CN106701730A - Alginate hydrogel microsphere carrier containing galactosyl chitosan molecule and application thereof - Google Patents
Alginate hydrogel microsphere carrier containing galactosyl chitosan molecule and application thereof Download PDFInfo
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Abstract
The invention relates to an alginate embedded type hydrogel microsphere carrier having the interior containing a galactosyl chitosan molecule and a preparation method thereof. A galactosyl group is grafted onto a chitosan molecule by an amidation reaction, and hepatocytes are cultured in microcapsules jointly formed by chitosan and alginic acid. Experiments prove that the microcapsules jointly prepared by the galactosyl-grafted chitosan and alginic acid are used for culturing primary hepatocytes of rats, and the activity and function of the hepatocytes are well maintained.
Description
Technical field
The present invention relates to a kind of alginate hydrogel microcarrier, gala is contained in specifically a kind of inside
The alginate hydrogel of glycosyl chitosan molecule is microsphere supported and its preparation and application.
Background technology
Material with bioactivity is encapsulated in and selectively pass through in film, the glomerate microcapsules of shape, referred to as
It is " bio-microcapsule " [Chang TMS.Hemoglobin corpuscles.Research report for
honours physiology.Medical Library,McGill University,1957].The nineties
Since, using bio-microcapsule as the immune isolation of cell and delivery vehicle, using gene-recombinated cell or original
Body physiological function is adjusted for the metabolite of cell, therapy-related disease turns into biomedical worker's
Study hotspot [Basic D, Vacek I, Sun A M.Microencapsulation and
transplantation of engineered cells：a new approach to somatic gene
therapy.Art Cells Blood Subs ImmobBiotechnol,1996,24(3)：219-255].
Alginic acid has excellent physical and chemical performance and biocompatible property because of it, as the main of microencapsulation
Material.Existing microencapsulation in culture hepatocyte, due to the growth characteristics of liver cell anchorage dependence,
Micro-capsule interior three-dimensional steric environment can not make the rapid Adaptable growth of liver cell.Galactolipin group is liver cell table
The ligands specific of face asialoglycoprotein receptor, being capable of specific recognition surface of hepatocytes asialoglycoprotein sugar
Protein receptor, therefore, the galactolipin group of Liver targeting is introduced on microencapsulated material, can induce and raising liver is thin
Born of the same parents' sticking inside microcapsules and propagation behavior [Jun Yang, Galactosylated alginate as a
scaffold for hepatocytes entrapment,Biomaterials,2002,23:471-479]。
Introducing galactolipin group inside micro-capsule at present can be by the blending on alginic acid or covalent modification.Contain half
Macromolecular substances [Seog-Jin Seo, the Yun-Jaie Choi, Alginate of lactose group
microcapsules prepared with xyloglucan as a synthetic extracellular
matrix for hepatocyte attachment,Biomaterials,2005,26:3607-3615]
Mainly played a role by preparing microcapsules with alginic acid blending, the material of blending easily occurs leakage therefore leads
Having caused the stability of this kind of microcapsules reduces；And the covalent modification for being directed to alginic acid occurs mainly in alginic acid
Carboxyl position, carboxyl site occupy so that alginic acid gelation reaction formed microcapsules when gel
The reduction of change degree, for the gel-forming property of not excessive influence alginic acid, galactosyl substitution value is restricted.
[Ivan Donati,AmedeoVetere,Galactose-Substituted Alginate:Preliminary
Characterization and Study of Gelling Properties,Biomacromolecules,
2003,4:624-631].Due to the limitation of microcapsule stability and galactosyl substitution value, either it is blended
Or covalent modification alginic acid, cannot introduce the half of higher concentration in the method for report inside microcapsules at present
Lactose group.
Shitosan is a kind of excellent natural biologic material of biocompatibility, practice have shown that alginate gathers with shell
The micro-capsule that sugar is made by electrostatic interaction, preparation method is gentle, simple, encystation speed is fast, its micro-capsule ball
Shape degree is good, medicine stability [Donati I, Holtan S, Morch YA, et al.New hypothes high
is on the role of alternating sequences in calcium-alginate gels,
Biomacromolecules,2005,6(2):1031-1040].So, alginate/shell gathers in recent years
The research of sugared micro-capsule achieves obvious progress.The current method for preparing alginate/chitosan microcapsules is main
There are following three kinds：Alginate instills shitosan with formation micro-capsule [Thomas in bivalent ions mixed solution
Chandy,Daniel L,Evaluation of Modified Alginate-Chitosan-Polyethylene
Glycol Microcapsules for Cell Encapsulation,Artificial Organs,1999]；
Shitosan instill alginate solution in formed micro-capsule [Sun-Hee Yu, Sung-Koo Kim,
Encapsulation of rat hepatocyte spheroids for the development of
Artificial liver, Biotechnology Techniques, 1999,13:609–614；X.L.
GUO,K.S.YANG,et al,Morphology and metabolism of
Ba-alginate-encapsulated hepatocytes with galactosylated chitosan and
poly(vinyl alcohol)as extracellular matrices,Journal of Biomaterials
Science, Polymer Edition, 2014,6：551-565]；Alginate forms bag in instilling calcium liquid
Type hydrogel microsphere carrier is buried, then the embedding type hydrogel microsphere carrier is immersed in chitosan solution further
Reaction film forming forms microcapsules, i.e. AC micro-capsules [TasimaHaque, In vitro study of alginate
–chitosan microcapsules:an alternative to liver cell transplants for
The treatment of liver failure, Biotechnology Letters, 2005,27：317-322；
Wujie Zhang, Shuting Zhao, A novel core-shell microcapsule for
Encapsulation and 3D culture of embryonic stem cells, Journal of Materials
Chemistry B, 2013,1：1002-1009].First two method there are problems that following, and one is
Because of the two kinds of macromolecular ivr limitations of shitosan and alginic acid, the microcapsule diameter of formation is in 1mm or so
Even more big, excessive micro-capsule is easily caused cell mass overgrowth, and centrocyte is limited out because mass transfer passes oxygen
Existing necrosis, influences cytoactive, and then influence the medical effect of cell secretory product；Two is macromolecular moment
Uneven with reference to that can be formed, the poor micro-capsule of sphericity have impact on the mechanical strength of micro-capsule, when in transplant
Easily there is inflammatory reaction that is damaged and causing body in micro-capsule；Three is no matter alginic acid or shitosan are used as gel
Bath, alginic acid can not be reacted completely with the shitosan being dissolved in calcium liquid or shitosan can not be completely combined marine alga
Acid, result in the waste of raw material.The third method, can by that can be formed in alginic acid instillation divalent salt solutions
Control size, sphericity and the good embedding type hydrogel microsphere carrier of mechanical strength, but type will be embedded again
When further reaction film forming forms traditional AC micro-capsules in hydrogel microsphere carrier immersion chitosan solution, shell
Glycan can't enter inside micro-capsule, and which forms with alginic acid as core, shitosan is the core-shell microcapsule of shell
Structure, shitosan does not contact the cell inside micro-capsule so that functional shitosan can not play due work
With.
The content of the invention
Regarding to the issue above, the present invention proposes a kind of alginate of the inside containing GC molecule
Embedding type hydrogel microsphere carrier, it is characterised in that：Alginate embedding type hydrogel microsphere carrier inside contains
There is GC molecule, wherein, GC molecule is intermolecular with alginate to form quiet
Electricity complexing.I.e. by the GC solution of covalent modification and alginate solution in the two equal not shape
After being sufficiently mixed uniformly under into the pH environment of precipitation, mixed liquor is formed into jet off field in high-pressure electrostatic, instilled
In the divalent salts coagulation bath of pH meta-acids, by shitosan and the Electrostatic complexation of alginate, internal containing is formed
The embedding type hydrogel microsphere carrier of galactolipin group.
Technical scheme
In the present invention, existed by the GC solution and alginate solution of covalent modification
After being sufficiently mixed under pH6.0-8.0 environment uniformly, mixed liquor is formed into jet off field in high-pressure electrostatic, instilled
In the divalent salts coagulation bath of pH5.0-6.9, form the internal embedding type hydrogel microsphere containing galactolipin group and carry
Body.Embedding type hydrogel microsphere carrier can continue to be reacted with polycation, be formed in microsphere supported surface complexation
Membrane structure, referred to as alginate/GC-polycation microcapsules；Wherein, polycation
Including it is following any one or it is two or more：Polyaminoacid class (such as polylysine, poly ornithine, poly- essence ammonia
Acid, polyhistidyl etc.), polyamine class (such as polyethyleneimine, polymethylene guanidine, poly N vinylcaprolactam,
Carboxy-propy-acrylamide copolymer, DEAE-dextran, amino-polyethyleneglycols etc.), shitosan etc..Should
Planting alginate/GC-polycation microcapsules can immerse in organic metal chelating agent solution,
Liquefaction microcapsules inside alginate；Participate in liquefaction reaction organic metal chelating agent solution be
The sodium citrate of 40-70mmol/L or the EDTA solution of 50-200mmol/L, microcapsules are chelated with organic metal
Agent solution volume range is 1:1~1:40, react 1-60 minutes, taking-up brine, now
To the microcapsules of interior liquid core.
Wherein, the GC is passed through by shitosan (Chitosan) and lactobionic acid (LA)
Amidation process generates GC (GC), and its grafting degree is in (every 100 shitosan monomers grafting
The percentage of lactobionic acid molecule) 1%-99%, reaction equation is as follows：
Lactobionic acid prepares the specific preparation process of GC material with shitosan by covalent modification, joins
Examine document as follows：TaekWoong Chung, Preparation of alginate/galactosylated
Chitosan scaffoldfor hepatocyteattachment, Biomaterials, 2002,23：
2827-2834。
Alginate/GC-polycation microcapsule product prepared by this method is particle diameter
100-1000 microns of spherical microcapsule；Microcapsule membrane thickness is at 1-100 microns；Gather sun in microcapsule membrane
Ionic material includes：Shitosan, its deacetylation is 60-98%, and molecular weight is 1kDa-800kDa (examples
Such as：1kDa-5kDa；10kDa-20kDa；50kDa-100kDa；100kDa-800kDa)；α-polylysine,
Molecular weight be 2kDa-500kDa (for example：2kDa-10kDa；70kDa-150kDa；150kDa-300kDa；
300kDa-500kDa)；Epsilon-polylysine, molecular weight be 2kDa-500kDa (for example：2kDa-50kDa；
50kDa-100kDa；100kDa-350kDa；350kDa-500kDa)；Poly arginine, molecular weight is
1kDa-500kDa is (for example：1kDa-100kDa；100kDa-350kDa；350kDa-500kDa)；Poly- bird ammonia
Acid, molecular weight be 1kDa-500kDa (for example：1kDa-50kDa；50kDa-100kDa；100kDa-350kDa；
350kDa-500kDa)；Polyhistidyl, molecular weight be 1kDa-500kDa (for example：1kDa-50kDa；
50kDa-100kDa；100kDa-350kDa；350kDa-500kDa).
Alginate is divalent metal calcium, barium or zinc hydrogel, alginate (molecule in the microcapsules
Amount 10KDa-10000kDa) solution for alginic acid sodium salt or potassium salt soln.GC solution is
Easily water-soluble GC (molecular weight 1KDa-800KDa) solution dissolved using sodium salt or sylvite.
The specific preparation process of product is：
1) lactobionic acid prepares galactosyl modification chitosan material with shitosan by amidation process, wherein,
The grafting rate of chitosan graft galactolipin group is 1%-99%；
2) difference compound concentration is that the GC solution and concentration of 1-60g/L are the sea of 10-30g/L
Alginate soln, both regulations pH environment is between 6.0-8.0, it is ensured that the two abundant dissolving.Wherein,
GC molecular weight is 1KDa-800KDa, deacetylation 80-98%, and alginic acid molecular weight is
10KDa-10000kDa；
3) with alginate solution it is 1 according to volume ratio by GC solution described in 2)：5-5：
1 ratio mixing, is stirred at room temperature 0.1-6h；
4) use 3) described in mixed solution pass through orifice extrusion molding, electrostatic drop generation, emulsion process, rotating pan
Etc. forming drop, pH is instilled in the divalent salts coagulation bath of 5.0-6.9, gel solidification 20-60 minutes, i.e.,
It is prepared into the internal embedding type hydrogel microsphere carrier containing GC molecule, referred to as A microballoons.
5) by step 4) in A microballoons immersion said polycation solution in, A microballoons and said polycation solution
The scope of volume ratio is 1:1-1:40, the reaction time be 1-60 minutes, reaction temperature at 0-37 DEG C,
Now obtain loading the embedding type hydrogel microsphere carrier of galactolipin group, i.e. alginate/galactolipin base enclosure to gather
Sugar-polycation microcapsules.
The compound method of said polycation solution is：
Shitosan is dissolved in the Acetic acid-sodium acetate buffer solution or 3-9g/L NaCl solutions that pH is 5.0-7.0,
Chitosan concentration is 0.1-15g/L；
Or, α-polylysine is dissolved in 3-9g/L NaCl solutions, α-polylysine concentration is 0.01-10g/L；
Or, epsilon-polylysine is dissolved in 3-9g/L NaCl solutions, epsilon-polylysine concentration is 0.01-10g/L；
Or, poly arginine is dissolved in 3-9g/L NaCl solutions, poly arginine concentration is 0.01-10g/L；
Or, poly ornithine is dissolved in 3-9g/L NaCl solutions, poly ornithine concentration is 0.01-10g/L；
Or, polyhistidyl is dissolved in 3-9g/L NaCl solutions, poly ornithine concentration is 0.01-10g/L；
The microcapsules can be used for the embedding of living cells, and GC solution is abundant with alginate solution
After mixing, mixed liquor mixes the microcapsules for preparing embedding living cells with the living cells of embedding is needed.Microencapsulation is thin
Born of the same parents' content is 105-2*107Individual/mL, cytoactive keeps more than 80%.The living cells is behaved or animal origin
In vitro liver cell, stem cell, the cell with hepatocyte function of stem cell differentiation, hepatic cell line is thin
Born of the same parents, the cell with hepatocyte function of transdifferentiation, endothelial cell, Kupffer cell, stellate cells,
Fibroblast, in mesenchymal stem cells MSCs one or two or more kinds.
Beneficial effects of the present invention
1st, this method ensure that the microcapsules to form that size is controllable, sphericity is good, it is to avoid microcapsules grain
Cell mass both central necrotic phenomenon caused by footpath is excessive.
2nd, alginate/GC-polycation microcapsules prepared by the method, galactolipin base enclosure
Glycan can enter inside microcapsules, and galactolipin can only be distributed in surface of microcapsule in solving traditional AC micro-capsules
Even if or become big into Microcapsules Size is also resulted in behind microcapsules inside, the problem that sphericity is deteriorated.
3rd, alginic acid is either instilled into GC solution or GC instillation is extra large
Alginic acid solution, in the alginate/GC hydrogel microsphere carrier of formation galactose content compared with
Low, the method in advance mixes alginic acid with GC in neutral conditions, therefore energy will be larger
The galactolipin of concentration is incorporated into inside microcapsules, is conducive to galactolipin group to be played inside microcapsules bigger
Function.
4th, directly be pre-mixed for two kinds of important macromolecular raw material alginic acids and GC by this method
After prepare microcapsules and can economize in raw materials to greatest extent, control cost.
5th, microcapsules prepared by this method maintain good mechanical strength while excellent sphericity is kept,
Can guarantee that the integrality as microcapsules in histocyte transplanting, cell culture application process.
6th, alginate/GC micro- glue of the microcapsules compared to core shell structure prepared by this method
Capsule, while keeping function of immune isolation, permeability is not affected, otherwise more preferably, is conducive to
Cell obtains more preferable mass transfer in the microcapsules and passes oxygen.
Specific embodiment
The method for preparing alginate/GC embedding type hydrogel microsphere carrier is electrostatic drop
Method (bibliography：In Vivo Culture of Encapsulated Endostatin-Secreting
Chinese Hamster Ovary Cells for Systemic Tumor Inhibition,Human Gene
Therapy.2007,18:474-481)。
Embodiment 1
1) sodium alginate soln is prepared：2.0g alginic acids are dissolved in 100mL physiological saline and are prepared into 20g/L
Sodium alginate soln, wherein, alginic acid molecular weight be 350kDa.
2) GC solution is prepared：GC is dissolved in physiological saline, is matched somebody with somebody respectively
Concentration processed is 0,15g/L, 20g/L, and 30g/L, pH are 7.0 GC solution.
3) coagulation bath solution is prepared：11g anhydrous calcium chlorides are dissolved in 1L deionized waters.
4) 2mL sodium alginate solns are taken, 2mL steps 2 are mixed respectively) in the various concentrations galactolipin that obtains
Base chitosan solution, is stirred at room temperature 3h, and now sodium alginate concentration is 10g/L, and GC is dense
Degree is followed successively by 0,7.5g/L, 10g/L, 15g/L, and calcium alginate/galactosyl is prepared using electrostatic drop generation
Chitosan imbedded type hydrogel microsphere carrier.
5) α-polylysin solution is prepared：α-polylysine is dissolved in physiological saline and is prepared into 5g/L's
α-polylysin solution.Wherein, α-polylysine molecule amount 30kDa.
6) calcium alginate/GC-α-polylysine microcapsules are prepared：By step 4) it is obtained
Embedding type hydrogel microsphere carrier immerse step 5) prepare α-polylysin solution in, embedding type hydrogel
Microballoon is 1 with α-polylysin solution volume ratio:10, react 10 minutes, brine is prepared into
Calcium alginate/GC-α-polylysine microcapsules.
7) by step 6) obtained in calcium alginate/GC-α-polylysine microcapsules, by micro-
Capsule：BSA=1：20 volume ratios are put into BSA (bovine serum albumin) solution of FITC marks, and laser is common
Real Time Observation under focusing microscope, each group microcapsules reached in 2h diffusion balance, show calcium alginate/
GC-α-polylysine microcapsules have good transparent performance.
8) by step 6) obtained in calcium alginate/GC-α-polylysine microcapsules and agate
Ball, physiological saline are positioned in triangular flask jointly, ball milling 24 are shaken at 37 degree, under the conditions of 170r/min small
When, the percentage of head rice of each group microcapsules shows calcium alginate/GC-α-poly- more than 90%
Lysine microcapsules have good mechanical strength.
Embodiment 2
1) sodium alginate soln is prepared：3.0g alginic acids are dissolved in 100mL physiological saline and are prepared into 30g/L
Sodium alginate soln, wherein, alginic acid molecular weight be 350kDa.
2) the GC solution of FITC marks is prepared：The GC that FITC is marked
It is dissolved in physiological saline, compound concentration is 10g/L, pH is 7.0 GC solution.
3) coagulation bath solution is prepared：11g anhydrous calcium chlorides are dissolved in 1L deionized waters.
4) 2mL sodium alginate solns are taken, 2mL steps 2 are mixed respectively) in the GC that obtains
Solution, is stirred at room temperature 3h, now sodium alginate concentration be 15g/L, GC concentration 5g/L,
Calcium alginate/GC embedding type hydrogel microsphere carrier is prepared using electrostatic drop generation.
Embodiment 3
1) sodium alginate soln is prepared：2.0g alginic acids are dissolved in 100mL physiological saline and are prepared into 20g/L
Sodium alginate soln, wherein, alginic acid molecular weight be 350kDa, filtration sterilization.
2) GC solution is prepared：0.4g GCs are dissolved in 10mL physiological saline
In be prepared into the chitosan solution of 40g/L, adjust pH value 7.2, filtration sterilization.
3) coagulation bath solution is prepared：11g anhydrous calcium chlorides are dissolved in 1L deionized waters, filtration sterilization.
4) α-polylysin solution is prepared：α-polylysine is dissolved in physiological saline and is prepared into 5g/L's
α-polylysin solution, filtration sterilization.Wherein, α-polylysine molecule amount 30kDa.
5) 3mL sodium alginate solns are taken, mixes 1mL GC solution, 1h is stirred at room temperature.Take
The 2mL mixed solutions, add 4*106Individual Rat Primary Hepatocytes, after being well mixed, use electrostatic drop generation
Prepare calcium alginate/GC mixing Rat Primary Hepatocytes embedding type hydrogel microsphere carrier.Its
In, form 350 μm or so of the particle size of embedding type hydrogel microsphere carrier.
6) calcium alginate/GC embedding type hydrogel that will be embedded with primary rat hepatocyte is micro-
Balloon borne body immerses step 4) in the polylysin solution for preparing, embedding type hydrogel microsphere carrier and polylysine
Liquor capacity ratio is 1:10, react 10 minutes, brine is prepared into calcium alginate/galactosyl
Shitosan-α-polylysine microcapsules.
7) primary rat hepatocyte in the microcapsules is cultivated and hepatocyte function is characterized, after one week,
Microcapsules keep intact form, and sphericity is good；Microcapsules inner cell activity keeps the 69% of initial activity, liver
Cell albumin secretory volume is 1.29 ± 0.33 μ g/10^6 cells, urea synthesizing amount for 349 ±
7 μ g/10^6 cells.
Comparative example
1) sodium alginate soln is prepared：2.0g alginic acids are dissolved in 100mL physiological saline and are prepared into 20g/L
Sodium alginate soln, wherein, alginic acid molecular weight be 350kDa, filtration sterilization.
2) coagulation bath solution is prepared：11g anhydrous calcium chlorides are dissolved in 1L deionized waters, filtration sterilization.
3) α-polylysin solution is prepared：α-polylysine is dissolved in physiological saline and is prepared into 5g/L's
α-polylysin solution, filtration sterilization.Wherein, α-polylysine molecule amount 30kDa.
4) 3mL sodium alginate solns are taken, mixes 1mL normal saline solutions, 1h is stirred at room temperature.Taking 2mL should
Mixed solution, adds 4*106Individual Rat Primary Hepatocytes, after being well mixed, sea are prepared using electrostatic drop generation
Calcium alginate mixing Rat Primary Hepatocytes embedding type hydrogel microsphere carrier.Wherein, embedding type hydrogel is formed
350 μm or so of microsphere supported particle size.
5) the calcium alginate embedded type hydrogel microsphere carrier immersion step 3 of primary rat hepatocyte will be embedded with)
In the α-polylysin solution of preparation, embedding type hydrogel microsphere carrier is with polylysin solution volume ratio
1:10, react 10 minutes, brine is prepared into calcium alginate-α-polylysine microcapsules.
6) primary rat hepatocyte in the microcapsules is cultivated and hepatocyte function is characterized, after one week,
Microcapsules keep intact form, and sphericity is good；Microcapsules inner cell activity keeps the 44% of initial activity, liver
Cell albumin secretory volume is 0.46 ± 0.05 μ g/10^6 cells, urea synthesizing amount for 231 ±
29 μ g/10^6 cells.
Claims (9)
1. alginate embedding type hydrogel microsphere carrier of a kind of inside containing GC molecule,
It is characterized in that：Alginate embedding type hydrogel microsphere carrier inside contains GC molecule,
Wherein, GC molecule and the intermolecular formation Electrostatic complexation of alginate；
The preparation of the alginate embedding type hydrogel microsphere carrier containing GC molecule inside this
Method is：
1) lactobionic acid prepares GC material with shitosan by covalent modification, wherein, shitosan
It is (every 100 shitosan monomers are grafted the percentage of lactobionic acid molecule) to be grafted the grafting rate of galactolipin group
1%-99%；
2) difference compound concentration is that the GC solution and concentration of 1-60g/L are 10-30g/L's
Alginate solution, two kinds of pH value of solution of regulation are between 6.0-8.0；
3) the GC solution by 2) middle preparation is according to volume ratio with alginate solution
1:5-5:1 ratio mixing, is stirred at room temperature 0.1-6h；
4) use 3) described in mixed solution pass through orifice extrusion molding, electrostatic drop generation, emulsion process or rotating disk
Method etc. forms drop, and pH is in the divalent salts coagulation bath of 5.0-6.9 for instillation, gel solidification 20-60 minutes,
It is prepared into the internal embedding type hydrogel microsphere carrier containing GC molecule.
2. according to the embedding type hydrogel microsphere carrier described in claim 1, it is characterised in that：The microballoon
Diameter of carrier is 100-1000 microns.
3. according to the embedding type hydrogel microsphere carrier described in claim 1, it is characterised in that：Embedding type water
Gel micro-ball can continue to be reacted with polycation, be complexed to form membrane structure in microsphere surface, i.e. microcapsules；Its
In, polycation include it is following any one or it is two or more：Polyaminoacid class (such as polylysine, poly- bird
Propylhomoserin, poly arginine, polyhistidyl etc.), polyamine class (such as polyethyleneimine, polymethylene guanidine, poly- N second
Alkenyl caprolactam, Carboxy-propy-acrylamide copolymer, DEAE-dextran, amino-polyethyleneglycols etc.),
Shitosan etc..
4. according to the embedding type hydrogel microcapsule described in claim 3, it is characterised in that：Described micro- glue
Capsule can be immersed in organic metal chelating agent solution, liquefaction microcapsules inside alginate；Participate in liquefaction anti-
The organic metal chelating agent solution answered is the sodium citrate of 40-70mmol/L or the EDTA of 50-200mmol/L
Solution, microcapsules are 1 with organic metal chelating agent solution volume range:1~1:40, react 1-60 minutes,
Taking-up brine, now obtains the microcapsules of interior liquid core.
5. according to the embedding type hydrogel microsphere carrier described in claim 1, it is characterised in that：The divalence
Salt coagulation bath is the one kind or two in mass concentration 3.3g/L-33g/L divalent metals calcium, barium or zinc ion solution
More than kind.
6. according to the embedding type hydrogel microsphere carrier described in claim 1, it is characterised in that：The water-setting
Alginate solution in glue support preparation method includes in sodium alginate, the potassium alginate that must contain
Kind or two kinds, do not contain or it is nonessential containing calcium alginate, barium alginate or alginic acid zinc in one kind or
More than two kinds of alginate, alginate mean molecule quantity is in 10kDa-10000kDa, the alginate middle ancient times
Lip river uronic acid monomer molar content is in 20-98%.
7. according to the embedding type hydrogel microsphere carrier described in claim 1, it is characterised in that：Shell used gathers
Sugar is easily water-soluble shitosan, including low-molecular weight chitoglycan (1KDa-10KDa), water soluble chitosan
The shitosan of (10KDa-800KDa), grafting hydrophilic group；Deacetylating degree of chitosan 60-98%, the half of formation
Lactose base enclosure glycan keeps water-soluble property.
8. the application of the embedding type hydrogel microsphere carrier described in a kind of claim 1, it is characterised in that：Institute
State the microsphere supported living cells that is mainly used in embed, GC solution is fully mixed with alginate solution
After conjunction, mixed liquor is mixed with the living cells of embedding is needed.
9. according to the application of the embedding type hydrogel microsphere carrier described in claim 8, it is characterised in that：Institute
State living cells behave or animal origin in vitro liver cell, stem cell, stem cell differentiation with liver cell
The cell of function, hepatic cell line cell, the cell with hepatocyte function of transdifferentiation, endothelial cell, liver
Kupffer's cells, stellate cells, fibroblast, in mesenchymal stem cells MSCs one or two or more kinds.
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| CN111375361A (en) * | 2020-03-17 | 2020-07-07 | 北京唐颐惠康生物医学技术有限公司 | Nano trehalose 3D microcapsule for large-scale stem cell culture |
| CN113017129A (en) * | 2019-12-24 | 2021-06-25 | 深圳波顿香料有限公司 | High water content capsule and preparation method thereof |
| CN114316309A (en) * | 2021-12-28 | 2022-04-12 | 上海瑞凝生物科技有限公司 | Polyethylene glycol-polylysine hydrogel microspheres and preparation method thereof |
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| CN108102915A (en) * | 2018-01-08 | 2018-06-01 | 大连大学 | A kind of mediate contact co-culture system for being engineered amplification |
| CN110201612A (en) * | 2019-05-20 | 2019-09-06 | 浙江大学 | A kind of core-shell structure microballoon and its application based on fluorescent microsphere monitoring myocyte's mechanical property and contraction frequency |
| CN113017129A (en) * | 2019-12-24 | 2021-06-25 | 深圳波顿香料有限公司 | High water content capsule and preparation method thereof |
| CN113017129B (en) * | 2019-12-24 | 2022-08-05 | 深圳波顿香料有限公司 | High water content capsule and preparation method thereof |
| CN111375361A (en) * | 2020-03-17 | 2020-07-07 | 北京唐颐惠康生物医学技术有限公司 | Nano trehalose 3D microcapsule for large-scale stem cell culture |
| CN114316309A (en) * | 2021-12-28 | 2022-04-12 | 上海瑞凝生物科技有限公司 | Polyethylene glycol-polylysine hydrogel microspheres and preparation method thereof |
| CN114316309B (en) * | 2021-12-28 | 2023-12-05 | 上海瑞凝生物科技有限公司 | Polyethylene glycol-polylysine hydrogel microsphere and preparation method thereof |
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