CN107930542A - One-step method continuously prepares the microflow control technique of calcium alginate microgel - Google Patents

Abstract

The present invention be based on miniflow drop technique realize one-step method continuously prepare can immobilized bioactive substance, multi-chamber calcium alginate microgel, can realize high throughput, continuous production microgel material.Parallel, steady flow condition can be formed first with different hydrogel prepolymer solutions in microchannel, so as to which the hydrogel prepolymer solution injection micro flow chip for forming microgel different chamber to be formed to the aqueous phase solution of multiphase parallel fluid, the microchannel then designed by T-shaped passage or fluid focus mixes it with the fluid (oil phase) not being blended, form water-in-oil emulsion drop, trigger the crosslinking of alginic acid in drop after droplet formation immediately, realize prepared by multi-chamber microgel；Then by realizing the cleaning of emulsion droplet on micro flow chip, so that microgel is quickly transported in water phase, realize that one-step method prepares the immobilized calcium alginate microgel for having bioactive substance, particularly suitable for commercial application.

Description

One-step method continuously prepares the microflow control technique of calcium alginate microgel

Technical field

The invention belongs to material micro Process field, biomaterial preparing technical field, organizational project and cell therapy field, It is more particularly to a kind of that the microgel for embedding immobilized active somatic cell or bioactive molecule medicine is prepared using micro fluidic device.

Background technology

In recent years, hydrogel is all able to extensive use in many biomedical sectors, especially in field of tissue engineering technology, water Gel has become very important biomaterial.Hydrogel is molecular by high score that is not soluble in water but being water-swellable Cross-linked network, it is similar with human tissue organ, it is to be made of the porous network rich in water, such loose structure is similar to human body Histoorgan, is especially advantageous for material and effectively exchanges.Meanwhile some have good biology by the molecular hydrogel of natural polymer Compatibility and biodegradability, and with the approximate mechanical performance of tissue, therefore, it can be used as tissue engineering bracket material Expect the growth and regeneration of sertoli cell.A branch in hydrogel material is made of large biological molecule performed polymer, its With good biocompatibility, gelation conditions are gentle, thus this kind of material is widely used in the three dimensional envelope of cell.Its In, many natural macromolecular material including collagens, fibrin, or alginate etc. belongs to such material, it is a large amount of Ground is used for the embedding of cell.Although hydrogel is as bio-medical material, in field of tissue engineering technology by numerous studies and use, But conventional hydrogels embedding cell technology organizational project application in still suffer from all urgent need to resolve the problem of：1) block hydrogel Size it is larger (>1 cm), macromolecule network aperture dimensions in nanoscale, large biological molecule due to diffusion rate and apart from low, because This is embedded in active somatic cell in block gel since nutriment and metabolite exchange efficiency are low and survival rate declines；2) water Gel passs the carrier released as bioactive substance and does not possess syringeability, can only carry out block implantation, and cannot utilize minimally invasive Intervening mode is implanted into；3) block hydrogel is used for during macromolecular or cell embedding, it is difficult to ensures load material solidifying It is uniformly distributed in colloid system.On the contrary, the carrier using microgel as the bioactive substance of micro-meter scale is asked for solution is above-mentioned Topic provides effective scheme.Because microgel scale is small, is conducive to material and quickly spreads, being encapsulated in cell therein can be with the external world Nutritional ingredient, signal factor and excreta etc. can realize effective exchange.Meanwhile micro-gel particles can be noted directly Penetrate, the direct intravenous injection transplanting for active macromolecules medicine and active somatic cell provides effective approach.Therefore, realize and carry carefully The high throughput of born of the same parents' microgel prepares the development and clinical practice for being beneficial to promote organizational project and cell therapy technology.

Emulsion technology is widely used in preparation encapsulating bioactive substance, and (such as bioactive macromolecule medicine or live body are thin Born of the same parents) microgel.This method fluid is not usually blended using water phase and an oil phase dispersed phase and continuous phase is formed under shearing force. Typically preparation method is：First, bioactive substance (such as bioactive macromolecule or active somatic cell) is dispersed in water-setting In the performed polymer aqueous solution of glue, which can form the single emulsion of Water-In-Oil with the blending of continuous oil phase, then induce polymerization Or cross-linking reaction makes the hydrogel performed polymer gelation in drop obtain cured microgel.These traditional emulsion methods exist with Lower problem：1) active somatic cell needs to be dispersed in for a long time in scattered micro emulsion, and cell can not be with the carry out nutrients outside drop The exchange of matter and gas, therefore the metabolic activity of cell gradually weakens, and can cause the drop of cytoactive in lotion for a long time It is low；2) it is the stable lotion of formation, generally requires to add surfactant in oil phase or water phase, and surfactant is often Destroy cell membrane and produce cytotoxicity, therefore there is also potential cytotoxicity to ask for contact surfactant for a long time for cell Topic；3) to trigger the polymerization of hydrogel or crosslinking to usually require to use cytotoxic crosslinking agent or cross-linking reaction initiator, And cell long-period contacts the problem of these components equally exist cytotoxicity.Therefore, it is immobilized micro- to prepare cell for traditional emulsion method Gel technique is difficult to maintain the high viability of cell and the metabolic activity of cell, and needs to carry out additionally time-consuming after immobilized The work of laborious oil phase cleaning, therefore it is not used to industrialization Continuous maching.

The a collection of micro-processing technology immobilized available for cell, including optical etching technology, micro- mould plate technique, breast are emerged in the recent period , all there is the problem of how maintaining immobilized cell survival rate in liquid technology etc., be accordingly difficult to really for the immobilized microgel of cell Industrial scale produces.Moreover, these conventional arts are all based on the production technology of mass, can not realize high-throughout continuous Prepare, and properties of product have differences between batch.

Microfluidic droplet technology is that the micro-processing technology of immiscible heterogeneous fluid, the skill are accurately controlled based on micro-fluidic chip Art can realize continuous sample introduction, quickly produce monodispersity, accurately control the microgel or microcapsules of size.Wrapped with traditional oil Water (W/ O) or oil-in-water (O/W) single emulsion drop technique are compared, and can pass through the miniflow with T-shaped runner or fluid focus structure Control device prepares the single emulsion drop with single-size.And it can be prepared as template by different polymerization methods single Scattered microgel.However, identical with conventional emulsion method, micro-fluidic single emulsion drop technique is not particularly suited for Continuous maching and carries carefully Born of the same parents' microgel.This is because during sample is prepared, can be for a long time exposed to oil phase, surface-active by immobilized cell Agent, crosslinking agent etc. and cause cytotoxicity.Therefore, it is necessary to interruption collection product and abolish lotion rapidly, cell is transferred to In the aqueous phase solution of biocompatibility, to keep the cell survival rate in the microgel prepared, process is time-consuming and laborious.Cause This, how to realize cell by it is immobilized enter microgel and quickly separation will greatly ensure cytoactive from oil phase, and can be real It is now continuous to prepare, improve production efficiency.

In addition, water-water (W/W) emulsion technology can also realize the preparation of microgel.This method uses aqueous two phase flow Body, drop can be formed using two alternate are not blended, and using drop as template, is realized and produced microgel particulate in aqueous. This method avoid it is extra the step of abolishing lotion, it can be achieved that one-step method cell embedding.However, this system be limited to it is specific not The combination of the aqueous solute of blending, such as glucan and polyethylene glycol, and aqueous solute concentration is high, so as to limit this method In the extensive use of the immobilized aspect of cell.

In conclusion although carry cell microgel technology has important application value in biomedical sector, but such as What realizes the immobilized survival rate for being embedded in microgel, and maintaining cell at the same time of cell, is still the skill of this field urgent need to resolve Art problem.This promotes researcher to develop new technology simpler, more efficient, that industrialization production can be achieved.It is although micro- solidifying Glue material has important application potentiality in biomedicine field, its function and structure is relatively single, it is difficult to carries out more functions Property extension.Therefore multi-chamber microgel material becomes the hot spot that biological medicine is applied and studied in the recent period.The microgel of multi-chamber The microgel material that grain is made of two or more different chamber, different chamber can be identical or different material.With tradition list Chamber microgel is compared, and multi-chamber microgel has multiplet, can assign different functions.Multi-chamber microgel is due to multiple Miscellaneous 26S Proteasome Structure and Function, it is immobilized and pass the field of releasing and have important application in biomedicine field, especially bioactive substance Potentiality.The existing method for preparing multi-chamber microgel material is typically by several different hydrogel monomers or the water of performed polymer Solution, emulsify and quickly triggers polymerization or crosslinking, so as to obtain the micro-gel particles of stable multi-chamber structure.It is common Prepare the material of multi-chamber microgel have can by the hydrogel performed polymer of photo-initiated crosslinking, or can fast ionic it is crosslinked Alginate etc..But these quick, violent cross-linking reactions usually have very strong bio-toxicity, can influence by immobilized material During bioactivity, especially embedding cell, the universal cytotoxicity of these methods is strong, hence in so that cell survival rate is decreased obviously, Limit its development and application in this field.Multi-chamber microgel is due to complicated 26S Proteasome Structure and Function, in biomedicine field, especially It is that bioactive substance is immobilized and pass the field of releasing and have important application potential.

The existing technology for preparing multi-chamber microgel includes microfluidic chip technology【Y.Du,E.Lo,S.Ali,A. Khademhosseini,P.Natl Acad.Sci.2008,105,9522-9527；S.Seiffert, Angew.Chem.Int.Ed 2013, 52,11462-11468】, fluid countermark technology【D.Dendukuri, D.C.Pregibon,J.Collins,T.A.Hatton,P.S. Doyle,Nat.Mater.2006,5,365-369；b) D.Dendukuri,S.S.Gu,D.C.Pregibon,T.A.Hatton,P.S. Doyle,Lab.Chip 2007,7,818- 828.】And centrifugation preparation process【K.Maeda,H.Onoe,M.Takinoue,S. Takeuchi,Adv.Mater.2012,24, 1340-1346】。

Fluid countermark technology【D.Dendukuri,D.C.Pregibon,J.Collins,T.A.Hatton,P.S.Doyle, Nat. Mater.2006,5,365-369；b)D.Dendukuri,S.S.Gu,D.C.Pregibon,T.A.Hatton, P.S.Doyle,Lab. Chip 2007,7,818-828.】It is the hydrogel using photopolymerization to prepare multi-chamber microgel technology The fluid solution of monomer or performed polymer as composition microgel different chamber, the structure of microgel is controlled by photomask structure, Aspherical micro-gel particles are usually prepared, which can only control multi-chamber structure in two-dimentional dimension, and cannot be in three-dimensional Space controls the dimension of microgel；And prepolymer solution, encapsulated medicine or loss cell are larger in prepared by this method, bag Envelope usually less than 50%.

Centrifuge preparation process【K.Maeda,H.Onoe,M.Takinoue,S.Takeuchi,Adv.Mater.2012,24, 1340-1346】It is to be added by regarding alginate aqueous solution as hydrogel performed polymer in the capillary of multichannel, utilizes centrifugation Technology injects liquid in capillary in the solution containing calcium ion.But the structure design due to multiple-pass capillary tube and it is processed into This height, therefore this method cost is higher；And since the performed polymer addition in capillary is limited, it is impossible to lasting sample-adding, therefore It can not realize Continuous maching, be unsuitable for industrialized production；Meanwhile the technology is difficult to use in cell encapsulating, this is because crosslinking sea Alginic acid needs to use high concentration calcium ion solution, is unfavorable for cell survival.

Microfluidic chip technology【Y.Du,E.Lo,S.Ali,A.Khademhosseini,P.Natl Acad.Sci.2008, 105, 9522-9527；S.Seiffert,Angew.Chem.Int.Ed 2013,52,11462-11468】Utilize fluid focus Micro flow chip design, by form emulsion droplet and trigger quick-gelatinizing reaction realize multi-chamber microgel prepare.The party Method usually requires violent gelation reaction, so that multi-chamber structure is quickly fixed, when being embedded applied to bioactive substance, Protein molecular easily loses bioactivity, and cell survival rate declines；Meanwhile existing process prepares microgel first with micro flow chip Particle, needs second step cleaning to elute oil phase, microgel is scattered in again in aqueous solution after collection, therefore can not realize continuous Prepared by sample introduction, be unsuitable for commercial application.

The content of the invention

The present invention is to realize that continuous prepare of one-step method can immobilized bioactive substance, multi-chamber sea based on miniflow drop technique Calcium alginate microgel, can realize high throughput, continuous production microgel material.It is pre- first with different hydrogels in microchannel Oligomer solution can form parallel, steady flow condition, so that the hydrogel prepolymer solution for forming microgel different chamber be noted Enter the aqueous phase solution that micro flow chip forms multiphase parallel fluid, the microchannel then designed by T-shaped passage or fluid focus It is mixed with the fluid (oil phase) not being blended, water-in-oil emulsion drop is formed, triggers seaweed in drop immediately after droplet formation The crosslinking of acid, realizes prepared by multi-chamber microgel；Then by realizing the cleaning of emulsion droplet on micro flow chip, so that micro- Gel is quickly transported in water phase, realizes that one-step method prepares the immobilized calcium alginate microgel for having bioactive substance.Should Preparation method good biocompatibility, the life for active material (such as bioactive macromolecule or active somatic cell) that can keep being embedded very well Thing activity, is high throughput, the continuous effective ways for preparing biological medicine microgel product.

The present invention discloses the microflow control technique that a kind of one-step method continuously prepares calcium alginate microgel, includes the following steps：

(1) solution is prepared

1. preparing seaweed aqueous acid in water by dissolution of raw material of water soluble algae hydrochlorate, then add cross-linking reaction and draw Agent is sent out, then bioactive substance and/or nano particle are scattered in above-mentioned aqueous solution and obtain hydrogel prepolymer solution, as system The aqueous phase solution of standby water-in-oil emulsion system, wherein：

The cross-linking reaction initiator is selected from chelate aqueous solution, the chelating of calcium-nitrilotriacetic acid of calcium-ethylenediamine tetra-acetic acid One or more of combination in thing aqueous solution, cium carbonate nanoparticles, sulfuric acid calcium nano, calcium phosphate nanoparticles；It is described The final concentration of cross-linking reaction initiator is denoted as 10-1000mM with calcium content；It is preferred that 20-500mM, more preferably 25-100mM；

2. fluorinated oil, fluorinated surfactant and acidic materials are blended, the first heavy oil as water-in-oil emulsion system Phase；

3. the mixed solution that fluorinated oil is obtained with perfluor alcohol or perfluor acid blending is the second heavy oil phase；

(2) aqueous phase solution for 1. obtaining step injects micro flow chip with the first flow velocity, with second flow speed by the first heavy oil Phase solution injects micro flow chip from the second input port, and aqueous phase solution and the first heavy oil are mutually blended through Over emulsfication passage, pass through emulsification The two-phase not being blended is blended passage, forms water-in-oil emulsion drop；Alginic acid in emulsification passages downstream, single emulsion drop Generation gelation reaction, rapid curing (time was at 0.00001-1 seconds) form alginic acid microgel；

(3) the second heavy oil is mutually injected by micro flow chip, the second heavy oil phase and step (2) by the 3rd input port with the 3rd flow velocity Gained lotion is fully blended in the hybrid channel for being arranged at emulsification passages downstream, is dispersed with the oil mixing with water of alginic acid microgel Thing flows out chip from output channel；The solution of output is inputted by conduit and collects phase aqueous solution, makes to be loaded with bioactive substance And/or the calcium alginate microgel dispersion of nano particle makes oil phase be mutually automatically separated with water, sinks in phase aqueous solution is collected The bottom of aqueous solution, alginic acid microgel are dispersed in aqueous solution automatically；Aqueous phase solution is collected up to finished product.

In technique described above scheme, specifically, there is the micro flow chip fluid focus structure, T-shaped to mix The microchannel of structure, concurrent ejector half or cross structure, micro flow chip also have at least 3 phase liquid inlets, and emulsification Passage and output channel.The 3 phase liquid inlets include：Aqueous phase solution input port, the first heavy oil phase input port and second Heavy oil phase input port；And the microchannel interior walls surface carries out hydrophobic treatment.

In technique described above scheme, specifically, the seaweed acid starting material is alginic acid, alginate, alginic acid The mixture of saline solution or alginic acid and water soluble polymer.The water soluble polymer is selected from collagen, gelatin, hyalomitome One or more of combinations in acid, polyethylene glycol, polyvinyl alcohol, polyacrylamide, glucan, chitosan, agarose.

In technique described above scheme, specifically, all seaweed acid starting material total concentrations are 0.1- in prepolymer solution 8w/v%.

In technique described above scheme, specifically, described in step (1) be embedded bioactive substance and/or Nano particle is selected from following one or more of combinations：Active somatic cell, water miscible activated protein drug molecule, nano particle； Those skilled in the art can make choice according to the difference for implementing purpose, such as：

The active somatic cell is generally in primary cultured cell, cultured cell line, cell line culture cell and heterozygote One or more；

The water miscible activated protein drug molecule be generally protein drug, polypeptide drug, enzyme drug and One or more in Porcine HGF.

Protein drug, such as：Seralbumin, gamma globulin, insulin；

Polypeptide drug, such as：Oxytocins, hyperglycemic factor；

Enzyme drug, such as：Digestive ferment (pepsin, pancreatin, malto-amylase), anti-inflammatory enzyme (lysozyme, tryptose Enzyme), treating cardiovascular disease enzyme (kallikrein expansion blood vessel blood pressure lowering) etc.；

Porcine HGF, such as：Interferon, interleukins, tumor necrosis factor, ostosis form albumen -2, bone Form protein-7, fibroblast growth factor, into angiogenesis factor etc.

The nano particle is generally：The metals such as nanogold, nano silver, nano-sized iron oxide or metal oxide nano The high molecular polymer nano particles such as grain, polyethylene, polypropylene, polystyrene, polymethyl methacrylate, polylactic acid, liposoluble Property vitamin, quinolones water-insoluble medicine nano particle, hydroxyapatite, silica, calcium phosphate, carbon nanotubes, stone One or more in black alkene inorganic non-metallic material nano particle.Nanoparticle size diameter 5-1000nm.

In technique described above scheme, specifically, the fluorinated oil is selected generally from following one or more of combinations： Perflenapent, perflexane, PF 5070, perfluoro butyl-methyl ether, perfluorooctane, Perfluorononane, perfluoro decane, perfluor ten One alkane, perfluor dodecane, perfluor tridecane, the perfluor tetradecane, perfluor pentadecane, perfluor hexadecane, perfluor heptadecane, perfluor naphthalene Alkane.

In technique described above scheme, specifically, the fluorinated surfactant is selected generally from following a kind of or several The combination of kind：Perfluoroether (perfluorinated ethers, PFPE), perfluoroalkyl acid perfluoroether-polyvinyl alcohol block copolymerization Thing, perfluoroether-polyvinyl alcohol-perfluoroether block copolymer surfactant；Wherein the concentration of fluorinated surfactant is 0.1- 10wt%, preferably 0.5-5wt%.

In technique described above scheme, specifically, the acidic materials are selected from following one or more of combinations：Sulphur Acid, nitric acid, hydrochloric acid, carbonic acid, phosphoric acid, acetic acid or citric acid, concentration of the acidic materials in mixed solution are 0.001- 20v/v%；

In technique described above scheme, specifically, perfluor alcohol is selected generally from following one in the second heavy oil phase Kind or several combinations：Seven fluoro- n-butyl alcohols of 22,33,444-, perfluor undecyl alcohol, 1H, 1H- perfluor-DODECANOL, 1-, 1H, 1H- Perfluor -1-heptanol, 1H, 1H, 2H, 2H- perfluorooctanols, 1H, 1H- perfluoro capryl -1- alcohol, 1H, 1H- perfluor -1 nonyl alcohol, 1H, 1H, 2H, 2H- perfluor -1- lauryl alcohols, 1H, 1H, 2H, 2H- perfluor -1- decyl alcohol, 1H, 1H- perfluors -1- 14 (alkane) alcohol, perfluor penta Alcohol, perfluor hexanol, perfluor enanthol, perfluorooctanol, perfluor nonyl alcohol, perfluor decyl alcohol；

The perfluor acid is selected generally from following one or more of combinations：Perfluor lauric acid/dodecanoic acid, positive perfluor valeric acid, 5H- are complete Fluorine valeric acid, perfluoro caprylic acid, perfluoro-pelargonic acid, perfluoro decanoate, perfluoro-heptanoic acid, perfluor caproic acid, perfluorobutyric acid, perfluorinated undecanoic acid；The The concentration of perfluor alcohol or perfluor acid in fluorinated oil is 1-100v/v%, preferably 10-50v/v% in double oil phase.

In technique described above scheme, specifically, the collection phase aqueous solution described in step (4) is acellular poison Property, suitable for cell culture, pH buffering ranges in the buffer solution of 6-8, can dilute and neutralize crosslinking agent, surface-active in water phase The solution of agent, those skilled in the art can be specifically chosen according to sample situation, under normal circumstances, selected from following one or more Combination：The buffer solutions such as HEPES buffer solution, cell culture medium, phosphate buffer (PBS), disodium hydrogen phosphate-potassium dihydrogen phosphate delay Fliud flushing, PBS buffer, disodium hydrogen phosphate-citrate buffer solution, citric acid-sodium hydroxide-hydrochloride buffer, citric acid-lemon Sour sodium buffer solution, potassium dihydrogen phosphate-sodium hydrate buffer solution, barbital sodium-hydrochloride buffer, Tris-hydrochloride buffer, boron Acid-borate buffer solution cell culture medium；Wherein, the ion concentration 10-2000mM of buffer solution, preferably 100-200mM.

In technique described above scheme, specifically, interior phase aqueous solution, the first heavy oil that the multiple fluid is formed It is respectively mutually mutually 5-2000 μ L/hr using the first flow velocity by micro pump or micro-syringe with the second heavy oil, second flow speed 200- 20000 μ L/hr, the 3rd flow velocity are that the flow velocity of 200-20000 μ L/hr is delivered in the respective microchannels of micro fluidic device, are formed The double lotions of monodispersed water/oil/water.

Preferable first flow velocity is 10-500 μ L/hr, more preferably 20-100 μ L/hr；

Preferable second flow speed is 500-3000 μ L/hr, more preferably 800-1000 μ L/hr；

Preferable 3rd flow velocity is 500-3000 μ L/hr, more preferably 800-1000 μ L/hr.

In technique described above scheme, specifically, the aqueous phase droplets of formation from upstream water phase and an oil phase cross passage to It is 0.1-30s, preferably 0.1-10s that channel time, which is mutually blended, in second heavy oil；To ensure that alginic acid performed polymer is sent out in chip channel Raw gelation obtains alginic acid microgel.Micro-gel particles collect the time of water-soluble liquid phase to injection after being mutually blended with the second heavy oil For 0.1-30s, preferably 1-10s；So as to ensureing that two-phase is sufficiently mixed, while when avoiding the micro-gel particles from being stopped under cross linking conditions Between it is long and cause cytotoxicity.

In technique described above scheme, specifically, the velocity ratio of the aqueous phase flow rate and the first heavy oil phase is 0.01 Between~1, preferably 0.1~0.5；First weight Oil phase flow rate and the second heavy oil phase velocity ratio are 1:0.5~50, preferably 1:0.5~ 10。

Another object of the present invention is to disclose one-step method described above and continuously prepare the micro- of calcium alginate microgel The microgel product that fluidics is prepared；A diameter of 5~1000 μm of the microgel；The coefficient of dispersion of particle diameter distribution exists 1~6%, microgel prepared by the method for the present invention is prepared into water-in-oil emulsion from micro flow chip and is dispersed in oil phase, to through output Aqueous solution is collected in mouth injection and thermoplastic polymer is in aqueous solution, and time of the microgel dispersion in oil phase is 1~60 second；When When active somatic cell is embedded with microgel, residence time was at 1~60 second in lotion for cell, the survival rate of cell>85%.

Another object of the present invention is to disclose one-step method and continuously prepare the micro- of multi-chamber calcium alginate micro-gel particles Fluidics, the difference of its microflow control technique for continuously preparing calcium alginate microgel with previously described one-step method are：Increase Add the quantity of water phase input port in the species and corresponding increase micro flow chip of hydrogel prepolymer solution；

Prepared by the microgel of usual single-chamber room need 3 input ports (1 water mutually inputs, 2 oil phase inputs)；Multi-chamber it is micro- Prepared by gel is the aqueous phase solution input port needed more than 2, other are constant.Specifically, the aqueous phase solution input port is used In input hydrogel prepolymer solution, therefore, when those skilled in the art are as needed, standby multi-chamber calcium alginate of drawing up is micro- solidifying When glue, then the species of increase hydrogel prepolymer solution is needed, correspondingly, being also required to increase aqueous phase solution input port quantity.

When preparing multi-chamber calcium alginate micro-gel particles, according to the method for step 1., multiple biological activities thing is selected Matter and/or nano particle are prepared to form the hydrogel prepolymer solution of microgel different chamber, different hydrogel pre-polymerizations Liquid solution is injected respectively as two dimension, three-dimensional or multidimensional parallel fluid by multiple water phase input ports on micro flow chip, is converged as water After phase solution, according to step 2., 3., (2), the method for (3) prepare multi-chamber micro-gel particles.

In the technical solution for preparing multi-chamber micro-gel particles described above, specifically, multiple input seaweed sour water Flow velocity summation between the aqueous phase stream body of gel performed polymer is 5-2000 μ L/hr；Difference between each phase performed polymer aqueous solution input flow velocity Different multiple is 1-100 times, and related with obtaining multi-chamber microgel chamber size.

In the technical solution for preparing multi-chamber micro-gel particles described above, in specific cross-linking process, contain in drop Various bioactivators and/or the seaweed acid solution of nano particle occur gelation reaction at the same time, rapid curing forms tool There is the alginic acid microgel of multi-chamber structure；Form the alginic acid in each phase hydrogel prepolymer solution of microgel different chamber Between concentration, its concentration difference multiple is 1-10 times (concentration difference of i.e. each indoor seaweed acid starting material of chamber).

Beneficial effect

1) realize prepared by the microgel one-step method of immobilized active somatic cell and bioactive substance, it is micro- can continuously to prepare multi-chamber Gel, suitable for commercial application.

2) this method is based on alginic acid microgel material, and good biocompatibility, cytotoxicity is low, and microgel is in lotion Residence time is less than 30 seconds, is just rapidly collected in the inner in aqueous solution, is conducive to be encapsulated the holding of substance bioactivity, immobilized thin The survival rate of born of the same parents is high.

3) preparation that complex three-dimensional structure microgel can be achieved is designed by micro flow chip passage, microgel chamber quantity can Reach more than 6；It can be achieved to carry cell or carry the continuous of medicine micro-gel particles, high throughput preparation.

4) being designed by micro flow chip, the size range of microgel is in 10-800 μm of regulation and control, and particle size distribution is narrow, from Divergence is less than 5%.Droplet size is decided by the scale of passage, and then drop is bigger greatly for channel dimension；Oily two velocity ratios of water at the same time Also droplet size is influenced, the more big then drop of interior phase flow velocity is bigger.

5) it is prepared by the microgel that hydrogel material difference crosslinking method can be achieved.

6) encapsulation efficiency of bioactive substance reaches 100%.

7) microgel can be used as extracellular matrix, and sertoli cell is survived in microgel and function.

8) micro-gel particles can be crosslinked to obtain the load cytoskeleton with macrostructure as basic structural unit, or It is used for the transplanting of cell as injectable materials.

Brief description of the drawings

Fig. 1 is the preparation principle figure that alginic acid microgel is continuously prepared by micro flow chip one-step method.5. refer in Fig. 1 Alginic acid in drop starts to be cross-linked to form micro-gel particles.

Fig. 2 is the structure diagram of the typical T-shaped hybrid channel described in embodiment 1.1 is the water containing bioactive substance Phase, 2 be the oil phase of continuous phase.

Fig. 3 is the structure diagram of the typical fluid focusing hybrid channel described in embodiment 1.1 is living containing biology Property material water phase, 2 be continuous phase oil phase.

Fig. 4 is micro- for the microgel being dispersed in aqueous phase solution is prepared by LAYER FLUID focusing micro flow chip one-step method Grain.(a) it is water phase that phase separation is formed after lotion is destroyed, wherein containing substantial amounts of microgel ball (c), is encapsulated in microgel There is the glucan (molecular weight 5kDa) of green fluorescent label, can be quickly permeable from microgel since molecular weight is smaller； (b) be continuous phase oil phase.

Fig. 5 is the Size Distribution of micro-gel particles prepared by 1 the method for embodiment.

Fig. 6 is the micro flow chip design principle figure that water-in-oil emulsion drop is prepared in comparative example 1.4. refer to liquid in Fig. 6 Alginic acid starts to be cross-linked to form micro-gel particles in drop.

Fig. 7 is the microgel being dispersed in oil phase for focusing on micro flow chip preparation described in comparative example 1 by a layer fluid Drop.(a) be using micro flow chip prepare emulsion droplet (since wherein aqueous phase densities are less than oil phase, in upper strata heap Product), contain crosslinked micro-gel particles in (c) drop, wherein being encapsulated with the glucan (molecular weight of green fluorescent label 5kDa)；(b) be continuous phase oil phase.

Fig. 8 is the micro-gel particles size different in flow rate than being formed.

Fig. 9 is micro- for the load cell being dispersed in aqueous phase solution is prepared by LAYER FLUID focusing micro flow chip one-step method Microgel particle.

Figure 10 is the survival rate that the immobilized cell of microgel is carried out by different micro flow chip methods.

Figure 11 is the seaweed acid gel prepared using different crosslinking agents.Wherein, A be using 1 the method for comparative example, with The alginic acid microgel that Ca-NTA is prepared as cross-linking reaction initiator, B are using 2 the method for comparative example, with CaCO₃Receive The seaweed acid gel that rice grain is prepared as cross-linking reaction initiator.

Figure 12 is the preparation principle figure that multi-chamber alginic acid microgel is continuously prepared by micro flow chip one-step method, wherein, 1st, 2,3,4, n- forms the hydrogel prepolymer solution of microgel different chamber, A- water phases, B- fluorinated oils, and C- contains perfluor alcohol Oil phase, 4D output channels, E- is that the alginic acid in drop starts to be cross-linked to form micro-gel particles；Hydrogel performed polymer excites Crosslinking, multiple interior phase blending area M1, forms the blending of water-in-oil emulsion drop and removes M2 and triple oil phase blending area M3.

Figure 13 is the micro-gel particles of two chambers prepared by preparation method described in embodiment 4.

Figure 14 is the micro-gel particles of three chambers prepared by preparation method described in embodiment 5.

Figure 15 is the micro-gel particles of four chambers prepared by preparation method described in embodiment 6.

Figure 16 is to be prepared to be dispersed in water by LAYER FLUID focusing micro flow chip one-step method in method shown in embodiment 7 Microgel particulate in phase solution.It is to use different two-phase alginic acid performed polymer aqueous solutions to pass through adjusting as input item to scheme A The alternate velocity ratio of two inputs realizes the control to preparing dual cavity microgel chamber size.It is figure A respectively to scheme a, b, c, d in B The fluorescent microscopy images for the micro-gel particles that corresponding difference preparation parameter obtains in curve.

Figure 17 is the micro-gel particles with two chamber structures that described in embodiment 8 prepared by method.

Figure 18 be 9 the method for embodiment realized using dual cavity microgel as template two kinds of different cells it is controllable, The three-dimensional immobilized and assembling of individual cell level.Figure A, C are the fluorescence micrographs of different amplification, and figure B, D are visible light fields Microphoto.Scale is 50 μm in figure.

Figure 19 is using the micro flow chip and preparation method and parameter described in embodiment 4, is prepared with two chambers The microgel of structure.

Embodiment

Subordinate's non-limiting example can make those of ordinary skill in the art be more fully understood the present invention, but not with Any mode limits the present invention.In addition, in following embodiments, unless otherwise specified, used experimental method is conventional side Method, material therefor, reagent etc. can be bought from biological or chemical Reagent Company.

Perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer surfactant purchase is certainly northern Jing Anxin micro ＆ nano technologies Co., Ltd；All chemical reagent are pointed out to buy from sigma companies as not special.

Embodiment 1

As shown in Figure 1, a kind of micro fluidic device, including 1. the first input channel, 2. the second input channel, 3. the 3rd inputs Passage, 4. output channel, 5. hydrogel performed polymer cross linking channels, M1 are two blending regions of water oil, and the two-phase not being blended passes through Microchannel with T-shaped structure (as shown in Figure 2) or fluid focus structure (as shown in Figure 3).M2 is prepared by upstream The microchannel that is mutually blended with the second heavy oil of lotion.The microchannel interior walls surface carries out hydrophobic treatment.

Sodium alginate, fluorescein-labeled dextran (molecular weight 10kDa) are dissolved in deionized water to configure to obtain sea The aqueous solution that alginic acid sodium content is 1w/v%, fluorescein-labeled dextran content is 0.01w/v%, then adds final dense Spend for the chelate aqueous solution of 100mM calcium ions-nitrilotriacetic acid (Ca-NTA)；Water-In-Oil is used as using the aqueous solution of above-mentioned configuration The water of lotion mutually enters 1. the first input channel.By perfluoroether (perfluorinated ethers, PFPE) surfactant and Acetic acid adds in perfluorooctane oil solution obtained mixed solution, and the first heavy oil using it as water-in-oil emulsion system mutually enters 2. the second input channel.Surfactant concentration is 2v/v% in above-mentioned oil phase, and the concentration of acetic acid is 1v/v%.By perfluor amylalcohol It is blended with perfluorooctane oil, obtains the second heavy oil that perfluor amylalcohol content is 10v/v% and mutually enter 3. the 3rd input channel.

Above-mentioned aqueous phase solution and the first heavy oil are mutually utilized into syringe respectively from the first and second input ports by constant flow pump (chip structure design is as shown in Figure 1) is injected in first layer " fluid focus " microchannel of micro fluidic device, by with fluid The microchannel (such as Fig. 3) of focusing structure, is mutually blended with the first heavy oil, and water is mutually cut into the homogeneous oil bag of Size Distribution by oil phase Water single emulsion drop.Wherein, the flow velocity of interior phase is 100ul/hr, and the first weight Oil phase flow rate is 1000ul/hr.With vinegar in oil phase Acid enters in aqueous phase droplets, causes the decline of pH in drop, makes the Ca for stablizing chelating with NTA molecules originally²⁺Ion is no longer stablized, Form the Ca of free state²⁺Ion, it further forms ionic bond with alginic acid macromolecular chain, triggers alginic acid and calcium ion crosslinking Form hydrogel.Therefore, micro-gel particles are formed in emulsion droplet, and since the presence of surfactant disperses with stabilizing it In oil phase.

Then, the second layer that the above-mentioned Water-In-Oil single emulsion for being dispersed with microgel flows through downstream has " fluid focus " structure Microchannel (such as Fig. 3), with perfluor amylalcohol content be 10v/v% perfluorooctane form the second heavy oil be mutually blended.Wherein The flow velocity of double oil phase is 1000ul/hr.After above-mentioned fluid-mixing is by U-shaped mixing microchannel gradually blending, the second heavy oil phase In perfluor amylalcohol will originally stablize water-oil interface perfluor ether surface active agent elution, make water-oil interface no longer stablize, water phase Drop (i.e. micro-gel particles) is difficult to continue to be stably dispersed in oil phase, therefore microgel is separated with oil phase.Obtain Carry the dispersion liquid of cell micro-gel particles in water.Be dispersed in water as shown in figure 4, micro-gel particles are prepared, and with oil Phase separation, by fluorescent microphoto as it can be seen that microgel dispersion in water, the fluorescein-labeled dextrorotation in initial aqueous phase solution Glucosides, since molecular weight is smaller, penetrates into aqueous solution quickly through the porous network of microgel.The ruler of gained micro-gel particles Very little distribution is homogeneous, as shown in Figure 5.Wherein, focused in first heavy fluid between microchannel and the second heavy fluid focusing microchannel Distance be 2 centimetres, after ensureing that alginic acid microgel is fully cured, the destruction of lotion is realized in downstream passage so that water oil Two-phase can realize separation in micro flow chip passage, realize one-step method cell embedding.

1 alginic acid two-step method of comparative example

Micro fluidic device as shown in Figure 6, is dissolved in deionized water, then by the sodium alginate for being grafted with gfp molecule The chelate aqueous solution of calcium-nitrilotriacetic acid (Ca-NTA) is added, wherein fluorescent marker sodium alginate concentration is 1wt%, Ca-NTA Concentration is 100mM；Water phase using the aqueous solution of above-mentioned configuration as water-in-oil emulsion.By perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer is added in perfluoro butyl-methyl ether as surfactant obtains mixing oil phase Oil phase of the solution as water-in-oil emulsion system.

Above-mentioned aqueous phase solution and oil-phase solution are noted from the first and second input ports respectively by constant flow pump using syringe Enter in the microchannel of micro fluidic device (such as Fig. 6), water is mutually cut into the homogeneous Water-In-Oil single emulsion drop of Size Distribution by oil phase. Wherein, the flow velocity of interior phase is 100ul/hr, Oil phase flow rate 1000ul/hr.As shown in fig. 7, since the density of aqueous phase droplets is small In oil phase, therefore oil phase upper strata is swum in, and fluorescence micrograph is shown, obtains the size uniform of aqueous phase droplets, and initial water Fluorescein-labeled dextran in phase is still wrapped in water-in-oil emulsion drop.

Then, acetic acid (final concentration 0.05vol%) is added in the lotion being prepared to the above method.Acetic acid expands from oil phase It is dissipated in aqueous phase droplets, causes the decline of pH in drop, makes the Ca for stablizing chelating with NTA molecules originally²⁺Ion is no longer stablized, shape Into the Ca of free state²⁺Ion, it further forms ionic bond with alginic acid macromolecular chain, forms hydrogel.Such emulsion droplet Middle formation micro-gel particles, and since what the presence of surfactant stabilized it is scattered in oil phase.In order to by microgel Grain is transformed into aqueous phase solution, it is necessary to which the micro-gel particles disperseed in lotion are cleaned.Into above-mentioned emulsion 30 after addition acetic acid Second, the lotion filtering that will be dispersed with microgel removes oil phase, then with substantial amounts of deionized water rinsing, so that by oil phase and surface Activating agent removes, and realizes microgel dispersion in aqueous solution.

Comparative example 2

The sodium alginate for being grafted with gfp molecule is dissolved in deionized water, then adds CaCO₃Calcium carbonate nano Grain simultaneously fully mixes, and wherein the sodium alginate concentration of fluorescent marker is 1wt%, and calcium carbonate granule concentration is 10mg/ml；With above-mentioned Water phase of the aqueous solution of configuration as water-in-oil emulsion.By perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) Block copolymer adds in perfluorooctane oil-phase solution obtained mixed solution as surfactant, and water in oil emulsion is used as using it The oil phase of liquid system.Surfactant concentration is 2v/v% in above-mentioned oil phase.Water in oil emulsion is prepared using 1 the method for comparative example Liquid drop, then to acetic acid (final concentration of 0.5v/v%) is added in lotion, after soaking 120 seconds, will be dispersed with the breast of microgel Liquid filtering removes oil phase, then with substantial amounts of deionized water rinsing, so that by oil phase and removal of surfactant, realizes microgel It is dispersed in aqueous solution.Figure 11 utilizes seaweed acid gel prepared by different crosslinking agents.Wherein, A is using side described in comparative example 1 Method, the alginic acid microgel prepared using Ca-NTA as cross-linking reaction initiator, B be using 2 the method for comparative example, with CaCO₃The seaweed acid gel that nano particle is prepared as cross-linking reaction initiator.The microphoto of microgel is shown, in microgel The high molecular skewness of fluorescent marker alginic acid, this is because cium carbonate nanoparticles are in sodium alginate prepolymer solution Skewness caused by.

The different cross-linking reaction initiators of comparative example 3

The sodium alginate for being grafted with gfp molecule is dissolved in deionized water, different cross-linking reactions is then added and triggers Agent：Calcium-nitrilotriacetic acid (Ca-NTA), calcium-ethylenediamine tetra-acetic acid (Ca-EDTA), CaCO₃Cium carbonate nanoparticles (average grain diameter About 300nm) and fully mix, wherein the sodium alginate concentration of fluorescent marker is 1wt%, Ca-NTA and Ca-EDTA concentration is 100mM, calcium carbonate granule concentration are 10mg/ml (calcium ion concentration 100mM)；Water-In-Oil is used as using the aqueous solution of above-mentioned configuration The water phase of lotion.Using perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer as surface-active Agent adds in perfluorooctane oil-phase solution obtained mixed solution, and the oil phase of water-in-oil emulsion system is used as using it.Above-mentioned oil phase Middle surfactant concentration is 2v/v%.Water-in-oil emulsion drop is prepared using 1 the method for comparative example, is added then in lotion Enter acetic acid (final concentration of 0.1v/v%), after soaking different time, the lotion filtering for being dispersed with microgel removed into oil phase immediately, Then with substantial amounts of deionized water rinsing, so that by oil phase and removal of surfactant, realize microgel dispersion in aqueous solution In.Then whether there is microgel to be formed by fluorescence microscope, think that the preparation parameter can not be formed if being formed without gel ball Microgel.The results are shown in Table 1, and Ca-NTA and Ca-EDTA can be crosslinked sea as cross-linking reaction initiator in very short time Alginates, obtain micro-gel particles, this mainly has benefited from the water soluble chelate compound of both calcium ions, can be water-based molten It is dispersed in liquid, and calcium ion dissociation reaction moment generation in the case where acid acts on, be conducive to alginic acid Quick cross-linking.Compare, CaCO₃The speed that nano particle dissociates calcium ion in the case where acid acts on is significantly slower, is being added into lotion phase in acetic acid 60 seconds all Microgel crosslinking is difficult to realize, when the soaking time in acid is more than 120 seconds, microgel is formed.The speed of crosslinking rate is straight Connect and be related to two very important applications：1st, when being prepared for carrying cell micro-gel particles, to ensure that microgel is formed, with Experimental group of the cium carbonate nanoparticles as cross-linking reaction initiator, because cell needs to stop for a long time under sour environment, Therefore cell survival rate is low (Figure 10).2nd, when using Ca-NTA and Ca-EDTA as cross-linking reaction initiator, alginic acid pre-polymerization Body quick-gelatinizing in emulsion droplet, this cleaning for realize emulsion droplet on chip, this is the present invention Realization continuously prepares one of key of micro-gel particles.

Microgel is formed using different alginic acid cross-linking reaction initiators and different crosslinking times described in 1 comparative example 3 of table Influence.

Embodiment 2 is to size Control

Sodium alginate is dissolved in deionized water to configure to obtain the aqueous solution that sodium alginate content is 2w/v%, by calcium-ammonia three Acetic acid (Ca-NTA) is dissolved in the aqueous solution that 100mM concentration is obtained in deionized water；Two above two solution blendings configure to obtain Aqueous solution, wherein sodium alginate final solution concentration are 1w/v%, and Ca-NTA concentration is 80mM, and are used as oil using this aqueous solution The interior phase aqueous solution of bag aqueous emulsion.By perfluoro butyl-methyl ether, perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG- Krytox) the mixed solution that the fluorinated surfactant of block copolymer and acetic acid are blended, water-in-oil emulsion is used as using it First heavy oil phase of system.Surfactant concentration is 1v/v% in above-mentioned oil phase, and the concentration of acetic acid is 0.1v/v%.Finally Perfluorooctanol and perfluoro butyl-methyl ether are blended, obtain the second heavy oil phase that perfluorooctanol content is 10v/v%.

Above-mentioned aqueous phase solution and the first heavy oil are mutually utilized into syringe respectively from the first and second input ports by constant flow pump Inject in first layer " fluid focus " microchannel of micro fluidic device, by with T-shaped structure or fluid focus structure Microchannel (such as Fig. 1), be mutually blended with the first heavy oil, oil phase water is mutually cut into Size Distribution it is homogeneous Water-In-Oil list breast Liquid drop.Wherein, the flow velocity of interior phase is 100ul/hr, changes the flow rate Qc/ of phase aqueous solution in the flow velocity of the first heavy oil phase Qd (oil：Water)<20, the flow velocity of the second heavy oil phase is identical with the first weight Oil phase flow rate.With in oil phase, acetic acid enters aqueous phase droplets In, cause the decline of pH in drop, be the Ca for stablizing chelating with NTA molecules originally²⁺Ion is no longer stablized, and forms free state Ca²⁺Ion, it further forms ionic bond with alginic acid macromolecular chain, and ionomer occurs and forms hydrogel.So lotion liquid Micro-gel particles are formed in drop, and since what the presence of surfactant stabilized it is scattered in oil phase.As a result such as Fig. 8 institutes Show.Reduction to dampening phase and the first heavy oil phase velocity ratio, the size of micro-gel particles reduce therewith.

3 active somatic cell of embodiment embeds

Cell culture：With NIH3T3 (CRL-1658^TM) exemplified by Fibroblast cell-culture, proliferated culture medium by DMEM, forms containing 10% hyclone (FBS, Gibco).Condition of culture is 37 DEG C, 95% relative humidity and 5%CO₂.Carefully Born of the same parents' culture medium is replaced afterwards every three days.Before use, cell is used into trypsase/EDTA solution with phosphate buffered saline (PBS) (PBS) Middle separation (0.25% trypsase/0.02%EDTA) 5 minutes, and be suspended in culture medium in case using.

The sodium alginate of green fluorescent label is dissolved in cell culture medium DMEM solution, configuration obtains the seaweed of 2w/v% Acid sodium aqueous solution.Cell suspension, sodium alginate aqueous solution and calcium-ethylenediamine tetra-acetic acid (Ca-EDTA) chelate aqueous solution are total to Mixed, it is 1w/v% to obtain sodium alginate concentration in mixed solution, and Ca-EDTA ultimate densities are 100mM, cell concentration 10⁶A/ ml.Interior phase aqueous solution using above-mentioned aqueous solution as water-in-oil emulsion.By perfluoro butyl-methyl ether, perfluoroether-polyvinyl alcohol- The mixing that the fluorinated surfactant and acetic acid of perfluoroether (Krytox-PEG-Krytox) block copolymer are blended is molten Liquid, the first heavy oil phase using it as water-in-oil emulsion system.Wherein surfactant concentration is 1w/v%, and acetate concentration is 0.1v/v%.Perfluorooctanol and perfluoro butyl-methyl ether are blended, obtain the second heavy oil that perfluorooctanol content is 10v/v% Phase.

Above-mentioned aqueous phase solution and the first heavy oil are mutually utilized into syringe respectively from the first and second input ports by constant flow pump Inject in first layer " fluid focus " microchannel of micro fluidic device, led to by the miniflow with T-shaped structure (Fig. 2) structure Road (such as Fig. 1), is mutually blended with the first heavy oil, and water is mutually cut into the homogeneous Water-In-Oil single emulsion drop of Size Distribution by oil phase.Its In, the flow velocity of interior phase is 100ul/hr, and the flow velocity of the first heavy oil phase is 1000ul/hr.Then, acetic acid enters water phase liquid in oil phase In drop, cause the decline of pH in drop, be the Ca for stablizing chelating with EDTA molecules originally²⁺Ion is no longer stablized, and forms free state Ca²⁺Ion, it further forms ionic bond with alginic acid macromolecular chain so that alginic acid occurs ionomer and forms water-setting Glue.Micro-gel particles are so formed in emulsion droplet, and since what the presence of surfactant stabilized it is scattered in oil phase.

Then, the second layer that the above-mentioned Water-In-Oil single emulsion for being dispersed with microgel flows through downstream has " fluid focus " is micro- to lead to Road (such as Fig. 3), blending is mutually formed with the second heavy oil.Wherein the flow velocity of the second heavy oil phase is 2000ul/hr.Above-mentioned fluid-mixing leads to Cross U-shaped mixing microchannel gradually (Fig. 1) after blending, the perfluorooctanol in the second heavy oil phase will stablize the table of water-oil interface originally Face activating agent elution, makes water-oil interface no longer stablize, aqueous phase droplets (i.e. micro-gel particles) are difficult to continue to be stably dispersed in oil phase In, therefore microgel is separated with oil phase.

DMEM cell culture mediums and buffer solution HEPES are blended, wherein buffer solution HEPES concentration is 10mM, will be above-mentioned molten Liquid is as collection aqueous solution.Output channel is blended by conduit with collecting aqueous solution, the blending water oil mixing exported in chip Thing is separated in aqueous solution is collected, and microgel is directly separated into aqueous phase solution, buffers the acid in drop, is preparation process Damage to cell is preferably minimized.

The cytotoxicity of gel rubber material is investigated by using life or death fluorescent staining (LIVE/DEAD assay).It is first First, gel is cleaned 30 minutes with sterile PBS before dyeing, adding 2mM calceins at room temperature, (Green fluorescent dye marks Living cells) and 4mM EtBrs dimer (red fluorescence dyestuff mark dead cell), and use confocal laser scanning microscope, CLSM Check.As a result as shown in the fluorescence micrograph of Fig. 9, it is seen that cell survival rate is about 85%；With directly on Tissue Culture Plate The control experiment group survival rate of the cell of culture is suitable, it was demonstrated that the method for the present invention has good biocompatibility, and (result is such as Shown in Figure 10).

4 conventional emulsion method (cell embedding) of comparative example

The two step method preparation process of microgel is prepared using comparative example 2, prepares and carries cell microgel.By green fluorescent label Sodium alginate be dissolved in cell culture medium DMEM solution, then add calcium-ethylenediamine tetra-acetic acid (Ca-EDTA) chelate it is water-soluble Liquid and cell dispersion liquid, wherein fluorescent marker sodium alginate concentration are 1wt%, and Ca-NTA concentration is 100mM, and cell concentration is 10⁶A/ml；Water phase using the aqueous solution of above-mentioned configuration as water-in-oil emulsion.By perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block copolymer is added in perfluoro butyl-methyl ether as surfactant obtains mixing oil phase Oil phase of the solution as water-in-oil emulsion system.

Above-mentioned aqueous phase solution and oil-phase solution are noted from the first and second input ports respectively by constant flow pump using syringe Enter in the microchannel of micro fluidic device (such as Fig. 6), water is mutually cut into the homogeneous Water-In-Oil single emulsion drop of Size Distribution by oil phase. Wherein, the flow velocity of interior phase is 100ul/hr, Oil phase flow rate 1000ul/hr.As shown in fig. 7, the density phase due to aqueous phase droplets For oil phase smaller, therefore oil phase upper strata is swum in, and fluorescence micrograph is shown, obtains the size uniform of aqueous phase droplets, and Fluorescein-labeled dextran in initial water phase is still wrapped in water-in-oil emulsion drop.

Then, acetic acid (final concentration 0.1vol%) is added in the lotion being prepared to the above method.Acetic acid expands from oil phase It is dissipated in aqueous phase droplets, causes the decline of pH in drop, makes the Ca for stablizing chelating with NTA molecules originally²⁺Ion is no longer stablized, shape Into the Ca of free state²⁺Ion, it further forms ionic bond with alginic acid macromolecular chain, forms hydrogel.Such emulsion droplet Middle formation micro-gel particles, and since what the presence of surfactant stabilized it is scattered in oil phase.In order to by microgel Grain is transformed into aqueous phase solution, it is necessary to which the micro-gel particles disperseed in lotion are cleaned.Into above-mentioned emulsion 300 after addition acetic acid Second, the lotion filtering that will be dispersed with microgel removes oil phase, then with substantial amounts of cell culture medium DMEM flushings, so that by oil phase And removal of surfactant, cell microgel will be carried and be finally dispersed in DMEM aqueous solutions.

Live although obtaining above-mentioned two-step method and preparing microgel technology available for the biology such as embedding cell or protein drug molecule Property material, but due to cell embedding and preparation process in it is too long with acid solution time of contact, seriously affect cell survival rate. As shown in Figure 10, the survival rate of the cell in cell micro-gel particles, gained microgel is prepared using two-step method described in comparative example It is extremely low, it is remarkably decreased compared with normal positive control (cell adhere-wall culture on two-dimentional culture plate).

It is prepared by 4 multi-chamber microgel of embodiment

As shown in figure 12, a kind of micro fluidic device for being used to prepare multi-chamber microgel, including the 1st, 2,3,4......n A input channel, converges to form interior phase input channel A jointly, and B is the first heavy oil phase input channel, and C mutually inputs for the second heavy oil Passage, D are output channel, and E is hydrogel performed polymer cross linking channels, and M1 is that composition microgel different chamber hydrogel performed polymer is molten The Mixed Zone of liquid, the characteristics of forming stable parallel fluid using fluid in microchannel, out of phase prepolymer solution input Formed after chip and stablize concurrent flow, the material between fluid spreads and exchanges limited.M2 is two blending regions of water oil, is not blended Two-phase passes through the microchannel with T-shaped structure (as shown in Figure 2) or fluid focus structure (as shown in Figure 3).M2 is The microchannel that lotion prepared by upstream is mutually blended with the second heavy oil.The microchannel interior walls surface carries out hydrophobic treatment.

Sodium alginate is dissolved in configuration in deionized water and obtains alginic acid hydrogel prepolymer solution；By nitrilotriacetic acid and hydrogen The chelate aqueous solution of configuration calcium-nitrilotriacetic acid (Ca-NTA) is blended in calcium oxide in deionized water.Above-mentioned two aqueous solution is total to It is mixed, the polystyrene nanoparticles (particle diameter 100nM) of red or green fluorescein mark are then added, it is uniformly divided Dissipate, obtain preparing the alginic acid hydrogel prepolymer solution of microgel.It is 1w/ to configure to obtain sodium alginate content with deionized water V%, fluorescein-labeled nano-particle content are 0.01w/v%, and it is 100mM calcium ions-three second of ammonia then to add ultimate density The chelate aqueous solution of sour (Ca-NTA)；Water phase using the aqueous solution of above-mentioned configuration as water-in-oil emulsion.By perfluoroether (perfluorinated ethers, PFPE) surfactant and acetic acid add in perfluorooctane oil-phase solution obtained mixing Solution, the first heavy oil phase using it as water-in-oil emulsion system.Surfactant concentration is 2v/v% in above-mentioned oil phase, acetic acid Concentration be 1v/v%.Perfluor amylalcohol and perfluorooctane oil phase are blended, obtain the second weight that perfluor amylalcohol content is 10v/v% Oil phase.

By the sodium alginate performed polymer for being dispersed with red fluorescence and green fluorescence nano particle respectively of above-mentioned preparation, pass through Microchannel structure as described in Figure 13 inputs chip from the 1st and 2 input channels, forms the concurrent flow of flow speed stability (as schemed 13), aqueous phase solution and the first heavy oil are mutually injected micro-fluidic from the first and second input ports respectively by constant flow pump using syringe In first layer " fluid focus " microchannel of device (chip structure design is as shown in figure 12), by with fluid focus structure Microchannel (such as Fig. 2), is mutually blended with the first heavy oil, and water is mutually cut into the homogeneous Water-In-Oil single emulsion liquid of Size Distribution by oil phase Drop.Wherein, for the flow velocity of phase to be respectively 100ul/hr, the first weight Oil phase flow rate is 1000ul/hr in two kinds.With vinegar in oil phase Acid enters in aqueous phase droplets, causes the decline of pH in drop, makes the Ca for stablizing chelating with NTA molecules originally²⁺Ion is no longer stablized, Form the Ca of free state²⁺Ion, it further forms ionic bond with alginic acid macromolecular chain, triggers alginic acid and calcium ion crosslinking Form hydrogel.Therefore, micro-gel particles are formed in emulsion droplet, and since what the presence of surfactant stabilized it disperses In oil phase.

Then, the second layer that the above-mentioned Water-In-Oil single emulsion for being dispersed with microgel flows through downstream has " fluid focus " is micro- to lead to Road (such as Fig. 2), is mutually blended with the second heavy oil of the perfluorooctane composition that perfluor amylalcohol content is 10v/v%.Wherein the second heavy oil The flow velocity of phase is 1000ul/hr.Above-mentioned fluid-mixing passes through (such as Figure 12) behind U-shaped mixing microchannel gradually blending, the second heavy oil The perfluor ether surface active agent that script is stablized water-oil interface by the perfluor amylalcohol in phase elutes, and water-oil interface is no longer stablized, water Phase drop (i.e. micro-gel particles) is difficult to continue to be stably dispersed in oil phase, therefore microgel is separated with oil phase.Pass through Above-mentioned mixing is exported into liquid by conduit connection output to collecting in aqueous solution, that is, obtains the microgel with two chambers structure Particle (such as Figure 13 B).

Embodiment 5

The sea for the nano particle for being dispersed with red fluorescence and green fluorescent label respectively is prepared using 4 the method for embodiment Alginic acid performed polymer aqueous solution, is distinguished three kinds of different interior phase aqueous solutions using the interior phase aqueous solution input structure in such as Figure 14 A Injection, forms the parallel fluid of phase aqueous solution in three kinds of differences.Wherein, the flow velocity of phase is is respectively 100ul/hr in three kinds, and the One weight Oil phase flow rate is 1500ul/hr.Microgel is further prepared by 4 the method for embodiment, is finally obtained with three The micro-gel particles (Figure 14 B) of different chamber.

Embodiment 6

The sea for the nano particle for being dispersed with red fluorescence and green fluorescent label respectively is prepared using 4 the method for embodiment Alginic acid performed polymer aqueous solution, is distinguished four kinds of different interior phase aqueous solutions using the interior phase aqueous solution input structure in such as Figure 15 A Injection, formed four kinds of differences in phase aqueous solution parallel fluid, wherein, in four kinds the flow velocity of phase for be respectively 50ul/hr, first Weight Oil phase flow rate is 1000ul/hr.Microgel is further prepared by 4 the method for embodiment, is finally obtained with four not With the micro-gel particles (Figure 15 B) of chamber.

Embodiment 7

The sea for the nano particle for being dispersed with red fluorescence and green fluorescent label respectively is prepared using 4 the method for embodiment Alginic acid performed polymer aqueous solution, is distinguished two kinds of different interior phase aqueous solutions using the interior phase aqueous solution input structure in such as Figure 13 A Injection, forms the parallel fluid of phase aqueous solution in two kinds of differences, wherein, the flow velocity of phase aqueous solution in two-phase is adjusted, is between the two Flow rate (Q1/Q2) is from 1 to 9, and the summation of two-phase flow velocity is 100ul/hr, and the first weight Oil phase flow rate is 1000ul/hr.Into One step prepares microgel by 4 the method for embodiment, finally obtains two different chambers with different chamber volume ratio Micro-gel particles (Figure 16).

The sodium alginate for being grafted with gfp molecule is dissolved in deionized water, then adds calcium-nitrilotriacetic acid (Ca- NTA chelate aqueous solution), wherein fluorescent marker sodium alginate concentration are 1wt%, and Ca-NTA concentration is 100mM；Will be non-fluorescence The sodium alginate of mark is dissolved in deionized water, then adds the chelate aqueous solution of calcium ion-nitrilotriacetic acid (Ca-NTA), wherein Fluorescent marker sodium alginate concentration is 1wt%, and Ca-NTA concentration is 100mM.Made with above two hydrogel performed polymer aqueous solution For phase in difference, according to 4 the method for embodiment, using if the interior phase aqueous solution input structure in Figure 13 A is by two kinds different Interior phase aqueous solution is injected separately into, and forms the parallel fluid of phase aqueous solution in two kinds of differences.Wherein, phase aqueous solution in two-phase is adjusted Flow velocity is 50ul/hr, and the summation of two-phase flow velocity is 100ul/hr, and the first weight Oil phase flow rate is 1000ul/hr.Further pass through 4 the method for embodiment prepares microgel, finally obtains the micro-gel particles (Figure 17) with two chambers.

Embodiment 8

Using 3 the method culture NIH3T3 of embodiment (CRL-1658^TM) fibroblast, cell is distinguished (ThermoFisher Scientific companies of the U.S., green is marked using green and red living cells tracer fluorescent dye With red living cells tracer dye CellTracker^TM).Connect respectively using red and Green fluorescent dye (Sigma Co., USA) Branch alginic acid (sigma companies of the U.S., medium-viscosity), the alginic acid for obtaining fluorescent marker are dissolved in cell culture medium DMEM solution In.

Configuration is dispersed with the hydrogel performed polymer of active somatic cell as water phase first：The sodium alginate of fluorescent marker is dissolved in In cell culture medium DMEM solution, configuration obtains the sodium alginate aqueous solution of 2w/v%.By cell suspension, sodium alginate aqueous solution It is blended with calcium-ethylenediamine tetra-acetic acid (Ca-EDTA) chelate aqueous solution, it is 1w/ to obtain sodium alginate concentration in mixed solution V%, Ca-EDTA ultimate density are 50mM.Wherein, green fluorescent label NIH3T3 active somatic cells are dispersed in red fluorescence mark In the seaweed aqueous acid of note, the alginic acid that red fluorescence mark NIH3T3 active somatic cells are dispersed in green fluorescent label is water-soluble In liquid, cell concentration is respectively 10⁶A/ml.Two kinds using above two cell dispersion liquid as preparation two chambers microgel Interior phase aqueous solution.Perfluoro butyl-methyl ether, perfluoroether-polyvinyl alcohol-perfluoroether (Krytox-PEG-Krytox) block is common The mixed solution that the fluorinated surfactant and acetic acid of polymers are blended, the first weight using it as water-in-oil emulsion system Oil phase.Wherein surfactant concentration is 1w/v%, acetate concentration 0.1v/v%.By perfluor amylalcohol and perfluoro butyl-methyl Ether is blended, and obtains the second heavy oil phase that perfluor amylalcohol content is 10v/v%.

It is according to 4 the method for embodiment, two kinds of cell dispersion liquids of above-mentioned preparation are micro- by as described in Figure 13 respectively Circulation road structure inputs chip from the 1st and 2 input channels, forms the concurrent flow (such as Figure 13) of flow speed stability, aqueous phase solution and the One heavy oil mutually injects the first layer " fluid of micro fluidic device from the first and second input ports respectively by constant flow pump using syringe Focus on " in microchannel (chip structure design is as shown in figure 12), by the microchannel (such as Fig. 2) with fluid focus structure, Mutually it is blended with the first heavy oil, water is mutually cut into the homogeneous Water-In-Oil single emulsion drop of Size Distribution by oil phase.Wherein, phase in two kinds Flow velocity for be respectively 50ul/hr, first weight Oil phase flow rate be 1000ul/hr.As acetic acid enters in aqueous phase droplets in oil phase, Cause the decline of pH in drop, make the Ca for stablizing chelating with EDTA molecules originally²⁺Ion is no longer stablized, and forms the Ca of free state²⁺ Ion, it further forms ionic bond with alginic acid macromolecular chain, triggers alginic acid to form hydrogel with calcium ion crosslinking.Cause This, forms micro-gel particles in emulsion droplet, and since what the presence of surfactant stabilized it is scattered in oil phase.

Then, the second layer that the above-mentioned Water-In-Oil single emulsion for being dispersed with microgel flows through downstream has " fluid focus " is micro- to lead to Road (such as Fig. 2), is mutually blended with the second heavy oil of perfluoro butyl-methyl ether composition that perfluor amylalcohol content is 10v/v%.Wherein The flow velocity of double oil phase is 1000ul/hr.After above-mentioned fluid-mixing is gradually blended by U-shaped mixing microchannel (such as Figure 12), the The perfluor ether surface active agent that script is stablized water-oil interface by the perfluor amylalcohol in double oil phase elutes, and makes water-oil interface no longer Stablize, aqueous phase droplets (i.e. micro-gel particles) are difficult to continue to be stably dispersed in oil phase, therefore with oil phase phase point occurs for microgel From.DMEM cell culture mediums and buffer solution HEPES (pH7.2-7.4) are blended, wherein buffer solution HEPES concentration is 10mM, will Above-mentioned solution is as collection aqueous solution.Output channel is blended by conduit with collecting aqueous solution, the blending water exported in chip Oil mixture is separated in aqueous solution is collected, and microgel is directly separated into aqueous phase solution, buffers the acid in drop, is system Standby damage of the process to cell is preferably minimized.Pass through the mixing for being dispersed with load cell dual cavity microgel for preparing the above method For solution by conduit connection output to collecting in aqueous solution, collection obtains the micro-gel particles with two chambers structure, and not There is different types of single active somatic cell with immobilized in chamber.As a result as shown in the fluorescence micrograph of Figure 18.

It is prepared by 9 multi-chamber microgel of embodiment

Using 4 the method for embodiment, configuration alginic acid hydrogel prepolymer solution (sodium alginate content is 1w/v%, Calcium-ethylenediamine tetra-acetic acid chelate concentration is 100mM), red fluorescence dye is separately added into above-mentioned hydrogel prepolymer solution Expect the polystyrene nanoparticles (average diameter 100nm) and superparamag-netic iron oxide (average diameter 100nm) of mark, obtain To the two-phase aqueous solution for preparing two chambers microgel, wherein nano-particle content is 0.01w/v%；With the scattered of above-mentioned configuration There is the alginic acid hydrogel performed polymer aqueous solution of different nano particles as two kinds of aqueous phase solutions for preparing two chambers microgel. Perfluoroether (perfluorinated ethers, PFPE) surfactant and acetic acid are added in perfluorooctane oil-phase solution and obtained The mixed solution arrived, the first heavy oil phase using it as water-in-oil emulsion system.Surfactant concentration is 1v/ in above-mentioned oil phase V%, the concentration of acetic acid is 1v/v%.Perfluor amylalcohol and perfluorooctane oil phase are blended, it is 10v/v% to obtain perfluor amylalcohol content The second heavy oil phase.Using the micro flow chip described in embodiment 4 and preparation method and parameter, it is prepared with two chambers knot The microgel of structure, as shown in figure 19, two of which chamber contain the pipe/polyhenylethylene nano of red fluorescence dyestuff mark respectively Grain (average diameter 100nm) and superparamag-netic iron oxide (black, average diameter 100nm).And two chamber microgel of gained With magnetic response, disperseing two chamber micro-gel particles in a liquid can be in displacement under attraction, and can be in magnetic field Middle ordered arrangement.

Claims (18)

1. one-step method continuously prepares the microflow control technique of calcium alginate microgel, it is characterised in that：Include the following steps：

(1) solution is prepared

1. preparing seaweed aqueous acid in water by dissolution of raw material of water soluble algae hydrochlorate, cross-linking reaction initiator is added, then Bioactive substance and/or nano particle are scattered in above-mentioned aqueous solution to obtain hydrogel prepolymer solution, as aqueous phase solution, Wherein：

The cross-linking reaction initiator is selected from chelate aqueous solution, the chelate water of calcium-nitrilotriacetic acid of calcium-ethylenediamine tetra-acetic acid One or more of combination in solution, cium carbonate nanoparticles, sulfuric acid calcium nano, calcium phosphate nanoparticles；The crosslinking The final concentration of reaction initiator is denoted as 10-1000mM with calcium content；

2. the first heavy oil phase is blended to obtain in fluorinated oil, fluorinated surfactant and acidic materials；

3. the second heavy oil phase is blended to obtain in fluorinated oil and perfluor alcohol or perfluor acid；

(2) aqueous phase solution for 1. obtaining step injects micro flow chip with the first flow velocity, and the first heavy oil is mixed with second flow speed Liquid injects micro flow chip from the second input port, and aqueous phase solution and the first heavy oil are mutually blended through Over emulsfication passage, form water in oil emulsion Liquid drop；

(3) the second heavy oil is mutually injected by micro flow chip, the second heavy oil phase and step (2) gained by the 3rd input port with the 3rd flow velocity After lotion is fully blended in the hybrid channel for being arranged at emulsification passages downstream, chip is flowed out from output channel；By the molten of output Phase aqueous solution is collected in liquid input, and the calcium alginate microgel dispersion for making to be loaded with bioactive substance and/or nano particle is being collected In phase aqueous solution, aqueous phase solution is collected up to finished product.

2. one-step method according to claim 1 continuously prepares the microflow control technique of calcium alginate microgel, it is characterised in that： The micro flow chip has the microchannel of fluid focus structure, T-shaped mixed structure, concurrent ejector half or cross structure, tool There are at least 3 phase liquid inlets, and emulsification passage and output channel.

3. the microflow control technique that one-step method according to claim 1 continuously prepares calcium alginate microgel is characterized in that： The seaweed acid starting material is alginic acid, alginate, alginate aqueous solution or the mixture of alginic acid and water soluble polymer.

4. one-step method according to claim 1 continuously prepares the microflow control technique of calcium alginate microgel, it is characterised in that： Seaweed acid starting material total concentration is 0.1-8w/v% wherein in prepolymer solution.

5. one-step method according to claim 1 continuously prepares the microflow control technique of calcium alginate microgel, it is characterised in that： Described in step (1) be embedded bioactive substance and/or nano particle is selected from following one or more of combinations：Live body is thin Born of the same parents, water miscible activated protein drug molecule, nano particle；

The active somatic cell is primary cultured cell, cultured cell line, cell line culture cell and heterozygote；

The water miscible activated protein drug molecule is protein drug, polypeptide drug, enzyme drug and cell growth The factor；

The nano particle is metal or the metal oxide nanoparticles such as nanogold, nano silver, nano-sized iron oxide, polyethylene, The high molecular polymer nano particle such as polypropylene, polystyrene, polymethyl methacrylate, polylactic acid, liposoluble vitamin, quinoline Promise ketone water-insoluble medicine nano particle, the non-gold of hydroxyapatite, silica, calcium phosphate, carbon nanotubes, graphene inorganic Belong to material nanoparticle；The nanoparticle size diameter is more than 5-1000nm.

6. one-step method according to claim 1 continuously prepares the microflow control technique of calcium alginate microgel, it is characterised in that： The fluorinated oil is selected from following one or more of combinations：Perflenapent, perflexane, PF 5070, perfluoro butyl-methyl It is ether, perfluorooctane, Perfluorononane, perfluoro decane, perfluor hendecane, perfluor dodecane, perfluor tridecane, the perfluor tetradecane, complete Fluorine pentadecane, perfluor hexadecane, perfluor heptadecane, perfluorodecalin.

7. one-step method according to claim 1 continuously prepares the microflow control technique of calcium alginate microgel, it is characterised in that： The fluorinated surfactant is selected from following one or more of combinations：Perfluoroether, perfluoroalkyl acid perfluoroether-polyvinyl alcohol are embedding Section copolymer, perfluoroether-polyvinyl alcohol-perfluoroether block copolymer surfactant；The wherein concentration of fluorinated surfactant For 0.1-10wt%.

8. the microflow control technique that one-step method according to claim 1 continuously prepares calcium alginate microgel is characterized in that：Institute State acidic materials and be selected from following one or more of combinations：Sulfuric acid, nitric acid, hydrochloric acid, carbonic acid, phosphoric acid, acetic acid or citric acid；It is described Concentration of the acidic materials in mixed solution is 0.001-20v/v%.

9. one-step method according to claim 1 continuously prepares the microflow control technique of calcium alginate microgel, it is characterised in that： Perfluor alcohol is selected from following one or more of combinations in the second heavy oil phase：Seven fluoro- n-butyl alcohols of 22,33,444-, perfluor ten One alcohol, 1H, 1H- perfluors-DODECANOL, 1-, 1H, 1H- perfluor -1-heptanol, 1H, 1H, 2H, 2H- perfluorooctanols, 1H, 1H- perfluors Octyl group -1- alcohol, 1H, 1H- perfluor -1 nonyl alcohol, 1H, 1H, 2H, 2H- perfluor -1- lauryl alcohols, 1H, 1H, 2H, the 2H- perfluor -1- last of the ten Heavenly stems Alcohol, 1H, 1H- perfluors -1- 14 (alkane) alcohol, perfluor amylalcohol, perfluor hexanol, perfluor enanthol, perfluorooctanol, perfluor nonyl alcohol, perfluor Decyl alcohol；The perfluor acid is selected from following one or more of combinations：Perfluor lauric acid/dodecanoic acid, positive perfluor valeric acid, 5H- perfluors valeric acid, Perfluoro caprylic acid, perfluoro-pelargonic acid, perfluoro decanoate, perfluoro-heptanoic acid, perfluor caproic acid, perfluorobutyric acid, perfluorinated undecanoic acid；Second heavy oil phase The concentration of middle perfluor alcohol or perfluor acid in fluorinated oil is 1-100v/v%.

10. one-step method according to claim 1 continuously prepares the microflow control technique of calcium alginate microgel, its feature exists In：Collection phase aqueous solution described in step (4) is no cytotoxicity, pH buffering ranges in the buffer solution of 6-8, buffer solution Ion concentration 10-2000mM.

11. one-step method according to claim 1 continuously prepares the microflow control technique of calcium alginate microgel, its feature exists In：First flow velocity is 5-2000 μ L/hr, second flow speed is 200-20000 μ L/hr, and the 3rd flow velocity is 200-20000 μ L/hr.

12. one-step method according to claim 1 continuously prepares the microflow control technique of calcium alginate microgel, its feature exists In：The aqueous phase droplets of formation cross from upstream water phase and an oil phase, and channel time is mutually blended to the second heavy oil is 0.1-30s to passage, micro- solidifying Glue particle arrives the water-soluble liquid phase of injection collection time after being mutually blended with the second heavy oil is 0.1-30s.

13. the microflow control technique that one-step method according to claim 1 continuously prepares calcium alginate microgel is characterized in that： The aqueous phase flow rate and be 0.01~1 with the velocity ratio of the first heavy oil phase；First weight Oil phase flow rate and the second heavy oil phase velocity ratio For 1:0.5~50.

What 14. the microflow control technique that one-step method according to claim 1 continuously prepares calcium alginate microgel was prepared Microgel；It is characterized in that：A diameter of 5~1000 μm of the microgel；The coefficient of dispersion of particle diameter distribution is micro- solidifying 1~6% The time that glue is dispersed in oil phase is 1~60 second；When being embedded with active somatic cell in microgel, cell stopped in lotion when Between at 1~60 second, the survival rate of cell>80%.

15. one-step method continuously prepares the microflow control technique of multi-chamber calcium alginate micro-gel particles, it is characterised in that：When step 1. Bioactive substance and/or nano particle for it is a variety of when, respectively according to step method 1. with different bioactive substances And/or nano particle prepares hydrogel prepolymer solution, injected respectively by multiple water phase input ports on micro flow chip, according still further to step Suddenly 2., 3., (2), the method for (3) prepare multi-chamber micro-gel particles.

16. one-step method according to claim 15 continuously prepares the micro-fluidic skill of multi-chamber calcium alginate micro-gel particles Art, it is characterised in that：The flow velocity summation of the aqueous phase stream body of multiple hydrogel performed polymers is 5-2000 μ L/hr.

17. one-step method according to claim 16 continuously prepares the micro-fluidic skill of multi-chamber calcium alginate micro-gel particles Art, it is characterised in that：Flow velocity fold differences between the aqueous phase stream body of multiple hydrogel performed polymers are 1-100 times.

18. one-step method according to claim 15 continuously prepares the micro-fluidic skill of multi-chamber calcium alginate micro-gel particles Art, it is characterised in that：When step bioactive substance 1. and/or nano particle for it is a variety of when, in hydrogel prepolymer solution Seaweed acid concentration, its fold differences be 1-10 times.