CN114276978B - Microfluidic system based on multifunctional cryogel and preparation method thereof - Google Patents
Microfluidic system based on multifunctional cryogel and preparation method thereof Download PDFInfo
- Publication number
- CN114276978B CN114276978B CN202111643982.2A CN202111643982A CN114276978B CN 114276978 B CN114276978 B CN 114276978B CN 202111643982 A CN202111643982 A CN 202111643982A CN 114276978 B CN114276978 B CN 114276978B
- Authority
- CN
- China
- Prior art keywords
- solution
- microfluidic system
- stock solution
- cryogel
- dimethylaminopropyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000495 cryogel Substances 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 239000011550 stock solution Substances 0.000 claims abstract description 42
- 239000000243 solution Substances 0.000 claims abstract description 41
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims abstract description 32
- IBVAQQYNSHJXBV-UHFFFAOYSA-N adipic acid dihydrazide Chemical compound NNC(=O)CCCCC(=O)NN IBVAQQYNSHJXBV-UHFFFAOYSA-N 0.000 claims abstract description 32
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 22
- 239000011259 mixed solution Substances 0.000 claims abstract description 22
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000010257 thawing Methods 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000008367 deionised water Substances 0.000 claims abstract description 18
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 18
- 239000000499 gel Substances 0.000 claims abstract description 18
- 238000005406 washing Methods 0.000 claims abstract description 18
- 238000003756 stirring Methods 0.000 claims abstract description 17
- 230000008014 freezing Effects 0.000 claims abstract description 16
- 238000007710 freezing Methods 0.000 claims abstract description 16
- 239000004205 dimethyl polysiloxane Substances 0.000 claims abstract description 15
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims abstract description 15
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims abstract description 15
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims abstract description 15
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229920002385 Sodium hyaluronate Polymers 0.000 claims abstract description 13
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 13
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 13
- 239000000661 sodium alginate Substances 0.000 claims abstract description 13
- 229940010747 sodium hyaluronate Drugs 0.000 claims abstract description 13
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims abstract description 13
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 11
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 239000007787 solid Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 10
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- 238000009792 diffusion process Methods 0.000 abstract description 3
- 235000015097 nutrients Nutrition 0.000 abstract description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 2
- 239000001301 oxygen Substances 0.000 abstract description 2
- 229910052760 oxygen Inorganic materials 0.000 abstract description 2
- 230000008467 tissue growth Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 22
- 239000000017 hydrogel Substances 0.000 description 10
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 5
- 238000000018 DNA microarray Methods 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000006399 behavior Effects 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 210000001665 muscle stem cell Anatomy 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 230000004791 biological behavior Effects 0.000 description 1
- 239000006177 biological buffer Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000003618 dip coating Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
Landscapes
- Medicinal Preparation (AREA)
- Materials For Medical Uses (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a preparation method of a microfluidic system based on multifunctional cryogel, which comprises the following steps: adding sodium alginate into 2- (N-morpholinyl) ethanesulfonic acid solution, adding sodium hyaluronate, stirring and dissolving to obtain mixed solution; adding adipic acid dihydrazide stock solution and N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution into the mixed solution, uniformly mixing, injecting into a micro-channel of a PDMS chip by using a syringe, freezing, thawing at room temperature, continuing freezing, repeatedly circulating, washing frozen gel obtained after thawing by using deionized water, treating by using sodium bicarbonate solution, and washing by using deionized water to prepare the microfluidic system. The cryogel has an interconnected macroporous network, is convenient for the diffusion of oxygen and nutrient substances, is beneficial to tissue growth, and has good stability and excellent mechanical property.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a microfluidic system based on multifunctional cryogel and a preparation method thereof.
Background
The microenvironment where the cells are located affects the biological behavior of the cells, and is mainly represented by the influence of two factors, namely chemical and physical factors on the growth and the activity of the cells. Physical factors mainly refer to the mechanical environment in which the cells grow, such as the hardness of the substrate. In recent years, a great deal of research has revealed that the hardness of the substrate has a great influence on cells growing in an anchored manner, and is expressed in various aspects such as cell morphology, skeleton assembly, gene expression and the like. Therefore, the magnitude of the environmental mechanical properties has a non-negligible impact on the cell biological behaviour. Numerous studies have shown that the mechanical signals transmitted by the extracellular matrix are closely related to many physiological and pathological processes. Along with the continuous research of the cell mechanical response behaviors, the cell mechanical response and the cell chemical response behaviors are combined, and the research on relevant mechanisms of various major tissue lesions is realized through interpenetration among subjects such as cell biology, biomechanics, immunology and the like, so that the method is an pioneering and significant work.
In the research of major tissue lesion mechanism, when cell tissues are cultured in vitro, the adopted in vitro culture matrix has a larger difference from the body environment in which the cells actually grow, and the actual growth environment of the cells cannot be really simulated. The hydrogel is a high molecular polymer material, can absorb water and swell and has a certain network structure. Because the hydrogel has the advantages of good tissue compatibility, higher water content, good elastic performance and the like, the hydrogel serving as a biological material is widely applied in the fields of biology, chemistry and the like.
Chinese patent CN202011598284.0 discloses a preparation method and application of a crosslinked hydrogel for muscle stem cell culture, wherein chitosan, alginate, dextran and Ca 2+ are crosslinked by a physical crosslinking method to form a double-network hydrogel with high mechanical strength, and then heparin and collagen are coated on the hydrogel by a dip coating method, so that the hydrogel can retain growth factors and adherent cells. Meanwhile, the extracted primary muscle stem cells were inoculated onto a hydrogel and cultured in a growth medium (79% dmem,10% fbs,1% diabody) for 24 hours. Then cultured in an incubator changed to a differentiation medium (97% dmem,2% horse serum, 1% diabody) for 7 days. With this hydrogel, the absorption of nutrients by muscle stem cells can be enhanced and the growth thereof can be facilitated. Although the hydrogels of the prior art described above can facilitate cell growth, in some cases, cell culture is subject to limitations of medium diffusion, which may prevent cell recombination.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the defects existing in the prior art, the invention provides a microfluidic system based on multifunctional frozen gel and a preparation method thereof, wherein sodium alginate and sodium hyaluronate are firstly subjected to double-crosslinking to prepare gel precursor solution, then the gel precursor solution is injected into a PDMS biochip, frozen polymerization and thawing are carried out, and the steps are repeated to prepare the microfluidic system compounded by the PDMS biochip and porous frozen gel, wherein the porous frozen gel has an interconnected macroporous network, is convenient for diffusion of oxygen and nutrient substances, is beneficial to tissue growth, and has good stability and excellent mechanical property.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method of preparing a microfluidic system based on a multifunctional cryogel, comprising the steps of:
(1) Adding sodium alginate into 2- (N-morpholinyl) ethanesulfonic acid solution, stirring until the solid is dissolved, adding sodium hyaluronate, and continuously stirring until the solid is dissolved to obtain a mixed solution;
(2) Adipic acid dihydrazide is dissolved in 2- (N-morpholinyl) ethanesulfonic acid solution to prepare adipic acid dihydrazide stock solution, and N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride is dissolved in 2- (N-morpholinyl) ethanesulfonic acid solution to prepare N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution;
(3) Adding adipic acid dihydrazide stock solution and N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution into the mixed solution, uniformly mixing, injecting into a micro-channel of a PDMS chip by using a syringe, freezing, thawing at room temperature, continuing freezing, repeating the cycle for 2-3 times, washing frozen gel obtained after thawing by using deionized water, treating by using sodium bicarbonate solution, and washing by using deionized water to obtain the microfluidic system.
As the preferable technical scheme, the concentration of the 2- (N-morpholino) ethane sulfonic acid solution is 45-55mmol/L, the concentration of sodium alginate in the mixed solution is 1-2wt% and the concentration of sodium hyaluronate is 1wt%.
As a preferable mode of the above technical scheme, the concentration of adipic acid dihydrazide in the adipic acid dihydrazide stock solution is 95-105mg/ml, and the concentration of N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride in the N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution is 95-105mg/ml.
As a preferable mode of the above technical scheme, the volume ratio of the mixed solution, adipic acid dihydrazide stock solution and N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution is 1: (0.01-0.015): (0.025-0.03).
As the preferable choice of the technical proposal, the temperature of the freezing treatment is between minus 20 ℃ and minus 80 ℃ and the time of the freezing treatment is between 20 hours and 24 hours.
As a preferable mode of the above technical scheme, the concentration of the sodium bicarbonate solution is 0.05-0.15mmol/L, and the treatment time is 15-25min.
As the preferable choice of the technical scheme, the diameter of the micro-channel in the PDMS chip is 0.8-0.9mm, and the length is 40-50mm.
Due to the adoption of the technical scheme, the invention has the beneficial effects that:
The sodium alginate and the sodium hyaluronate have good cell compatibility and biocompatibility, the invention takes 2- (N-morpholino) ethane sulfonic acid solution as a biological buffer, takes adipic acid dihydrazide and N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride as a cross-linking agent, carries out covalent cross-linking at low temperature, and then thawing, and the reaction treatment is carried out to prepare the frozen gel with a communicated porous structure, which has good mechanical property, the frozen gel is distributed in a micro-channel of the PDMS biochip, thereby facilitating fluid flow and realizing cell fixation, further forming 3D culture of cells, and the frozen gel is degraded along with proliferation of the cells, but the 3D cell structure is kept stable. The micro-fluidic system provided by the invention provides a 3D micro-environment, is convenient for cell perfusion culture and convenient to operate, and enables 3D tissue culture of the whole biochip to be possible.
Detailed Description
The invention is further illustrated below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The diameter of the micro-channel of the PDMS chip used in the following examples was 0.8-0.9mm and the length was 40-50mm.
Example 1
(1) Adding 1g of sodium alginate into 100ml of 2- (N-morpholinyl) ethanesulfonic acid solution with the concentration of 45mmol/L, stirring until the solid is dissolved, adding 1g of sodium hyaluronate, and continuously stirring until the solid is dissolved to prepare a mixed solution;
(2) Adipic acid dihydrazide stock solution was prepared by dissolving 4.75g of adipic acid dihydrazide in 50ml of a 45 mmol/L2- (N-morpholino) ethanesulfonic acid solution, and N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride stock solution was prepared by dissolving 4.75g N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride in 50ml of a 45 mmol/L2- (N-morpholino) ethanesulfonic acid solution;
(3) Adding 1ml of adipic acid dihydrazide stock solution and 2.5ml of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution into 100ml of mixed solution, uniformly mixing, injecting into a micro-channel of a PDMS chip by using a syringe, freezing at-20 ℃ for 20 hours, thawing at room temperature, continuing to freeze at-20 ℃ for 20 hours, repeatedly circulating for 2-3 times, washing frozen gel obtained after thawing by using deionized water, treating for 15 minutes by using sodium bicarbonate solution with the concentration of 0.05mmol/L, and finally washing by using deionized water to obtain the micro-fluidic system.
Example 2
(1) Adding 2g of sodium alginate into 100ml of 2- (N-morpholinyl) ethanesulfonic acid solution with the concentration of 55mmol/L, stirring until the solid is dissolved, adding 1g of sodium hyaluronate, and continuously stirring until the solid is dissolved to prepare a mixed solution;
(2) Adipic acid dihydrazide stock solution was prepared by dissolving 5.25g of adipic acid dihydrazide in 50ml of 2- (N-morpholinyl) ethanesulfonic acid solution with a concentration of 55mmol/L, and N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride stock solution was prepared by dissolving 5.25g N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride in 50ml of 2- (N-morpholinyl) ethanesulfonic acid solution with a concentration of 55 mmol/L;
(3) Adding 1.5ml of adipic acid dihydrazide stock solution and 3ml of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution into 100ml of mixed solution, uniformly mixing, injecting into a micro-channel of a PDMS chip by using a syringe, freezing at-20 ℃ for 24 hours, thawing at room temperature, continuing to freeze at-20 ℃ for 24 hours, repeatedly circulating for 2-3 times, washing frozen gel obtained after thawing by using deionized water, treating for 25 minutes by using sodium bicarbonate solution with the concentration of 0.15mmol/L, and finally washing by using deionized water to obtain the microfluidic system.
Example 3
(1) Adding 1.5g of sodium alginate into 100ml of 2- (N-morpholinyl) ethanesulfonic acid solution with the concentration of 45mmol/L, stirring until the solid is dissolved, adding 1g of sodium hyaluronate, and continuously stirring until the solid is dissolved to prepare a mixed solution;
(2) Adipic acid dihydrazide stock solution was prepared by dissolving 4.75g of adipic acid dihydrazide in 50ml of a 45 mmol/L2- (N-morpholino) ethanesulfonic acid solution, and N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride stock solution was prepared by dissolving 5.25g N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride in 50ml of a 45-55 mmol/L2- (N-morpholino) ethanesulfonic acid solution;
(3) Adding 1ml of adipic acid dihydrazide stock solution and 2.5ml of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution into 100ml of mixed solution, uniformly mixing, injecting into a micro-channel of a PDMS chip by using a syringe, freezing at-20 ℃ for 24 hours, thawing at room temperature, continuing to freeze at-20 ℃ for 24 hours, repeatedly circulating for 2-3 times, washing frozen gel obtained after thawing by using deionized water, treating for 25 minutes by using sodium bicarbonate solution with the concentration of 0.05mmol/L, and finally washing by using deionized water to obtain the micro-fluidic system.
Example 4
(1) Adding 2g of sodium alginate into 100ml of 2- (N-morpholinyl) ethanesulfonic acid solution with the concentration of 50mmol/L, stirring until the solid is dissolved, adding 1g of sodium hyaluronate, and continuously stirring until the solid is dissolved to prepare a mixed solution;
(2) Adipic acid dihydrazide stock solution was prepared by dissolving 5g of adipic acid dihydrazide in 50ml of a 50 mol/L2- (N-morpholinyl) ethanesulfonic acid solution, and N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride stock solution was prepared by dissolving 5g N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride in 50ml of a 50 mmol/L2- (N-morpholinyl) ethanesulfonic acid solution;
(3) Adding 1.2ml of adipic acid dihydrazide stock solution and 2.6ml of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution into 100ml of mixed solution, uniformly mixing, injecting into a micro channel of a PDMS chip by using a syringe, freezing at-20 ℃ for 21 hours, thawing at room temperature, continuing to freeze at-20 ℃ for 21 hours, repeatedly circulating for 2-3 times, washing frozen gel obtained after thawing by using deionized water, treating for 15 minutes by using sodium bicarbonate solution with the concentration of 0.07mmol/L, and washing by using deionized water to obtain a microfluidic system.
Example 5
(1) Adding 1.5g of sodium alginate into 100ml of 2- (N-morpholinyl) ethanesulfonic acid solution with the concentration of 50mmol/L, stirring until the solid is dissolved, adding 1g of sodium hyaluronate, and continuously stirring until the solid is dissolved to prepare a mixed solution;
(2) Adipic acid dihydrazide stock solution was prepared by dissolving-5.25 g of adipic acid dihydrazide in 50ml of 2- (N-morpholinyl) ethanesulfonic acid solution with the concentration of 50mmol/L, and N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride stock solution was prepared by dissolving 5.25g N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride in 50ml of 2- (N-morpholinyl) ethanesulfonic acid solution with the concentration of 50 mmol/L;
(3) Adding 1.3ml of adipic acid dihydrazide stock solution and 2.7ml of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution into 100ml of mixed solution, uniformly mixing, injecting into a micro channel of a PDMS chip by using a syringe, freezing at-20 ℃ for 22 hours, thawing at room temperature, continuing to freeze at-20 ℃ for 22 hours, repeatedly circulating for 2-3 times, washing frozen gel obtained after thawing by using deionized water, treating for 20 minutes by using sodium bicarbonate solution with the concentration of 0.1mmol/L, and washing by using deionized water to obtain a microfluidic system.
Example 6
(1) Adding 1.52g of sodium alginate into 100ml of 2- (N-morpholinyl) ethanesulfonic acid solution with the concentration of 50mmol/L, stirring until the solid is dissolved, adding 1g of sodium hyaluronate, and continuously stirring until the solid is dissolved to prepare a mixed solution;
(2) Adipic acid dihydrazide stock solution was prepared by dissolving 5.25g of adipic acid dihydrazide in 50ml of a 45 mmol/L2- (N-morpholino) ethanesulfonic acid solution, and N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride stock solution was prepared by dissolving 4.75g N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride in 50ml of a 45 mmol/L2- (N-morpholino) ethanesulfonic acid solution;
(3) Adding 1.4ml of adipic acid dihydrazide stock solution and 2.7ml of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution into 100ml of mixed solution, uniformly mixing, injecting into a micro channel of a PDMS chip by using a syringe, freezing at-20 ℃ for 23 hours, thawing at room temperature, continuing to freeze at-20 ℃ for 23 hours, repeatedly circulating for 2-3 times, washing frozen gel obtained after thawing by using deionized water, treating for 25 minutes by using sodium bicarbonate solution with the concentration of 0.055mmol/L, and washing by using deionized water to obtain a microfluidic system.
The young's modulus and pore size of the cryogels prepared in the above examples are shown in table 1:
TABLE 1
| Young's modulus kGa | Average pore size, μm | |
| Example 1 | 8.0 | 110 |
| Example 2 | 8.7 | 115 |
| Example 3 | 8.2 | 115 |
| Example 4 | 8.5 | 113 |
| Example 5 | 8.7 | 114 |
| Example 6 | 8.7 | 115 |
From the test results, the Young's modulus of the frozen gel prepared by the invention is similar to that of healthy liver tissue, has a porous structure, and is beneficial to cell culture.
Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (7)
1. A method for preparing a microfluidic system based on a multifunctional cryogel, comprising the steps of:
(1) Adding sodium alginate into 2- (N-morpholinyl) ethanesulfonic acid solution, stirring until the solid is dissolved, adding sodium hyaluronate, and continuously stirring until the solid is dissolved to obtain a mixed solution;
(2) Adipic acid dihydrazide is dissolved in 2- (N-morpholinyl) ethanesulfonic acid solution to prepare adipic acid dihydrazide stock solution, and N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride is dissolved in 2- (N-morpholinyl) ethanesulfonic acid solution to prepare N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution;
(3) Adding adipic acid dihydrazide stock solution and N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution into the mixed solution, uniformly mixing, injecting into a micro-channel of a PDMS chip by using a syringe, freezing, thawing at room temperature, continuing freezing, repeating the cycle for 2-3 times, washing frozen gel obtained after thawing by using deionized water, treating by using sodium bicarbonate solution, and washing by using deionized water to prepare a microfluidic system; the volume ratio of the mixed solution to the adipic acid dihydrazide stock solution to the N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution is 1: (0.01-0.015): (0.025-0.03).
2. A method of preparing a multi-functional cryogel-based microfluidic system according to claim 1, wherein: the concentration of the 2- (N-morpholinyl) ethanesulfonic acid solution is 45-55mmol/L, the concentration of sodium alginate in the mixed solution is 1-2wt% and the concentration of sodium hyaluronate is 1wt%.
3. A method of preparing a multi-functional cryogel-based microfluidic system according to claim 1, wherein: the concentration of adipic acid dihydrazide in the adipic acid dihydrazide stock solution is 95-105mg/ml, and the concentration of N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide hydrochloride in the N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride stock solution is 95-105mg/ml.
4. A method of preparing a multi-functional cryogel-based microfluidic system according to claim 1, wherein: the temperature of the freezing treatment is-20 to-80 ℃, and the time of the freezing treatment is 20-24 hours.
5. A method of preparing a multi-functional cryogel-based microfluidic system according to claim 1, wherein: the concentration of the sodium bicarbonate solution is 0.05-0.15mmol/L, and the treatment time is 15-25min.
6. A method of preparing a multi-functional cryogel-based microfluidic system according to claim 1, wherein: the diameter of the micro-channel in the PDMS chip is 0.8-0.9mm, and the length is 40-50mm.
7. A microfluidic system based on a multifunctional cryogel, characterized in that: prepared by the method of any one of claims 1 to 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111643982.2A CN114276978B (en) | 2021-12-30 | 2021-12-30 | Microfluidic system based on multifunctional cryogel and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111643982.2A CN114276978B (en) | 2021-12-30 | 2021-12-30 | Microfluidic system based on multifunctional cryogel and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114276978A CN114276978A (en) | 2022-04-05 |
| CN114276978B true CN114276978B (en) | 2024-06-11 |
Family
ID=80878403
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111643982.2A Active CN114276978B (en) | 2021-12-30 | 2021-12-30 | Microfluidic system based on multifunctional cryogel and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114276978B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106146912A (en) * | 2016-06-30 | 2016-11-23 | 华中科技大学 | A kind of calcium alginate hydrogel with three-dimensional micro-pattern and preparation method thereof |
| CN109652310A (en) * | 2017-10-12 | 2019-04-19 | 中国科学院大连化学物理研究所 | A kind of simple controllable method for preparing of the groove microfilament based on microflow control technique |
| KR20190102345A (en) * | 2018-02-26 | 2019-09-04 | 강원대학교산학협력단 | Hydrogel membrane fixed vertically in microfluidic chip and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2011573B1 (en) * | 2007-11-05 | 2010-07-21 | Agilent Technologies, Inc. | Freezing a microfluidic chip |
-
2021
- 2021-12-30 CN CN202111643982.2A patent/CN114276978B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106146912A (en) * | 2016-06-30 | 2016-11-23 | 华中科技大学 | A kind of calcium alginate hydrogel with three-dimensional micro-pattern and preparation method thereof |
| CN109652310A (en) * | 2017-10-12 | 2019-04-19 | 中国科学院大连化学物理研究所 | A kind of simple controllable method for preparing of the groove microfilament based on microflow control technique |
| KR20190102345A (en) * | 2018-02-26 | 2019-09-04 | 강원대학교산학협력단 | Hydrogel membrane fixed vertically in microfluidic chip and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Hongbin Zhang等.Physically crosslinked hydrogels from polysaccharides prepared by freeze–thaw technique.Reactive and Functional Polymers.2013,第73卷(第7期),第924页第3节第1段. * |
| Physically crosslinked hydrogels from polysaccharides prepared by freeze–thaw technique;Hongbin Zhang等;Reactive and Functional Polymers;第73卷(第7期);第924页第3节第1段 * |
| 壳聚糖类水凝胶的制备及其作为吸附材料的应用研究进展;狄莹莹等;合成材料老化与应用;第47卷(第6期);第74-83页 * |
| 表面复合微/纳拓扑化丝素/壳聚糖/沙蒿籽胶水凝胶的制备及对雪旺细胞的影响;韩琦等;生物医学工程研究;第40卷(第2期);第149-156页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114276978A (en) | 2022-04-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108794771B (en) | Double-network cross-linked cellulose/silk fibroin high-strength hydrogel and preparation and application thereof | |
| CN109762182B (en) | Preparation method and application of high-strength porous polyvinyl alcohol-tannic acid hydrogel | |
| CN113416326B (en) | Injectable hydrogel and preparation method and application thereof | |
| CN111632198A (en) | Self-crosslinking hyaluronic acid and gelatin composite hydrogel injection and preparation method and application thereof | |
| CN102274545A (en) | Galactosylated chitosan scaffold material for bioartificial liver and preparation method thereof | |
| CN105802916B (en) | Preparation and application methods of chitosan hydrogel three-dimensional cell culture medium | |
| Sazhnev et al. | Preparation of chitosan cryostructurates with controlled porous morphology and their use as 3D-scaffolds for the cultivation of animal cells | |
| Zhang et al. | Injectable conductive micro-cryogel as a muscle stem cell carrier improves myogenic proliferation, differentiation and in situ skeletal muscle regeneration | |
| CN109705359B (en) | Modified polyethylene glycol-polysebacic acid (PEGS) injectable biological elastomer and preparation method and application thereof | |
| CN103819694B (en) | A kind of silk fibroin hydrogel with cell compatibility and preparation method thereof | |
| CN104587525A (en) | Scaffold containing platelets and hyaluronic acid and preparation method of scaffold | |
| CN114276978B (en) | Microfluidic system based on multifunctional cryogel and preparation method thereof | |
| Concaro et al. | Effect of cell seeding concentration on the quality of tissue engineered constructs loaded with adult human articular chondrocytes | |
| CN107982578B (en) | Preparation method of nano hydroxyapatite/cyclodextrin-based polyurethane composite porous bone tissue engineering scaffold material | |
| CN106750416A (en) | A kind of injection aquagel for possessing self-healing and pH response performances and its preparation method and application | |
| CN107936267A (en) | A kind of preparation method and expansion of stem cells and collection method of two ionic starch base expansion of stem cells hydrogel | |
| CN112111162B (en) | Rapidly-curable double-network hydrogel and preparation method and application thereof | |
| CN111286483A (en) | Porous nano-scale soft colloid and method for transporting cell or tissue culture by using same | |
| CN116240166A (en) | Self-assembly method and application of osteochondral organoids | |
| CN115369072A (en) | Hydrogel fiber for realizing formation and passage of multicellular spheroids and preparation method thereof | |
| CN109851834B (en) | Polyethylene-hydroxyapatite micron nano multilevel structure composite microsphere material and application | |
| CN109824934B (en) | Polyethylene-titanium oxide micron nano multilevel structure composite microsphere material and application | |
| CN106075599A (en) | A kind of can cell in-situ cladding porous congeal glue and preparation method thereof | |
| CN113018517A (en) | 3D printing skin stent and preparation method and application thereof | |
| Ohya et al. | Material design for an artificial extracellular matrix: cell entrapment in poly (N-isopropylacrylamide)(PNIPAM)-grafted gelatin hydrogel |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |