CN105664248B - A kind of protein scaffolds preparation method based on piezo jet India side formula - Google Patents
A kind of protein scaffolds preparation method based on piezo jet India side formula Download PDFInfo
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- CN105664248B CN105664248B CN201610028686.4A CN201610028686A CN105664248B CN 105664248 B CN105664248 B CN 105664248B CN 201610028686 A CN201610028686 A CN 201610028686A CN 105664248 B CN105664248 B CN 105664248B
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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
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Abstract
The present invention proposes a kind of protein scaffolds preparation method based on piezo jet India side formula, is related to cell from the stem cell culture of patient autologous tissue, two kinds of protein scaffolds solution are configured by suffering from body fibrin and heat shock protein.Piezoelectric ink jet printing technique of the present invention, print heat shock protein white, fibrin layer successively according to model, then denaturation is set to form the protein scaffolds solidified using heating lamp tube irradiation fibrin, cell is printed again on the holder, and so on, albumin layer and cellular layer printing alternate, ultimately form the biological support with certain three-dimensional structure.Cell and albumen used in the present invention, which derive from, suffers from body; repel small; high temperature makes fibrin denaturation be physical modification; fibrin is not influenced provides nutriment for cell growth; heat shock protein white protects cellular layer not by the high temperature injury of heating lamp tube simultaneously, drives parameter expeditiously to print high molecular protein solution and cell liquid from the piezo jets of low level development by setting.
Description
Technical field
The present invention relates to a kind of construction methods for the molding biological support of cell three-dimensional, especially a kind of to be based on piezoelectricity
The protein scaffolds preparation method of spray printing mode.
Background technology
3D printing technique is most advanced one of the technology in the current whole world, with popularizing and cross discipline for 3D printing technique
Constantly fusion, application of the technology in biological manufacturing field obtain great development.In terms of medical treatment, the production model of 3D printing
The existing treatment flow of doctor can be changed, gradually the opening up of the Continuous Innovation of technology and application can extensively provide wide for clinician
Application prospect.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 organs.
Research in relation to Organ printing technology attracts wide public concern, and reason is that the field is handed over apparent subject
The characteristics of fork is merged with infiltration, it is in life science and rapid shaping technique, Biotechnology, bioscience and material supply section
Joint, it breaks through the limitation of two dimension research for organizational engineering, is accurately controlled on three dimension scale and tissue
Or the research in terms of the similar three-dimensional tectosome of organ provides a kind of new thinking.
In recent years, the feasibility of cell printing is constantly verified, and new Method of printing is constantly proposed, common printing
Mode has, ink-jet cell printing technology, injection cell printing technology, Laser Cell printing technique, acoustic control cell printing technology etc.,
Printing type respectively has quality.The Method of printing of piezoelectric type has its unique advantage, efficient and high resolution, but difficult printing is high viscous
Degree liquid and easily blocking are that it is maximum insufficient.
With scientific development, scientific research is conceived to cell survival rate, and damage of the cell in print procedure is efficiently controllable
Method of printing, in the researchs such as three-dimensional tiling method.Wherein the three-dimensional packing of cell usually uses hydrogel as biological support, will
Cell liquid is blended in printing or both separately printing in hydrogel, ultimately forms the type-script with stereochemical structure.Hydrogel has
There is macromolecule network system, property is soft, can keep certain shape, porous, can store a large number of nutrients, is that culture is thin
The ideal holder of born of the same parents；But the preparation process of non-protein based aquagel is sufficiently complex, and technology stability is poor.
Invention content
To solve problem of the prior art, the present invention proposes a kind of protein scaffolds preparation based on piezo jet India side formula
Method.It is big to print by the way that driving voltage effective time and driving voltage in print frequency and each pulse is arranged in this method
The cell liquid and protein solution of molecule；Suffer from the protein setting rear as holder of body itself using being derived from, both solves allosome
Exclusive problem, protein scaffolds are more advantageous to cell growth breeding, can be absorbed by cell, also solve complicated preparation and ask
Topic, can be printed to cell liquid and protein liquid on carrier by way of printing successively.
Have a large amount of research and experiments have shown that high temperature can be such that protein denaturation solidifies, based on this principle, this method makes
Protein liquid and cell liquid are printed successively with piezo jets, solve the problems, such as inefficient protein layer covering and cell seeding；
Secondly this method has used the heat shock protein white of resistance to 70 degree of high temperature or more to protect intracellular protein unaffected, and controls
The time of heating lamp tube irradiation makes fibrin layer cure and heat shock protein white is influenced or unaffected by smaller.
This method suffers from the fibrin and heat shock protein of body itself by extraction, is beaten using the piezo jets of parameter design
Two layers of albumin layer is printed off, so that fibrin denaturation in surface layer is formed by curing holder with heating lamp tube irradiation, is then printed on holder
Cellular layer, cellular layer as above and albumin layer printing alternate, to form the biological support with certain stereochemical structure.
Specifically technical solution is：
A kind of protein scaffolds preparation method based on piezo jet India side formula, it is characterised in that：Include the following steps：
Step 1：The cell to be printed for obtaining patient itself, to cell culture to be printed and induces differentiation, cell is made and beats
Print liquid；The fibrin and heat shock protein for obtaining patient itself, it is molten by fibrin is prepared in fibrin addition culture solution
Heat shock protein is added in culture solution and prepares heat shock protein solution by liquid；
Step 2：Cell printing liquid, fibrinogen solution and heat shock protein solution are respectively charged into biological 3D printing equipment
Correspondence liquid storage cylinder in, each liquid storage cylinder connects corresponding piezo jets；In the control system of the biology 3D printing equipment
It is stored with the hierarchical mode of patient tissue 3D models to be printed；
Step 3：The spray orifice of three nozzles of adjustment and the initial distance of glass slide are 5mm, and injection diameter is 50 μm, and nozzle drives
Dynamic voltage is 28-35v, and frequency 2-4khz, nozzle spray printing speed is 5.0m/s；
Step 4：Biological 3D printing equipment is stored up according to the hierarchical mode described in step 2 using connection heat shock protein solution
The nozzle of sap cavity prints one layer of heat shock protein white on glass slide, and nozzle movement speed is 0.015m/s, heat shock protein white
Thickness is 25um-35um；
Step 5：Biological 3D printing equipment uses connection fibrinogen solution liquid storage according to the hierarchical mode described in step 2
The nozzle of chamber prints one layer of fibrin layer in heat shock protein white, and nozzle movement speed is 0.015m/s, fibrin layer
Thickness is 25um-35um；
Step 6：Fibrin layer is irradiated at fibrin layer 50mm using heating lamp tube 10 seconds, with postcooling 20-
30s；
Step 7：Biological 3D printing equipment uses connection cell printing liquid liquid storage cylinder according to the hierarchical mode described in step 2
Nozzle cell liquid is printed on fibrin layer, then wait for 20-30s；
Step 8：Step 4~step 7 is repeated, until obtaining the albumen branch consistent with patient tissue 3D models to be printed
Frame.
Further preferred embodiment, a kind of protein scaffolds preparation method based on piezo jet India side formula, feature
It is：Cell printing liquid concentration in step 1 is not less than 104cells/ml, a concentration of 12mg/ml of fibrinogen solution, and heat is stopped
Gram a concentration of 20mg/ml of protein solution.
Further preferred embodiment, a kind of protein scaffolds preparation method based on piezo jet India side formula, feature
It is：The piezo jets model Sai Er Xaar128.
Advantageous effect
Compared with prior art, the present invention has remarkable advantage：
(1) present invention utilizes the nozzle printing of piezoelectric type, can be expeditiously according to importing model printing protein liquid and thin
Cytosol is accurately obtained the biological support of fixed point printing cell.
(2) for the albumen holder raw material that the present invention uses from body itself is suffered from, minimum repulsion is degradable to be provided for cell culture
Nutriment.
(3) the holder curing that the present invention uses is thermal denaturation, and cellular layer is protected using heat-resisting heat shock protein white,
It can be to make the solidification of shelf layer protein denaturation without the cellular layer under injuring in the short time.
(4) present invention is by being arranged piezo jets driving frequency, driving voltage, single pulse effective driving voltage time
Method prints the cell liquid and protein liquid containing macromolecular substances.
Description of the drawings
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 printing heat shocks
The nozzle of albumen, the nozzle of 7 printing fibrins, the nozzle of 8 printing cells, the liquid storage cylinder of 9 dress heat shock proteins, 10 dress blood fibres
The liquid storage cylinder of albumen, the liquid storage cylinder of 11 dress cells, 12 cellular layers, 13 fibrin layers, 14 heat shock protein white, the movement of 15 nozzles
Holder, 16 hoistable platforms.
Specific implementation mode
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to 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, beaten for cell
Print or Organ printing provide a kind of new biological support preparation method.Cell to be printed, heat shock protein bletilla blood fibre in this method
Albumen, which is taken from, suffers from body；The piezoelectricity printing type one layer of heat shock protein white of printing set using bottom, one layer of fibrin layer,
After heating lamp tube irradiates, fibrin is made to be denaturalized to form cured protein scaffolds, and heat shock protein white is unaffected；
Then pinpoint designated cell is printed on protein scaffolds；Cellular layer and albumin layer are and so on printed, is eventually formed
Biological support with certain three-dimensional structure.
The present embodiment after to carrying out CT scan at injury, by repairing and reconstructing, is built using skin injury patient as object
Vertical patient tissue 3D models to be printed, then successively separation is carried out to 3D models and is parsed, obtaining biological 3D printing equipment can know
Other hierarchical mode.
It is as follows：
Step 1：The cell to be printed for obtaining patient itself, to cell culture to be printed and induces differentiation, cell is made and beats
Print liquid；The fibrin and heat shock protein for obtaining patient itself, it is molten by fibrin is prepared in fibrin addition culture solution
Heat shock protein is added in culture solution and prepares heat shock protein solution by liquid.
The process that skin progenitor cell liquid is prepared in the present embodiment is：
Suffer from one fritter of body skin of foreskin 1. taking, then shred skin histology, with penicillin containing 2000u/L, 200mg/L chains
Mycin and 2.5mg/L amphotericin Bs rinse 3 times repeatedly without calcium ions and magnesium ions PBS liquid；2. the skin graft tissue rinsed well is placed in
20min is digested in 0.25% neutral proteinase II, then is placed in 0.25% trypsase and 0.02%EDTA, 37 DEG C of digestion
30min, 200 mesh metal mesh filters harvest cell suspension, and 1000r/min centrifuges 5~10min, then the Hanks liquid with no calcium and magnesium
Washing centrifugation 2 times；3. the cell after digestion is washed is added containing 15% fetal calf serum, 4mmol/L glutamine, 0.4 μ g/mL
Hydrocortisone, 5 μ g/mL transferrins, 5 μ g/mL insulin, 10ng/mL epidermal growth factor, 1.8 × 10-4mmol/L glands
In DMEM and F12 (V: V=3: 1) culture medium of purine, 100u/mL penicillin and 100 μ g/mL streptomysins, culture bottle bottom is with IV
Collagen Type VI is coated with, and is incubated in 37 DEG C, 5% carbon dioxide incubator, and culture solution and non-attached cell is sucked out in next day, is added new
Fresh culture solution, changes liquid for 2-3 days 1 time, and cell takes out cell when growing to 70%~80%, and PSB buffer solutions are added, are configured to 104-
The cell liquid of 107cells/ml.
The process that protein solution is prepared in the present embodiment is：
The very thin born of the same parents of bleeding are extracted in body body from suffering from, and cultivate number generation, it is for use to extract fibrin.It is extracted from suffering from body body
Go out heat-shocked cell, cultivate in vitro, expresses heat shock protein with thermostimulation stimulation heat-shocked cell, extraction heat shock protein waits for
With.DMEM culture solutions are added in two kinds of albumen respectively, the protein solution of a concentration of 12mg/ml and 20mg/ml is made.
Step 2：Cell printing liquid, fibrinogen solution and heat shock protein solution are respectively charged into biological 3D printing equipment
Correspondence liquid storage cylinder in, each liquid storage cylinder connects corresponding piezo jets, and piezo jets are Sai Er Xaar128；The biology
The hierarchical mode of patient tissue 3D models to be printed is stored in the control system of 3D printing equipment.
Step 3：The spray orifice of three nozzles of adjustment and the initial distance of glass slide are 5mm, nozzle and nozzle spacing 10mm, spray
Bore dia is 50 μm, and nozzle driving voltage is 28-35v, and frequency 2-4khz, nozzle spray printing speed is 5.0m/s.
Start printing when, first input printer model data to driving plate, driving plate be layered after single layer print data,
Then single layer printing is carried out according to the process of below step 4 to step 7：
Step 4：Biological 3D printing equipment is stored up according to the hierarchical mode described in step 2 using connection heat shock protein solution
The nozzle of sap cavity prints one layer of heat shock protein white on glass slide, and nozzle movement speed is 0.015m/s, and duplicate printing is primary,
Heat shock protein layer thickness is 25um-35um.
Step 5：Biological 3D printing equipment uses connection fibrinogen solution liquid storage according to the hierarchical mode described in step 2
The nozzle of chamber prints one layer of fibrin layer in heat shock protein white, and nozzle movement speed is 0.015m/s, duplicate printing one
Secondary, fibrin layer thickness is 25um-35um.
Step 6：After the completion of printing, nozzle group is removed, and 100 DEG C of control heating lamp tube surface temperature constant temperature uses fever lamp
Pipe irradiates fibrin layer 10 seconds at fibrin layer 50mm, then removes heating lamp tube, cooling 20-30s.
Step 7：Biological 3D printing equipment uses connection cell printing liquid liquid storage cylinder according to the hierarchical mode described in step 2
Nozzle print cell liquid in designated position on fibrin layer, then wait for 20-30s.
Step 8：After completing one layer of printing, print platform moves down 100um, repeats step 4~step 7, until obtaining
The albumen holder consistent with patient tissue 3D models to be printed.Holder higher slice is attached to the cell of designated position, cellular layer
Between there is abundant nutriment, albumen holder to have and import the consistent shape of model, there is elasticity and supportive.
The obtained biological support comprising skin progenitor cell and protein scaffolds is cultivated into a period of time in vitro, is implanted into skin
Skin injury region.
The skin progenitor cell holder that the present embodiment is produced out not only has the characteristics that expeditiously, and almost without repulsion, makes
It uses oneself protein matter to be more advantageous to cell growth breeding as biological support, can also constantly degrade with the breeding of cell, be implanted into
Safety has prodigious advantage compared with hydrogel, and uses the cell printing mode of piezoelectric type, it is ensured that very high printing speed
Rate.The method for being different from other biological supports, this method are made using piezo jets printing protein liquid and cell liquid with protein
For holder, while using of short duration hot setting holder, cell is protected with heat safe heat shock protein, is a kind of effective
Manufacture invention.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case of can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
Claims (3)
1. a kind of protein scaffolds preparation method based on piezo jet India side formula, it is characterised in that：Include the following steps：
Step 1：The cell to be printed for obtaining patient itself, to cell culture to be printed and induces differentiation, cell printing liquid is made；
Fibrin is added in culture solution and prepares fibrinogen solution by the fibrin and heat shock protein for obtaining patient itself, will
Heat shock protein is added in culture solution and prepares heat shock protein solution；
Step 2：Cell printing liquid, fibrinogen solution and heat shock protein solution are respectively charged into pair of biological 3D printing equipment
It answers in liquid storage cylinder, each liquid storage cylinder connects corresponding piezo jets；Storage in the control system of the biology 3D printing equipment
There is the hierarchical mode of patient tissue 3D models to be printed；
Step 3：The spray orifice of three nozzles of adjustment and the initial distance of glass slide are 5mm, and injection diameter is 50 μm, nozzle driving electricity
Pressure is 28-35v, and frequency 2-4khz, nozzle spray printing speed is 5.0m/s；
Step 4：Biological 3D printing equipment uses connection heat shock protein solution liquid storage cylinder according to the hierarchical mode described in step 2
Nozzle print one layer of heat shock protein white on glass slide, nozzle movement speed is 0.015m/s, heat shock protein layer thickness
For 25um-35um；
Step 5：Biological 3D printing equipment uses connection fibrinogen solution liquid storage cylinder according to the hierarchical mode described in step 2
Nozzle prints one layer of fibrin layer in heat shock protein white, and nozzle movement speed is 0.015m/s, fibrin layer thickness
For 25um-35um；
Step 6：Fibrin layer is irradiated at fibrin layer 50mm using heating lamp tube 10 seconds, with postcooling 20-30s；
Step 7：Biological 3D printing equipment uses the spray of connection cell printing liquid liquid storage cylinder according to the hierarchical mode described in step 2
Head prints cell liquid on fibrin layer, then waits for 20-30s；
Step 8：Step 4~step 7 is repeated, until obtaining the albumen holder consistent with patient tissue 3D models to be printed.
2. a kind of protein scaffolds preparation method based on piezo jet India side formula according to claim 1, it is characterised in that：Step
Cell printing liquid concentration in rapid 1 is not less than 104Cells/ml, a concentration of 12mg/ml of fibrinogen solution, heat shock protein are molten
A concentration of 20mg/ml of liquid.
3. a kind of protein scaffolds preparation method based on piezo jet India side formula according to claim 1, it is characterised in that：Institute
State piezo jets model Sai Er Xaar128.
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|---|---|---|---|---|
| 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 |
| CN104287875A (en) * | 2014-03-05 | 2015-01-21 | 青岛尤尼科技有限公司 | Multifunctional bioprinting system and tissue engineering organ preparation method based on bioprinting system |
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| AU2008262331B2 (en) * | 2007-06-07 | 2013-10-17 | Wake Forest University Health Sciences | Inkjet gene printing |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104287875A (en) * | 2014-03-05 | 2015-01-21 | 青岛尤尼科技有限公司 | Multifunctional bioprinting system and tissue engineering organ preparation method based on bioprinting system |
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