embodiments.Everyly belong to the row that conspicuous variation that technical scheme of the present invention extends out or change still are in protection scope of the present invention.
Claims (8)
1. the islet cells of external evoked differentiation of stem cells forms the method for pancreatic islet-like structures, it is characterized in that: with the islet cells of differentiation of stem cells through in the cultivation vessel of silane treatment, use cell culture medium, by the mode inducing cell formation insulin-like cell structure of suspension culture.
2. the islet cells of external evoked differentiation of stem cells according to claim 1 forms the method for pancreatic islet-like structures, it is characterized in that: described cell culture medium is any substratum that is suitable for cell cultures, and adds 20% foetal calf serum, the glucose of 4.5g/L, the Ca of 1～2mmol/L
2+, 0.01～2mmol/L Triphosaden, 2mmol/L glutamine, 100U/ml penicillin and 100U/ml Streptomycin sulphate.
3. the islet cells of external evoked differentiation of stem cells according to claim 1 and 2 forms the method for pancreatic islet-like structures, it is characterized in that: in the described cell culture medium, also contain and the similar extracellular matrix components of natural pancreas islet.
4. the islet cells of external evoked differentiation of stem cells according to claim 3 forms the method for pancreatic islet-like structures, it is characterized in that: the similar extracellular matrix components of described and natural pancreas islet comprises ln 0.1～20 μ g/ml, IV Collagen Type VI 0.1～20 μ g/ml and fibronectin 0.1～20 μ g/ml.
5. the islet cells of external evoked differentiation of stem cells according to claim 3 forms the method for pancreatic islet-like structures, it is characterized in that: the similar extracellular matrix components of described and natural pancreas islet comprises ln 0.5～10 μ g/ml, IV Collagen Type VI 0.5～10 μ g/ml and fibronectin 0.5～10 μ g/ml.
6. the islet cells of external evoked differentiation of stem cells according to claim 3 forms the method for pancreatic islet-like structures, it is characterized in that: the similar extracellular matrix components of described and natural pancreas islet comprises ln 1～5 μ g/ml, IV Collagen Type VI 2～8 μ g/ml and fibronectin 1～5 μ g/ml.
7. the islet cells of external evoked differentiation of stem cells according to claim 4 forms the method for pancreatic islet-like structures, and it is characterized in that: the part by weight of described ln, IV Collagen Type VI and fibronectin is 2: 5: 3.
8. the islet cells of external evoked differentiation of stem cells according to claim 1 forms the method for pancreatic islet-like structures, and it is characterized in that: described suspension culture is at 37 ℃, 5%CO
2With carry out 24～72 hours cultivation in the incubator of 95% air, and in culturing process per 10～14 hours gently rotational oscillation once, each 1～3 minute.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102517248A (en) * | 2011-12-30 | 2012-06-27 | 中日友好医院 | A method for inducing the formation of islet-like structures in vitro |
| CN111269875A (en) * | 2020-03-24 | 2020-06-12 | 山东兴瑞生物科技有限公司 | Method for directionally differentiating into islet cells by using autoimmune cells |
| CN113583937A (en) * | 2021-07-02 | 2021-11-02 | 武汉大学 | Culture medium and culture method for differentiating pluripotent mammalian stem cells into definitive endoderm cells |
-
2007
- 2007-08-22 CN CNA2007101206251A patent/CN101139573A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102517248A (en) * | 2011-12-30 | 2012-06-27 | 中日友好医院 | A method for inducing the formation of islet-like structures in vitro |
| CN111269875A (en) * | 2020-03-24 | 2020-06-12 | 山东兴瑞生物科技有限公司 | Method for directionally differentiating into islet cells by using autoimmune cells |
| CN111269875B (en) * | 2020-03-24 | 2022-04-08 | 山东兴瑞生物科技有限公司 | Method for directionally differentiating into islet cells by using autoimmune cells |
| CN113583937A (en) * | 2021-07-02 | 2021-11-02 | 武汉大学 | Culture medium and culture method for differentiating pluripotent mammalian stem cells into definitive endoderm cells |
| CN113583937B (en) * | 2021-07-02 | 2023-02-10 | 深圳市北科生物科技有限公司 | Culture medium and culture method for differentiating pluripotent mammalian stem cells into definitive endoderm cells |
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