CN103877613A - System and method for constructing injected three-dimensional cell microenvironment based on microfrozen gel - Google Patents

System and method for constructing injected three-dimensional cell microenvironment based on microfrozen gel Download PDF

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CN103877613A
CN103877613A CN201210553882.5A CN201210553882A CN103877613A CN 103877613 A CN103877613 A CN 103877613A CN 201210553882 A CN201210553882 A CN 201210553882A CN 103877613 A CN103877613 A CN 103877613A
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carrier
cell
ice glue
ice
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CN103877613B (en
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杜亚楠
刘伟
李雅倩
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Beijing Hua Ting Biotechnology Co., Ltd.
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Tsinghua University
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Abstract

The invention discloses a system and a method for constructing an injected three-dimensional cell microenvironment based on microfrozen gel. A microfrozen gel three-dimensional microenvironment vector is made of the frozen gel and in micro scale size. Experiments demonstrate that the prepared microfrozen gel three-dimensional microenvironment vector has the following characteristics that mechanical properties are good; whole morphology can be kept after being injected; the vector can load cells effectively; compared with conventional large-scale frozen gel, the microfrozen gel three-dimensional microenvironment vector can absorb the cells uniformly and has good material transmission performance; the microfrozen gel three-dimensional microenvironment vector has a protection effect for loaded cells in an injection process; and the microfrozen gel loaded with the cells can be injected into an animal body at specific sites and keep original functions and activities in in-vivo cells. The microfrozen gel three-dimensional microenvironment vector plays an important role in the field of cell injection therapy, and has wide application prospects in animal model establishment and the drug screening field.

Description

Build the system and method for injection-type three-dimensional cell microenvironment based on micro-ice glue
Technical field
The invention provides a kind of system and method that builds injection-type three-dimensional cell microenvironment based on micro-ice glue.
Background technology
Regenerative medicine taking cell therapy as representative becomes the important directions of development of clinical medicine gradually, for the complicated major disease that reply Drug therapy is difficult to take effect has been brought new hope.Cell therapy is to utilize patient's's autologous (or allosome) one-tenth somatic cell or stem cell to tissue, the Therapeutic Method that organ is repaired.Utilize at present the existing a large amount of reports of research of the pertinacious diseases such as cell therapy cardiovascular disease, diabetes, parkinson disease, keratopathy, cartilaginous tissue damage and tumor, and in clinical practice, obtained Preliminary Results.Particularly the research based on stem-cell therapy and application have obtained 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, for example neurocyte, myocardial cell, vascular endothelial cell, nephrocyte and hepatocyte etc.Utilize stem cell to have the pluripotency characteristic changing to various cell differentiations, treat tissue degeneratiaon, damage, necrosis disease clinically, thereby reach tissue regeneration, reproduce, the objects such as replacement, have realized taking stem-cell therapy technology as basic regenerative medicine theory.
At present, continue to bring out for the clinical technology of cell therapy, mainly comprise following several diverse ways:
1, directly by allosome or autologously isolatedly there is biological function and learn the one-tenth somatic cell of characteristic or stem cell direct injection in damaged tissues place, be more conventional effective clinical cell therapy means.The method is easy and simple to handle, but after injection, lower cell survival rate and cell enters the uncontrollability in body, seriously restricting 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 is found, only being less than 3% transplanting stem cell and playing tissue repair effect at damaged part, there is the dead and free phenomenon of losing in all the other most of transplanted cells.After direct injection causes transplanting stem cell to enter in body, the reason of mortality and loss 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, has reduced the survival ability of cell.(2) external free stem cell enters in the interior damaged tissues of body region, in this process, the residing microenvironment of cell changes, and the microenvironment in focus region is unfavorable for cell survival and growth, stem cell can not adapt to focus microenvironment of living in, thereby has reduced the survival rate of cell.(3) direct injection stem cell is in body, different due to therapentic part and injection point, part injection site is due to the reasons such as pressure difference inside and outside mechanical movement or tissue site (disease model as outstanding in vertebra shroud, cartilaginous tissue damage model, the models such as cardiopulmonary repairing transplant), cause the cell that is injected into target spot tissue to overflow from pin hole along with extracting of syringe, after causing the free stem cell transplantation of a large amount of cell losses (4) to enter in body, cell can be along with body fluid be to organizing surrounding non-directional free, cannot initiatively assemble, and then cause a large amount of transplanted cells to run off, cannot carry out the fixed point treatment of cell.
2, the great number of issues producing in the face of directly transplanting cell, based on the structure of biomaterial accessory cell implant system, for the histoorgan recovery technique in regenerative medicine field has been brought new breakthrough.The technology that this biomaterial accessory cell is transplanted, can use tissue engineering technique on the one hand, in conjunction with the characteristic of seed cell, somatomedin and biomaterial, carry out in vitro histoorgan cultivation, ripe In vitro culture organ is replaced to damaged tissues, thereby maintain, recover even to promote the function of damaged tissues.(DJ Mooney et al.PNAS 2008) designed a kind of stem cell transplantation support, modified VEGF (VEGF) and endothelial progenitor cells (EPC) is sticked to internal stent at internal stent.Through In vitro culture after a while, to be transplanted to ischemic tissue inside in connection with the timbering material that has somatomedin and functional stem cell, thereby to carry out reparation and the regeneration of blood vessel, effect is very remarkable.But, the immunologic rejection that large volume allosome material is transplanted, the problems such as the healing of the traumatic and postoperative wound of operation process, are restricting the paces that this technology is advanced to clinical practice.
3, the common issue in the face of running in operation transplantation process, design is a kind of non-invasive, reduces rejection, and stem-cell therapy method easy and simple to handle becomes the focus that researcheres are paid close attention to.Wait noinvasive or Wicresoft's mode that the cell with certain functional characteristic is transferred in body by injection, the regenerative medicine treatment theory that realizes 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 impact, utilize bioreactor, the cell microsphere of preparing the stem cell adipose-derived by a large amount of people (hADSCs) gathering and form, the cell microsphere that preparation is completed arrives mice lower limb ischemia position by injector to inject, there is a large amount of new vesselses in lower limb ischemia position, therapeutic effect is obvious.But cell microsphere form prepared by the method size heterogeneity, plasticity is poor, in injection process, still cannot avoid the damage of cells of superficial layer.(PX Maetal.Nat Mater 2011) designed 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; realize again the method for injectable type cell therapy; and due to the existence of cell carrier; thereby provide supportive protection effect to stem cell in injection process; the transplanting survival rate that has improved cell, repair of damaged tissues effect is comparatively obvious.But this self assembly carrier is comparatively complicated on synthetic making, a large amount of cell carrier form homogeneity of making are poor, the ability of the autonomous adherent cell of carrier a little less than, cell can not all enter into carrier inside and partly stick to carrier surface, thereby affects cell transmission and the efficiency for the treatment of.(K.L.Fujimoto et al.Biomaterials 2009) utilizes a kind of degradable temperature-sensitive hydrogel as cell transmission carrier, in the time that temperature is lower, cell suspension is in liquid hydrogel solution, by injection means fixed-point injection behind damaged tissues region, the hydrogel solution that is suspended with cell forms immediately hydrogel when temperature environment in body, and gel is in injection areas, prevents the loss of cell, thereby reach the effect that cell fixed point is treated.But in this design, the three dimension system of hydrogel exists the problems such as mass transfer on the one hand, affect the normal survival of cell, the method cannot be avoided the damage of injection machinery power to 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, thereby have a strong impact on the clinical therapeutic efficacy of cell transplantation.
The development of medical treatment, health industry needs efficient disease treatment scheme, for realizing the simple and easy to do for the treatment of technology, improves therapeutic efficiency, reduces patient's pain, and Noninvasive therapy technology has advantageous advantage.Noninvasive therapy technology (injectable type cell carrier treatment technology) has been avoided the problems such as the traumatic and postoperative wound healing of cell operation transplantation, for patient reduced huge at heart and physiological pain, for medical worker provides Therapeutic Method very simply and easily, reduce the risk of operative treatment, realize more and having optimized, 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 to the difference of the microenvironment of surviving in microenvironment that cell cultivates in vitro and body.Traditional bidimensional cell culture (based on business-like culture dish or porous plate) technical development history of existing 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 is found the survival condition of cell under the culture environment of bidimensional gradually, cellular morphology and its are far apart under condition of culture of true environment in vivo.Therefore the three-dimensional microenvironment in body can not well be simulated and reappear to the bidimensional microenvironment of this simplification.And depend on the stem cell of cultivating under conventional two-dimensional environment, in cellular replacement therapy process, due to the great change of inside and outside living environment, cause the reduction of stem cell survival and function, reparation is gone down with regeneration function, thereby has restricted the effect of stem-cell therapy in clinical practice.Research shows, cultivates than 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 cell and the internal milieu state of In vitro culture more close.Therefore three-dimensional cell culture technique is receiving much concern in recent years, and has obtained significantly development.
Build the dimensional culture model of cells in vitro, biomaterial used is because needs directly contact with tissue in vivo, therefore must possess certain particular/special requirement: as the biological activity (adhesion sites, inducement signal etc.) of the physical and chemical performance (chemical compatibility, mechanical performance, degradability, architectural characteristic etc.) of the source of matrix of materials (natural or synthetic material), matrix of materials, matrix of materials, equipment, service condition, the scope of application that dimensional culture technology is required, the suction type of cell, adhesion mode, training method, detection mode etc.Desirable three-dimensional cell culture systems can realize in cell seeding, cultivation, the process of breeding, go down to posterity and follow-up imaging 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 multidisciplinary knowledge fork such as materialogy, select optimum biomaterial, design the most simple and efficient cell carrier processing technology, thereby reach extensive simple and easy manufacture, the form homogeneity and the absorbability that ensure cell carrier are particularly important, simultaneously also for solid foundation has been established in the clinical practice that advances cell therapy.
In sum, in the face of complexity, the challenge of cell therapy clinical practice and all difficulties that run into, need a kind of processing technology of design badly easy, the cell three-dimensional microenvironment carrier can be mass-produced, operational approach and the condition (as the standard of the syringe of clinical practice and 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 three-dimensional microenvironment carrier of a kind of micro-ice glue and preparation method thereof and application.
Technical scheme of the present invention is described below respectively:
One, structure and the composition of the three-dimensional microenvironment carrier of micro-ice glue.
The three-dimensional microenvironment carrier of micro-ice glue provided by the present invention is as follows: the material that forms this carrier is ice glue (cryogel), and this carrier size belongs to micron order.
Described micron order is 1 μ m-999 μ m, is specially 50 μ m-999 μ m, then is specially 100 μ m-800 μ m.
Above-mentioned ice glue generally all has several holes, and is communicated with between hole.
The aperture of above-mentioned ice glue mesopore can be 1 μ m-500 μ m, and pitch of holes can be 1 μ m-999 μ m.Object lesson of the present invention, pore size 10 μ m-150 μ m, pitch of holes 1 μ m-999 μ m, porosity is 94.2%.
Above-mentioned ice glue can be prepared as follows: the slow plastic under low temperature environment of the material for ice making glue is formed to ice crystal pore simultaneously, and its plastic process completes jointly with the process that forms ice crystal, finally obtains ice glue.Concrete preparation method can comprise the step of crosslinked and adjustment aperture.Described cross-linking method can be following at least one: chemical crosslink technique, physical crosslinking method, crosslinking with radiation method, enzyme-catalyzed cross-linking method.The method in the micro-ice glue of described adjusting 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.
Be crosslinkable material for the preparation of the material of above-mentioned ice glue.Crosslinkable material can be biomaterial and/or the natural biologic material of synthetic; The biomaterial of described synthetic be following at least one: Polyethylene Glycol, polyethyleneglycol derivative, polyethyleneglycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alcohol acid copolymer, polydimethylsiloxane, poly-anhydride, poly-acid esters, polyamide, polyamino acid, polyacetals, polybutylcyanoacrylate, polyurethanes, polypyrrole, polyester, polymethacrylates, polyethylene, Merlon and polyethylene glycol oxide; Described natural biologic material be following at least one: gelatin, gelatine derivative, alginate, alginate derivant, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, layer connect albumen and fibronectin.
The crosslinked condition that present inventor gropes is alone that reaction raw materials is reacted and exceedes 1h at-80 DEG C to 0 DEG C.
The ice glue that the present inventor gropes is alone prepared as follows and obtains: taking polyethyleneglycol diacrylate as crosslinkable material, with Ammonium persulfate. and N, N, N', N '-tetramethyl diethylamine, as cross-linking agent, reacts 20h at-20 DEG C, makes ice glue.
Further concrete example is, by 5～50g polyethyleneglycol diacrylate and 0.5g Ammonium persulfate. and 0.025 ~ 0.2g N, and N, N', the preparation of arranging in pairs or groups of N '-tetramethyl diethylamine.
Further preferred example is again, by 10g polyethyleneglycol diacrylate and 0.5g Ammonium persulfate. and 0.05gN, N, N', N '-tetramethyl diethylamine mixes, 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.
The three-dimensional microenvironment carrier of above-mentioned micro-ice glue can be any shape, if cross-sectional area is circle, ellipse, polygon (triangle, square etc.), heart, Herba Galii Bungei shape etc.Specifically can be as column type granule, the diameter of its cross-sectional area can be 1-999 μ m, 50-999 μ m or 100-800 μ m, and cylindrical height can be 1-999 μ m, specifically can be 300 μ m.Those skilled in the art according to actual needs, can select any shape.
Those skilled in the art can according to actual needs, select any size in micron order.
Two, the preparation method of the three-dimensional microenvironment carrier of above-mentioned micro-ice glue.
The method of the three-dimensional microenvironment carrier of the above-mentioned micro-ice glue of preparation provided by the present invention, comprise the steps: the material mixing for making ice glue, then mixture is divided into micron order size, the process conditions of recycling ice making glue are processed, and obtain the three-dimensional microenvironment carrier of micro-ice glue.
Described micron order is 1 μ m-999 μ m, is specially 50 μ m-999 μ m, then is specially 100 μ m-800 μ m.
The above-mentioned method that mixture is divided into micron order size is die methods or emulsion process.
Above-mentioned emulsion process is to carry out emulsifying with mixture, obtains object size and object shape.
Above-mentioned die methods comprises the steps: described mixture to be placed in the pit-holes of 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 μ m-999 μ m, is specially 50 μ m-999 μ m, then is specially 100 μ m-800 μ m.
In above-mentioned mould the size of pit-holes and shape all with size and the shape corresponding (or identical) of the three-dimensional microenvironment carrier of the micro-ice glue that will obtain.Can design according to actual needs the size and shape of hole, controlled to realize shape size.
Pit-holes in above-mentioned mould can be arrayed, and array distribution can be 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 preparation in batches.
The material of preparing above-mentioned mould can be existing common used material, also can oneself grope to obtain.What inventor groped alone is to adopt PMMA to prepare mould.
The object lesson of a mould of the present invention is one to have the flat board of pit-holes; Pit-holes is arrayed, the array distribution of 45x14, and each pit-holes is column type, and being column type cross section is that diameter is the circle of 100-800um, and the center of circle spacing of each circle is 1.5mm, and the height of pit-holes is 300 μ m, 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.
Be crosslinkable material for the preparation of the material of ice glue.Crosslinkable biomaterial can be biomaterial and/or the natural biologic material of synthetic; The biomaterial of described synthetic be following at least one: Polyethylene Glycol, polyethyleneglycol derivative, polyethyleneglycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alcohol acid copolymer, polydimethylsiloxane, poly-anhydride, poly-acid esters, polyamide, polyamino acid, polyacetals, polybutylcyanoacrylate, polyurethanes, polypyrrole, polyester, polymethacrylates, polyethylene, Merlon and polyethylene glycol oxide; Described natural biologic material be following at least one: gelatin, gelatine derivative, alginate, alginate derivant, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, layer connect albumen and fibronectin.
Can be prepared as follows ice glue: the slow plastic under low temperature environment of the material for ice making glue is formed to ice crystal pore simultaneously, and its plastic process completes jointly with the process that forms ice crystal, finally obtains ice glue.Concrete preparation method can comprise the step of crosslinked and adjustment aperture.Described cross-linking method can be following at least one: chemical crosslink technique, physical crosslinking method, crosslinking with radiation method, enzyme-catalyzed cross-linking method.The method in the micro-ice glue of described adjusting 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: taking polyethyleneglycol diacrylate as crosslinkable material, with Ammonium persulfate. and N, N, N', N'-tetramethyl diethylamine is as cross-linking agent.Further concrete, by 5～50g polyethyleneglycol diacrylate and 0.5g Ammonium persulfate. and 0.025 ~ 0.2g N, N, N', the preparation of arranging in pairs or groups of N'-tetramethyl diethylamine.Further preferred example is again, 10g polyethyleneglycol diacrylate and 0.5g Ammonium persulfate. and 0.05g N, N, N', N'-tetramethyl diethylamine.Further concrete, above-mentioned material mixes 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 are alone that at-80 DEG C to 0 DEG C, reaction exceedes 1h, and further concrete condition is to react 20h at-20 DEG C.
The process conditions of above-mentioned ice making glue also comprise: after completion of the reaction, and in air drying or lyophilization.Specifically can be under the condition of-50 DEG C and 20pa dry 30min.
In the preparation method of the three-dimensional microenvironment carrier of above-mentioned micro-ice glue, also comprise from the step of the three-dimensional microenvironment carrier of the micro-ice glue of enrichment.
The method of the three-dimensional microenvironment carrier of the micro-ice glue of described enrichment be following at least one: micro-processing top board method, vacuum method, centrifuging etc.
The enrichment method that the present inventor gropes is alone following 1) or 2):
1) top board is buckled on described mould, utilizes the bulge-structure on top board that three-dimensional the micro-ice glue in the pit-holes of described mould microenvironment carrier is peeled off out; Described top board is one to have the flat board of bulge-structure, and the position of bulge-structure on flat board is corresponding with the pit-holes position in described mould;
2) mould that contains the three-dimensional microenvironment carrier of micro-ice glue in pit-holes is placed in to liquid (as water), then the three-dimensional microenvironment carrier of micro-ice glue dissociates out automatically owing to expanding from mould, just obtain the three-dimensional microenvironment carrier of micro-ice glue 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 is 0.5mm.
The present invention gropes alone, and the material of preparing above-mentioned top board is polydimethylsiloxane (PDMS).
In above-mentioned enriching step, after peeling off, further can collect the three-dimensional microenvironment carrier of described micro-ice glue to culture dish by drainage screen.
Three, the application of the three-dimensional microenvironment carrier of above-mentioned micro-ice glue.
The three-dimensional microenvironment carrier of above-mentioned micro-ice glue is following 1) to 3) in arbitrary described application also belong to protection scope of the present invention:
1) application of the three-dimensional microenvironment carrier of above-mentioned micro-ice glue in the carrier for the preparation of biological sample injection;
2) the three-dimensional microenvironment carrier of above-mentioned micro-ice glue has the application in the carrier of following function in preparation: for biological sample injection and make not to be free on injection position after biological sample injection;
3) application of the three-dimensional microenvironment carrier of above-mentioned micro-ice glue in the carrier of transplanting clinical injection treatment for the preparation of biological sample.
Described biological sample can be following a)-arbitrary described material in d): a) any in various molecular substances (as micromolecular compound, medicine, nucleic acid, albumen etc.) or appoint several mixture; B) any in various natural and synthetic materials (as extracellular matrix, macromolecular material, microballon etc.) or appoint several mixture; C) any in various cells and microorganism (as eucaryon/prokaryotic cell, virus, microorganism etc.) or several mixture; D) a)-c) in appoint several mixture.
Four, the method that the three-dimensional microenvironment carrier of above-mentioned micro-ice glue adsorbs biological sample in vitro.
The method that the three-dimensional microenvironment carrier of micro-ice glue provided by the present invention adsorbs biological sample in vitro, comprises the steps: biological sample solution to mix with the matrix solution of attachment point is provided for biological sample, obtains mixed liquor; Carry out again following a) or step b):
A) prepare the three-dimensional microenvironment carrier of micro-ice glue by method in above-mentioned two, before the three-dimensional microenvironment carrier of the micro-ice glue of enrichment (being that the three-dimensional microenvironment carrier of micro-ice glue is also in mould), described mixed liquor is added drop-wise on the three-dimensional microenvironment carrier of micro-ice glue, drive mixed liquor slowly to scrape back and forth uniformly with slide, described mixed liquor is autonomous to be sucked in the three-dimensional microenvironment carrier of micro-ice glue;
B) prepare the three-dimensional microenvironment carrier of micro-ice glue by method in above-mentioned two, after the three-dimensional microenvironment carrier of the micro-ice glue of enrichment (being that the three-dimensional microenvironment carrier of micro-ice glue is free from mould), three-dimensional micro-ice glue microenvironment carrier is collected in container, form the three-dimensional microenvironment carrier layer of micro-ice glue, described mixed liquor is added drop-wise in the three-dimensional microenvironment carrier layer of micro-ice glue, and described mixed liquor is autonomous to be sucked in the three-dimensional microenvironment carrier of micro-ice glue.
Describedly can from prior art, select for biological sample provides the substrate of attachment point, also can oneself grope.What in prior art, commonly use for biological sample provides the substrate of attachment point is: gelatin, gelatine derivative, alginate, alginate derivant, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, layer connect albumen and fibronectin.
Also can take following method to make the three-dimensional microenvironment carrier of micro-ice glue draw biological sample: to rely on static draw (static-seeding) such as gravity, utilize the bioreactor of rotation to draw (rotated bioreactor), cellular layer pack (cell sheet wrapping), in conjunction with vacuum, centrifugal and mobile Synergistic method (the synergisticeffects of vacuum, centrifugal force and flow), utilize magnetic attraction method (magnetic forceseeding), surface acoustic wave method (surface acoustic wave-driven seeding).）
Described biological sample can be following a)-arbitrary described material in d): a) any in various molecular substances (as micromolecular compound, medicine, nucleic acid, albumen etc.) or appoint several mixture; B) any in various natural and synthetic materials (as extracellular matrix, macromolecular material, microballon etc.) or appoint several mixture; C) any in various cells and microorganism (as eucaryon/prokaryotic cell, virus, microorganism etc.) or several mixture; D) a)-c) in appoint several mixture.
The concrete example of the present invention is that, with the three-dimensional microenvironment carrier adsorption of micro-ice glue cell, used is alginate (as sodium alginate) hydrogel or 1 Collagen Type VI hydrogel for cell provides the substrate of attachment point.
Beneficial effect of the present invention is as follows:
(1) the three-dimensional microenvironment carrier of micro-ice glue has following features:
(1) syringeability: the three-dimensional microenvironment carrier of micro-ice glue of the present invention, its size belongs to micron level, the design of this micro-meter scale has ensured the syringeability of cell carrier, add the porous connectedness of the three-dimensional microenvironment carrier of the micro-ice glue of the present invention, make it have extremely strong mechanical performance and scalability, not cracky, guarantee to load the injector for medical purpose that the micro-ice glue carrier of cell can use by routine clinical smoothly in injection, and can strictly control injection thrust, make in its safety range that remains on clinical regulation, thereby meet the requirement of clinical cell injection for curing.
(2) non-invasive: by three-dimensional micro-ice glue of the present invention microenvironment carrier, for the treatment of cell transplantation clinical injection, that has avoided operative therapy has problems such as wound property and wound healing, simple to operation, low-risk, safety is higher, and the suitability is strong.
(3) cell three-dimensional microenvironment carrier pattern: the present invention is in the design of cell carrier, original and different, take the external three-dimensional cell that current field of tissue engineering technology is widely praised highly to cultivate theory---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) by autonomous type, for cell provides a large amount of adhesion space and sites.In the process of cultivating in vitro, cell is the growth of the 3 D stereo on implementation space in carrier, in addition the existence of particular substrate material and active factors, the real microenvironment of simulating in vitro cells in vivo growth, thereby cell is avoided because of the different inadaptabilities that produce of inside and outside existence microenvironment after being expelled in body, improve the survival rate of cell, maintained the multifunctionality of cell.
(4) protect supportive: the cell transplantation carrier the present invention relates to; can be by cell micro-environment component (cell; host material and active factors) be adsorbed in carrier inside; make cell in the porous air of carrier inside, carry out three dimensional growth on the one hand; carrier itself can be used as the protective of cell in injection process on the other hand; by its powerful mechanical performance and scalability, cushion the damage of injection machinery shearing force to cell, thereby improved the survival rate of cell.
(5) initiatively aggregation (the anti-free property lost) of fixed point in body: the cell carrier the present invention relates to is micro-meter scale rank, after carrier enters in body by syringe, due to micron order volume and corresponding gravity effect, carrier can be realized fixed point gathering and avoid omnidirectional free with liquid, thereby reach the local fixed point of injection point enrichment effect, prevent the disperse of cell around injection point, more effective effect that reaches repair of damaged tissues.
(6) anti-spilled property: the micro-ice glue of the cell carrier that the porous the present invention relates to is communicated with, there is stronger mechanical performance, scalability is stronger, in the time that the carrier of loading cell passes through syringe, the form of carrier can be shunk and diminish because of syringe internal diameter size and shearing force, cushions the damage of shearing force to carrier inside cell.Enter after body tissue when carrying the carrier of cell, due to the disappearance of outside shearing force, form restores to the original state and is greater than syringe needle diameter, the moment of therefore getting pin after injection, cell and carrier can not overflow from pin hole along with the taking-up of syringe needle, reduce the loss of cell after injection, improved the effect of cell therapy.
(2) preparation method of the three-dimensional microenvironment carrier of micro-ice glue has following features:
(1) technological process simple, can automated production, can realize large-scale production.
(2) can prepare according to actual needs the three-dimensional microenvironment carrier of micro-ice glue of various shapes, if cross-sectional area is circle, ellipse, polygon etc., can be applicable to various fields.
(3) the three-dimensional microenvironment carrier size of prepared micro-ice glue homogeneous, (>1 μ m) all can realize various minute yardstick ranks, therefore makes carrier material to be more widely used.
(4) easily enrichment: used a kind of novel micro-processing top board method to carry out enrichment to micro-ice glue in preparation method of the present invention.Micro-processing top board, prepares easyly, 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) utilize the method for the three-dimensional microenvironment carrier adsorption of micro-ice glue cell to there is following features:
(1) multiple autonomous type adherent cell and In vitro culture mode: the present invention relates to the method for two kinds of micro-ice glue autonomous type adherent cells, be respectively thin layer and scrape 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 application.
(2) various kinds of cell training method: the microarray formula cell absorption culture method relating in the present invention, contribute to micro-ice glue controllability and operability to aspects such as carrier number, number of cells and array arrangements in cell adhesion processes, quantitative culture and the array arrangement of cell be can carry out, multiple medical research and clinical treatment application are applicable to.The another kind of cell culture processes of directly drawing, simple and efficient to handle, can realize few cells absorption object, reduce the unnecessary loss that cell and culture fluid etc. produce in adsorption process, improve the efficiency of In vitro culture research and the clinical treatment application of cell.
Above-mentioned three-dimensional cell microenvironment systematic research application is extensive, includes but not limited to: the chip of molecule/material/cell is used 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 diagnosis treatment etc.
Brief description of the drawings
Fig. 1 is that system design is always schemed.Wherein, A is that designing mould and micro-processing method are prepared 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 that separates autonomous adherent cell from micro-processing mold; E is micro-ice glue that enrichment absorption has cell; F be by the micro-ice glue that is mounted with cell after enrichment by injector to inject in Mice Body, the cell therapy of fixing a point.
Fig. 2 is that chemical crosslink technique is prepared micro-ice glue and separation and enrichment.A is for preparing the array ice glue that declines by micro-processing technology: wherein, A1 is that micro-ice glue of micro-processing technology manufacture is prepared mould (mould can be processed into difformity according to demand, as circle, ellipse, polygon and Herba Galii Bungei shape); A2 is the mould by micro-processing and manufacturing, 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 shapes.B is the enrichment process of micro-ice glue.B1 is the array ice glue that declines; B2 is the PDMS top board for the micro-ice glue of enrichment that utilizes 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 being enriched in culture dish; B4 is the diagram of 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 large more aperture 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 traditional hydrogel.
Fig. 4 is that injection schematic diagram and the injection property of micro-ice glue detects.A is detection method and the device that detects micro-ice glue injectable performance.A1 is the checkout gear that detects micro-ice glue injectable performance.A2 is the injection process of micro-ice glue; A3 is the pictorial diagram that micro-ice glue injects 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 detecting 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 injection; D is the cartogram of percentage of head rice after the micro-ice glue of same volume and hydrogel injection.
Fig. 5 is that cell seeding detects in vigor and the uniformity of large scale ice glue.A is that yardstick is other large scale ice glue absorption of bold and unconstrained meter level NIH3T3 cell process 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 the three-dimensional microenvironment system of cell construction and the autonomous adherent cell ability of micro-ice glue.A is micro-ice glue is scraped suction method absorption cell on microarray Fluirescence observation figure by thin layer.A1 is the NIH3T3 cell of micro-ice glue autonomous absorption RFP3T3(transfection RFP red fluorescent protein on microarray) 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 are independently loaded into the qualitative and quantitative analysis in micro-ice glue.A is after absorption is with isodensity mescenchymal stem cell (hMSCs), observe hMSC long term survival state and multiplication capacity (4x is 4 times of mirror visuals field under fluorescence microscope, and 10x is the lower 10 times of mirror visuals field of fluorescence microscope) under the three-dimensional microenvironment of micro-ice glue formation; B is the cartogram that directly absorption method detects micro-ice cellula adhesiae absorbability.C is the rate of increase of hMSC cell in micro-ice glue.
Fig. 8 is free cell and is loaded in the vitality test of micro-ice glue inner cell before and after injection.The detection (utilize live & dead survival colouring method to carry out qualitative analysis, wherein green fluorescence represents living cells, and red fluorescence represents dead cell) that A is people source mescenchymal stem cell (hMSC) cell survival before and after injection; B is that NIH3T3 and hMSC are loaded in the detection (being shown as cell in figure after live & dead fluorescence staining, the synthetic diagram of green fluorescence figure and red fluorescence figure) of cell survival before and after injection in micro-ice glue.
Fig. 9 be micro-ice glue of 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 result figure under rear 4 power microscopes of HE dyeing is carried out in the sample section of blank group by E; F is the result figure under local 10 power microscopes that amplify in E figure; The result figure under 4 power microscopes after HE dyeing is carried out in the sample section of experimental group by G; H is the result figure under local 10 power microscopes that amplify of G figure.
Figure 10 is that cross-sectional diameter is the aspect graph under the microscope of the three-dimensional microenvironment carrier of micro-ice glue of 100 microns, 200 microns, 800 microns.
Detailed description of the invention
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
The cell using in following embodiment is:
RFP-3T3 cell: the NIH3T3 cell of transfection RFP red fluorescent protein.
NIH3T3 cell: collect center (ATCC) purchased from US mode strain.
HMSC cell: the in vitro fatty tissue separation and Extraction providing from cosmetic surgery hospital.
The Macrogol 4000 diacrylate (PEGDA4000) using in following embodiment can synthesize according to existing method: under condition of nitrogen gas, 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, lower floor's dichloromethane mixed liquor collected.Dichloromethane mixed liquor is added drop-wise in 500ml absolute ether solvent and is precipitated, filter, collect white powder, then drying at room temperature, finally obtains white polyethyleneglycol diacrylate pressed powder.
Embodiment 1, the three-dimensional microenvironment carrier of micro-ice glue and preparation method thereof
1, for the preparation of the preparation of the mould (being called for short micro-ice sealing rubber die) of the three-dimensional microenvironment carrier of micro-ice glue
Micro-ice sealing rubber die structure: be one to there is the flat board of pit-holes; Pit-holes is arrayed, the array distribution of 45x14, and each pit-holes is column type, and being column type cross section is that diameter is the circle of 400um, and the center of circle spacing of each circle is 1.5mm, and the height of pit-holes is 300 μ m.Plate material is PMMA.
Adopt laser cutting method, detailed process is as follows:
The present embodiment is chosen polymethyl methacrylate (PMMA) the plate making mould that thickness is about 300 μ m;
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 μ m, 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, water cleans, and dries.
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 to 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 one to there is the flat board of column type projection, column type projection 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).
With soft etching technique making.Detailed process is as follows:
The PMMA mould for the preparation of top board that is 0.5mm with laser engraving machine carve thickness.
PDMS is mixed homogeneously with mass ratio 10:1 ratio with sclerosing agent according to host, and make bubble floating in mixed liquor to surface and break by the mode of evacuation.
One side of PMMA mould is cemented with adhesive tape, and be put in the PDMS groove that the set degree of depth is 2-3mm, make to be stained with under a side direction of adhesive tape.To mix bubble-free PDMS mixed liquor 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 to afterwards in PMMA aperture and between PMMA and glass and exist without bubble.
Whole device is put in to 60 DEG C of bakings, after 3h, takes out, with icking tool, by the microscope slide that hardens and be sticked together, PDMS and PMMA scale off in PDMS groove; PMMA mould is peeled carefully, and there is the kick corresponding with PMMA mould pit-holes on visible PDMS surface.See Fig. 2 B2.
3, the preparation of micro-ice glue
The preparation of reaction solution: by being dissolved in DPBS solution under PEGDA4000 powder room temperature, obtain pre-gathering solutions; Again by pre-gathering solutions and Ammonium persulfate. and N, N, N', N'-tetramethyl diethylamine mixes, and fully mixes, and obtains 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; N, N, N', the concentration of N'-tetramethyl diethylamine in reaction solution is 0.05g/100ml.
The decline preparation of ice glue of array: micro-ice sealing rubber die of making is positioned over to ice chest top, gets the reaction solution that 200ul prepares and drip uniformly on PMMA mould, the reactant liquor on die surface is slowly blown in the hole in mould uniformly with coverslip.By the mould of populated PEGDA reactant liquor freezing 20h under-20 DEG C of conditions, after finishing, reaction proceeds to the dry 30min of freezer dryer (50 DEG C, 20pa), obtain the micro-ice glue of white porous.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 three-dimensional microenvironment carrier of micro-ice glue obtaining has hole, pore size 10 μ m-150 μ m, pitch of holes 1 μ m-999 μ m; 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.Traditional hydrogel in Fig. 3 C is the hydrogel that PEGDA forms under normal-temperature reaction condition by chemical crosslinking.
The preparation of large scale ice glue: raw materials used, preparation condition is same as described above, difference is: mould is standard 384 orifice plates, 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 the three-dimensional microenvironment carrier of micro-ice glue, utilize the bulge-structure on top board that three-dimensional the micro-ice glue in pit-holes microenvironment carrier is peeled off out, be collected in culture dish or orifice plate with drainage screen, by washed with de-ionized water 2-3 time, put into again freezing 1h under-20 DEG C of conditions, take out postlyophilization machine (50 DEG C, 20Pa) inner drying 30min, obtain the three-dimensional microenvironment carrier of the free micro-ice glue existing.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.
The three-dimensional microenvironment carrier of the difform micro-ice glue pictorial diagram of preparation is shown in Fig. 2 A2 and A3.
The Performance Detection of embodiment 2, the three-dimensional microenvironment carrier of micro-ice glue
Below in experiment, the three-dimensional microenvironment carrier of micro-ice glue used all refers to the three-dimensional microenvironment carrier of the micro-ice glue of gained in embodiment 1.
One, the three-dimensional microenvironment carrier of micro-ice glue keeps complete in injection process, and its good mechanical property is described
Method: inject experiment with micro-injection pump, whether have clogging in thrust when observing integrity, the injection of the three-dimensional microenvironment carrier of micro-ice glue, 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 three-dimensional micro-ice glue 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 required injection standard of 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 is arranged on to syringe and pushes away between handle and syringe pump, fully contact, the thrust of mensuration 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 the syringe needle of 1ml asepsis injector is taken off, be evenly suspended with the cmc soln of the three-dimensional microenvironment carrier of micro-ice glue with syringe absorption 500ul, reinstall afterwards back stitching head, syringe is arranged on micro-injection pump.Start micro-injection pump according to the injection standard of having debugged, collect the cmc soln that is evenly suspended with the three-dimensional microenvironment carrier of micro-ice glue injecting at the syringe needle place of syringe, and the real-time thrust curve figure of Real Time Observation digital display type push-pull effort ergometer drafting.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: inject the required thrust of micro-ice glue and be no more than 10N, meet 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.
Microscopic examination figure before and after micro-ice glue and hydrogel injection asks for an interview Fig. 4 C, shows: after injection, hydrogel is almost completely broken, and the maintenance of micro-ice glue form is complete, shows compared with hydrogel, and micro-ice glue has better mechanical performance and plasticity.Hydrogel: be the hydrogel that PEGDA obtains under room temperature reflection condition by chemical crosslinking.
After the micro-ice glue of same volume and hydrogel injection, Fig. 4 D is asked for an interview in the statistical result of percentage of head rice.
Two, the three-dimensional microenvironment carrier of micro-ice glue can efficient loading cell
The three-dimensional microenvironment carrier of micro-ice glue itself is acellular can adhesion sites, need to use can provide cell adhesion site again crosslinkable biomaterial cell is wrapped up, then could realize the loading of cell on micro-ice glue three-dimensional microenvironment carrier.Can provide cell adhesion site again crosslinkable biomaterial can be following as long as a kind of: gelatin, gelatine derivative, alginate, alginate derivant, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, layer connect albumen, fibronectin, sodium alginate.
Detect the cell loading performance of the three-dimensional microenvironment carrier of micro-ice glue by following two kinds of modes:
(1), thin layer is scraped suction method and is made the autonomous load material of micro-ice glue, cell
In this experiment, prepare as stated above the three-dimensional microenvironment carrier of micro-ice glue, before the three-dimensional microenvironment carrier of the micro-ice glue of enrichment (being that the three-dimensional microenvironment carrier of micro-ice glue is also in mould), the mixed liquor of cell and substrate is added drop-wise on the three-dimensional microenvironment carrier of micro-ice glue, drive mixed liquor slowly to scrape back and forth uniformly with slide, described mixed liquor is autonomous to be sucked in the three-dimensional microenvironment carrier of micro-ice glue.And then peel off with top board.
This experiment is using RFP-3T3 cell as the example of loading cell.Cell adsorbing medium is taked Sodium Alginate Hydrogel Films.
(1) qualitative detection
Making concentration according to the ratio of cell suspension: 2% sodium alginate=1:1 is 1% sodium alginate cell suspension.Draw 100ul sodium alginate cell suspension, be placed on the three-dimensional microenvironment carrier of micro-ice glue, ensure that each micro-ice glue draws cell.There is the mould of sodium alginate cell suspension to be immersed in the CaCl of 100mmol/L suction 2in solution, after 10min, take out; Then be immersed in PBS solution and make Ca 2+micro-ice glue dissociates.Utilize the method for filtering that three-dimensional the micro-ice glue after adherent cell microenvironment carrier is taken out.Fluorescence microscope.
Result is as Fig. 6 A, shows that micro-ice glue can evenly draw cell.
(2) detection by quantitative
Prepare the sodium alginate cell suspension of different cell concentrations: 5x10 6/ ml, 1x10 7/ ml, 1.5x10 7/ ml, 2x10 7/ ml, 2.5x10 7/ ml.Each concentration is drawn respectively 100ul sodium alginate cell suspension, for absorption.All the other methods are all consistent with (1).There is micro-ice glue of different cell concentrations to be placed in 96 orifice plates suction.Each concentration is established respectively 3 micro-ice offset plates.
The sodium alginate cell suspension of getting respectively the above-mentioned different cell densities of 20ul, joins in 96 orifice plates, and adds 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.After 4h, measure its fluorescent value at 560nm/590nm.
In standard curve formulation and micro-ice glue, cell number calculates.Formulate the fluorescent value standard curve corresponding with cell number according to the fluorescent value of the Sodium Alginate Hydrogel Films of different cell densities.Calculate the cell number in micro-ice glue according to standard curve.Result is as Fig. 6 B and 6C.
(2), directly drawing method is independently loaded into material, cell in micro-ice glue
Prepare as stated above the three-dimensional microenvironment carrier of micro-ice glue, after the three-dimensional microenvironment carrier of the micro-ice glue of enrichment, (with top board, three-dimensional micro-ice glue microenvironment carrier is peeled off from mould), three-dimensional micro-ice glue microenvironment carrier is collected in culture dish, forms the single thin layer of the three-dimensional microenvironment carrier of micro-ice glue; The mixed liquor of cell and substrate is added drop-wise on the single thin layer of the three-dimensional microenvironment carrier of micro-ice glue, and described mixed liquor is autonomous to be sucked in the three-dimensional microenvironment carrier of micro-ice glue.
This experiment is using RFP-3T3 cell as the example of loading cell.Cell adsorbing medium is 1 Collagen Type VI hydrogel.
(1) qualitative detection
1 Collagen Type VI of the 10mg/ml of BD company 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.Use again this collagen working solution suspension cell, make cell collagen suspension.Draw 40-100 μ l cell collagen suspension, drop on the three-dimensional microenvironment carrier of micro-ice glue thin layer.The three-dimensional microenvironment carrier of the micro-ice glue thin layer of drawing cell is put in to 37 DEG C, 3h plastic.After 3h, add culture fluid, and piping and druming makes micro-ice glue thin layer be separated into single micro-ice glue gently.Be put in CO 2in incubator, cultivate.Fluorescence microscope.Result as shown in Figure 7 A.
(2) detection by quantitative
The collagen hydrogel cell suspension of different cell densities: 2x10 6/ ml, 5x10 6/ ml, 1x10 7/ ml, 1.5x10 7/ ml, 2x10 7/ ml.The cell collagen suspension of drawing respectively 60ul different densities is 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.After 4h, carry out fluoremetry, wavelength is 560nm/590nm.Result is as Fig. 7 B.
Three,, compared with existing large scale ice glue, the three-dimensional microenvironment carrier of micro-ice glue can evenly be drawn cell and have good mass transfer
The cell adsorbing medium of this experiment is Sodium Alginate Hydrogel Films, and cell is NIH3T3 cell.Carry out the difference between comparison large scale ice glue and micro-ice glue by dye distribution and the survival rate of observation of cell of live/dead.
The preparation of large scale ice glue: raw materials used, preparation condition is identical with the preparation of the three-dimensional microenvironment carrier of micro-ice glue, and difference is: mould is standard 384 orifice plates, and the diameter in each hole is 2.7mm, and plate height is 5.4mm
Large scale ice mucilage binding carries cell method: draw 60 μ l sodium alginate cell suspension and be added drop-wise on large scale ice glue.There are the large scale ice glue of sodium alginate cell suspension and micro-ice offset plate to be immersed in the CaCl of 100mmol/L suction 2in solution, after 10min, take out; Then be immersed in PBS solution and make Ca 2+micro-ice glue dissociates.
After adherent cell, cultivate with DMEM culture fluid respectively.Cultivate and carry out live/dead detection in the 2nd, 9 days, testing result is shown in Fig. 5.
Result shows, the survival of large scale ice glue superficial cell is good, and inner most cells 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 the three-dimensional microenvironment carrier of micro-ice glue 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 to be uniformly distributed (asking for an interview Fig. 6 A, 7A and 8B).
Four,, compared with not using the three-dimensional microenvironment carrier of micro-ice glue, in injection process, use the three-dimensional microenvironment carrier of micro-ice glue to there is protective effect to the cell loading
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 mixes, and 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.There is micro-ice offset plate of cell Sodium Alginate Hydrogel Films suspension to be immersed in the CaCl of 100mmol/L suction 2in solution, after 10min, take out; After taking-up, be immersed in PBS solution and make Ca 2+micro-ice glue dissociates.
Micro-ice glue is divided into two groups, and one group is non-injection group, and another group is injection group.Non-injection group: three-dimensional the micro-ice glue after adherent cell microenvironment carrier is peeled off from mould, and resuspended with 500 μ l1% carboxymethyl celluloses, density is 1000/ml, then discards after carboxymethyl cellulose, is put in 48 orifice plates and cultivates.Injection group: three-dimensional the micro-ice glue after adherent cell microenvironment carrier is peeled off from mould, and resuspended with 500 μ l1% carboxymethyl celluloses, density is 1000/ml; Draw 500 μ l with 1ml syringe and hang the carboxymethyl cellulose that has 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, after injection, discard carboxymethyl cellulose, collect micro-ice glue and cultivate in 48 orifice plates.
Adopt live/dead method to measure the survival condition of cell.
Result as shown in Figure 8 B.Show, the NIH3T3 cell before and after injection in the three-dimensional microenvironment carrier of micro-ice glue is without obvious death condition.HMSC cell is also like this.
Matched group: taking 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, the three-dimensional microenvironment carrier of micro-ice glue has good protective effect to cell.
Five, the three-dimensional microenvironment carrier of micro-ice glue is being injected all well sustenticular cell adhesions of front and back, is being sprawled and breed
The present embodiment is taking 1 Collagen Type VI (collagen) hydrogel as cell adhesion substrate material, and selected human adipose mesenchymal stem cells (hMSCs) is as object of study.
Collagen solution is prepared.The method providing according to BD company is prepared collagen solution, and working concentration is 1mg/ml, is obtained with the dilution of hMSCs culture fluid by the collagen solution of 10mg/ml.
The collagen solution preparation of parcel cell.By trypsinization centrifugal collection for hMSCs, resuspended with the collagen solution preparing in step 2, cell density is 3 × 10 6/ ml.
Load cell.Draw human mesenchymal stem cell 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 discards after 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 that has 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, after injection, discard carboxymethyl cellulose, collect micro-ice glue and cultivate in 48 orifice plates.
Cellular morphology and proliferation assay.Select live/dead dyeing to observe cell, measure cell proliferation rate by alamar blue.As shown in Fig. 7 A and Fig. 7 C, hMSC can well adhere to and sprawl in micro-ice glue, and compared with 0 day, cell has had significant proliferation.As shown in Figure 8 B, after injection, the hMSC in micro-ice glue keeps good vigor.
Six, the micro-ice glue that is mounted with cell can also be fixed a point to induce new vessels to generate by locating injection in Mice Body
The cell adsorbing medium of this experiment is sodium alginate, and cell is RFP3T3, is expelled to the formation of the subcutaneous observation induction of mice new vessels.
Cell Sodium Alginate Hydrogel Films suspension: according to cell suspension: 2% sodium alginate=1:1 mixes, and making cell density is 2 × 10 7the cell Sodium Alginate Hydrogel Films suspension of/ml.
Micro-ice mucilage binding carries cell: cell Sodium Alginate Hydrogel Films suspension is loaded on micro-ice offset plate, and resuspended with carboxymethyl cellulose after collecting, mix homogeneously, density is 1000/ml.Meanwhile, another is organized micro-offset plate and only loads sodium alginate, resuspended with carboxymethyl cellulose after collecting, mix homogeneously, and density is 1000/ml.
Mouse subcutaneous injection: by mice Avertin(0.12ml/10g) after anesthesia, be expelled to mice with the resuspended good micro-ice glue of 1ml syringe extraction subcutaneous, be divided into matched group and experimental group.After injection, normally raise mice 21 days.Matched group: the three-dimensional microenvironment carrier of micro-ice glue of injection only adsorbs sodium alginate, experimental group: the three-dimensional microenvironment carrier adsorption of the micro-ice glue cell Sodium Alginate Hydrogel Films suspension of injection.
Paraffin section and HE dyeing are observed.After 21 days, mouse anesthesia is put to death, the micro-ice glue of taking-up injection point, carries out routine paraffin wax section and HE dyeing is observed.
Result is as Fig. 9.Show, compared with matched group, RFP3T3 obviously induces generation new vessels, illustrates that cell has good function in micro-ice glue.
Prepare the three-dimensional microenvironment carrier of micro-ice glue according to method described in embodiment 1, method is substantially the same manner as Example 1, the varying in size of pit-holes that different is in mould, and specifically the diameter of pit-holes cross section is respectively 100 microns, 200 microns, 800 microns.The three-dimensional microenvironment carrier of the micro-ice glue of result gained form is under the microscope as Figure 10.Three-dimensional micro-gained ice glue microenvironment carrier is carried out respectively testing one in embodiment 2, detect its mechanical performance.As a result, in the process of the micro-ice glue of injection, the three-dimensional microenvironment carrier of micro-ice glue, smoothly by the entry needle of 27G, occurs without clogging, and after injection in percentage of head rice and embodiment 1 the micro-ice glue three-dimensional of gained microenvironment carrier without significant difference.

Claims (10)

1. the three-dimensional microenvironment carrier of micro-ice glue, the material that forms this carrier is ice glue, this carrier size belongs to micron order.
2. prepare the method for the three-dimensional microenvironment carrier of micro-ice glue for one kind, comprise the steps: the material mixing for making ice glue, then mixture is divided into micron order size, the process conditions of recycling ice making glue are processed, and obtain the three-dimensional microenvironment carrier of micro-ice glue.
3. method according to claim 2, it is characterized in that: the described method that mixture is divided into micron order size comprises the steps: described mixture to be placed in the pit-holes of mould, described mould structure is the flat board with pit-holes, and the size of described pit-holes is micron order.
4. it is characterized in that according to the method in claim 2 or 3: described is crosslinkable material for making the material of ice glue;
Biomaterial and/or natural biologic material that described crosslinkable material is synthetic;
The biomaterial of described synthetic be following at least one: Polyethylene Glycol, polyethyleneglycol derivative, polyethyleneglycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alcohol acid copolymer, polydimethylsiloxane, poly-anhydride, poly-acid esters, polyamide, polyamino acid, polyacetals, polybutylcyanoacrylate, polyurethanes, polypyrrole, polyester, polymethacrylates, polyethylene, Merlon and polyethylene glycol oxide;
Described natural biologic material be following at least one: gelatin, gelatine derivative, alginate, alginate derivant, agar, matrigel, collagen, proteoglycan, glycoprotein, hyaluronic acid, layer connect albumen and fibronectin;
Concrete, described is polyethyleneglycol diacrylate and Ammonium persulfate. and N for making the material of ice glue, N, N', N'-tetramethyl diethylamine, its proportioning is 5-50g polyethyleneglycol diacrylate: 0.5g Ammonium persulfate.: 0.025-0.2gN, N, N', N'-tetramethyl diethylamine.
5. according to arbitrary described method in claim 2-4, it is characterized in that: the process conditions of described ice making glue comprise: be that under the condition of-80 DEG C to 0 DEG C, reaction exceedes 1h in temperature.
6. according to arbitrary described method in claim 2-5, it is characterized in that: the process conditions of described ice making glue also comprise: after completion of the reaction, in air drying or lyophilization.
7. according to arbitrary described method in claim 2-6, it is characterized in that: described method also comprises the step of the three-dimensional microenvironment carrier of the micro-ice glue of enrichment.
8. according to arbitrary described method in claim 2-7, it is characterized in that: the method for the three-dimensional microenvironment carrier of the micro-ice glue of described enrichment is following 1) or 2):
1) top board is buckled on described mould, utilizes the bulge-structure on top board that three-dimensional the micro-ice glue in the pit-holes of described mould microenvironment carrier is peeled off out; Described top board is one to have the flat board of bulge-structure, and the position of bulge-structure on flat board is corresponding with the pit-holes position in described mould;
2) mould that contains the three-dimensional microenvironment carrier of micro-ice glue in pit-holes is placed in to liquid, then the three-dimensional microenvironment carrier of micro-ice glue dissociates out automatically owing to expanding from mould, just obtains the three-dimensional microenvironment carrier of micro-ice glue after collection.
9. described in claim 1 following 1 of the three-dimensional microenvironment carrier of micro-ice glue) to 3) in arbitrary described application:
1) application of the three-dimensional microenvironment carrier of micro-ice glue in the carrier for the preparation of biological sample injection described in claim 1;
2) described in claim 1, the three-dimensional microenvironment carrier of micro-ice glue has the application in the carrier of following function in preparation: after biological sample injection, be not free on injection position for injecting biological sample and making;
3) application of the three-dimensional microenvironment carrier of micro-ice glue in the carrier of transplanting clinical injection treatment for the preparation of biological sample described in claim 1.
10. utilize the method that the three-dimensional microenvironment carrier of micro-ice glue adsorbs biological sample in vitro described in claim 1, comprise the steps: biological sample solution to mix with the matrix solution of attachment point is provided for biological sample, obtain mixed liquor; Carry out again following a) or step b):
A) prepare the three-dimensional microenvironment carrier of micro-ice glue according to arbitrary described method in claim 2-8, before the three-dimensional microenvironment carrier of the micro-ice glue of enrichment, described mixed liquor is added drop-wise on the three-dimensional microenvironment carrier of micro-ice glue, drive mixed liquor slowly to scrape back and forth uniformly with slide, described mixed liquor is autonomous to be sucked in the three-dimensional microenvironment carrier of micro-ice glue;
B) prepare the three-dimensional microenvironment carrier of micro-ice glue according to arbitrary described method in claim 2-8, after the three-dimensional microenvironment carrier of the micro-ice glue of enrichment, three-dimensional micro-ice glue microenvironment carrier is collected in container, form the three-dimensional microenvironment carrier layer of micro-ice glue, described mixed liquor is added drop-wise in the three-dimensional microenvironment carrier layer of micro-ice glue, and described mixed liquor is autonomous to be sucked in the three-dimensional microenvironment carrier of micro-ice glue.
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