CN109957539A - A kind of artificial islet tissue and its preparation and application - Google Patents
A kind of artificial islet tissue and its preparation and application Download PDFInfo
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- CN109957539A CN109957539A CN201711341949.8A CN201711341949A CN109957539A CN 109957539 A CN109957539 A CN 109957539A CN 201711341949 A CN201711341949 A CN 201711341949A CN 109957539 A CN109957539 A CN 109957539A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/39—Pancreas; Islets of Langerhans
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic cells
- C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
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Abstract
The present invention relates to a kind of artificial islet tissues, and specifically, including by 3D biometric print technology biologically active artificial islet tissue is made in biocompatible scaffold and islet tissue by biocompatible scaffold, islet cells and mixed-culture medium.The culture and preservation of mixed-culture medium of the invention for islet cells and tissue.Biocompatible scaffold is made with 3D printing method.Artificial islet tissue of the invention can be used as pathophysiological mechanism research and the treatment tool of diabetes.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to artificial islet tissue.
Background technique
Pancreas islet is the endocrine portion of pancreas, and of different sizes, elliposoidal cell cluster, the pancreas of secretion are presented in pancreas
Island element and glucagon directly participate in the adjusting of body glycometabolism.Islet cells be broadly divided into α cell, β cell, gamma cells and
PP cell etc..Wherein β cell accounts for the 60%-70% of islet tissue, excreting insulin.Insulin can act on liver, muscle, rouge
The target organs such as fat, accelerate the storage and metabolism of glucose, and inhibit the generation of glucose, are the intracorporal unique hypoglycemic hormones of people.
Hypoinsulinism leads to the generation of insulin-dependent diabetes mellitus (type-1 diabetes mellitus).
The method that there is no radical cure for diabetes at present, for the patient that insulin relies on, only way is exactly pancreas islet
Plain drug injection, however this mode are directed not only to injection at regular time and quantity daily, moreover, also inevitable pharmacological dependence with
And the toxic side effect of drug, it can be seen that, it is false to develop a kind of simple disposable islet tissue with biological capacity of self-regulation
Body will be a kind of promising treating diabetes means, and therefore, the present invention is based on biological 3D printing techniques, develop a kind of people
Islet tissue is made, artificial constructed biologically active 3D printing islet tissue not only facilitates diabetes in vitro or in vivo and grinds
Study carefully model, and provides new therapeutic strategy for insulin-dependent diabetes mellitus.
Summary of the invention
Mentioned above in order to solve the problems, such as, the present invention is on the basis of islet cell culture, in conjunction with Method of Tissue Engineering
With currently advanced 3D printing technique, external printing has the 3D islet tissue of biological function, islet cells is seeded in liquid
State timbering material is co-cultured, or directly mixing printing type forms three-dimensional islet tissue spline structure.
A kind of artificial islet tissue passes through 3D biometric print mode system by bioavailability bracket material, islet cells
At.
In the inventive solutions, pass through 3D biometric print mode are as follows:
Biocompatible scaffold is made by 3D biometric print in bioavailability bracket material, then will be containing islet cells
Mixed-culture medium injects in biocompatible scaffold；Or
Bioavailability bracket material is mixed with the mixed-culture medium containing islet cells, is made up of 3D biometric print
Artificial islet tissue.
In technical solution of the present invention, mixed-culture medium is the mobility liquid for cultivating islet cells；
It preferably, include animal blood serum and antibiotic in mixed-culture medium, it is highly preferred that the animal blood serum is selected from
RPMI1640、FBS。
In the inventive solutions, bioavailability bracket material selects gelatin, collagen, sodium alginate, fine jade
One of lipolysaccharide, matrigel, hyaluronic acid, chitosan, glucan or several any combination；Preferably, the collagen egg
It is white selected from fiber collagen, more preferably I-type collagen, II collagen type, type III collagen, XI collagen type,
One of XXIV collagen type, XXVII collagen type or several any combination.
In the inventive solutions, being made up on artificial islet tissue of 3D biometric print has gap, gap
Diameter is no more than 1.5mm, more preferably no more than 1mm.
Another aspect of the invention provides the preparation method of artificial islet tissue comprising following steps:
1) bioavailability bracket material is prepared,
2) mixed-culture medium is prepared；
3) islet cells is separated, and is cultivated respectively with mixed-culture medium；
4) biocompatible scaffold is made by 3D biometric print in bioavailability bracket material, then islet cells will be contained
Mixed-culture medium injection biocompatible scaffold on；Or
4) bioavailability bracket material is mixed with the mixed-culture medium containing islet cells, passes through 3D biometric print system
At artificial islet tissue.
In the inventive solutions, the preparation method of artificial islet tissue can also include step 5), will be prepared into
To artificial islet tissue cultivated in mixed-culture medium.
Another aspect of the present invention provides artificial islet tissue for treating type-1 diabetes mellitus, type-2 diabetes mellitus, height
Glucohemia, postprandial hyperglycemia, impaired glucose tolerance, diabetic complication, obesity, Impaired Glucose Tolerance Treated, insulin resistance, pancreas
Island hyperfunction, islet tissue physically or chemically damage in purposes.
Another aspect of the present invention provides artificial islet tissue for treating type-1 diabetes mellitus, type-2 diabetes mellitus, height
Glucohemia, postprandial hyperglycemia, impaired glucose tolerance, diabetic complication, obesity, Impaired Glucose Tolerance Treated, insulin resistance, pancreas
The purposes in medical instrument that island hyperfunction, islet tissue physically or chemically damage.
Another aspect of the present invention provides islet tissue and pancreas islet of the artificial islet tissue in preparation for research
Purposes in the directly related disease of secreting hormone or the medical model of indirect correlation disease.
The directly related disease or indirect correlation disease of islet secretion hormone are preferably type-1 diabetes mellitus, type-2 diabetes mellitus, height
Glucohemia, postprandial hyperglycemia, impaired glucose tolerance, diabetic complication, obesity, Impaired Glucose Tolerance Treated, insulin resistance, pancreas
Island hyperfunction, islet tissue physically or chemically damage.
Another aspect of the present invention provides artificial islet tissue in preparation for studying islet tissue and pancreas islet point
Purposes in the medical model of krinin.
Specific summary of the invention is as follows:
1. timbering material selects
In the inventive solutions, bioavailability bracket material selects gelatin, collagen, sodium alginate, fine jade
One of lipolysaccharide, hyaluronic acid, matrigel, glucan, chitosan or several any combination；Preferably, of the invention one
Using gelatin as timbering material in a preferred embodiment；
2. culture medium selects
Mixed-culture medium is the mobility made of culture medium and the bioactive substance for promoting islet cells growth required
Liquid；
Islet tissue mixed-culture medium :+1% streptomysin of RPMI1640+10%FBS+1% penicillin.Islet function detection
Culture solution (KRBH buffer) ingredient: 118.5mM NaCl, 4.74mM KCl, 25mM NaHCO3、1.19mM MgSO4、2.54mM
CaCl2、1.19mM KH2PO4, 10mM HEPES, 0.1%BSA.
3. islet cells selects
In the inventive solutions, the islet cells can be single islet cells, or include single pancreas islet
The cell mixing of cell, perhaps islet cells cluster or islet tissue fragment comprising multiple islet cells, of the present invention
Islet cells is the cell or commercially available cell in subject source, can also be from experimental animal etc..
In a technical solution of the invention, the islet cells is selected from mouse, through collagenase perfusion and density gradient
Centrifugal process separation obtains, and selection volume is moderate after staying overnight, structural integrity, and coating clear smooth pancreas islet in edge is further disappeared
Change until forming unicellular mixed liquor.In addition to this, the source of islet cells can also pass through commercially available acquisition.
4. printing type
The present invention provides a kind of artificial islet tissues, by bioavailability bracket material, islet cells and mixing
Culture solution is made up of 3D biometric print mode；
In the solution of the present invention, bracket is made by 3D biometric print in bioavailability bracket material, then pancreas will be contained
On the mixed-culture medium injection bracket of island cell；Or
After bioavailability bracket material is mixed with islet cells mixed-culture medium, people is made by 3D biometric print
Make islet tissue；
5. print parameters select
In the inventive solutions, being made up on artificial islet cells of 3D biometric print has gap, gap
Diameter is no more than 1mm.
In the inventive solutions, the size in the gap has a kind or more of size, preferably has 2-3 kind ruler
It is very little.
In a preferred technical solution, the 3D printing size of biocompatible scaffold of the present invention are as follows: pore-size
(R): 50 μm~800 μm；Lines accumulation angle: 0-90 °.
Pore-size (R) is preferably 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μ
m,550μm,600μm,650μm,700μm,750μm；
It is preferably 5 °, 10 °, 15 °, 20 °, 30 °, 40 °, 50 °, 60 °, 70 °, 80 °, 90 ° that lines, which accumulate angle,.
6.3D prints the preparation process of artificial islet tissue
The present invention provides the preparation methods of artificial islet tissue comprising following steps:
1) bioavailability bracket material is prepared；
2) mixed-culture medium is prepared；
3) islet cells is separated, and is cultivated respectively with mixed-culture medium；
4) first by bioavailability bracket material, three-dimensional rack is made by 3D biometric print, then will to contain islet tissue thin
Artificial islet tissue is made in the mixing thing liquid injection bracket of born of the same parents；Or
4) bioavailability bracket material is mixed with islet tissue cell mixture, and artificial pancreas is made by 3D biometric print
Island tissue；
5) islet tissue being prepared is carried out to culture to save backup in mixed-culture medium.
7. artificial islet tissue application
1) external hormone test: ELISA method is used, the insulin secretion water of the artificial islet tissue of in vitro culture is detected
It is flat；Or
2) internal transplantation experiments: by the pancreatin islet tissue transplantation prepared in diabetic mice, mouse is then detected
Blood glucose level and level of insulin secretion.
Beneficial effect
1) artificial islet tissue of the invention can be used as the directly related disease of glycometabolism caused by any factor or indirect phase
The treatment tool of related disorders；
2) artificial islet tissue of the invention can be used as the research model of diabetes.
Detailed description of the invention
Fig. 1: artificial islet tissue schematic diagram.
Fig. 2: influence of the islet tissue cell to level of insulin secretion under different concentration of glucose.
Fig. 3: islet tissue Histological Study.
Specific embodiment
The following example describes specific aspect of the invention to explain the present invention and to mention as those skilled in the art
For the description in relation to the method for the present invention.These embodiments are not applied to limit the present invention, because these embodiments provide only use
In the specific method for understanding and implementing the present invention and its various aspects.
Embodiment 1: the configuration of mixed-culture medium
The ingredient of mixed-culture medium are as follows:+1% streptomysin of RPMI1640+10%FBS+1% penicillin, culture solution are put into 37
DEG C, 5%CO2Incubator inner equilibrium 2-4 hours.
Embodiment 2: islet cells obtains
Disconnected neck puts to death mouse, ligatures choledochus duodenum arrival end, collagenase P 2.4mg/ is perfused only in choledochus, to pancreas
Careful complete taking-up after gland is full；The complete pancreas of taking-up is put into pre-cooling 6ml HBSS buffer, 37 DEG C of concussion digestion
12min；Pre-cooling 10%FBS RPMI1640 culture medium is added after digestion and terminates digestion, rocks 4 DEG C of 800rpm after 15s energetically
Centrifugation 4min removes supernatant；50 mesh metallic sieves filter after precipitating is resuspended in 10%FBS RPMI1640 culture medium, 4 DEG C of 800rpm of filtrate
Centrifugation 4min removes supernatant；10ml histopaque 1077, which is resuspended, to be precipitated and is slowly added to 10ml serum-free RPMI1640 culture medium
Making two solution, there are clear layering interfaces；20 DEG C of 2400rpm are centrifuged 20 minutes, draw histocyte between two layers of solution interface
Group, 1640 culture medium of 10%FBS RPMI are washed 1-2 times, and 800rpm is centrifuged 2min；2ml10%FBS RPMI 1640 cultivates base weight
Pancreas islet is obtained by filtration in outstanding precipitating, 75um cell sieve, pancreas islet is moved into 6cm culture dish, RPMI1640+10%FBS+1% mould
Plain+1% streptomysin overnight incubation.Selection volume is moderate after overnight, structural integrity, and coating clear smooth pancreas islet in edge is as 3D
Print raw material.And the preparation (as shown in Figure 1) of the artificial islet tissue of 3D is completed in 1 day.
The preparation of embodiment 3:3D printing islet tissue
1. the design and preparation of biological support
(1) CAD (Computer aided design, CAD) is combined, is constructed using three-dimensional printing technology
Bracket, wherein shape, constituent and internal structure all have good designability；
(2) shape of three-dimensional islet tissue is designed like the shape of pancreas, and guarantees internal structure connection, has certain
Pore structure；
(3) timbering material selection gelatin compatibility printing.
(4) three-dimensional islet tissue internal structure parameter constructed by: pore-size (R) :=0.2mm；Lines accumulate angle:
90°。
(5) branch prepared -80 DEG C are placed on to store for future use.
2. preparing the artificial islet tissue of 3D
Contain 600-4000 islet tissue in every mice pancreatic, in each islet tissue containing 1000 or so pancreas islet
Cell is made three-dimensional rack (such as: 2mm × 2mm × 1mm) by 3D biometric print when printing, then will contain islet tissue cell (about
Containing 1.5 × 106A islet cells) mixing thing liquid injection bracket in artificial islet tissue is made.
And it is put into short term spare in+1% streptomysin culture solution of RPMI1640+10%FBS+1% penicillin.
Embodiment 4: the external hormone test of artificial islet tissue and histology
After the hungry artificial islet tissue 1h of the KRBH of glucose containing 2.8mM buffer, it is utilized respectively containing 2.8mM/16.7mM
The artificial islet tissue 4h of glucose KRBH buffer processing, takes supernatant, passes through the artificial pancreas using ELISA method detection in vitro culture
Island tissue insulin secretion level and glucose stimulated insulin secrete sensibility.The experiment proved that islet cells used has
There is excreting insulin ability, compared with low sugar condition, the stimulation of high sugar can effectively improve the content of insulin in culture solution, tool
The standby ability for adjusting blood glucose level in vivo, concrete outcome is referring to fig. 2, wherein 16.7mM glucose group insulin content (3.2ng/
Ml it) is significantly improved compared with 2.8mM glucose group (2.3ng/ml).Islet tissue cellular morphology is observed by histotomy, it is specific to try
It is as shown in Figure 3 to test result.
Embodiment 5: transplantation experiments in artificial islet tissue body
Artificial islet tissue prepared by the present invention can be used for transplant experiment, and the type 1 diabetes model that will specially grow up is small
After mouse anesthesia, by the pancreatin islet tissue transplantation prepared in mouse subcutaneously or at kidney packing.
Because various changes, meaning can be carried out to above-mentioned theme in the case where not departing from the scope of the invention and essence
Figure be include in foregoing description or appended claims defined in all themes be construed to the description of this invention and say
It is bright.Many modifications and variations can be carried out to the present invention according to the above instruction.
Claims (10)
1. a kind of artificial islet tissue of 3D printing passes through the biometric print side 3D by bioavailability bracket material, islet cells
Formula is made.
2. the artificial islet tissue of 3D printing according to claim 1, is prepared by the following:
Biocompatible scaffold is made by 3D biometric print in bioavailability bracket material, then by the mixing containing islet cells
Culture solution injects in biocompatible scaffold；Or
Bioavailability bracket material is mixed with the mixed-culture medium containing islet cells, is made up of 3D biometric print artificial
Islet tissue.
3. the preparation method of the artificial islet tissue of 3D printing according to claim 1 or 2 comprising following steps:
1) bioavailability bracket material is prepared,
2) mixed-culture medium is prepared；
3) islet cells is separated, and is cultivated respectively with mixed-culture medium；
4) biocompatible scaffold is made by 3D biometric print in bioavailability bracket material, then will be mixed containing islet cells
It closes in culture solution injection biocompatible scaffold；Or
4) bioavailability bracket material is mixed with the mixed-culture medium containing islet cells, and people is made by 3D biometric print
Make islet tissue.
4. preparation method according to claim 3 can also include step 5), the artificial islet tissue being prepared is existed
It is cultivated in mixed-culture medium.
5. the artificial islet tissue of 3D printing according to claim 1 or 2 or the preparation side according to claim 3-4
Method, the 3D printing size of biocompatible scaffold are as follows: pore-size (R): 50 μm~800 μm；Lines accumulation angle: 0-90 °.
6. the artificial islet tissue of 3D printing according to claim 1 or 2 or the preparation side according to claim 3-4
Method, bioavailability bracket material select collagen, sodium alginate, gelatin, agarose, matrigel, hyaluronic acid, shell poly-
One of sugar, glucan or several any combination；
Preferably, the collagen is selected from into fiber collagen, more preferably I-type collagen, II collagen type, type III
One of collagen, XI collagen type, XXIV collagen type, XXVII collagen type or several any combination.
7. the artificial islet tissue of 3D printing according to claim 1 or 2 or the preparation side according to claim 3-4
Method, wherein be single islet cells, cell mixing perhaps comprising single islet cells or include multiple islet cells
Islet cells cluster or islet tissue fragment, it is preferable that the islet cells derives from subject.
8. the artificial islet tissue of the described in any item 3D printings of according to claim 1-2,4-7 is for treating type-1 diabetes mellitus, II
Patients with type Ⅰ DM, hyperglycemia, postprandial hyperglycemia, impaired glucose tolerance, diabetic complication, obesity, Impaired Glucose Tolerance Treated, pancreas
Insulin resistance, hyperinsulinism, islet tissue physically or chemically damage in purposes.
9. the artificial islet tissue of the described in any item 3D printings of according to claim 1-2,4-7 is for treating type-1 diabetes mellitus, II
Patients with type Ⅰ DM, hyperglycemia, postprandial hyperglycemia, impaired glucose tolerance, diabetic complication, obesity, Impaired Glucose Tolerance Treated, pancreas
The purposes in medical instrument that insulin resistance, hyperinsulinism, islet tissue physically or chemically damage.
10. pancreas islet of the artificial islet tissue of the described in any item 3D printings of according to claim 1-2,4-7 in preparation for research
Purposes in the medical model of the directly related disease or indirect correlation disease of tissue and islet secretion hormone.
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| CN201711341949.8A CN109957539A (en) | 2017-12-14 | 2017-12-14 | A kind of artificial islet tissue and its preparation and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110464880A (en) * | 2019-09-27 | 2019-11-19 | 中国医学科学院生物医学工程研究所 | A kind of artificial langerhans ' islet or artificial pancreas and preparation method thereof |
| CN114376569A (en) * | 2022-01-19 | 2022-04-22 | 浙江大学 | Glucagon-carrying wearable device for treating hypoglycemia |
| CN114870093A (en) * | 2022-05-07 | 2022-08-09 | 四川大学 | 3D printing tissue engineering pancreatic islet based on digital light processing and preparation method and application thereof |
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| CN110464880A (en) * | 2019-09-27 | 2019-11-19 | 中国医学科学院生物医学工程研究所 | A kind of artificial langerhans ' islet or artificial pancreas and preparation method thereof |
| CN114376569A (en) * | 2022-01-19 | 2022-04-22 | 浙江大学 | Glucagon-carrying wearable device for treating hypoglycemia |
| CN114376569B (en) * | 2022-01-19 | 2023-10-13 | 浙江大学 | Glucagon-carrying wearable device for treating hypoglycemia |
| CN114870093A (en) * | 2022-05-07 | 2022-08-09 | 四川大学 | 3D printing tissue engineering pancreatic islet based on digital light processing and preparation method and application thereof |
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