CN105664248A - Preparation method of protein scaffold based on piezoelectric jet printing mode - Google Patents
Preparation method of protein scaffold based on piezoelectric jet printing mode Download PDFInfo
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- CN105664248A CN105664248A CN201610028686.4A CN201610028686A CN105664248A CN 105664248 A CN105664248 A CN 105664248A CN 201610028686 A CN201610028686 A CN 201610028686A CN 105664248 A CN105664248 A CN 105664248A
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- heat shock
- cell
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
Abstract
The invention provides a preparation method of a protein scaffold based on piezoelectric jet printing mode, cells related to the method are cultivated from stem cells of autologous tissue of patients, and two kinds of protein scaffold solutions are prepared from fibrin and heat shock protein of patients. A heat shock protein layer and a fibrin layer are printed in order according to models by using the piezoelectric ink-jet printing technology, fibrin is irradiated by a heating lamp tube for denaturation and a solidified protein scaffold is formed, cells are printed on the scaffold, the steps are repeated, a protein layer and a cell layer are alternatively printed, and finally the biological scaffold with a certain three dimensional structure is formed. The cells and protein are derived from the patients, so that rejection reaction is less; high temperature ensures fibrin denaturation which is physical denaturation and does not influence the fibrin which provides nutrients for cell growth, at the same time the heat shock protein layer prevents the cell layer from high temperature damage due to the heating lamp tube, and a driving parameter of a piezoelectric nozzle which is developed from a low level is set for efficiently printing a high-molecule protein solution and cell liquid.
Description
Technical field
The present invention relates to the construction method of a kind of biological support for cell three-dimensional molding, especially a kind of protein scaffolds preparation method based on piezo jet India side formula.
Background technology
3D printing technique is one of technology that the whole world is most advanced at present, and along with the continuous fusion of the universal of 3D printing technique and cross discipline, the application in biology manufacture field of this technology obtains great development. In medical treatment, the production model that 3D prints can change the existing treatment flow process of doctor, and opening up gradually of the Continuous Innovation of technology and application extensively can provide wide application prospect for clinicist. Biological 3 D-printing is to carry out 3 D-printing with biomaterial or living cells, to build complex biological three dimensional structure, such as personalized implant, renewable artificial bone, cell in vitro three-dimensional structure, artificial organ etc.
Cause about the research of Organ printing technology and pay close attention to widely, its reason is in that this field has the advantages that obvious subject crossing merges with infiltration, it is in the joint of life sciences and rapid shaping technique, Biotechnology, bioscience and material science, it breaks through the limitation of two dimension research for tissue engineering, accurately controls the research in the three-dimensional tectosome similar to tissue or organ and provide a kind of new thinking on three dimension scale.
In recent years, the feasibility of cell printing is constantly verified, new Method of printing is constantly proposed, and common printing type has, ink-jet cell printing technology, injection cell printing technology, Laser Cell printing technique, acoustic control cell printing technology etc., printing type is respectively arranged with quality. The Method of printing of piezoelectric type has the advantage of its uniqueness, and efficiency is high and resolution is high, but difficult printing high viscosity liquid and easily blocking are its maximum deficiencies.
In the researchs such as along with scientific development, scientific research is conceived to cell survival rate, cell damage in print procedure, efficiently controlled Method of printing, three-dimensional tiling method. Wherein the three-dimensional packing of cell is typically by hydrogel as biological support, is blended in by Cell sap in hydrogel and prints or both separately printings, ultimately forms the type-script with stereochemical structure.Hydrogel has macromolecule network system, and character is soft, can keep certain shape, porous, it is possible to store a large number of nutrients, is cultivate the support that cell is ideal; But the preparation technology of non-protein based aquagel is sufficiently complex, and technology stability is poor.
Summary of the invention
For solving problem of the prior art, the present invention proposes a kind of protein scaffolds preparation method based on piezo jet India side formula. In the method, print Cell sap and the protein solution of macromole by arranging driving voltage effective time and driving voltage in print frequency and each pulse; Adopt take from suffer from body self protein setting after as support, both allosome exclusive problem had been solved, protein scaffolds is more beneficial for Growth of Cells breeding, can by Cell uptake, also the preparation problem of complexity is solved, it is possible to Cell sap and protein liquid are printed to successively on carrier by the mode printed.
Existing substantial amounts of research and experiment prove that high temperature can make protein denaturation solidify, and based on this principle, this method uses piezo jets protein liquid and Cell sap to be printed successively, and the protein layer solving poor efficiency covers and cell seeding problem; Secondly this method employs the heat shock protein white of more than resistance to 70 degree high temperature and protects intracellular protein unaffected, and controls that the time chien shih fibrin layer that heating lamp tube irradiates solidifies and heat shock protein white is subject to less impact or unaffected.
This method is by extracting fibrin and the heat shock protein suffering from body self, the piezo jets utilizing parameter design prints two-layer albumin layer, irradiating with heating lamp tube makes top layer fibrin degeneration solidify to form support, then on support, print cellular layer, as above cellular layer and albumin layer printing alternate, thus forming the biological support with certain stereochemical structure.
Concrete technical scheme is:
Described a kind of protein scaffolds preparation method based on piezo jet India side formula, it is characterised in that: comprise the following steps:
Step 1: obtain the cell to be printed of patient self, cell to be printed cultivated and induces differentiation, making cell printing liquid; Obtain fibrin and the heat shock protein of patient self, fibrin is added in culture fluid and prepare fibrinogen solution, heat shock protein is added in culture fluid and prepare heat shock protein solution;
Step 2: cell printing liquid, fibrinogen solution and heat shock protein solution being respectively charged in the corresponding liquid storage cylinder of biological 3D printing device, each liquid storage cylinder connects the piezo jets of correspondence; The Installed System Memory that controls of described biological 3D printing device contains the hierarchical mode of patient tissue 3D model to be printed;
Step 3: adjusting the spray orifice of three shower nozzles with the initial distance of microscope slide is 5mm, and injection diameter is 50 μm, and shower nozzle driving voltage is 28-35v, and frequency is 2-4khz, and shower nozzle spray printing speed is 5.0m/s;
Step 4: biological 3D printing device hierarchical mode according to step 2, the shower nozzle connecting heat shock protein solution liquid storage cylinder is used to print one layer of heat shock protein white on microscope slide, shower nozzle translational speed is 0.015m/s, and heat shock protein layer thickness is 25um-35um;
Step 5: biological 3D printing device hierarchical mode according to step 2, the shower nozzle connecting fibrinogen solution liquid storage cylinder is used to print one layer of fibrin layer in heat shock protein white, shower nozzle translational speed is 0.015m/s, and fibrin layer thickness is 25um-35um;
Step 6: use heating lamp tube distance fibrin layer 50mm place to irradiate fibrin layer 10 seconds, cool down 20-30s subsequently;
Step 7: biological 3D printing device hierarchical mode according to step 2, uses the shower nozzle connecting cell printing liquid liquid storage cylinder to print Cell sap on fibrin layer, then waits 20-30s;
Step 8: repeat step 4～step 7, until obtaining albumen support model consistent with patient tissue 3D to be printed.
Further preferred version, described a kind of protein scaffolds preparation method based on piezo jet India side formula, it is characterized in that: the cell printing liquid concentration in step 1 is not less than 104cells/ml, and fibrinogen solution concentration is 12mg/ml, and heat shock protein solution concentration is 20mg/ml.
Further preferred version, described a kind of protein scaffolds preparation method based on piezo jet India side formula, it is characterised in that: described piezo jets model is Sai Er Xaar128.
Beneficial effect
Compared with prior art, the present invention has a significant advantage that
(1) present invention utilizes the nozzle printing of piezoelectric type, it is possible to expeditiously according to importing model printing protein liquid and Cell sap, is accurately obtained fixed point and prints the biological support of cell.
(2) the albumen support raw material that the present invention uses is from suffering from body self, minimum repulsion, and degradable is cultivated for cell and provided nutrient substance.
(3) the support curing that the present invention uses is thermal denaturation, utilizes heat-resisting heat shock protein white protection cellular layer, it is possible to make shelf layer protein denaturation solidify and not injure beneath cellular layer in the short time.
(4) present invention by arranging piezo jets driving frequency, driving voltage, the method for individual pulse effective driving voltage time print the Cell sap containing macromolecular substances and protein liquid.
Accompanying drawing explanation
Fig. 1 is the operating diagram of biological piezoelectricity printing shaping machine.
In figure: 1 print platform, 2 piezo jets groups, 3 liquid storage cylinder groups, 4 biological supports, 5 heating lamp tubes, 6 print the shower nozzle of heat shock protein, 7 shower nozzles printing fibrin, 8 print the shower nozzle of cell, the liquid storage cylinder of 9 dress heat shock proteins, the liquid storage cylinder of 10 dress fibrins, the liquid storage cylinder of 11 dress cells, 12 cellular layers, 13 fibrin layer, 14 heat shock protein white, 15 shower nozzle traversing carriages, 16 hoistable platforms.
Detailed description of the invention
Being described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has the element of same or like function from start to finish. The embodiment described below with reference to accompanying drawing is illustrative of, it is intended to is used for explaining the present invention, and is not considered as limiting the invention.
It is an object of the invention to propose a kind of protein scaffolds preparation method based on piezo jet India side formula, provide a kind of new biological support preparation method for cell printing or Organ printing. In the method, cell to be printed, heat shock protein Pseudobulbus Bletillae (Rhizoma Bletillae) fibrin are taken from suffering from body; Utilize the piezoelectricity printing type that bottom sets to print one layer of heat shock protein white, one layer of fibrin layer, after heating lamp tube irradiates, make fibrin degeneration form the protein scaffolds solidified, and heat shock protein white is unaffected; Then on protein scaffolds, print designated cell pinpoint; Print cellular layer and albumin layer and so forth, eventually form the biological support with certain three dimensional structure.
The present embodiment is with skin injury patient for object, after injury place is carried out CT scan, by repairing and reconstructing, sets up patient tissue 3D model to be printed, then 3D model is successively separated parsing, obtains the hierarchical mode that biological 3D printing device is capable of identify that.
Specifically comprise the following steps that
Step 1: obtain the cell to be printed of patient self, cell to be printed cultivated and induces differentiation, making cell printing liquid; Obtain fibrin and the heat shock protein of patient self, fibrin is added in culture fluid and prepare fibrinogen solution, heat shock protein is added in culture fluid and prepare heat shock protein solution.
The process preparing skin progenitor cell liquid in the present embodiment is:
1. take and suffer from body skin of foreskin one fritter, then skin histology is shredded, repeatedly rinse 3 times with the liquid of PBS without calcium ions and magnesium ions containing 2000u/L penicillin, 200mg/L streptomycin and 2.5mg/L amphotericin B, 2. the skin graft tissue rinsed well is placed in 0.25% neutral protease II and digests 20min, it is placed in 0.25% trypsin and 0.02%EDTA again, 37 DEG C of digestion 30min, 200 order metal mesh filters, harvesting suspension, 1000r/min is centrifuged 5～10min, then washs centrifugal 2 times with the Hanks liquid without calcium and magnesium, 3. the cell after digestion washing is added containing 15% hyclone, 4mmol/L glutamine, 0.4 μ g/mL hydrocortisone, 5 μ g/mL transferrinss, 5 μ g/mL insulins, 10ng/mL epidermal growth factor, 1.8 × 10-4mmol/L adenine, in DMEM and F12 (V: V=3: the 1) culture medium of 100u/mL penicillin and 100 μ g/mL streptomycins, it is coated with type Ⅳ collagen at the bottom of culture bottle, in 37 DEG C, 5% carbon dioxide incubator is hatched, sucking-off next day culture fluid and non-attached cell, add fresh medium, within 2-3 days, change liquid 1 time, cell is taken out when cell length is to 70%～80%, add PSB buffer, it is configured to the Cell sap of 104-107cells/ml.
The process preparing protein solution in the present embodiment is:
Extract hemorrhage very thin born of the same parents in body body from suffering from, cultivate number generation, extract fibrin stand-by. Extract heat-shocked cell from suffering from body body, cultivate in vitro, express heat shock protein with thermostimulation irritation fever shock cell, extract heat shock protein stand-by. Respectively two kinds of albumen are added DMEM culture fluid, make the protein solution that concentration is 12mg/ml and 20mg/ml.
Step 2: cell printing liquid, fibrinogen solution and heat shock protein solution being respectively charged in the corresponding liquid storage cylinder of biological 3D printing device, each liquid storage cylinder connects the piezo jets of correspondence, and piezo jets is Sai Er Xaar128; The Installed System Memory that controls of described biological 3D printing device contains the hierarchical mode of patient tissue 3D model to be printed.
Step 3: adjusting the spray orifice of three shower nozzles with the initial distance of microscope slide is 5mm, shower nozzle and sprinkler spacing 10mm, injection diameter is 50 μm, and shower nozzle driving voltage is 28-35v, and frequency is 2-4khz, and shower nozzle spray printing speed is 5.0m/s.
When starting to print, first input printer model data are to driving plate, the monolayer print data after driving plate to be layered, then according to the process of below step 4 to step 7 carries out monolayer printing:
Step 4: biological 3D printing device hierarchical mode according to step 2, the shower nozzle connecting heat shock protein solution liquid storage cylinder is used to print one layer of heat shock protein white on microscope slide, shower nozzle translational speed is 0.015m/s, and once, heat shock protein layer thickness is 25um-35um to duplicate printing.
Step 5: biological 3D printing device hierarchical mode according to step 2, the shower nozzle connecting fibrinogen solution liquid storage cylinder is used to print one layer of fibrin layer in heat shock protein white, shower nozzle translational speed is 0.015m/s, and once, fibrin layer thickness is 25um-35um to duplicate printing.
Step 6: after having printed, jet head sets is removed, and controls heating lamp tube surface temperature constant temperature 100 DEG C, uses heating lamp tube distance fibrin layer 50mm place to irradiate fibrin layer 10 seconds, removes heating lamp tube subsequently, cool down 20-30s.
Step 7: biological 3D printing device hierarchical mode according to step 2, uses the shower nozzle connecting cell printing liquid liquid storage cylinder to specify position to print Cell sap on fibrin layer, then waits 20-30s.
Step 8: complete one layer print after, print platform moves down 100um, repeats step 4～step 7, until obtaining albumen support model consistent with patient tissue 3D to be printed. Support higher slice is attached to the cell specifying position, and cell interlayer has abundant nutrient substance, and albumen support has and imports model consistent shape, has elasticity and supportive.
The biological support comprising skin progenitor cell and protein scaffolds obtained is cultivated a period of time in vitro, implants cutaneous lesion.
The skin progenitor cell support that the present embodiment is produced out, not only there is feature expeditiously, and almost without repulsion, oneself protein matter is used to be more beneficial for Growth of Cells breeding as biological support, also constantly can degrade along with the breeding of cell, implant safety and have very big advantage than hydrogel, and use the cell printing mode of piezoelectric type, it is ensured that significantly high printing speed. The method being different from other biological support; this method uses piezo jets to print protein liquid and Cell sap, with protein as support, while using of short duration hot setting support; protect cell with resistant to elevated temperatures heat shock protein, be a kind of effective manufacture invention.
Although above it has been shown and described that embodiments of the invention, it is understandable that, above-described embodiment is illustrative of, being not considered as limiting the invention, above-described embodiment can be changed when without departing from principles of the invention and objective, revises, replace and modification by those of ordinary skill in the art within the scope of the invention.
Claims (3)
1. the protein scaffolds preparation method based on piezo jet India side formula, it is characterised in that: comprise the following steps:
Step 1: obtain the cell to be printed of patient self, cell to be printed cultivated and induces differentiation, making cell printing liquid; Obtain fibrin and the heat shock protein of patient self, fibrin is added in culture fluid and prepare fibrinogen solution, heat shock protein is added in culture fluid and prepare heat shock protein solution;
Step 2: cell printing liquid, fibrinogen solution and heat shock protein solution being respectively charged in the corresponding liquid storage cylinder of biological 3D printing device, each liquid storage cylinder connects the piezo jets of correspondence; The Installed System Memory that controls of described biological 3D printing device contains the hierarchical mode of patient tissue 3D model to be printed;
Step 3: adjusting the spray orifice of three shower nozzles with the initial distance of microscope slide is 5mm, and injection diameter is 50 μm, and shower nozzle driving voltage is 28-35v, and frequency is 2-4khz, and shower nozzle spray printing speed is 5.0m/s;
Step 4: biological 3D printing device hierarchical mode according to step 2, the shower nozzle connecting heat shock protein solution liquid storage cylinder is used to print one layer of heat shock protein white on microscope slide, shower nozzle translational speed is 0.015m/s, and heat shock protein layer thickness is 25um-35um;
Step 5: biological 3D printing device hierarchical mode according to step 2, the shower nozzle connecting fibrinogen solution liquid storage cylinder is used to print one layer of fibrin layer in heat shock protein white, shower nozzle translational speed is 0.015m/s, and fibrin layer thickness is 25um-35um;
Step 6: use heating lamp tube distance fibrin layer 50mm place to irradiate fibrin layer 10 seconds, cool down 20-30s subsequently;
Step 7: biological 3D printing device hierarchical mode according to step 2, uses the shower nozzle connecting cell printing liquid liquid storage cylinder to print Cell sap on fibrin layer, then waits 20-30s;
Step 8: repeat step 4～step 7, until obtaining albumen support model consistent with patient tissue 3D to be printed.
2. a kind of protein scaffolds preparation method based on piezo jet India side formula according to claim 1, it is characterized in that: the cell printing liquid concentration in step 1 is not less than 104cells/ml, fibrinogen solution concentration is 12mg/ml, and heat shock protein solution concentration is 20mg/ml.
3. a kind of protein scaffolds preparation method based on piezo jet India side formula according to claim 1, it is characterised in that: described piezo jets model is Sai Er Xaar128.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107234804A (en) * | 2017-06-23 | 2017-10-10 | 大连理工大学 | The electric jet Method of printing that a kind of nanometer of point infiltration is focused on |
| CN108379659A (en) * | 2018-05-06 | 2018-08-10 | 西北工业大学 | A kind of more gradient artificial cartilage preparation methods of cell density |
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| US20150299730A1 (en) * | 2007-06-07 | 2015-10-22 | Wake Forest University Health Sciences | Inkjet gene printing |
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| US20150299730A1 (en) * | 2007-06-07 | 2015-10-22 | Wake Forest University Health Sciences | Inkjet gene printing |
| CN103341989A (en) * | 2013-07-08 | 2013-10-09 | 上海大学 | Regeneration bone scaffold forming system and method based on comprehensive 3D printing formation |
| CN103468635A (en) * | 2013-08-23 | 2013-12-25 | 浙江大学 | Three-dimensional cell scaffold printing method based on cell state feedback |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107234804A (en) * | 2017-06-23 | 2017-10-10 | 大连理工大学 | The electric jet Method of printing that a kind of nanometer of point infiltration is focused on |
| CN108379659A (en) * | 2018-05-06 | 2018-08-10 | 西北工业大学 | A kind of more gradient artificial cartilage preparation methods of cell density |
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