CN104548208A - Preparation method and application of cell-loading three-dimensional support - Google Patents
Preparation method and application of cell-loading three-dimensional support Download PDFInfo
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- CN104548208A CN104548208A CN201510045823.0A CN201510045823A CN104548208A CN 104548208 A CN104548208 A CN 104548208A CN 201510045823 A CN201510045823 A CN 201510045823A CN 104548208 A CN104548208 A CN 104548208A
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Abstract
The invention provides a preparation method of a cell-loading three-dimensional support. The preparation method of the cell-loading three-dimensional support is characterized by comprising the following steps: uniformly mixing autologous cells with a high temperature intermittently sterilized hydrogel solution to obtain mixed liquor, injecting the mixed liquor into a crosslinking agent solution, standing, then removing the crosslinking agent solution, and rinsing, so that cell-loading microspheres are obtained; mixing the cell-loading microspheres with a matrix material to obtain cell blend; and extruding the cell blend onto a three-dimensional micro-positioning platform carrying a sterile glass base plate by adopting a biological printer, and overlaying layer by layer, so that the cell-loading three-dimensional support is formed. The preparation method of the cell-loading three-dimensional support has the advantages that damage on cellular activity and bioactivity in a cell printing process is reduced, and high-activity cell printing is guaranteed.
Description
Technical field
The invention belongs to cell printing and technical field of biological materials, be specially adapted to the preparation of biomedical tissue engineering scaffold material, be specially 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.Cell three-dimensional printing technique that is theoretical and organizational project theory is manufactured, for new theory and technology space has been expanded in tissue engineering, pathological model structure, drug screening and the field such as detection, RESEARCH ON CELL-BIOLOGY based on advanced person.
Compared with traditional Method of Tissue Engineering, the advantage of cell printing mainly contains: (1) is built with bioactive two dimension or three-dimensional " cell/material " system simultaneously; (2) the different types of cell of accurate deposition over time and space; (3) three-dimensional microenvironment needed for cell is built.Cell printing export technique mainly comprises inkjet printing (piezoelectricity volume drive-type and hot bubble type), laser direct-writing, induced with laser shifts, electrostatic spraying, concentration ultrasonic sprays, micro-ly to extrude, the feasibility that conventional cell prints and reliability aspect are verified all, but how keeping also having limitation in cytoactive: during printing, most of mammalian cell is comparatively fragile, be vulnerable to the impact of environment, and in print procedure the maximum temperature of nozzle more than 300 DEG C, and there is larger shear stress, instantaneous high-temperature is produced in cell printing process, high pressure or instantaneously strong electrostatic field cause injury to cell.Therefore preparation there is appropriate bore gap structure and thickness is greater than the load cells three-dimensional rack of 300 μm time, in print procedure, keep cytoactive, reducing the adverse effect of its biological characteristics is problem demanding prompt solution.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of method preparing load cells three-dimensional rack based on cell printing export technique is provided, have the advantages that to keep cytoactive.
A preparation method for load cells three-dimensional rack, is mixed homogeneously with the hydrogel solution of high temperature discontinuous sterilization by autogenous cell to obtain mixed liquor, is ejected in cross-linking agent solution, leaves standstill, then removes cross-linking agent solution, obtain load cells microsphere after rinsing; Load cells microsphere and host material are mixed to get cell blend; Adopt biometric print machine to be extruded on three-dimensional micromotion platform by cell blend, be layering, form load cells three-dimensional rack.
Further, described hydrogel solution is that hydrogel monomer and hydrogel solvent mix homogeneously are formulated, and concentration is 0.05-10 wt%; Described hydrogel monomer is the mixture of one or more in collagen, gelatin, hyaluronic acid, chitosan or Na-alginate; 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 10
5-10
7/ 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 CaCl
2solution, concentration is 0.1-20 wt%.
Further, described autogenous cell is the one or more combination in mesenchymal stem cells MSCs, fat stem cell, chondrocyte, osteoblast.
Further, described rinsing first uses PBS solution rinsing 3 times, then uses DMEM culture fluid rinsing 3 times.
Further, the preparation method of described host material is: prepare recombinant collagen solution and sodium alginate soln respectively, 1:1-5 mixing in mass ratio, 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 CaCl
2solution is 7:3 mix homogeneously by volume, obtains host material.
Further, in described cell blend, the concentration of load cells microsphere is 10
5-10
7/ mL.
Described load cells three-dimensional rack is for the preparation of tissue engineering bracket.
Compared with prior art, advantage of the present invention and good effect are: the present invention wraps up cell with the similar hydrogel material of extracellular matrix components by adopting, and effectively can reduce the damage that machinery in print procedure and thermal environment cause cell.The present invention is by being ejected into CaCl by cell and hydrogel mixed solution
2method in solution, prepares the microsphere of load cells, and the parcel of hydrogel material can form protection to cell, alleviates the infringement that cell printing process causes cytoactive and biological activity, ensures highly active cell printing.The present invention's seed cell used is autogenous cell, without immunologic rejection.The present invention can according to the different needs of tissue or organ, and adjustment print parameters, directly can prepare the three-dimensional rack with various structure (porous, tubulose) and mechanical property, study for organizational project and regenerative medicine.The inventive method is simple, and be easy to operation, cost is low, and the three-dimensionally shaped support with complicated form of energy, can meet clinical needs, be suitable for the preparation of engineering rack.
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 load cells microsphere of the present invention;
Fig. 3. load cells three dimensional scaffold structure figure of the present invention;
Fig. 4. in the present invention, support cytoactive described in embodiment 1 detects coloration result.
Detailed description of the invention
Below in conjunction with detailed description of the invention, technical scheme of the present invention is described in further detail.
A kind of load cells three-dimensional rack, be utilize own cells to prepare to contain the microsphere supported of cell, be mixed to get cell blend with host material, utilize cell printing export technique to be layering by cell blend, form load cells three-dimensional rack, concrete steps are as follows:
1) load cells microsphere is prepared: mixed homogeneously with the hydrogel solution of high temperature discontinuous sterilization by autogenous cell and obtain mixed liquor, in described mixed liquor, autogenous cell concentration is 10
5-10
7/ mL; Be ejected in cross-linking agent solution according to the injection conditions of pressure 10-200 kPa, jet diameters 500-1000 μm by mixed liquor, the microlayer model containing cell ejected is cross-linked in cross-linking agent, and form load cells microsphere, described cross-linking agent solution is CaCl
2solution, concentration is 0.1-20 wt%, leave standstill 2-60 min, cross-linking agent solution is absorbed with pipettor, then PBS buffer solution rinsing 3 times are used, use DMEN (Dulbecco's Modified Eagle Media) cell culture fluid rinsing 3 times, obtain load cells microsphere, as shown in Figure 2.
Described autogenous cell can be the one or more combination in mesenchymal stem cells MSCs, fat stem cell, chondrocyte, osteoblast.
Described hydrogel sol is that hydrogel monomer and hydrogel solvent mix homogeneously are mixed with, and mass concentration is 0.05-10 wt%; Described hydrogel monomer is the mixture of one or more in collagen, gelatin, hyaluronic acid, chitosan or Na-alginate; Phosphate (PBS) buffer solution of described hydrogel solvent to be water or pH value be 5.7-8.0.The parcel of hydrogel material can form protection to cell, alleviates the infringement that cell printing process causes cytoactive and biological activity, ensures highly active cell printing.
2) prepare host material: prepare recombinant collagen solution and sodium alginate soln respectively, 1:1-5 mixing in mass ratio, 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 CaCl
2solution is 7:3 mix homogeneously by volume, obtains host material.
Described recombinant collagen is recombined collagen, also referred to as recombination human collagen, has the advantage of machinability, virus-free hidden danger, low rejection, deformation temperature raising.
Described host material, as cell carrier, has three dimensional structure and the constituent of natural extracellular matrix, to realize identical physiological function.Desirable host material has avirulence, has good biocompatibility, certain formability.The current host material for cell assembling, based on natural biologic material, comprises gelatin, fibrin, sodium alginate, chitosan etc.These natural materials are from organism, similar to the composition of extracellular matrix, strong with cellular affinity, and the factor containing the vital movement such as Promote cell's growth, propagation.Based on the feature of the analysis required for host material and natural high molecular substance, the present invention have selected there is good biocompatibility, degradability and cellular affinity recombination human collagen and sodium alginate as host material.
3) cell blend preparation: by medical three-way valve by load cells microsphere and host material mixing, wherein the concentration of load cells microsphere is 10
5-10
7/ mL.
4) load cells three-dimensional rack is printed: adopt biometric print machine to be extruded by material on the three-dimensional micromotion platform being loaded with sterilizing glass film plates, under the cad model Direct driver with synusia information, form different aspects, staggered superposition layer by layer again between different aspects, form three-dimensional structure, with aseptic 1-20 wt % CaCl
2solution crosslinking 2-20 min, obtained load cells three-dimensional rack.
5) the 5th step, support is cross-linked, by the aseptic 2 wt % CaCl of load cells three-dimensional rack
2solution soaking, crosslinked 2 min.
6) with PBS solution rinsing load cells three-dimensional rack 3 times, after DMEM culture fluid rinsing 3 times, put routine in vitro in six orifice plates and cultivate, change liquid every other day and routine observation.
The load cells three-dimensional rack utilizing the inventive method to prepare is suitable for the preparation of tissue engineering bracket.
Embodiment 1
Prepare a method for load cells three-dimensional rack based on cell printing export technique, comprise the following steps:
The first step, prepares load cells microsphere, and mixed homogeneously with 1 wt % sodium alginate aqueous solution of high temperature discontinuous sterilization by chondrocyte, in mixed liquor, chondrocyte concentration is 2.2 × 10
5/ mL, according to pressure 120 kPa, mixed liquor is ejected into the cross-linking agent CaCl that concentration is 2 wt% by the injection conditions that jet diameters is 730 μm
2in solution, leave standstill 10min, absorb CaCl
2solution, with PBS solution rinsing 3 times, DMEN cell culture fluid rinsing 3 times, obtains load cells microsphere, for subsequent use.
Second step, prepares host material, and compound concentration is the recombinant collagen solution of 7wt% and concentration is respectively the sodium alginate soln of 7wt%, 1:1 mixing in mass ratio, high temperature discontinuous sterilization.Configure the CaCl of 0.5 wt %
2solution, by recombinant collagen-sodium alginate mixed liquor and CaCl
2solution is 7:3 mix homogeneously by volume, for subsequent use.
3rd step, prepared by cell blend, by medical three-way valve, load cells microsphere and host material are mixed to get cell blend, in blend, the concentration of load cells microsphere is 1.2 × 10
5/ mL.
4th step, print load cells three-dimensional rack, adopt biometric print machine, getting 2 mL cell blends adds in the barrel that volume is 5 mL, adopt the manufacturing process that step motor drive plunger is extruded, material is extruded on the three-dimensional micromotion platform being loaded with sterilizing glass film plates, under the cad model Direct driver with synusia information, form different aspects, between different aspects, staggered superposition bonds layer by layer again, and the three-dimensional structure (as shown in Figure 3) forming 15 mm × 2, mm × 15 mm sizes obtains load cells three-dimensional rack.
5th step, by the aseptic 2 wt % CaCl of above-mentioned load cells three-dimensional rack
2solution soaking, crosslinked 2 min.
6th step, with PBS solution rinsing load cells three-dimensional rack 3 times, after DMEM culture fluid rinsing 3 times, puts routine in vitro in six orifice plates and cultivates, change liquid every other day and routine observation.
7th step, in load cells three-dimensional rack, cytoactive detects, and carries out testing by operating instruction with work-dead cell stain test kit (Invitrogen).Concrete steps are: get the load cells three-dimensional rack PBS solution rinsing of In vitro culture after 3 days 3 times, contain 2 μMs of calcein-AM(Calcein-AM at 3 mL) and 4 μMs of ethidium bromides (EthD-1) dyeing liquor in incubated at room 30min, PBS solution rinsing 3 times, fluorescence microscope record coloration result, as shown in Figure 4, in Fig. 4, a figure is rack surface testing result, b figure is support chi structure place testing result (i.e. the testing result of three-dimensional rack inside), wherein most of region in green fluorescent label living cells 1(figure), circles mark region in red fluorescence labelling dead cell 2(figure), dead cell quantity is shown few in figure, illustrate that carrying out cell printing by the mode of microsphere load cells effectively can reduce the damage that machinery in print procedure and thermal environment cause cell, ensure cell survival rate.
Embodiment 2
Prepare a method for load cells three-dimensional rack based on cell printing export technique, comprise the following steps:
The first step, prepares load cells microsphere, and mixed homogeneously with 2.5 wt % sodium alginate aqueous solution of high temperature discontinuous sterilization by mesenchymal stem cells MSCs, in mixed liquor, chondrocyte concentration is 5 × 10
5/ mL, according to pressure 150 kPa, mixed liquor is ejected into the cross-linking agent CaCl that concentration is 5 wt% by the injection conditions that jet diameters is 500 μm
2in solution, leave standstill 10 min, absorb CaCl
2solution, with PBS solution rinsing 3 times, DMEN cell culture fluid rinsing 3 times, obtains load cells microsphere, for subsequent use.
Second step, prepares host material, respectively the sodium alginate soln of compound concentration to be the recombinant collagen solution of 3 wt% and concentration be 5 wt%, 1:3 mixing in mass ratio, high temperature discontinuous sterilization.Configure the CaCl of 2 wt %
2solution, by recombinant collagen-sodium alginate mixed liquor and CaCl
2solution is 7:3 mix homogeneously by volume, for subsequent use.
3rd step, prepared by cell blend, by medical three-way valve, load cells microsphere and host material are mixed to get cell blend, in blend, the concentration of load cells microsphere is 2.5 × 10
5/ mL.
4th step, print load cells three-dimensional rack, adopt biometric print machine, getting 2 mL cell blends adds in the barrel that volume is 5 mL, adopt the manufacturing process that step motor drive plunger is extruded, material is extruded on the three-dimensional micromotion platform being loaded with sterilizing glass film plates, under the cad model Direct driver with synusia information, form different aspects, between different aspects, staggered superposition bonds layer by layer again, form 10 mm × 10 mm × 3mm size three-dimensional structures, obtain load cells three-dimensional rack.
5th step, by the aseptic 2 wt % CaCl of load cells three-dimensional rack
2solution soaking, crosslinked 5 min.
6th step, with PBS solution rinsing load cells three-dimensional rack 3 times, after DMEM culture fluid rinsing 3 times, puts routine in vitro in six orifice plates and cultivates, change liquid every other day and routine observation.
Above embodiment is only several in the several preferred implementation of the present invention, it should be pointed out that and the invention is not restricted to above-described embodiment; For the person of ordinary skill of the art, still the technical scheme described in previous embodiment can be modified, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (10)
1. a preparation method for load cells three-dimensional rack, is characterized in that, is to be mixed homogeneously with the hydrogel solution of high temperature discontinuous sterilization by autogenous cell to obtain mixed liquor, be ejected in cross-linking agent solution, leave standstill, then remove cross-linking agent solution, after rinsing, obtain load cells microsphere; Load cells microsphere and host material are mixed to get cell blend; Adopt biometric print machine to be extruded on three-dimensional micromotion platform by cell blend, be layering, form load cells three-dimensional rack.
2. the preparation method of a kind of load cells three-dimensional rack according to claim 1, is characterized in that, described hydrogel solution is that hydrogel monomer and hydrogel solvent mix homogeneously are formulated, and concentration is 0.05-10 wt%; Described hydrogel monomer is the mixture of one or more in collagen, gelatin, hyaluronic acid, chitosan or Na-alginate; Described hydrogel solvent is water or pH value is the phosphate buffered solution of 5.7-8.0.
3. the preparation method of a kind of load cells three-dimensional rack according to claim 1, is characterized in that, in described mixed liquor, the concentration of autogenous cell is 10
5-10
7/ mL.
4. the preparation method of a kind of load cells three-dimensional rack according to claim 1, is characterized in that, the pressure of described injection is 10-200 kPa, and jet diameters is 500-1000 μm.
5. the preparation method of a kind of load cells three-dimensional rack according to claim 1, is characterized in that, described cross-linking agent solution is CaCl
2solution, concentration is 0.1-20 wt%.
6. the preparation method of a kind of load cells three-dimensional rack according to claim 1, is characterized in that, described autogenous cell is the one or more combination in mesenchymal stem cells MSCs, fat stem cell, chondrocyte, osteoblast.
7. the preparation method of a kind of load cells three-dimensional rack according to claim 1, is characterized in that, described rinsing first uses PBS solution rinsing 3 times, then uses DMEM culture fluid rinsing 3 times.
8. the preparation method of a kind of load cells three-dimensional rack according to claim 1, it is characterized in that, the preparation method of described host material is: prepare recombinant collagen solution and sodium alginate soln respectively, 1:1-5 mixing in mass ratio, 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 CaCl
2solution is 7:3 mix homogeneously by volume, obtains host material.
9. the preparation method of a kind of load cells three-dimensional rack according to claim 1, is characterized in that, in described cell blend, the concentration of load cells microsphere is 10
5-10
7/ mL.
10. the load cells three-dimensional rack for preparing of method according to claim 1 is for the preparation of tissue engineering bracket.
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| CN201510045823.0A CN104548208B (en) | 2015-01-29 | 2015-01-29 | A kind of preparation method and application of load cells three-dimensional rack |
| AU2015101219A AU2015101219A4 (en) | 2015-01-29 | 2015-09-03 | Method for preparing cell-laden 3D scaffolds and application |
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Cited By (12)
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| CN105012060A (en) * | 2015-07-08 | 2015-11-04 | 上海大学 | Method for preparing three-dimensional multi-dimensioned vascularization support |
| CN105056302A (en) * | 2015-08-26 | 2015-11-18 | 上海市肺科医院 | Method for preparing biological composite artificial trachea and application thereof |
| 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 |
| CN108079384A (en) * | 2015-04-07 | 2018-05-29 | 四川蓝光英诺生物科技股份有限公司 | A kind of biological brick comprising endothelial cell and application thereof |
| 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 |
| CN108340569A (en) * | 2018-01-04 | 2018-07-31 | 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 |
| CN108795687A (en) * | 2018-05-10 | 2018-11-13 | 太原理工大学 | A kind of 3D biometric print machines 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 |
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| 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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| CN108079384A (en) * | 2015-04-07 | 2018-05-29 | 四川蓝光英诺生物科技股份有限公司 | A kind of biological brick comprising endothelial cell 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 |
| CN105012060A (en) * | 2015-07-08 | 2015-11-04 | 上海大学 | Method for preparing three-dimensional multi-dimensioned vascularization support |
| CN105056302A (en) * | 2015-08-26 | 2015-11-18 | 上海市肺科医院 | Method for preparing biological composite artificial trachea and application thereof |
| 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 |
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| CN108340569A (en) * | 2018-01-04 | 2018-07-31 | 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 |
| CN108795687A (en) * | 2018-05-10 | 2018-11-13 | 太原理工大学 | A kind of 3D biometric print machines of detectable cell activity |
| 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 |
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| CN113274554A (en) * | 2021-05-14 | 2021-08-20 | 清华大学 | Gel microsphere-based 3D printing biological ink and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2015101219A4 (en) | 2015-10-08 |
| CN104548208B (en) | 2016-10-26 |
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