CN103877613B - The system and method for injection-type three-dimensional cell microenvironment is built based on micro-ice glue - Google Patents
The system and method for injection-type three-dimensional cell microenvironment is built based on micro-ice glue Download PDFInfo
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- CN103877613B CN103877613B CN201210553882.5A CN201210553882A CN103877613B CN 103877613 B CN103877613 B CN 103877613B CN 201210553882 A CN201210553882 A CN 201210553882A CN 103877613 B CN103877613 B CN 103877613B
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Abstract
The invention discloses a kind of system and method building injection-type three-dimensional cell microenvironment based on micro-ice glue.This micro-ice glue three-dimensional microenvironment carrier, the material forming this carrier is ice glue, and this carrier size belongs to micron order.Experiment proves, micro-ice glue three-dimensional microenvironment carrier that the present invention obtains has following characteristics: good mechanical property, can also keep intact form after injection; Energy efficient loading cell; Compared with existing large scale ice glue, micro-ice glue three-dimensional microenvironment carrier evenly can be drawn cell and have good mass transfer; In injection process, micro-ice glue three-dimensional microenvironment carrier can have protective effect to the cell loaded; Be mounted with cell micro-ice glue can locating injection in animal body, and cell still keeps original function and activity in vivo.Gained of the present invention micro-ice glue three-dimensional microenvironment carrier will play a significant role in cell infusion treatment field, all will have broad application prospects at Animal Model and drug screening field.
Description
Technical field
The invention provides a kind of system and method building injection-type three-dimensional cell microenvironment based on micro-ice glue.
Background technology
Take cell therapy as the important directions that the regenerative medicine of representative becomes development of clinical medicine gradually, the complicated major disease being difficult to take effect for reply Drug therapy brings new hope.Cell therapy utilizes the adult cell of autologous patient (or allosome) or stem cell to tissue, and organ carries out the Therapeutic Method repaired.Utilize the existing a large amount of report of the research of the pertinacious diseases such as cell therapy cardiovascular disease, diabetes, parkinson disease, keratopathy, cartilage tissue damage and tumor at present, and achieve Preliminary Results in clinical practice.Research and apply particularly based on stem-cell therapy achieves multinomial breakthrough.Stem cell is the pluripotent cell that a class has the of self-replication capacity, through the induction of felicity condition, can be divided into the cell of several functions, such as neurocyte, myocardial cell, vascular endothelial cell, nephrocyte and hepatocyte etc.The pluripotency characteristic utilizing stem cell to have to change to various cell differentiation, treats tissue degeneratiaon, damage, necrosis disease clinically, thus reaches tissue regeneration, reproduce, the objects such as replacement, achieve the regenerative medicine theory based on stem-cell therapy technology.
At present, the clinical technology for cell therapy continues to bring out, and mainly comprises following several diverse ways:
1, directly by allosome or autologous isolated there is biological function characteristic adult cell or stem cell direct injection in damaged tissues place, be more conventional effective clinical cytology treatment means.The method is easy and simple to handle, but after injection, lower cell survival rate and cell enter the uncontrollability in body, seriously govern based on the application clinically of direct injection stem-cell therapy.Research shows, adopt direct injection to carry out stem-cell therapy, after injection, cell survival rate in vivo only has 50%-60%, and long-term observation finds, the transplanting stem cell being only less than 3% plays tissue repair effect at damaged part, and dead and free loss phenomenon appears in all the other most of transplanted cells.The reason that direct injection causes transplanting stem cell to enter mortality and loss after in body has four kinds: (1) stem cell is in injection transplantation process, due to the impact of mechanical shear stress in syringe, breaking appears in part cell, and cell enters the apoptosis phase, reduces the survival ability of cell.(2) external free stem cell to enter in body in damaged tissue region, microenvironment in this process residing for cell changes, and the microenvironment of focal area is unfavorable for cell survival and growth, stem cell can not adapt to residing focus microenvironment, thus reduces the survival rate of cell.(3) direct injection stem cell is in body, different due to therapentic part and injection point, partial syringe position is because the reasons such as pressure difference inside and outside mechanical movement or tissue site are (as disease model given prominence to by vertebra shroud, cartilage tissue damage model, the models such as cardiopulmonary repairing transplant), cause the cell being injected into target tissue can overflow from pin hole along with extracting of syringe, cause a large amount of cells to lose (4) free stem cell transplantation to enter after in body, cell can dissociate to organizing surrounding non-directional along with body fluid, cannot initiatively assemble, and then cause a large amount of transplanted cells to run off, the fixed point treatment of cell cannot be carried out.
2, in the face of the great number of issues that directly transplanting cell produces, based on the structure of biomaterial accessory cell implant system, for the histoorgan recovery technique in regenerative medicine field brings new breakthrough.The technology that this biomaterial accessory cell is transplanted, tissue engineering technique can be used on the one hand, in conjunction with the characteristic of seed cell, somatomedin and biomaterial, carry out histoorgan cultivation in vitro, the In vitro culture organ of maturation is replaced damaged tissues, thus maintains, recover even to promote the function of damaged tissues.(DJ Mooney et al.PNAS 2008) devises a kind of stem cell transplantation support, has modified VEGF (VEGF) and endothelial progenitor cells (EPC) is sticked to internal stent at internal stent.Through In vitro culture after a while, the timbering material being combined with somatomedin and functional stem cell is transplanted to ischemic tissue inside, thus carries out reparation and the regeneration of blood vessel, effect is very remarkable.But the immunologic rejection of large volume allosome implant material, the problems such as the healing of the traumatic and postoperative wound of operation process, govern the paces that this technology is advanced to clinical practice.
3, the common issue run in operation transplantation process, design is a kind of non-invasive, and reduce rejection, stem-cell therapy method easy and simple to handle becomes the focus that researcheres are paid close attention to.To have the cell delivery of certain functional characteristic in body by the noinvasive such as injection or invasive manner, the regenerative medicine treatment concept realizing the reparation of damaged tissues organ demonstrates its advantage and good application prospect gradually.(Byung-Soo Kim et al.Biomaterials 2011) is for avoiding operation wound to affect, utilize bioreactor, prepare the cell microsphere formed by adipose-derived stem cell (hADSCs) gathering of a large amount of people, by the cell microsphere prepared by injector to inject to mice lower limb ischemia position, there is a large amount of new vesselses in lower limb ischemia position, therapeutic effect is obvious.But cell microsphere form size heterogeneity prepared by the method, plasticity is poor, still cannot avoid in injection process to the damage of cells of superficial layer.(PX Maetal.Nat Mater 2011) devises a kind of cell carrier of hollow self assembling type injectable type, by injecting method by stem cell transplantation in damaged tissues, carry out tissue repair and regeneration.This design concept; what both solved that operation transplantation brings has wound property and the problem such as postoperative wound healing is difficult; achieve again the method for injectable type cell therapy; and due to the existence of cell carrier; thus supportive protection effect is provided to stem cell in injection process; improve the transplanting survival rate of cell, repair of damaged tissues effect is comparatively obvious.But this self assembly carrier is comparatively complicated on synthesis makes, the a large amount of cell carrier morphological homogeneity made are poor, the ability of the autonomous adherent cell of carrier is more weak, cell all can not enter into carrier inside and be partly adhered to carrier surface, thus affects the efficiency of cell delivery and treatment.(K.L.Fujimoto et al.Biomaterials 2009) utilizes a kind of degradable temperature-sensitive hydrogel as cell transmission carrier, when temperature is lower, cell suspension is in the hydrogel solution of liquid state, by injection means fixed-point injection behind damaged tissue region, the hydrogel solution being suspended with cell forms hydrogel when being in temperature environment in body immediately, and gel is in injection areas, prevent the loss of cell, thus reach the effect of cell fixed point treatment.But in this design, the three dimension system of hydrogel also exists the problems such as mass transfer on the one hand, affect the survive of cell, the method cannot avoid injection machinery power to the damage of cell on the other hand, reduce the survival rate of cell, and in injection process, cell will participate in the process of plastic, also can cause certain infringement to cell, thus have a strong impact on the clinical therapeutic efficacy of cell transplantation.
The development of medical treatment, plumbing industry needs efficient disease treatment scheme, and for realizing the simple and easy to do for the treatment of technology, improve therapeutic efficiency, reduce patient pain, Noninvasive therapy technology has advantageous advantage.Noninvasive therapy technology (injectable type cell carrier treatment technology) avoids the problems such as the traumatic of cell operation transplantation and postoperative wound healing, for patient decrease huge at heart and physiological pain, for medical worker provides Therapeutic Method very simply and easily, reduce the risk of operative treatment, achieve and more optimize, convenient, painless, the cell clinic application of safety.
Along with biomedicine, materialogy, mechanics, the fast development of the cross disciplines such as engineering, increasing people pays close attention in microenvironment and body that cell cultivates in vitro the difference of microenvironment of surviving.Traditional bidimensional cell culture (based on business-like culture dish or porous plate) technical development has the history of over one hundred year, has extensive use in fields such as life sciences basic research, medical researches.But along with the fast development of micro-imaging technique, researcher finds the survival condition of cell under the culture environment of bidimensional gradually, far apart under the condition of culture of cellular morphology and its true environment in vivo.Therefore the bidimensional microenvironment of this simplification well can not be simulated and reappear the three-dimensional microenvironment in body.And the stem cell cultivated under depending on conventional two-dimensional environment, in cellular replacement therapy process, due to the great change of inside and outside living environment, result in the reduction of stem cell survival and function, reparation is gone down with regeneration function, thus constrains the effect of stem-cell therapy in clinical practice.Research shows, cultivates compared to the bidimensional of cell, and the simulation of external three-dimensional microenvironment is conducive to increment and the differentiation of cell, keeps cell dryness, makes the cell under the cell of In vitro culture and internal milieu state more close.Therefore Three-dimensional cell culture technology is receiving much concern in recent years, and obtains and significantly develop.
Build the dimensional culture model of cells in vitro, biomaterial used due to needs in vivo with tissue directly contact, therefore certain particular/special requirement must be possessed: as the source (natural or synthetic material) of matrix of materials, the physical and chemical performance (chemical compatibility, mechanical performance, degradability, architectural characteristic etc.) of matrix of materials, the biological activity (attachment sites, inducement signal etc.) of matrix of materials, equipment needed for dimensional culture technology, service condition, the scope of application, the suction type, adherent fashion, training method, detection mode etc. of cell.Desirable Three-dimensional cell culture system can realize imaging in cell seeding, cultivation, propagation, succeeding generations and follow-up with qualitative on than traditional bidimensional training method more close to interior state, and simple and easy to do equally with conventional two-dimensional cell culture mode in operational means.
In conjunction with biology, medical science, engineering, the multi-crossed disciplines knowledge such as materialogy, select optimum biomaterial, design cell carrier processing technology the most simple and efficient, thus reach extensive simple and easy manufacture, ensure that morphological homogeneity and the absorbability of cell carrier are particularly important, simultaneously also for advancing the clinical practice of cell therapy to establish solid foundation.
In sum, in the face of the complexity of cell therapy clinical practice, challenge and all difficulties of running into, need a kind of processing technology of design badly easy, the cell three-dimensional microenvironment carrier that can be mass-produced, operational approach and the condition (as the syringe of clinical practice and the standard of injection volume) that need not change conventional cell treatment can conveniently realize.
Summary of the invention
An object of the present invention is to provide a kind of micro-ice glue three-dimensional microenvironment carrier and preparation method thereof and application.
Technical scheme of the present invention is described below respectively:
One, the structure of micro-ice glue three-dimensional microenvironment carrier and composition.
Micro-ice glue three-dimensional microenvironment carrier provided by the present invention is as follows: the material forming this carrier is ice glue (cryogel), and this carrier size belongs to micron order.
Described micron order is 1 μm-999 μm, is specially 50 μm-999 μm, then is specially 100 μm-800 μm.
Above-mentioned ice glue generally all has several holes, and is be communicated with between hole.
The aperture of above-mentioned ice glue mesopore can be 1 μm-500 μm, and pitch of holes can be 1 μm-999 μm.The present invention's object lesson, pore size 10 μm-150 μm, pitch of holes 1 μm-999 μm, porosity is 94.2%.
Above-mentioned ice glue can be prepared as follows: by the material being used for ice making glue at low ambient temperatures slowly plastic form ice crystal pore simultaneously, its plastic process completes jointly with the process forming ice crystal, finally obtains ice glue.Concrete preparation method can comprise step that is crosslinked and adjustment aperture.Described cross-linking method can be following at least one: chemical crosslink technique, physical crosslinking method, radiation cross-linking process, enzyme-catalyzed cross-linking method.The method in described adjustment micro-ice glue aperture can be following at least one: porogen (porogen) filtering method, phase separation method, Emulsion freeze-drying, solvent evaporated method, gas foam method, fiber Method for bonding etc.
Material for the preparation of above-mentioned ice glue is crosslinkable material.Crosslinkable material can be biomaterial and/or the natural biologic material of synthetic; The biomaterial of described synthetic is following at least one: Polyethylene Glycol, polyethyleneglycol derivative, polyethyleneglycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymers, polydimethylsiloxane, condensing model, poly-acid esters, polyamide, polyamino acid, polyacetals, polybutylcyanoacrylate, polyurethanes, polypyrrole, polyester, polymethacrylates, polyethylene, Merlon and polyethylene glycol oxide; Described natural biologic material is following at least one: gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, layer connect albumen and fibronectin.
The cross linking conditions that present inventor gropes alone is reacted more than 1h at-80 DEG C to 0 DEG C by reaction raw materials.
The ice glue that the present inventor gropes alone is prepared as follows and obtains: be crosslinkable material with polyethyleneglycol diacrylate, with Ammonium persulfate. and N, N, N', N '-tetramethyl diethylamine, as cross-linking agent, reacts 20h at-20 DEG C, obtained ice glue.
Example concrete is further, by 5 ~ 50g polyethyleneglycol diacrylate and 0.5g Ammonium persulfate. and 0.025 ~ 0.2g N, N, N', N '-tetramethyl diethylamine carries out collocation preparation.
Preferred example is further again, by 10g polyethyleneglycol diacrylate and 0.5g Ammonium persulfate. and 0.05gN, N, N', N '-tetramethyl diethylamine mixing, at-20 DEG C, react 20h, above-mentioned reaction is carried out in solvent (as DPBS solution), and the final concentration of polyethyleneglycol diacrylate in reaction system is 10g/100ml.
In above-mentioned example, in described polyethyleneglycol diacrylate, Polyethylene Glycol is Macrogol 2000, Macrogol 4000 or polyethylene glycol 6000.
Above-mentioned micro-ice glue three-dimensional microenvironment carrier can be any shape, if cross-sectional area is circle, ellipse, polygon (triangle, square etc.), heart, Herba Galii Bungei shape etc.Specifically can as column type granule, the diameter of its cross-sectional area can be 1-999 μm, 50-999 μm or 100-800 μm, and cylindrical height can be 1-999 μm, specifically can be 300 μm.Those skilled in the art according to actual needs, can select any shape.
Those skilled in the art according to actual needs, can select any size in micron order.
Two, the preparation method of above-mentioned micro-ice glue three-dimensional microenvironment carrier.
The method of the above-mentioned micro-ice glue three-dimensional microenvironment carrier of preparation provided by the present invention, comprise the steps: the material mixing by being used for making ice glue, then mixture is divided into micron order size, the process conditions of recycling ice making glue process, and namely obtain micro-ice glue three-dimensional microenvironment carrier.
Described micron order is 1 μm-999 μm, is specially 50 μm-999 μm, then is specially 100 μm-800 μm.
The above-mentioned method of micron order size that is divided into by mixture is die methods or emulsion process.
Above-mentioned emulsion process carries out emulsifying by with mixture, obtains object size and object shape.
Above-mentioned die methods comprises the steps: the pit-holes described mixture being placed in mould, and described mould structure is the flat board with pit-holes, and the size of described pit-holes is micron order.Described micron order is 1 μm-999 μm, is specially 50 μm-999 μm, then is specially 100 μm-800 μm.
In above-mentioned mould the size of pit-holes and shape all with size and the shape corresponding (or identical) of micro-ice glue three-dimensional microenvironment carrier that will obtain.The size and shape of hole can be designed according to actual needs, controlled to realize shape size.
Pit-holes in above-mentioned mould can be arrayed, and array distribution can according to required arbitrary arrangement, and the spacing between each pit-holes can be arbitrary size, and the height of pit-holes can define as required, so that extensive batch preparation.
The material preparing above-mentioned mould can be existing common used material, also can oneself grope to obtain.What inventor groped alone is adopt PMMA to prepare mould.
The object lesson of a mould of the present invention is a flat board with pit-holes; Pit-holes is arrayed, the array distribution of 45x14, and each pit-holes is column type, and the circle of to be diameter in column type cross section be 100-800um, the center of circle spacing of each circle is 1.5mm, and the height of pit-holes is 300 μm, and plate material is PMMA.
Above-mentioned mould can adopt following micro-processing technology to make: laser ablation (laser cutting); Machine drilling (mechanical drilling); Photolithography (photolithography), micro-contact printing technique (microcontact printing), microfluid patterning technique (microfluidic patterning), laminar flow patterning technique (laminar flow patterning), reticle pattern technology (stencil patterning), imprint lithography techniques (Imprint lithography), fluid photoetching technique (flow lithography) etc.
Material for the preparation of ice glue is crosslinkable material.Crosslinkable biomaterial can be biomaterial and/or the natural biologic material of synthetic; The biomaterial of described synthetic is following at least one: Polyethylene Glycol, polyethyleneglycol derivative, polyethyleneglycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymers, polydimethylsiloxane, condensing model, poly-acid esters, polyamide, polyamino acid, polyacetals, polybutylcyanoacrylate, polyurethanes, polypyrrole, polyester, polymethacrylates, polyethylene, Merlon and polyethylene glycol oxide; Described natural biologic material is following at least one: gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, layer connect albumen and fibronectin.
Ice glue can be prepared as follows: by the material being used for ice making glue at low ambient temperatures slowly plastic form ice crystal pore simultaneously, its plastic process completes jointly with the process forming ice crystal, finally obtains ice glue.Concrete preparation method can comprise step that is crosslinked and adjustment aperture.Described cross-linking method can be following at least one: chemical crosslink technique, physical crosslinking method, radiation cross-linking process, enzyme-catalyzed cross-linking method.The method in described adjustment micro-ice glue aperture can be following at least one: porogen (porogen) filtering method, phase separation method, Emulsion freeze-drying, solvent evaporated method, gas foam method, fiber Method for bonding etc.
The material of what the present inventor groped alone prepare ice glue is: be crosslinkable material with polyethyleneglycol diacrylate, with Ammonium persulfate. and N, N, N', N'-tetramethyl diethylamine as cross-linking agent.Further particularly, by 5 ~ 50g polyethyleneglycol diacrylate and 0.5g Ammonium persulfate. and 0.025 ~ 0.2g N, N, N', N'-tetramethyl diethylamine carries out collocation preparation.Further preferred example is again, 10g polyethyleneglycol diacrylate and 0.5g Ammonium persulfate. and 0.05g N, N, N', N'-tetramethyl diethylamine.Further particularly, above-mentioned material is mixing in solvent (as DPBS solution), and the final concentration of polyethyleneglycol diacrylate in reaction system is 10g/100ml.In above-mentioned example, in described polyethyleneglycol diacrylate, Polyethylene Glycol is Macrogol 2000, Macrogol 4000 or polyethylene glycol 6000.
The process conditions of the ice making glue that present inventor gropes alone are that at-80 DEG C to 0 DEG C, reaction is more than 1h, and condition concrete further reacts 20h at-20 DEG C.
The process conditions of above-mentioned ice making glue also comprise: after completion of the reaction, in atmosphere dry or lyophilization.Specifically can under the conditions of-50 DEG C and 20pa dry 30min.
In the preparation method of above-mentioned micro-ice glue three-dimensional microenvironment carrier, also comprise the step from enrichment micro-ice glue three-dimensional microenvironment carrier.
The method of described enrichment micro-ice glue three-dimensional microenvironment carrier is following at least one: micro Process top board method, vacuum method, centrifuging etc.
The enrichment method that the present inventor gropes alone is following 1) or 2):
1) top board is buckled on described mould, utilizes the bulge-structure on top board to be stripped out by the micro-ice glue three-dimensional microenvironment carrier in the pit-holes of described mould; Described top board is a flat board with bulge-structure, and the position of bulge-structure on flat board is corresponding with the pit-holes position in described mould;
2) mould containing micro-ice glue three-dimensional microenvironment carrier in pit-holes is placed in liquid (as water), then micro-ice glue three-dimensional microenvironment carrier dissociates out automatically owing to expanding from mould, just obtains micro-ice glue three-dimensional microenvironment carrier after collection.
The shape of the bulge-structure on above-mentioned top board and large I designed, designed according to actual needs, an object lesson of the present invention is, bulge-structure is column type, highly for 0.5mm.
The present invention gropes alone, and the material preparing above-mentioned top board is polydimethylsiloxane (PDMS).
In above-mentioned enriching step, after stripping, collect described micro-ice glue three-dimensional microenvironment carrier in culture dish by drainage screen further.
Three, the application of above-mentioned micro-ice glue three-dimensional microenvironment carrier.
Above-mentioned micro-ice glue three-dimensional microenvironment carrier is following 1) to 3) in arbitrary described application also belong to protection scope of the present invention:
1) application of above-mentioned micro-ice glue three-dimensional microenvironment carrier in the carrier injected for the preparation of biological sample;
2) above-mentioned micro-ice glue three-dimensional microenvironment carrier is preparing the application that has in the carrier of following function: be not free on injection position for biological sample injection after biological sample is injected;
3) above-mentioned micro-ice glue three-dimensional microenvironment carrier is transplanting the application in the carrier of clinical injection treatment for the preparation of biological sample.
Described biological sample can be following a)-d) in arbitrary described material: a) in various molecular substance (as micromolecular compound, medicine, nucleic acid, albumen etc.) any one or appoint several mixture; B) various natural with any one in synthetic material (as extracellular matrix, macromolecular material, microballon etc.) or appoint several mixture; C) in various cell and microorganism (as eucaryon/prokaryotic cell, virus, microorganism etc.) any one or appoint several mixture; D) a)-c) in appoint several mixture.
Four, above-mentioned micro-ice glue three-dimensional microenvironment carrier adsorbs the method for biological sample in vitro.
Micro-ice glue three-dimensional microenvironment carrier provided by the present invention adsorbs the method for biological sample in vitro, comprises the steps: biological sample solution to mix with providing the matrix solution of attachment point for biological sample, obtains mixed liquor; Carry out again following a) or b) shown in step:
A) micro-ice glue three-dimensional microenvironment carrier is prepared by method in above-mentioned two, before enrichment micro-ice glue three-dimensional microenvironment carrier (namely micro-ice glue three-dimensional microenvironment carrier is also in mould), described mixed liquor is added drop-wise on micro-ice glue three-dimensional microenvironment carrier, drive mixed liquor slowly to scrape back and forth uniformly with slide, described mixed liquor independently sucks in micro-ice glue three-dimensional microenvironment carrier;
B) micro-ice glue three-dimensional microenvironment carrier is prepared by method in above-mentioned two, after enrichment micro-ice glue three-dimensional microenvironment carrier (namely micro-ice glue three-dimensional microenvironment carrier is free from mould), micro-ice glue three-dimensional microenvironment carrier is collected in container, form micro-ice glue three-dimensional microenvironment carrier layer, be added drop-wise to by described mixed liquor in micro-ice glue three-dimensional microenvironment carrier layer, described mixed liquor independently sucks in micro-ice glue three-dimensional microenvironment carrier.
Describedly provide the substrate of attachment point can select from prior art for biological sample, but also oneself is groped.What commonly use in prior art for biological sample provides the substrate of attachment point is: gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, layer connect albumen and fibronectin.
Following method also can be taked to make micro-ice glue three-dimensional microenvironment carrier draw biological sample: rely on the quiescent draw (static-seeding) such as gravity, the bioreactor rotated is utilized to draw (rotated bioreactor), cellular layer pack (cell sheet wrapping), in conjunction with vacuum, centrifugal and flow Synergistic method (the synergisticeffects of vacuum, centrifugal force and flow), magnetic force is utilized to attract method (magnetic forceseeding), surface acoustic wave method (surface acoustic wave-driven seeding).）
Described biological sample can be following a)-d) in arbitrary described material: a) in various molecular substance (as micromolecular compound, medicine, nucleic acid, albumen etc.) any one or appoint several mixture; B) various natural with any one in synthetic material (as extracellular matrix, macromolecular material, microballon etc.) or appoint several mixture; C) in various cell and microorganism (as eucaryon/prokaryotic cell, virus, microorganism etc.) any one or appoint several mixture; D) a)-c) in appoint several mixture.
The concrete example of the present invention is, with micro-ice glue three-dimensional microenvironment carrier adsorption cell, used provides the substrate of attachment point to be alginate (as sodium alginate) hydrogel or 1 Collagen Type VI hydrogel for cell.
Beneficial effect of the present invention is as follows:
(1) micro-ice glue three-dimensional microenvironment carrier has following features:
(1) syringeability: micro-ice glue three-dimensional microenvironment carrier of the present invention, its size belongs to micron level, the design of this micro-meter scale ensure that the syringeability of cell carrier, the porous of adding the present invention's micro-ice glue three-dimensional microenvironment carrier is connective, make it have extremely strong mechanical performance and scalability, not cracky, ensure that load micro-ice glue carrier of cell can smoothly by injector for medical purpose that routine clinical uses in injection, and can strictly control to inject thrust, make in its safety range remaining on clinical regulation, thus meet the requirement of clinical cytology injection for curing.
(2) non-invasive: micro-ice glue three-dimensional microenvironment carrier of the present invention to be used for the treatment of cell transplantation clinical injection, what avoid operative therapy has the problems such as wound property and wound healing, and simple to operation, low-risk, safety is higher, and the suitability is strong.
(3) cell three-dimensional microenvironment carrier mode: the present invention is in the design of cell carrier, original and different, takes the external Three-dimensional cell culture theory that current field of tissue engineering technology is widely praised highly---build the external three-dimensional microenvironment model of cell.Cell carrier based on micro-ice glue builds, and its inside has porous and high connectivity, can load cell micro-environment component (cell, host material and active factors), for cell provides a large amount of adhesion spaces and site by autonomous type.In the process of cultivating in vitro, the 3 D stereo growth of cell in carrier on implementation space, in addition the existence of particular substrate material and active factors, the real microenvironment simulating cells in vivo growth in vitro, thus make cell being expelled to the inadaptability avoiding after in body and produce because inside and outside existence microenvironment is different, improve the survival rate of cell, maintain the multifunctionality of cell.
(4) protect supportive: the cell transplantation carrier that the present invention relates to; can by cell micro-environment component (cell; host material and active factors) be adsorbed in carrier inside; cell is made to carry out three dimensional growth in the porous air of carrier inside on the one hand; carrier itself can be used as the protective of cell in injection process on the other hand; the mechanical performance powerful by it and scalability, cushioned the damage of injection machinery shearing force to cell, thus improve the survival rate of cell.
(5) fixed point initiatively aggregation (the anti-free property lost) in body: the cell carrier that the present invention relates to is micro-meter scale rank, carrier is entered after in body by syringe, due to micron order volume and corresponding gravity effect, carrier can realize fixed point gathering and avoid with liquid omnidirectional free, thus reach injection point local fixed point enrichment effect, prevent the disperse of cell around injection point, more effective effect reaching repair of damaged tissues.
(6) anti-spilled property: cell micro-ice glue carrier that the porous that the present invention relates to is communicated with, there is stronger mechanical performance, scalability is stronger, when the carrier loading cell passes through syringe, the form of carrier can be shunk because of syringe internal diameter size and shearing force and be diminished, and buffering shearing force is to the damage of carrier inside cell.When after the carrier ingress engine soma that carry cell, due to the disappearance of External shearing forces, form restores to the original state and is greater than needle diameter, therefore after injection, get the moment of pin, cell and carrier can not overflow from pin hole along with the taking-up of syringe needle, decrease the loss of the rear cell of injection, improve the effect of cell therapy.
(2) preparation method of micro-ice glue three-dimensional microenvironment carrier has following features:
(1) technological process simple, can automated production, can large-scale production be realized.
(2) micro-ice glue three-dimensional microenvironment carrier of various shape can be prepared according to actual needs, if cross-sectional area is circle, ellipse, polygon etc., can be applicable to various field.
(3) the micro-ice glue three-dimensional microenvironment carrier size obtained by is homogeneous, and various micro-scale level (>1 μm) all can realize, and therefore makes carrier material to be more widely used.
(4) easily enrichment: employ a kind of novel micro Process top board method in preparation method of the present invention and enrichment is carried out to micro-ice glue.Micro Process top board, prepares easy, can design different protruding moulds according to the difformity of enriched sample, and plasticity is strong, and recycling rate of waterused is high, simple and quick, and the suitability is strong.
(3) method of micro-ice glue three-dimensional microenvironment carrier adsorption cell is utilized to have following features:
(1) multiple autonomous type adherent cell and In vitro culture mode: the method that the present invention relates to two kinds of micro-ice glue autonomous type adherent cells, is respectively thin layer and scrapes suction method and direct absorption method.The mode of these two kinds of autonomous adherent cells, loss cell is few, swift to operate easy, is applicable to multiple research and clinical treatment is applied.
(2) various kinds of cell training method: the microarray formula cell adsorption culture method related in the present invention, contribute to micro-ice glue in cell adhesion processes to the controllability in carrier number, number of cells and array arrangement etc. and operability, quantitative culture and the array arrangement of cell can be carried out, be applicable to multiple medical research and clinical treatment application.Another kind directly draws cell culture processes, simple and efficient to handle, few cells absorption object can be realized, decrease the unnecessary loss that cell and culture fluid etc. produce in adsorption process, improve the In vitro culture research of cell and the efficiency of clinical treatment application.
Above-mentioned three-dimensional cell microenvironment systematic research application is extensive, includes but not limited to: the chip of molecule/material/cell is for studying cell therapy molecule/cell, material/cell, cell/cell interaction; External model builds; Cell carrier; Cell therapy; Organizational project; Regenerative medicine; Cosmetics industry; Pathological study; Clinical conditions etc.
Accompanying drawing explanation
Fig. 1 is that system design is always schemed.Wherein, A is that designing mould and micro Process legal system are for mould; B is that the micro-ice glue of preparation is sent out in chemical crosslinking; C is autonomous type adherent cell and builds its microenvironment; D is the micro-ice glue being separated autonomous adherent cell from micro Process mould; E is micro-ice glue that enrichment is adsorbed with cell; F be by be mounted with cell after enrichment micro-ice glue by injector to inject in Mice Body, carry out fixed point cell therapy.
Fig. 2 is that chemical crosslink technique prepares micro-ice glue and separation and enrichment.A to decline ice glue for preparing array by micro-processing technology: wherein, and A1 is that mould (mould can be processed into difformity according to demand, as circle, oval, polygon and Herba Galii Bungei shape) prepared by micro-ice glue of micro-processing technology manufacture; A2 is the mould manufactured by micro Process, adopts chemical crosslink technique to be prepared into difform micro-ice glue; A3 is under microscope, the aspect graph of the micro-ice glue of various shape.B is the enrichment process of micro-ice glue.B1 is that array declines ice glue; B2 is the PDMS top board for the micro-ice glue of enrichment utilizing micro-processing method to manufacture, and top board surface presents the projection of corresponding micro-ice sealing rubber die plasticity pore size; B3 is the micro-ice glue be enriched in culture dish; B4 is the diagram utilizing filter screen separating liquid and micro-ice glue.
Fig. 3 is sign and the performance test of micro-ice glue.A is micro-ice glue Electronic Speculum figure.Wherein, A1 is the overall diagram of micro-ice glue under scanning electron microscope; A2 is the high power lens visual field of micro-ice glue under scanning electron microscope, can see aperture more more and have good connectedness; B is the comparison and detection of the imbibition ability of micro-ice glue and larger ice glue.C is the comparative determination of the mechanics elastic modelling quantity of micro-ice glue and conventional hydrogels.
Fig. 4 is injection schematic diagram and the injection property detection of micro-ice glue.A is the detection method and the device that detect micro-ice glue injectable performance.A1 is the checkout gear detecting micro-ice glue injectable performance.A2 is the injection process of micro-ice glue; A3 is the pictorial diagram that micro-ice glue is injected from pin hole by 1ml syringe; The enlarged drawing that the micro-ice glue of A4 injects from syringe pin hole; A5 is the micro-ice glue by the enrichment of 1ml injector to inject; The enlarged drawing of micro-ice glue that A6 injects.B is the thrust numerical value in the micro-ice glue injection process detected by micro-injection pump and thrust ergometer; C is the microscopic examination figure before and after the micro-ice glue of same volume and hydrogel are injected; D is the cartogram of percentage of head rice after the micro-ice glue of same volume and hydrogel are injected.
To be cell seeding detect at the vigor of large scale ice glue and the uniformity Fig. 5.A is yardstick is that other large scale ice glue of bold and unconstrained meter level adsorbs NIH3T3 cell through long-term cultivation, is adsorbed onto the fluorescence microscope figure of the cell mortality in centre; B is the fluorescence microscope figure that large scale ice glue absorption NIH3T3 cell causes absorption profiles inhomogeneity.
Fig. 6 is that thin layer is scraped suction method and made micro-ice glue independently load the detection of cell construction three-dimensional microenvironment system and the autonomous adherent cell ability of micro-ice glue.A is that micro-ice glue scrapes by thin layer the Fluirescence observation figure that suction method draws cell on the micro-array.A1 is the NIH3T3 cell that micro-ice glue independently adsorbs RFP3T3(transfection RFP red fluorescent protein on the micro-array) after fluorescent scanning figure (utilizing gene chip scanning instrument); A2 is micro-ice glue enlarged drawing of adherent cell; B is the fluorogram of micro-ice glue absorption different densities cell; C is the numerical statistic figure of micro-ice glue adherent cell ability.
Fig. 7 directly draws method material, cell to be independently loaded into Qualitative and quantitative analysis in micro-ice glue.A is after absorption is with isodensity mescenchymal stem cell (hMSCs), to observe under the three-dimensional microenvironment that hMSC forms at micro-ice glue long term survival state and multiplication capacity (4x be fluorescence microscope under 4 times of mirror visuals field, 10x is the lower 10 times of mirror visuals field of fluorescence microscope); B directly draws the cartogram that method detects micro-ice cellula adhesiae absorbability.C is the rate of increase of hMSC cell in micro-ice glue.
Fig. 8 be free cell and be loaded in micro-ice glue inner cell before the injection after vitality test.A for people source mescenchymal stem cell (hMSC) before the injection after cell survival detection (utilize live & dead survive colouring method carry out qualitative analysis, wherein green fluorescence represents living cells, and red fluorescence represents dead cell); B is the detection (being shown as cell in figure after live & dead fluorescence staining, the synthesis diagram of green fluorescence figure and red fluorescence figure) that NIH3T3 and hMSC is loaded in cell survival before and after injection in micro-ice glue.
Fig. 9 be the micro-ice glue being mounted with cell be expelled to mice subcutaneous after, form pictorial diagram and the HE colored graph of new vessels.A-B is blank group, subcutaneous mice, injects blank micro-ice glue (unloaded cell), the pictorial diagram after 21 days.C-D is experimental group, is mounted with micro-ice glue of RFP3T3 cell, the pictorial diagram after 21 days at mouse subcutaneous injection.The sample slice of blank group is carried out the result figure after HE dyeing under 4 power microscopes by E; F is the result figure under 10 power microscopes of partial enlargement in E figure; G is the result figure carried out by the sample slice of experimental group under 4 power microscopes after HE dyeing; H is the result figure under 10 power microscopes of G figure partial enlargement.
Figure 10 to be cross-sectional diameter be micro-ice glue three-dimensional microenvironment carrier aspect graph under the microscope of 100 microns, 200 microns, 800 microns.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The cell used in following embodiment is:
RFP-3T3 cell: the NIH3T3 cell of transfection RFP red fluorescent protein.
NIH3T3 cell: purchased from American Type Culture Collection (ATCC).
HMSC cell: the isolated adipose tissue separation and Extraction provided from cosmetic surgery hospital.
The Macrogol 4000 diacrylate (PEGDA4000) used in following embodiment can synthesize according to existing method: under a nitrogen atmosphere, 5g Polyethylene Glycol (PEG4000) powder is dissolved in 50ml dichloromethane solution while stirring, dropwise slowly add the acryloyl chloride solution of 0.47ml and the triethylamine solution of 0.76ml, at room temperature stirring reaction 24h.Then, add the solution of potassium carbonate of 100ml2mol/L with washing, and stratification, collect lower floor's dichloromethane mixed liquor.Dichloromethane mixed liquor is added drop-wise in 500ml absolute ether solvent and precipitates, filter, collect white powder, then drying at room temperature, finally obtain white polyethyleneglycol diacrylate pressed powder.
Embodiment 1, micro-ice glue three-dimensional microenvironment carrier and preparation method thereof
1, for the preparation of the preparation of the mould (being called for short micro-ice sealing rubber die) of micro-ice glue three-dimensional microenvironment carrier
Micro-ice sealing rubber die structure: be a flat board with pit-holes; Pit-holes is arrayed, the array distribution of 45x14, and each pit-holes is column type, and the circle of to be diameter in column type cross section be 400um, the center of circle spacing of each circle is 1.5mm, and the height of pit-holes is 300 μm.Plate material is PMMA.
Adopt laser cutting method, detailed process is as follows:
The present embodiment chooses polymethyl methacrylate (PMMA) the plate making mould that thickness is about 300 μm;
Utilize AutoCAD software to draw, draw out long 75cm, the rectangle of wide 25cm, and in this rectangle, draw the circle that diameter is 400um, arrange according to the array distribution of 45x14, the center of circle spacing of each circle is that 1.5mm(is as Figure 1A and Fig. 2 A1).
According to the legend of AutoCAD Software on Drawing, utilize Rayjet laser engraving machine, on the PMMA plate of 300 μm, engraving cuts out required mould; Being set to of laser engraving machine: engraving energy 20, speed 30, engraving number of times 1 time; Ablation energy 70, speed 3, cutting times 2 times;
After engraving cutting, mould is taken out, with water cleaning, and dry.
Clean mould is placed in PLASMA CLEANER processor, carries out surface-active-treatment 1min, take out PMMA mould, for future use.
Also the pore design in mould can be become other shape, if cross section is ellipse, triangle, Herba Galii Bungei shape (as Fig. 2 A1).
2, the preparation of top board
The structure of top board: be a flat board with column type projection, column type is protruding is arrayed on flat board, corresponding with the pit-holes position in above-mentioned micro-ice sealing rubber die, and the height of this column type projection is 0.5mm, the cross-sectional area of this column type projection is less than the cross-sectional area of pit-holes in above-mentioned micro-ice sealing rubber die.Plate material is polydimethylsiloxane (PDMS).
Make by soft etching technique.Detailed process is as follows:
With the PMMA mould for the preparation of top board that laser engraving machine carve thickness is 0.5mm.
PDMS is mixed homogeneously with mass ratio 10:1 ratio with sclerosing agent according to host, and makes the bubble floating in mixed liquor to surface by the mode of evacuation and break.
The side adhesive tape of PMMA mould is cemented, and is put in PDMS groove that the set degree of depth is 2-3mm, make the side being stained with adhesive tape downward.Bubble-free PDMS mixed liquor will be mixed and pour PMMA mould into, and ensure that PMMA surface has certain thickness PDMS to exist; Then place a microscope slide on surface, be evacuated in PMMA aperture afterwards and bubble-free existence between PMMA and glass.
Whole device is put in 60 DEG C of bakings, takes out after 3h, will harden and the microscope slide be sticked together with icking tool, PDMS and PMMA scales off in PDMS groove; Peeled carefully by PMMA mould, there is the kick corresponding with PMMA mold indentions hole on visible PDMS surface.See Fig. 2 B2.
3, the preparation of micro-ice glue
The preparation of reaction solution: be dissolved under PEGDA4000 powder room temperature in DPBS solution, obtain pre-gathering solutions; Again pre-gathering solutions and Ammonium persulfate. and N, N, N', N'-tetramethyl diethylamine are mixed, fully mix, obtain reaction solution;
The concentration of PEGDA4000 in reaction solution is 10g/100ml, and the concentration of Ammonium persulfate. in reaction solution is 0.5g/100ml; The concentration of N, N, N', N'-tetramethyl diethylamine in reaction solution is 0.05g/100ml.
Array declines the preparation of ice glue: be positioned over above ice chest by the micro-ice sealing rubber die made, and gets the reaction solution that 200ul prepares and drips uniformly on PMMA mould, slowly to be blown into uniformly by the reactant liquor on die surface in the hole in mould with coverslip.By the mould of populated PEGDA reactant liquor freezing 20h under-20 DEG C of conditions, proceed to freezer dryer (-50 DEG C, 20pa) dry 30min after reaction terminates, obtain the micro-ice glue of white porosity.According to the method described above, prepare micro-ice glue, its pictorial diagram is shown in Fig. 2 A2.The electron-microscope scanning figure of micro-ice glue is as Fig. 3 A.The micro-ice glue three-dimensional microenvironment carrier obtained has hole, pore size 10 μm-150 μm, pitch of holes 1 μm-999 μm; Porosity is 94.2%, connective good; Water absorption is 10-20 times of theoretical volume, sees Fig. 3 B; Micro-ice glue satisfactory mechanical property is shown in Fig. 3 C.Conventional hydrogels in Fig. 3 C is that PEGDA is by the hydrogel of chemical crosslinking under normal-temperature reaction condition.
The preparation of large scale ice glue: raw materials used, preparation condition is same as described above, difference is: mould is standard 384 orifice plate, and the diameter in each hole is 2.7mm, and plate height is 5.4mm.
4, the enrichment of micro-ice glue
Top board is buckled on the above-mentioned mould with micro-ice glue three-dimensional microenvironment carrier, the bulge-structure on top board is utilized to be stripped out by the micro-ice glue three-dimensional microenvironment carrier in pit-holes, be collected in culture dish or orifice plate with drainage screen, by washed with de-ionized water 2-3 time, freezing 1h under putting into-20 DEG C of conditions again, take out postlyophilization machine (-50 DEG C, 20Pa) inner drying 30min, obtain the free micro-ice glue three-dimensional microenvironment carrier existed.Enrichment process is shown in Fig. 2 B, and the micro-ice glue pictorial diagram after enrichment is shown in Fig. 2 B3 and B4.
Difform micro-ice glue three-dimensional microenvironment carrier pictorial diagram of preparation is shown in Fig. 2 A2 and A3.
The Performance Detection of embodiment 2, micro-ice glue three-dimensional microenvironment carrier
Below in experiment, micro-ice glue three-dimensional microenvironment carrier used all refers to gained micro-ice glue three-dimensional microenvironment carrier in embodiment 1.
One, micro-ice glue three-dimensional microenvironment carrier keeps complete in injection process, and its good mechanical property is described
Whether method: carry out injection experiment with micro-injection pump, by thrust when integrity, the injection of observing micro-ice glue three-dimensional microenvironment carrier, have clogging in injection process, study the mechanical performance of micro-ice glue.Under specific implementation method is shown in:
The preparation of microactuator suspension ice glue injection: by resuspended with the normal saline solution of 1% carboxymethyl cellulose for micro-ice glue three-dimensional microenvironment carrier, the concentration of prepared microactuator suspension ice glue is 1000/ml.
The debugging of micro-injection pump: first injection standard is adjusted to the injection standard needed for 1ml asepsis injector: internal diameter 4.53mm, injection flow velocity 1000 μ l/min, injection range 500ul; Then digital display type push-pull effort ergometer being arranged on syringe pushes away between handle and syringe pump, fully contacts, and measures the thrust of syringe pump pushing syringe under certain flow rate.Digital display type push-pull effort ergometer connects computer, draws real-time thrust curve figure.
The injection of micro-ice glue: after being taken off by the syringe needle of 1ml asepsis injector, draws with syringe the cmc soln that 500ul even suspension has micro-ice glue three-dimensional microenvironment carrier, reinstalls back stitching head afterwards, be arranged on micro-injection pump by syringe.Micro-injection pump is started according to the injection standard of having debugged, the cmc soln that the even suspension injected has micro-ice glue three-dimensional microenvironment carrier is collected at the syringe needle place of syringe, and the real-time thrust curve figure that Real Time Observation digital display type push-pull effort ergometer is drawn.Injection flow process is shown in Fig. 4 A.
Result: the real-time thrust curve figure that push-pull effort ergometer is drawn is shown in Fig. 4 B, show: the thrust of injecting needed for micro-ice glue is no more than 10N, meets clinical demand, show to inject in the process of micro-ice glue simultaneously, micro-ice glue, smoothly by the entry needle of 27G, occurs without clogging.
The microscopic examination figure that front and back injected by micro-ice glue and hydrogel asks for an interview Fig. 4 C, shows: after injection, and hydrogel is almost completely broken, and micro-ice glue form keeps complete, and show compared with hydrogel, micro-ice glue has better mechanical performance and plasticity.Hydrogel: be the hydrogel that PEGDA is obtained under room temperature reflection condition by chemical crosslinking.
Fig. 4 D is asked for an interview in the statistical result that rear percentage of head rice injected by the micro-ice glue of same volume and hydrogel.
Two, micro-ice glue three-dimensional microenvironment carrier can efficient loading cell
Micro-ice glue three-dimensional microenvironment carrier itself is acellular can attachment sites, need use can provide cell attachment sites again crosslinkable biomaterial by cell encapsulation, then could realize the loading of cell on micro-ice glue three-dimensional microenvironment carrier.As long as can provide cell attachment sites again crosslinkable biomaterial can be following a kind of: gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, layer connect albumen, fibronectin, sodium alginate.
The loading cells performance of micro-ice glue three-dimensional microenvironment carrier is detected by the following two kinds mode:
(1), thin layer is scraped suction method and is made the autonomous load material of micro-ice glue, cell
Micro-ice glue three-dimensional microenvironment carrier is prepared as stated above in this experiment, before enrichment micro-ice glue three-dimensional microenvironment carrier (namely micro-ice glue three-dimensional microenvironment carrier is also in mould), the mixed liquor of cell and substrate is added drop-wise on micro-ice glue three-dimensional microenvironment carrier, drive mixed liquor slowly to scrape back and forth uniformly with slide, described mixed liquor independently sucks in micro-ice glue three-dimensional microenvironment carrier.And then peel off with top board.
This experiment is using RFP-3T3 cell as the example of loading cell.Cell adsorption medium takes Sodium Alginate Hydrogel Films.
(1) qualitative detection
According to cell suspension: the ratio of 2% sodium alginate=1:1 makes the sodium alginate cell suspension that concentration is 1%.Draw 100ul sodium alginate cell suspension, be placed on micro-ice glue three-dimensional microenvironment carrier, ensure that cell drawn by each micro-ice glue.The mould of sodium alginate cell suspension is had to be immersed in the CaCl of 100mmol/L suction
2in solution, take out after 10min; Then be immersed in PBS solution and make Ca
2+dissociate micro-ice glue.The method of filtering is utilized to be taken out by the micro-ice glue three-dimensional microenvironment carrier after adherent cell.Fluorescence microscope.
Result, as Fig. 6 A, shows that micro-ice glue evenly can draw cell.
(2) detection by quantitative
Prepare the sodium alginate cell suspension of different cell concentration: 5x10
6/ ml, 1x10
7/ ml, 1.5x10
7/ ml, 2x10
7/ ml, 2.5x10
7/ ml.Each concentration draws 100ul sodium alginate cell suspension respectively, for absorption.All the other methods are all consistent with (1).Micro-ice glue of different cell concentration is had to be placed in 96 orifice plates suction.Each concentration establishes 3 micro-ice offset plates respectively.
Get the sodium alginate cell suspension of the above-mentioned different cell density of 20ul respectively, join in 96 orifice plates, and add the CaCl of 100ul100mmol/L
2solution, makes it directly form hydrogel; Then be immersed in PBS solution and make Ca
2+dissociate, obtain Sodium Alginate Hydrogel Films.
Alamar blue measures: in each hole of micro-ice glue and Sodium Alginate Hydrogel Films, add 40ul alamar blue solution, hatch 4h for 37 DEG C.Its fluorescent value at 560nm/590nm is measured after 4h.
Standard curve is formulated and in micro-ice glue, cell number calculates.The fluorescent value standard curve corresponding with cell number is formulated according to the fluorescent value of the Sodium Alginate Hydrogel Films of different cell density.The cell number in micro-ice glue is calculated according to standard curve.Result is as Fig. 6 B and 6C.
(2), directly material, cell are independently loaded in micro-ice glue by absorption method
Prepare micro-ice glue three-dimensional microenvironment carrier as stated above, (namely with top board, micro-ice glue three-dimensional microenvironment carrier is peeled off from mould) after enrichment micro-ice glue three-dimensional microenvironment carrier, micro-ice glue three-dimensional microenvironment carrier is collected in culture dish, forms the single thin layer of micro-ice glue three-dimensional microenvironment carrier; Be added drop-wise on the single thin layer of micro-ice glue three-dimensional microenvironment carrier by the mixed liquor of cell and substrate, described mixed liquor independently sucks in micro-ice glue three-dimensional microenvironment carrier.
This experiment is using RFP-3T3 cell as the example of loading cell.Cell adsorption medium is 1 Collagen Type VI hydrogel.
(1) qualitative detection
1 Collagen Type VI of BD company 10mg/ml is diluted to 1mg/ml, with the acetic acid in the 1mol/lNaOH of 0.023 times of original content collagen volume and in collagen, obtains collagen working solution.Again with this collagen working solution suspension cell, make cell collagen suspension.Draw 40-100 μ l cell collagen suspension, drop on micro-ice glue three-dimensional microenvironment carrier thin layer.The micro-ice glue three-dimensional microenvironment carrier thin layer drawing cell is put in 37 DEG C, 3h plastic.Add culture fluid after 3h, and piping and druming makes micro-ice glue thin layer be separated into single micro-ice glue gently.Be put in CO
2cultivate in incubator.Fluorescence microscope.Result as shown in Figure 7 A.
(2) detection by quantitative
The collagen hydrogel cell suspension of different cell density: 2x10
6/ ml, 5x10
6/ ml, 1x10
7/ ml, 1.5x10
7/ ml, 2x10
7/ ml.The cell collagen suspension drawing 60ul different densities is respectively added drop-wise on the thin layer of micro-ice glue, after drawing, is put in 37 DEG C, 3h plastic.After 3h, dispel as single micro-ice glue.Micro-ice glue is transferred in 96 orifice plates, adds 200ul culture fluid and 40ulalamar blue reagent, hatch 4h for 37 DEG C.Carry out fluoremetry after 4h, wavelength is 560nm/590nm.Result is as Fig. 7 B.
Three, compared with existing large scale ice glue, micro-ice glue three-dimensional microenvironment carrier evenly can be drawn cell and have good mass transfer
The cell adsorption medium of this experiment is Sodium Alginate Hydrogel Films, and cell is NIH3T3 cell.Difference between large scale ice glue and micro-ice glue is compared by the dye distribution of observation of cell and survival rate of live/dead.
The preparation of large scale ice glue: raw materials used, preparation condition is identical with the preparation of micro-ice glue three-dimensional microenvironment carrier, difference is: mould is standard 384 orifice plate, and the diameter in each hole is 2.7mm, and plate height is 5.4mm
Large scale ice mucilage binding carries cellular processes: draw 60 μ l sodium alginate cell suspension and be added drop-wise on large scale ice glue.The large scale ice glue of sodium alginate cell suspension and micro-ice offset plate is had to be immersed in the CaCl of 100mmol/L suction
2in solution, take out after 10min; Then be immersed in PBS solution and make Ca
2+dissociate micro-ice glue.
After adherent cell, cultivate with DMEM culture fluid respectively.Cultivate and carry out live/dead detection on the 2nd, 9 day, testing result is shown in Fig. 5.
Result shows, the survival of large scale ice glue superficial cell is good, and inner most cell dead is as Fig. 5 A; In addition, large scale ice cell that glue is inhaled can not be uniformly distributed as Fig. 5 B.
And micro-ice glue three-dimensional microenvironment carrier of the present invention, no matter in carrier inside or at carrier surface, the equal well-grown of cell, and inside does not have cell death, cell is uniformly distributed (asking for an interview Fig. 6 A, 7A and 8B).
Four, with do not use compared with micro-ice glue three-dimensional microenvironment carrier, in injection process, use micro-ice glue three-dimensional microenvironment carrier can have protective effect to the cell loaded
This tests cell used is NIH3T3 cell, hMSC cell, and adsorbing medium is respectively sodium alginate and collagen hydrogel.
Experimental group:
According to cell suspension: 2% sodium alginate=1:1 mixing, making cell density is 2 × 10
7the cell Sodium Alginate Hydrogel Films suspension of/ml.
Draw 100 μ l cell Sodium Alginate Hydrogel Films suspensions, softly scrape uniformly on micro-ice offset plate with thin microscope slide.Micro-ice offset plate of cell Sodium Alginate Hydrogel Films suspension is had to be immersed in the CaCl of 100mmol/L suction
2in solution, take out after 10min; Be immersed in PBS solution after taking-up and make Ca
2+dissociate micro-ice glue.
Micro-ice glue is divided into two groups, and one group is non-injection group, and another group is injection group.Non-injection group: peeled off from mould by the micro-ice glue three-dimensional microenvironment carrier after adherent cell, resuspended with 500 μ l1% carboxymethyl celluloses, density is 1000/ml, then after discarding carboxymethyl cellulose, is put in 48 orifice plates and cultivates.Injection group: peeled off from mould by the micro-ice glue three-dimensional microenvironment carrier after adherent cell, resuspended with 500 μ l1% carboxymethyl celluloses, density is 1000/ml; Draw 500 μ l with 1ml syringe and hang the carboxymethyl cellulose having micro-ice glue, syringe is placed on micro-injection pump, to inject flow velocity 1000 μ l/min, the condition injection of injection range 500 μ l, discard carboxymethyl cellulose after injection, collect micro-ice glue and cultivate in 48 orifice plates.
Live/dead method is adopted to measure the survival condition of cell.
Result as shown in Figure 8 B.Show, the NIH3T3 cell before and after injection in micro-ice glue three-dimensional microenvironment carrier is without obvious death condition.HMSC cell is also like this.
Matched group: take hMSC as experimental cell, after suspending with normal saline, to inject flow velocity 1000 μ l/min, the condition injection of injection range 500 μ l, the cell after injection is cultivated in 48 orifice plates, adopts live/dead method to measure the survival condition of cell.Result as shown in Figure 8 A, shows, visible cell mortality rate half nearly after injection.
This description of test, in injection process, micro-ice glue three-dimensional microenvironment carrier has good protective effect to cell.
Five, micro-ice glue three-dimensional microenvironment carrier before the injection after all can well sustenticular cell adhere to, sprawl and breed
The present embodiment is cell adhesion matrix material with 1 Collagen Type VI (collagen) hydrogel, and selected human adipose mesenchymal stem cells (hMSCs) is as object of study.
Collagen solution prepares.The method provided according to BD company prepares collagen solution, and working concentration is 1mg/ml, is namely diluted with hMSCs culture fluid by the collagen solution of 10mg/ml and obtains.
The collagen solution preparation of parcel cell.By hMSCs trypsinization and collected by centrifugation, resuspended with the collagen solution prepared in step 2, cell density is 3 × 10
6/ ml.
Load cell.Human mesenchymal stem cell is drawn according to direct absorption method.
The injection of micro-ice glue.Micro-ice glue is divided into two groups, and one group is non-injection group, and another group is injection group.Non-injection group is resuspended with 500 μ l1% carboxymethyl celluloses, and density is 1000/ml, then after discarding carboxymethyl cellulose, is put in 48 orifice plates and cultivates.Injection group is resuspended with 500 μ l1% carboxymethyl celluloses, and density is 1000/ml.Draw 500 μ l with 1ml syringe and hang the carboxymethyl cellulose having micro-ice glue, syringe is placed on micro-injection pump, to inject flow velocity 1000 μ l/min, the condition injection of injection range 500 μ l, discard carboxymethyl cellulose after injection, collect micro-ice glue and cultivate in 48 orifice plates.
Cellular morphology and proliferation assay.Select live/dead to dye to observe cell, measure cell proliferation rate by alamar blue.As shown in Fig. 7 A and Fig. 7 C, hMSC well can adhere to and sprawl in micro-ice glue, and compared with 0 day, cell has had significant proliferation.As shown in Figure 8 B, the hMSC after injection in micro-ice glue keeps good vigor.
Six, the micro-ice glue being mounted with cell can locating injection also fixed point induction new vessels generation in Mice Body
The cell adsorption medium of this experiment is sodium alginate, and cell is RFP3T3, is expelled to the subcutaneous formation observing induction new vessels of mice.
Cell Sodium Alginate Hydrogel Films suspension: according to cell suspension: 2% sodium alginate=1:1 mixing, making cell density is 2 × 10
7the cell Sodium Alginate Hydrogel Films suspension of/ml.
Micro-ice mucilage binding carries cell: be loaded into by cell Sodium Alginate Hydrogel Films suspension on micro-ice offset plate, collect rear carboxymethyl cellulose resuspended, mix homogeneously, density is 1000/ml.Meanwhile, another is organized micro-offset plate and only loads sodium alginate, collects rear carboxymethyl cellulose resuspended, mix homogeneously, and density is 1000/ml.
Mouse subcutaneous injection: by mice Avertin(0.12ml/10g) after anesthesia, extracting resuspended good micro-ice glue with 1ml syringe, to be expelled to mice subcutaneous, is divided into matched group and experimental group.After injection, normal raising mice 21 days.Matched group: micro-ice glue three-dimensional microenvironment carrier of injection only adsorbs sodium alginate, experimental group: micro-ice glue three-dimensional microenvironment carrier adsorption cell Sodium Alginate Hydrogel Films suspension of injection.
Paraffin section and HE dyeing are observed.After 21 days, by mouse anesthesia execution, take out the micro-ice glue of injection point, carry out routine paraffin wax section and HE dyeing observation.
Result is as Fig. 9.Show, compared with matched group, the obvious inductive formation new vessels of RFP3T3, illustrates that cell has good function in micro-ice glue.
Prepare micro-ice glue three-dimensional microenvironment carrier according to method described in embodiment 1, method is substantially the same manner as Example 1, and unlike varying in size of pit-holes in mould, specifically the diameter of pit-holes cross section is respectively 100 microns, 200 microns, 800 microns.Result gained micro-ice glue three-dimensional microenvironment carrier form is under the microscope as Figure 10.Micro-for gained ice glue three-dimensional microenvironment carrier is carried out respectively in embodiment 2, test one, detect its mechanical performance.As a result, in the process of the micro-ice glue of injection, micro-ice glue three-dimensional microenvironment carrier, smoothly by the entry needle of 27G, occurs without clogging, and injection afterwards in percentage of head rice and embodiment 1 gained micro-ice glue three-dimensional microenvironment carrier without significant difference.
Claims (9)
1. a micro-ice glue three-dimensional microenvironment carrier, the material forming this carrier is ice glue, and this carrier size belongs to micron order;
The preparation method of described micro-ice glue three-dimensional microenvironment carrier, comprise the steps: the material mixing by being used for making ice glue, then mixture is divided into micron order size, the process conditions of recycling ice making glue process, and namely obtain micro-ice glue three-dimensional microenvironment carrier;
The described pit-holes being divided into by mixture the method for micron order size to comprise the steps: described mixture to be placed in mould, described mould structure is the flat board with pit-holes, and the size of described pit-holes is micron order;
The preparation method of described micro-ice glue three-dimensional microenvironment carrier also comprises the step of enrichment micro-ice glue three-dimensional microenvironment carrier;
The method of described enrichment micro-ice glue three-dimensional microenvironment carrier is:
1) top board is buckled on described mould, utilizes the bulge-structure on top board to be stripped out by the micro-ice glue three-dimensional microenvironment carrier in the pit-holes of described mould; Described top board is a flat board with bulge-structure, and the position of bulge-structure on flat board is corresponding with the pit-holes position in described mould;
2) mould containing micro-ice glue three-dimensional microenvironment carrier in pit-holes is placed in liquid, then micro-ice glue three-dimensional microenvironment carrier dissociates out automatically owing to expanding from mould, just obtains micro-ice glue three-dimensional microenvironment carrier after collection.
2. prepare the method for micro-ice glue three-dimensional microenvironment carrier for one kind, comprise the steps: the material mixing by being used for making ice glue, then mixture is divided into micron order size, the process conditions of recycling ice making glue process, and namely obtain micro-ice glue three-dimensional microenvironment carrier;
The described pit-holes being divided into by mixture the method for micron order size to comprise the steps: described mixture to be placed in mould, described mould structure is the flat board with pit-holes, and the size of described pit-holes is micron order;
The preparation method of described micro-ice glue three-dimensional microenvironment carrier also comprises the step of enrichment micro-ice glue three-dimensional microenvironment carrier;
The method of described enrichment micro-ice glue three-dimensional microenvironment carrier is:
1) top board is buckled on described mould, utilizes the bulge-structure on top board to be stripped out by the micro-ice glue three-dimensional microenvironment carrier in the pit-holes of described mould; Described top board is a flat board with bulge-structure, and the position of bulge-structure on flat board is corresponding with the pit-holes position in described mould;
2) mould containing micro-ice glue three-dimensional microenvironment carrier in pit-holes is placed in liquid, then micro-ice glue three-dimensional microenvironment carrier dissociates out automatically owing to expanding from mould, just obtains micro-ice glue three-dimensional microenvironment carrier after collection.
3. method according to claim 2, is characterized in that: the described material for making ice glue is crosslinkable material;
Described crosslinkable material is biomaterial and/or the natural biologic material of synthetic;
The biomaterial of described synthetic is following at least one: Polyethylene Glycol, polyethyleneglycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polylactic acid alkyd copolymers, polydimethylsiloxane, polyamide, polyamino acid, polybutylcyanoacrylate, polyurethanes, polypyrrole, polymethacrylates, polyethylene, Merlon and polyethylene glycol oxide;
Described natural biologic material is following at least one: gelatin, gelatine derivative, alginate, alginate derivative, agar, matrigel, collagen, proteoglycan, glycoprotein.
4. method according to claim 3, it is characterized in that: the described material for making ice glue is polyethyleneglycol diacrylate and Ammonium persulfate. and N, N, N', N'-tetramethyl diethylamine, its proportioning is 5-50g polyethyleneglycol diacrylate: 0.5g Ammonium persulfate.: 0.025-0.2g N, N, N', N'-tetramethyl diethylamine.
5. according to described method arbitrary in claim 2-4, it is characterized in that: the process conditions of described ice making glue comprise: be react more than 1h under the condition of-80 DEG C to 0 DEG C in temperature.
6. according to described method arbitrary in claim 2-4, it is characterized in that: the process conditions of described ice making glue also comprise: after completion of the reaction, dry or lyophilization in atmosphere.
7. micro-ice glue three-dimensional microenvironment carrier described in claim 1 following 1) or 2) described application:
1) application of micro-ice glue three-dimensional microenvironment carrier in the carrier injected for the preparation of biological sample described in claim 1;
2) micro-ice glue three-dimensional microenvironment carrier described in claim 1 is transplanting the application in the carrier of clinical injection treatment for the preparation of biological sample.
8. application according to claim 7, is characterized in that: the described 1) application of micro-ice glue three-dimensional microenvironment carrier in the carrier injected for the preparation of biological sample described in claim 1, is not free on injection position after biological sample injection.
9. utilize micro-ice glue three-dimensional microenvironment carrier described in claim 1 to adsorb the method for biological sample in vitro, comprise the steps: biological sample solution to mix with providing the matrix solution of attachment point for biological sample, obtain mixed liquor; Carry out again following a) or b) shown in step:
A) micro-ice glue three-dimensional microenvironment carrier is prepared according to described method arbitrary in claim 2-6, before enrichment micro-ice glue three-dimensional microenvironment carrier, described mixed liquor is added drop-wise on micro-ice glue three-dimensional microenvironment carrier, drive mixed liquor slowly to scrape back and forth uniformly with slide, described mixed liquor independently sucks in micro-ice glue three-dimensional microenvironment carrier;
B) micro-ice glue three-dimensional microenvironment carrier is prepared according to described method arbitrary in claim 2-6, after enrichment micro-ice glue three-dimensional microenvironment carrier, micro-ice glue three-dimensional microenvironment carrier is collected in container, form micro-ice glue three-dimensional microenvironment carrier layer, be added drop-wise to by described mixed liquor in micro-ice glue three-dimensional microenvironment carrier layer, described mixed liquor independently sucks in micro-ice glue three-dimensional microenvironment carrier.
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| Interconnected Macroporous Poly(Ethylene Glycol) Cryogels as a Cell Scaffold for Cartilage Tissue Engineering;Yongsung Hwang et al;《TISSUE ENGINEERING: Part A》;20101231;第16卷(第10期);3033-3040 * |
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