CN104548208B - A kind of preparation method and application of load cells three-dimensional rack - Google Patents
A kind of preparation method and application of load cells three-dimensional rack Download PDFInfo
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- CN104548208B CN104548208B CN201510045823.0A CN201510045823A CN104548208B CN 104548208 B CN104548208 B CN 104548208B CN 201510045823 A CN201510045823 A CN 201510045823A CN 104548208 B CN104548208 B CN 104548208B
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Abstract
The invention provides the preparation method of a kind of load cells three-dimensional rack, it is characterised in that be to mix homogeneously the hydrogel solution of autogenous cell with high temperature discontinuous sterilization to obtain mixed liquor, it is ejected in cross-linking agent solution, stand, then remove cross-linking agent solution, after rinsing, obtain load cells microsphere;Load cells microsphere and host material are mixed to get cell blend;Use biometric print machine to be extruded on the three-dimensional micromotion platform being loaded with sterilizing glass film plates by cell blend, be layering, form load cells three-dimensional rack.The present invention is by alleviating the infringement that cytoactive and biological activity are caused by cell printing process, it is ensured that highly active cell printing.
Description
Technical field
The invention belongs to cell printing and technical field of biological materials, be particularly well-suited to biomedical tissue engineering rack material
The preparation of material, a kind of method preparing load cells three-dimensional rack.
Background technology
Life sciences, information science combine with manufacturing science, are the important trend of 21st century development in science and technology.Based on
Advanced person manufactures theoretical and that organizational project is theoretical cell three-dimensional printing technique, for tissue engineering, pathological model structure, drug sieve
New theory and technology space has been expanded in the fields such as choosing and detection, RESEARCH ON CELL-BIOLOGY.
Compared with tradition Method of Tissue Engineering, the advantage of cell printing mainly has: (1) is built with bioactive simultaneously
Two dimension or three-dimensional " cell/material " system;(2) the different types of cell of accurate deposition over time and space;(3) build carefully
Three-dimensional microenvironment needed for born of the same parents.Cell printing export technique mainly includes inkjet printing (piezoelectricity volume drive-type and thermal
Formula), laser direct-writing, induced with laser transfer, electrostatic spraying, the injection of focusing ultrasound wave, micro-extrusion etc., it is feasible that conventional cell prints
Property and reliability aspect be the most verified, but in terms of how keeping cytoactive, also have limitation: during printing, great majority
Mammalian cell is the most fragile, is vulnerable to the impact of environment, and in print procedure the maximum temperature of nozzle 300 DEG C with
On, and there is bigger shear stress, during cell printing, produce instantaneous high-temperature, high pressure or moment strong electrostatic field to carefully
Born of the same parents cause injury.Therefore prepare when there is appropriate bore gap structure and thickness more than the load cells three-dimensional rack of 300 μm,
Keeping cytoactive in print procedure, reducing the adverse effect to its biological characteristics is problem demanding prompt solution.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of negative based on the preparation of cell printing export technique
The method carrying cell three-dimensional support, has the advantages that to keep cytoactive.
The preparation method of a kind of load cells three-dimensional rack, is by the hydrogel solution of autogenous cell Yu high temperature discontinuous sterilization
Mix homogeneously obtains mixed liquor, is ejected in cross-linking agent solution, stands, then removes cross-linking agent solution, loaded after rinsing
Cell microspheres;Load cells microsphere and host material are mixed to get cell blend;Use biometric print machine by cell
Blend extrusion, on three-dimensional micromotion platform, is layering, and forms load cells three-dimensional rack.
Further, described hydrogel solution be hydrogel monomer and hydrogel solvent mix homogeneously formulated, concentration
For 0.05-10 wt%;Described hydrogel monomer is the one in collagen, gelatin, hyaluronic acid, chitosan or Na-alginate or several
The mixture planted;Described hydrogel solvent is water or pH value is the phosphate buffered solution of 5.7-8.0.
Further, in described mixed liquor, the concentration of autogenous cell is 105-107/mL。
Further, the pressure of described injection is 10-200 kPa, and jet diameters is 500-1000 μm.
Further, described cross-linking agent solution is CaCl2Solution, concentration is 0.1-20 wt%.
Further, described autogenous cell is that mesenchymal stem cells MSCs, fat stem cell, chondrocyte, skeletonization are thin
One or more combination in born of the same parents.
Further, described rinsing is first to rinse 3 times by PBS solution, then uses DMEM culture fluid to rinse 3 times.
Further, the preparation method of described host material is: preparation recombinant collagen solution and sodium alginate soln respectively,
1:1-5 in mass ratio mixes, and high temperature discontinuous sterilization, the concentration of described recombinant collagen solution is 2-10 wt %, described sodium alginate
The concentration of solution is 2-10 wt %;Configuration concentration is the CaCl of 0.05-2 wt %2Solution, mixes recombinant collagen-sodium alginate
Close liquid and CaCl2Solution 7:3 mix homogeneously by volume, obtains host material.
Further, in described cell blend, the concentration of load cells microsphere is 105-107/mL。
Described load cells three-dimensional rack is used for preparing tissue engineering bracket.
Compared with prior art, advantages of the present invention with good effect is: the present invention is by using to become with extracellular matrix
As classification, cell is wrapped up by hydrogel material, it is possible to cell is made by the mechanically and thermally environment effectively reduced in print procedure
The damage become.The present invention is by being ejected into CaCl by cell and hydrogel mixed solution2Method in solution, prepares load
The microsphere of cell, the parcel of hydrogel material can form protection to cell, alleviate cell printing process to cytoactive and life
The infringement that thing activity causes, it is ensured that highly active cell printing.Seed cell used by the present invention is autogenous cell, without immunity row
Scold.The present invention can adjust print parameters according to tissue or the different needs of organ, can directly prepare and have various structure
(porous, tubulose) and the three-dimensional rack of mechanical property, study for organizational project and regenerative medicine.The inventive method is simple, easily
In operation, low cost, the three-dimensionally shaped support with complicated form of energy, can meet clinical needs, be suitable for the system of engineering rack
Standby.
Accompanying drawing explanation
Fig. 1. the preparation method flow chart of load cells three-dimensional rack of the present invention;
Fig. 2. the microexamination figure of the load cells microsphere of the present invention;
Fig. 3. the load cells three dimensional scaffold structure figure of the present invention;
Fig. 4. support cytoactive detection coloration result described in embodiment 1 in the present invention.
Detailed description of the invention
Below in conjunction with detailed description of the invention, technical scheme is described in further detail.
A kind of load cells three-dimensional rack, is to utilize own cells preparation to contain the microsphere supported, with host material of cell
It is mixed to get cell blend, utilizes cell printing export technique to be layering by cell blend, form load cells three-dimensional
Support, specifically comprises the following steps that
1) load cells microsphere is prepared: mixed homogeneously by the hydrogel solution of autogenous cell with high temperature discontinuous sterilization and obtain
Mixed liquor, in described mixed liquor, autogenous cell concentration is 105-107/mL;According to pressure 10-200 kPa, jet diameters 500-
Mixed liquor is ejected in cross-linking agent solution by the injection conditions of 1000 μm, makes the microlayer model containing cell ejected in crosslinking
Cross-linking in agent, form load cells microsphere, described cross-linking agent solution is CaCl2Solution, concentration is 0.1-20 wt%, stands 2-
60 min, absorb cross-linking agent solution with pipettor, then use PBS buffer solution to rinse 3 times, use DMEN (Dulbecco's
Modified Eagle Media) cell culture fluid rinse 3 times, obtain load cells microsphere, as shown in Figure 2.
Described autogenous cell can be in mesenchymal stem cells MSCs, fat stem cell, chondrocyte, osteoblast
One or more combination.
Described hydrogel sol is hydrogel monomer and hydrogel solvent mix homogeneously is configured to, and mass concentration is
0.05-10 wt%;Described hydrogel monomer is one or more in collagen, gelatin, hyaluronic acid, chitosan or Na-alginate
Mixture;Described hydrogel solvent is water or phosphate (PBS) buffer solution that pH value is 5.7-8.0.Hydrogel material
Parcel can form protection to cell, alleviates the infringement that cytoactive and biological activity are caused by cell printing process, it is ensured that high alive
The cell printing of property.
2) preparing host material: preparation recombinant collagen solution and sodium alginate soln respectively, 1:1-5 in mass ratio mixes,
High temperature discontinuous sterilization, the concentration of described recombinant collagen solution is 2-10 wt %, and the concentration of described sodium alginate soln is 2-10
wt %.Configuration concentration is the CaCl of 0.05-2 wt %2Solution, by recombinant collagen-sodium alginate mixed liquor and CaCl2Solution is pressed
Volume ratio 7:3 mix homogeneously, obtains host material.
Described recombinant collagen is recombined collagen, also referred to as recombination human collagen, has machinability, virus-free
The advantage that hidden danger, low rejection, deformation temperature improve.
Described host material, as cell carrier, has three dimensional structure and the constituent of natural extracellular matrix, with reality
Existing identical physiological function.Preferably host material has avirulence, has good biocompatibility, certain formability.Mesh
The front host material for cell assembling is based on natural biologic material, including gelatin, fibrin, sodium alginate, chitosan
Deng.These natural materials are from organism, similar to the composition of extracellular matrix, strong with cellular affinity, and thin containing promoting
The factor of the vital movement such as intracellular growth, propagation.Based on the analysis required for host material and the feature of natural high molecular substance,
The present invention have selected recombination human collagen and the alginic acid with good biocompatibility, degradability and cellular affinity
Sodium is as host material.
3) prepared by cell blend: load cells microsphere and host material is mixed by medical three-way valve, wherein bears
The concentration carrying cell microspheres is 105-107/mL。
4) load cells three-dimensional rack is printed: use biometric print machine that material extrusion is being loaded with the three of sterilizing glass film plates
On dimension micromotion platform, under the cad model with synusia information directly drives, form different aspects, between different aspects
Interlock superposition the most layer by layer, forms three-dimensional structure, with aseptic 1-20 wt % CaCl2Solution crosslinking 2-20 min, prepares load
Cell three-dimensional support.
5) the 5th step, support cross-links, by load cells three-dimensional rack with aseptic 2 wt % CaCl2Solution soaking, cross-links 2
min。
6) rinse load cells three-dimensional rack 3 times by PBS solution, after DMEM culture fluid rinses 3 times, put in six orifice plates conventional
In vitro culture, changes liquid routine observation every other day.
The load cells three-dimensional rack utilizing the inventive method to prepare is suitable for the preparation of tissue engineering bracket.
Embodiment 1
A kind of method preparing load cells three-dimensional rack based on cell printing export technique, comprises the following steps:
The first step, prepares load cells microsphere, by 1 wt % sodium alginate water of chondrocyte Yu high temperature discontinuous sterilization
Solution mix homogeneously, in mixed liquor, chondrocyte concentration is 2.2 × 105/ mL, according to pressure 120 kPa, jet diameters 730 μm
Injection conditions mixed liquor is ejected into the cross-linking agent CaCl that concentration is 2 wt%2In solution, stand 10min, absorb CaCl2Molten
Liquid, rinses 3 times by PBS solution, and DMEN cell culture fluid rinses 3 times, obtains load cells microsphere, standby.
Second step, prepares host material, and compound concentration is the recombinant collagen solution of 7wt% and concentration is the sea of 7wt% respectively
Solution of sodium alginate, 1:1 in mass ratio mixes, high temperature discontinuous sterilization.Configure the CaCl of 0.5 wt %2Solution, by recombinant collagen-sea
Sodium alginate mixed liquor and CaCl2Solution 7:3 mix homogeneously by volume, standby.
3rd step, prepared by cell blend, load cells microsphere and host material are mixed by medical three-way valve
To cell blend, in blend, the concentration of load cells microsphere is 1.2 × 105/mL。
4th step, prints load cells three-dimensional rack, uses biometric print machine, takes 2 mL cell blends addition volumes and is
In the barrel of 5 mL, use the manufacturing process of step motor drive plunger extrusion, material extrusion is being loaded with sterilizing glass film plates
Three-dimensional micromotion platform on, under the cad model with synusia information directly drives, form different aspects, different aspects
Between interlock the most layer by layer superposition bonding, form the three-dimensional structure (as shown in Figure 3) of 15 mm × 2, mm × 15 mm sizes
To load cells three-dimensional rack.
5th step, by above-mentioned load cells three-dimensional rack with aseptic 2 wt % CaCl2Solution soaking, cross-links 2 min.
6th step, rinses load cells three-dimensional rack 3 times by PBS solution, after DMEM culture fluid rinses 3 times, puts six orifice plates
Middle routine in vitro is cultivated, and changes liquid routine observation every other day.
7th step, cytoactive detection in load cells three-dimensional rack, with work-dead cell stain test kit
(Invitrogen) carry out testing by operating instruction.Concretely comprise the following steps: take In vitro culture load cells three-dimensional after 3 days and prop up
Frame PBS solution rinses 3 times, contains 2 μMs of calcein-AM(Calcein-AM at 3 mL) and 4 μMs of ethidium bromides (EthD-1)
Incubated at room 30min in dyeing liquor, PBS solution rinses 3 times, fluorescence microscope record coloration result, as shown in Figure 4, Fig. 4
Middle a figure is rack surface testing result, and b figure is (the i.e. detection knot within three-dimensional rack of testing result at support chi structure
Really), wherein major part region in green fluorescent label living cells 1(figure), circles mark in red fluorescence labelling dead cell 2(figure
Region), figure showing, dead cell quantity is few, illustrating to carry out cell printing by the way of microsphere load cells can be effective
Reduce the damage that cell is caused by the mechanically and thermally environment in print procedure, it is ensured that cell survival rate.
Embodiment 2
A kind of method preparing load cells three-dimensional rack based on cell printing export technique, comprises the following steps:
The first step, prepares load cells microsphere, by 2.5 wt % of mesenchymal stem cells MSCs Yu high temperature discontinuous sterilization
Sodium alginate aqueous solution mix homogeneously, in mixed liquor, chondrocyte concentration is 5 × 105/ mL, according to pressure 150 kPa, shower nozzle is straight
Mixed liquor is ejected into the cross-linking agent CaCl that concentration is 5 wt% by the injection conditions of footpath 500 μm2In solution, stand 10 min, absorb
CaCl2Solution, rinses 3 times by PBS solution, and DMEN cell culture fluid rinses 3 times, obtains load cells microsphere, standby.
Second step, prepares host material, and compound concentration is the recombinant collagen solution of 3 wt% and concentration is 5 wt%'s respectively
Sodium alginate soln, 1:3 in mass ratio mixes, high temperature discontinuous sterilization.Configure the CaCl of 2 wt %2Solution, by recombinant collagen-sea
Sodium alginate mixed liquor and CaCl2Solution 7:3 mix homogeneously by volume, standby.
3rd step, prepared by cell blend, load cells microsphere and host material are mixed by medical three-way valve
To cell blend, in blend, the concentration of load cells microsphere is 2.5 × 105/mL。
4th step, prints load cells three-dimensional rack, uses biometric print machine, takes 2 mL cell blends addition volumes and is
In the barrel of 5 mL, use the manufacturing process of step motor drive plunger extrusion, material extrusion is being loaded with sterilizing glass film plates
Three-dimensional micromotion platform on, under the cad model with synusia information directly drives, form different aspects, different aspects
Between interlock the most layer by layer superposition bonding, form 10 mm × 10 mm × 3mm size three-dimensional structure, obtain load cells three-dimensional
Support.
5th step, by load cells three-dimensional rack with aseptic 2 wt % CaCl2Solution soaking, cross-links 5 min.
6th step, rinses load cells three-dimensional rack 3 times by PBS solution, after DMEM culture fluid rinses 3 times, puts six orifice plates
Middle routine in vitro is cultivated, and changes liquid routine observation every other day.
Above example is only several in the several preferred implementation of the present invention, it is noted that on the invention is not restricted to
State embodiment;For the person of ordinary skill of the art, still the technical scheme described in previous embodiment can be entered
Row amendment, or wherein portion of techniques feature is carried out equivalent;And these amendments or replacement, do not make appropriate technical solution
Essence depart from claimed technical solution of the invention spirit and scope.
Claims (9)
1. the preparation method of a load cells three-dimensional rack, it is characterised in that by autogenous cell and high temperature discontinuous sterilization
Hydrogel solution mix homogeneously obtains mixed liquor, is ejected in cross-linking agent solution, stands, then removes cross-linking agent solution, rinsing
After obtain load cells microsphere;Load cells microsphere and host material are mixed to get cell blend;Biology is used to beat
Cell blend extrusion on three-dimensional micromotion platform, is layering by print machine, forms load cells three-dimensional rack;Described substrate material
The preparation method of material is: preparation recombinant collagen solution and sodium alginate soln respectively, and 1:1-5 in mass ratio mixes, high temperature interval
Sterilizing, the concentration of described recombinant collagen solution is 2-10 wt %, and the concentration of described sodium alginate soln is 2-10 wt %;Configuration
Concentration is the CaCl of 0.05-2 wt %2Solution, by recombinant collagen-sodium alginate mixed liquor and CaCl2Solution 7:3 by volume
Mix homogeneously, obtains host material.
The preparation method of a kind of load cells three-dimensional rack the most according to claim 1, it is characterised in that described water-setting
Sol solution be hydrogel monomer and hydrogel solvent mix homogeneously formulated, concentration is 0.05-10 wt%;Described hydrogel list
Body is the mixture of one or more in collagen, gelatin, hyaluronic acid, chitosan or Na-alginate;Described hydrogel solvent
It is the phosphate buffered solution of 5.7-8.0 for water or pH value.
The preparation method of a kind of load cells three-dimensional rack the most according to claim 1, it is characterised in that described mixed liquor
The concentration of middle autogenous cell is 105-107/mL。
The preparation method of a kind of load cells three-dimensional rack the most according to claim 1, it is characterised in that described injection
Pressure be 10-200 kPa, jet diameters is 500-1000 μm.
The preparation method of a kind of load cells three-dimensional rack the most according to claim 1, it is characterised in that described crosslinking
Agent solution is CaCl2Solution, concentration is 0.1-20 wt%.
The preparation method of a kind of load cells three-dimensional rack the most according to claim 1, it is characterised in that described is autologous
Cell is the one or more combination in mesenchymal stem cells MSCs, fat stem cell, chondrocyte, osteoblast.
The preparation method of a kind of load cells three-dimensional rack the most according to claim 1, it is characterised in that described rinsing is
First rinse 3 times by PBS solution, then use DMEM culture fluid to rinse 3 times.
The preparation method of a kind of load cells three-dimensional rack the most according to claim 1, it is characterised in that described cell is altogether
In mixed thing, the concentration of load cells microsphere is 105-107/mL。
The load cells three-dimensional rack that method the most according to claim 1 prepares is for preparing tissue engineering bracket.
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CN108079384B (en) * | 2015-04-07 | 2021-07-06 | 四川蓝光英诺生物科技股份有限公司 | Biological brick containing endothelial cells and application thereof |
CN105012060B (en) * | 2015-07-08 | 2017-03-15 | 上海大学 | The method for preparing three-dimensional multi-scale vascularizing scaffold |
CN105056302B (en) * | 2015-08-26 | 2017-12-26 | 上海市肺科医院 | A kind of preparation method and applications of biological combined artificial tracheae |
CN106474558A (en) * | 2016-11-10 | 2017-03-08 | 广东泰宝医疗科技股份有限公司 | A kind of cartilage repair material with biologically active and preparation method thereof |
CN107029294A (en) * | 2017-03-21 | 2017-08-11 | 湖南未名三胞转化医学科技有限公司 | The method that a kind of the human adipose mesenchymal stem cells blend printed for three dimensional biological and three dimensional biological print body bone tissue |
CN108340569B (en) * | 2018-01-04 | 2019-11-08 | 艾伯尔三氐打印技术(重庆)有限公司 | A kind of 3D printing method of three-dimensional cell hydrogel composite construction |
CN108144114A (en) * | 2018-01-05 | 2018-06-12 | 浙江省医学科学院 | For the 3D printing material of organizational project and the preparation method of Biodegradable scaffold material |
CN108159492A (en) * | 2018-03-27 | 2018-06-15 | 山东隽秀生物科技股份有限公司 | A kind of tissue engineering artificial ligament and preparation method thereof |
CN108795687B (en) * | 2018-05-10 | 2021-10-26 | 太原理工大学 | 3D bioprinter of detectable cell activity |
CN110772669A (en) * | 2019-11-04 | 2020-02-11 | 中南大学湘雅三医院 | Biological ink for 3D printing of artificial skin |
CN112140538A (en) * | 2020-09-15 | 2020-12-29 | 广州仁麦生物科技有限公司 | Biological 3D printing apparatus |
CN113274554A (en) * | 2021-05-14 | 2021-08-20 | 清华大学 | Gel microsphere-based 3D printing biological ink and application thereof |
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CN103120806B (en) * | 2013-01-16 | 2015-04-15 | 西北工业大学 | Preparation method of cartilage framework based on PVA (Polyvinyl Acetate) hydrogel |
CN103893818B (en) * | 2014-03-14 | 2015-07-01 | 华南理工大学 | Osteochondral three-dimensional stent with regular interpenetrating network structure and preparation method thereof |
CN104147641B (en) * | 2014-07-11 | 2016-08-31 | 深圳职业技术学院 | A kind of for personalized bone renovating material and its preparation method |
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