CN102172498B - Three-dimensional porous chitosan/gelatin microsphere, preparation method thereof and application thereof in hepatocyte culture - Google Patents
Three-dimensional porous chitosan/gelatin microsphere, preparation method thereof and application thereof in hepatocyte culture Download PDFInfo
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Abstract
The invention discloses a three-dimensional porous chitosan/gelatin microsphere, a preparation method thereof and application thereof in hepatocyte culture. The preparation method comprises the steps of mixing chitosan and gelatin B, dripping liquid nitrogen into the mixture through a high-pressure pulse microcapsule forming instrument to be rapidly frozen into spheres, crosslinking and curing the microspheres subjected to primary freeze-drying by using a saturated tripolyphosphate-85% ethanol solution, performing secondary freeze-drying, fully hydrating the microspheres obtained by the secondary freeze-drying, adding a crosslinking agent carbodiimide/N-hydroxysuccinimide, modifying the microspheres by using gelatin A, performing water bath, performing dark reaction, washing off unreacted carbodiimide/N-hydroxysuccinimide and gelatin A, performing tertiary freeze-drying to obtain the three-dimensional porous chitosan/gelatin microspheres with the diameter of 300-. The three-dimensional porous chitosan/gelatin microspheres obtained by the invention can be used for culturing high-density and high-activity liver cells in vitro.
Description
Technical field
the present invention relates to a kind of three-dimensional porous loaded Chitosan/Gelatin Microspheres and preparation method thereof and the application in hepatocyte cultures.Relate to more specifically the large pore property that utilizes gelatin and the gelatin characteristic as good extracellular matrix, in conjunction with generator for microcapsules-electrostatic method, ion condensation method and three desivacs, prepare a kind of three-dimensional porous loaded Chitosan/Gelatin Microspheres and the application in hepatocyte cultures.
Background technology
Shitosan is a kind of boiomacromolecule obtained through deacetylation by chitin; it is the alkaline polysaccharide of the unique positively charged of nature found up to now; nontoxic; there is good biocompatibility and biodegradable, all be widely used at aspects such as food, environmental protection, agricultural, medicine.Collagen is the rich in protein of animal body intensive amount, and gelatin, as the hydrolysate of collagen, mainly is comprised of unique amino acid sequence Gly-Pro-carboxyl proline, these sequences make gelatin fixedly hydrone form helical structure.Shitosan and gelatin can be according to the arbitrary proportion blend by intermolecular hydrogen bonding.Utilize the large pore property of gelatin and the gelatin characteristic as good extracellular matrix, generator for microcapsules-electrostatic method, ion condensation method and desivac combine and can make satisfactory three-dimensional porous loaded Chitosan/Gelatin Microspheres, for hepatocellular external a large amount of amplifications, simulation liver cell three-dimensional environment in vivo, rebuild hepatocellular polarity, keep hepatocellular high activity, the biosynthetic metabolism function.
Prepare according to the method described above a kind of three-dimensional porous loaded Chitosan/Gelatin Microspheres with shitosan, gelatin, and used it for hepatocyte cultures.Although both at home and abroad the existing article of preparing microballoon is delivered, material used, prepare the method for microballoon and the microballoon made and the present invention and all be very different.At first be on material is selected, selected shitosan and gelatin as the main material for preparing microballoon, although also useful shitosan or (with) the gelatin article of preparing microballoon delivers, but the method adopted is mainly that emulsion-crosslinking method (utilizes atoleine or edible oil as oil phase, shitosan or (with) gelatin or sodium alginate etc. be as water, stir, the size of controlling liquid drop forms oil-in-water or Water-In-Oil suspension, adding glutaraldehyde is main crosslinking agent reaction balling-up, washing, obtain microballoon after drying, the microballoon obtained mostly is solid microsphere, add the loose structure that the microballoon that obtains after pore-foaming agent neither be real, but the hole of internal independence, with the external world, do not communicate, and the easy adhesion of the microballoon that emulsion process obtains, and crosslinking agent glutaraldehyde itself is poisonous, also increased certain risk factor, the microballoon that at present emulsion process is prepared is mainly used in the research of microsphere drug slowly-releasing aspect) and the cohesion/precipitation method (utilize shitosan be insoluble to alkaline solution characteristics or (with) the both sexes characteristic of gelatin, shitosan or shitosan/gelatin solution are splashed in alkaline solution, as NaOH, NaOH-methyl alcohol etc., by centrifugal, filtration is carried out separation and purification to microballoon, obtain microballoon after washing, but the microsphere surface that this method obtains is shrinkage forms groove, surface does not communicate with inside, be blind hole).Although the bibliographical information of desivac is also arranged, mostly be directly and will after the solution mixing, pour in mould and directly after freeze-drying, prepare membranaceous or cylindric support; Or NaOH, the glutaraldehyde etc. for support of freeze-drying is crosslinked, but due to the shitosan after freeze-drying or (with) the gelatin materials space structure changes, very easily water-soluble, can not well keep original shape after crosslinked, therefore be generally used for the membranaceous support of preparation for skin tissue engineering or prepare mould rack for bone tissue engineer, there are no spherical, diameter communicates with inner aperture on 50-200um, surface at 300-800um, surface apertures and the report of larger three-dimensional porous microballoon the closer to aperture, microballoon inside.
The present invention directly splashes into snap frozen balling-up in liquid nitrogen by shitosan/gelatin B microballoon by high-voltage pulse microcapsules forming instrument, by adjusting shitosan, the concentration of gelatin and mixing match, the size of syringe pin hole, syringe needle point and metal guide wire loop distance, metal guide wire loop and liquid nitrogen container mouth distance, high-voltage pulse microcapsules forming instrument voltage, frequency, pulsewidth, the size of the control shitosan/gelatin B solution droplets such as Portable-type micro-injection pump speed is controlled the initial size of microballoon, and in subsequent step by adjusting the crosslinking agent tripolyphosphate, concentration and the pH value of carbodiimide/N-hydroxyl succinyl, crosslinking time, freezing temperature, three freeze-drying modes obtain keeping spherical-like morphology, enlarged aperture, surperficial and inner aperture communicates, transparent, be beneficial to the microballoon of cell adhesion.Therefore, in concentration and the proportioning of the selection of material, material, the application facet for preparing the concentration, crosslinking time of use, the crosslinking agent of two kinds of crosslinking agents in method (controlling the factor of microballoon size) that microballoon uses, preparation process, freezing temperature, freeze-drying mode, three-dimensional porous microballoon etc. all should be the claimed content of the present invention.
Summary of the invention
One of purpose of the present invention is to provide a kind of three-dimensional porous loaded Chitosan/Gelatin Microspheres.
Two of purpose of the present invention is to provide the preparation method of above-mentioned a kind of three-dimensional porous loaded Chitosan/Gelatin Microspheres.
Three of purpose of the present invention is to provide the above-mentioned application process of a kind of three-dimensional porous loaded Chitosan/Gelatin Microspheres in hepatocyte cultures.
Be the composition of the present invention according to three-dimensional microcosmic structure and the liver cell epimatrix of liver leaflet, according to bionics principle, prepare a kind of three-dimensional porous microballoon with biocompatible materials, microsphere surface is connected with inside, make liver cell can be attached on equably microsphere surface and internal void, three-dimensional environment in analogue body, reconstituted cell polarity, reach the hepatocellular purpose of external a large amount of amplification, prepare highly active liver cell, the performance hepatocyte function, to cell high density in bioartificial liver (BAL) and hepatocyte transplantation, high activity, large-scale culture provides certain theoretical foundation and experimental basis, promote its clinical practice at BAL and development.
Technical scheme of the present invention
Make three-dimensional porous loaded Chitosan/Gelatin Microspheres by generator for microcapsules-electrostatic method, ion condensation method and three desivacs.
The preparation method of above-mentioned three-dimensional porous loaded Chitosan/Gelatin Microspheres, as shown in Figure 2, it specifically comprises the following steps the process flow diagram of its preparation process:
(1), the preparation of a freeze-drying microballoon
1.5% shitosan acetic acid solution and 2% gelatin B (are taken to the ox-hide skin, sigma company) aqueous solution is 1.5% shitosan acetic acid solution by volume: the 2% gelatin B aqueous solution is that 5:1 mixes mutually, in the syringe that the suction aperture size is 0.7mm, syringe is arranged on Portable-type micro-injection pump, the syringe needle end connects the positive pole of high-voltage pulse microcapsules forming instrument, the metal guide wire loop of syringe needle below connects the negative pole of high-voltage pulse microcapsules forming instrument, the conductor loop below is liquid nitrogen container, open successively high-voltage pulse microcapsules forming instrument, the Portable-type micro-injection pump switch, under the high-pressure electrostatic effect, make the drop of formation splash into liquid nitrogen in the snap frozen balling-up, as Figure 1-1,
Control syringe needle point and metal guide wire loop in freezing balling-up process apart from 0mm-5mm, metal guide wire loop and liquid nitrogen container mouth, apart from 5mm-10mm, arrange high-voltage pulse microcapsules forming instrument voltage 60, frequency 90, and pulsewidth 6, Portable-type micro-injection pump speed is 90mm/h;
Carry out freeze-drying after balling-up, controlling freeze temperature is-60 ℃ to-80 ℃, obtains the loaded Chitosan/Gelatin Microspheres 1 of a freeze-drying after freeze-drying 24-48h again;
Described gelatin B takes from the ox-hide skin, by being that alkaline process obtains, and has more carboxyl on described gelatin B strand, and its isoelectric point is in pH=4.7～5;
(2), the preparation of secondary freeze-drying microballoon
The ethanolic solution that microballoon 1 use that step (1) is made contains saturated tripolyphosphate (TPP) 85%, hierarchy of control pH value is at 5-6, again after crosslinking curing 24-72h, stop cross-linking reaction, enter-20 ℃ of refrigerator overnight, freeze dryer on next day, controlling freeze temperature is-60 ℃ to-80 ℃, obtain the microballoon 2 of secondary freeze-drying after freeze-drying 24～48h, utilize the gelatin strong characteristic that absorbs water to prepare the macropore loaded Chitosan/Gelatin Microspheres 2 that aperture has enlarged after than a freeze-drying;
The proportioning of the amount of the loaded Chitosan/Gelatin Microspheres 1 of a freeze-drying and saturated tripolyphosphate (TPP) 85% ethanolic solution is by the amount of microballoon 1: with saturated tripolyphosphate (TPP) 85% ethanolic solution be 0.01g:50ml;
(3), the preparation of three three-dimensional porous loaded Chitosan/Gelatin Microspheres of freeze-drying
The abundant aquation of microballoon 2 that step (2) is made; add 2% water-soluble cross-linker carbodiimide/N-hydroxyl succinyl (EDC/NHS) and 2% gelatin A (to take from porcine skin; sigma company); after 38 ℃ of water-baths, lucifuge reaction 24～48h; wash away unreacted EDC/NHS and gelatin A; enter-20 ℃ of refrigerator overnight, carry out freeze-drying for the third time next day, controlling freeze temperature is to obtain three-dimensional porous loaded Chitosan/Gelatin Microspheres 3 of the present invention after-60 ℃ to-80 ℃ freeze-drying 24-48h;
The abundant aquation of microballoon 2 is at the bottom of microballoon is sink to test tube, the amount non-correlation of the water yield used and microballoon;
The proportioning of microballoon 2, carbodiimide/N-hydroxyl succinyl (EDC:NHS mass ratio=4:1) and gelatin, by the amount of microballoon 2: carbodiimide/N-hydroxyl succinyl (EDC:NHS mass ratio=4:1) aqueous solution volume: 2% gelatin A aqueous solution volume is 0.01g:40ml:10ml;
Described gelatin A takes from porcine skin, by acid system, obtains, and described gelatin A strand contains more amino, and isoelectric point is in pH=7～9.
Beneficial effect of the present invention
The three-dimensional porous loaded Chitosan/Gelatin Microspheres diameter that the present invention makes is at 300-800um, average diameter 642.49um, and surface apertures 50-200um, microsphere surface is connected with inner aperture.Utilize the large pore property of gelatin to prepare macroporous microsphere under the prerequisite that does not change microsphere diameter, microballoon after modifying with this good extracellular matrix of gelatin provides efficient binding site for liver cell, can promote and instruct the hepatocellular direction of sticking and stick.Size by regulating the syringe pin hole, syringe needle point and metal guide wire loop distance, metal guide wire loop and liquid nitrogen container mouth are apart from the concentration of the parameter (as voltage, frequency, pulsewidth) of, high-voltage pulse microcapsules forming instrument, speed, crosslinking agent tripolyphosphate and the carbodiimide of Portable-type micro-injection pump/N-hydroxyl succinyl, pH value, crosslinked time, freezing temperature, the mode of freeze-drying etc. can be controlled size and the aperture of microballoon.
In addition, the three-dimensional porous loaded Chitosan/Gelatin Microspheres of gained of the present invention, owing to having increased specific area, improved the density that cell is cultivated, thereby improved the oxygen supply of cell and the exchange of metabolite; Growth of Cells, in the microsphere surface hole of loose structure, has reduced the damage of shearing force to cell while cultivating that suspend; Three-dimensional porous characteristic, make liver cell pass through the 3 D stereo growing environment of lobuli hepatis in the span analogue body, and reconstituted cell polarity maintains form and the specific function of liver cell in vivo the time, reaches and turns out in vitro high density, highly active liver cell.Can be used for cell quantity and quality are had bioartificial liver, the hepatocyte transplantation of requirement for height.
The accompanying drawing explanation
Fig. 1 be the present invention's structural representation of preparing microballoon under the high-pressure electrostatic effect (in figure: 1 for Portable-type micro-injection pump, 2 for syringe, 3 for shitosan/gelatin mixed liquor, 4 for syringe needle, 5 for the metal guide wire loop, 6 for liquid nitrogen container, 7 for high-voltage pulse microcapsules forming instrument, 8 for the positive pole of high-voltage pulse microcapsules forming instrument, 9 be the negative pole of high-voltage pulse microcapsules forming instrument);
Fig. 2 is the process flow diagram of the preparation process of three-dimensional porous loaded Chitosan/Gelatin Microspheres;
Fig. 3 is the scanning electron microscope (SEM) photograph of a loaded Chitosan/Gelatin Microspheres after freeze-drying;
Fig. 4-1 is the scanning electron microscope (SEM) photograph of three three-dimensional porous loaded Chitosan/Gelatin Microspheres after freeze-drying;
Fig. 4-2 are scanning electron microscope (SEM) photographs of three three-dimensional porous loaded Chitosan/Gelatin Microspheres after freeze-drying;
Fig. 5 observes the growth conditions figure of HepG2 cell lines on three-dimensional porous loaded Chitosan/Gelatin Microspheres under light microscope;
Fig. 6 observes the growth conditions figure of HepG2 cell lines on three-dimensional porous loaded Chitosan/Gelatin Microspheres under the fluorescence inverted microscope;
Fig. 7 is the scanning electron microscope (SEM) photograph after HepG2 cell lines and three-dimensional porous loaded Chitosan/Gelatin Microspheres mix suspending are cultivated;
Fig. 8 is the transmission electron microscope picture after HepG2 cell lines and three-dimensional porous loaded Chitosan/Gelatin Microspheres mix suspending are cultivated;
Fig. 9, Figure 10 be respectively after HepG2 cell lines and three-dimensional porous loaded Chitosan/Gelatin Microspheres HE dyeing under light microscope 40 times and 100 times of enlarged drawings;
Figure 11 is control group (plane cultivation group k) and experimental group (Mixed culture group g) real-time fluorescence quantitative PCR albumin gene expression result.
The specific embodiment
below by embodiment, also the invention will be further described by reference to the accompanying drawings, but do not limit the present invention.
The present invention's HepG2 cell lines used is taken from the Shanghai Chinese Academy of Sciences.
Middle viscosity shitosan (is taken from the crab shell; Molecular weight: 400,000; 84%～89%), gelatin A (A type gelatin, take from porcine skin, Powdered, cell cultivate level), gelatin B(B type gelatin deacetylation:, take from the ox-hide skin), carbodiimide/N-hydroxyl succinyl (EDC/NHS) is purchased from sigma;
Tripolyphosphate (Alfa Aesar);
Contain the complete medium (RP-1640) of 10% calf serum (Gibco);
Poly HEMA (poly (2-hydroxyethyl Methacrylate), poly-HEMA) (sigma);
Acridine orange (sigma);
Acetic acid (glacial acetic acid is analyzed pure AR);
The equipment that the present invention is used:
High-voltage pulse microcapsules forming instrument (Shanghai University of Science and Technology);
Portable-type micro-injection pump (Angel 5805 types, the clean electronics of Town in Shanghai);
Vacuum freeze drier (VIRTIS, BT3.3EL);
Pure water instrument (Mili-Q50 type) (Millipore);
Surgical operation microscope (TOPCON, MS-XY03);
Light microscope (joining fluorescence) (OLYMPUS, IX71);
Stainless (steel) wire (200 order) (ancient cooking vessel state biology);
SEM (Scanning electron microscopy, SEM, PHILIPS XL30 FEG); Automatic clinical chemistry analyzer (BECKMAN-UniCel DxC 800);
The universal decolorization swinging table of TS-92 (QILINBEIER, TS-92).
A kind of preparation of three-dimensional porous loaded Chitosan/Gelatin Microspheres
(1), the preparation of a freeze-drying microballoon
Prepare respectively 1.5% shitosan (with 1% acetic acid preparation) and 2% gelatin B (preparing with distilled water), after mixing, be 1.5% shitosan acetic acid solution by volume: the 2% gelatin B aqueous solution is that 5:1 mixes mutually, after removing bubble, suck in the 5ml syringe that the syringe needle internal diameter is 0.7mm, connect generator for microcapsules-electrostatic equipment (as Fig. 1 shows), (voltage U=60 under the high-pressure electrostatic environment, frequency F=90, pulsewidth PW=6: high-voltage pulse microcapsules forming instrument, Shanghai University of Science and Technology) splash in liquid nitrogen, after at the bottom of microballoon is sink to liquid nitrogen container, microballoon is shifted out and puts on the iron screen cloth of crossing through immersing in liquid nitrogen, upper freeze dryer, controlling freeze temperature is-60 ℃ to-80 ℃, closing freeze dryer after 24-48 hour takes out, obtain the loaded Chitosan/Gelatin Microspheres 1 after freeze-drying one time,
(2), the preparation of secondary freeze-drying microballoon
The microballoon that step (1) is made uses the 85% ethanolic solution crosslinking curing that contains tripolyphosphate (TPP) after 24-72 hour, controlling the pH value is 5-6, remove the crosslinking agent on upper strata, add water and fully wash, microballoon is moved in culture dish, siphon away as much as possible excessive moisture, enter-20 ℃ of refrigerator overnight, freeze dryer on next day, controlling freeze temperature is-60 ℃ to-80 ℃, obtains the microballoon 2 after the secondary freeze-drying after 24-48 hour;
(3), the preparation of three freeze-drying novel three-dimensional porous chitosan/gelatine microspheres
The abundant aquation of microballoon that step (2) is made, the liquid on upper strata exhausts as far as possible, add 2% water-soluble cross-linker carbodiimide/N-hydroxyl succinyl (EDC/NHS) and 2% gelatin A(2%EDC/NHS:2% gelatin A=4:1(v/v)), lucifuge, in 38 ℃ of water-baths, reaction is after 24-48 hour, add washing and remove unreacted EDC/NHS and gelatin A, move in culture dish, siphon away excessive moisture as far as possible, enter-20 ℃ of refrigerator overnight, freeze dryer on next day, controlling freeze temperature is-60 ℃ to-80 ℃, obtains three-dimensional porous loaded Chitosan/Gelatin Microspheres of the present invention after 24-48 hour.
Fig. 1 prepares the structural representation of microballoon under the high-pressure electrostatic effect in step (1)
.
Fig. 3 is the scanning electron microscope (SEM) photograph of the loaded Chitosan/Gelatin Microspheres 1 after a freeze-drying of step (1) gained,
Microsphere diameter is at 300-500um, and its aperture is 10um-45um.
Fig. 4-1st, the scanning of the three-dimensional porous loaded Chitosan/Gelatin Microspheres after three freeze-drying of step (3) gained
Electronic Speculum figure, microsphere diameter is at 300-800um, and its aperture is 50um-200um.
Fig. 4-2 are scanning electron microscope (SEM) photographs (Fig. 4-1 enlarged drawing) of three three-dimensional porous loaded Chitosan/Gelatin Microspheres after freeze-drying, and aperture is 151um.
The aperture that can find out microballoon after three freeze-drying from Fig. 3, Fig. 4-1, Fig. 4-2 has after than a freeze-drying
Significantly enlarge, its aperture is 4.4 times～5 times after a freeze-drying.
application Example 1
A kind of three-dimensional porous loaded Chitosan/Gelatin Microspheres and HepG2 cell lines Mixed culture
(1), the recovery of HepG2 cell lines
Take out cryopreservation tube from liquid nitrogen container after, put into 38 ℃ of water bath vibrations, within 1 minute, thaw, the complete medium (RP-1640) that adds immediately 1ml to contain 10% calf serum (GIBCO) mixes, slowly add again the 9ml nutrient solution, after mixing piping and druming, centrifugal (1000rpm/min, 5min), after supernatant discarded, add appropriate nutrient solution in pellet, after mixing, be seeded in culture dish.Change every other day liquid and remove not adherent cell and metabolite, reach 2-3 for rear for subsequent experimental;
(2), the preparation of three-dimensional porous loaded Chitosan/Gelatin Microspheres
By above-mentioned after three freeze-drying after the abundant aquation of three-dimensional porous loaded Chitosan/Gelatin Microspheres (be at the bottom of microballoon is sink to test tube prompting aquation complete) of gained, suck water, add 75% ethanol disinfection 30min, with aseptic double-distilled water, wash away ethanol, add nutrient solution RP-1640 in aseptic microballoon and soak standby;
The consumption of nutrient solution RP-1640 is placed in the shared volume of test tube by three-dimensional porous loaded Chitosan/Gelatin Microspheres: RP-1640 nutrient solution volume is that 1:5 calculates;
(3), three-dimensional porous loaded Chitosan/Gelatin Microspheres and HepG2 cell lines Mixed culture
The trypsinization HepG2 cell lines, collecting cell, centrifugal (1000rpm/min, 5min), abandon supernatant, adds the 5mlRP-1640 nutrient solution in pellet, and counting is adjusted cell concentration 1 * 10
5individual/ml; Add the three-dimensional porous loaded Chitosan/Gelatin Microspheres after nutrient solution soaks in 12 orifice plates (coating poly-HEMA) of anti-adherent processing, confluent cultures ware bottom 50%, the nutrient solution that as far as possible exhausts, add cell suspension 1ml (cell concentration 1 * 10 in every hole
5individual/ml, TCS is 1 * 10
5individual), suspend and cultivate 1 hour on shaking table, static cultivation 1 hour, suspend on shaking table and cultivate 1 hour (so repeating 3 times), after every Kong Xu adds the 1ml fresh medium, low speed (30-40rpm/min) suspension is cultivated, and cultivates after 24 hours and changes nutrient solution, after this maintaining low speed (30-40rpm/min) suspension cultivates, within every 24 hours, change liquid, low speed (30-40rpm/min) suspends and cultivates, and in the time of 7 days, cell density is 4.2 * 10
5individual, in the time of 13 days, cell density is 2.8 * 10
6individual (and under the condition of culture of plane, 12 orifice plate adhere-wall culture, the cell initial concentration is identical with experiment suspension cultivation group, and in the time of the 5th day, cell is because of the growing space deficiency, it is floating that cell starts to pile up, then cell death).
Fig. 5 observes the growth conditions figure of HepG2 cell lines on three-dimensional porous loaded Chitosan/Gelatin Microspheres under light microscope, as can be seen from the figure three-dimensional porous loaded Chitosan/Gelatin Microspheres is transparent spherical, visible cell is grown on three-dimensional porous loaded Chitosan/Gelatin Microspheres, cell is bright is spherical, and after birth is complete.
Fig. 6 observes the growth conditions figure of HepG2 cell lines on three-dimensional porous loaded Chitosan/Gelatin Microspheres under the fluorescence inverted microscope, as can be seen from the figure cell is spherical after acridine orange dyeing, can indirectly demonstrate the profile of three-dimensional porous loaded Chitosan/Gelatin Microspheres after cell dyeing simultaneously, it is spherical that microballoon is, and local cells forms sphere aggregates.
Fig. 7 (Fig. 7-1,7-2) is the scanning electron microscope (SEM) photograph after HepG2 cell lines and three-dimensional porous loaded Chitosan/Gelatin Microspheres mix suspending are cultivated, the as can be seen from the figure spheric profile of three-dimensional porous loaded Chitosan/Gelatin Microspheres, the surface hole defect of local visible microballoon, cell is obvious at the microsphere surface clustering phenomena, cell keeps spherical distribution, to the hole growth inside, visible abundant microvillus (Fig. 7-2 show) between cell and cell.
Fig. 8 is the transmission electron microscope picture after HepG2 cell lines and three-dimensional porous loaded Chitosan/Gelatin Microspheres mix suspending are cultivated, and many irregular microvillus appear in the surface contacted with nutrient solution as seen in figure, and quantity is abundant, the sinus hepaticus face in analog.Liver plasma membrane is complete, and nucleus is similar round, is positioned at central authorities or slightly is partial to certain side, and caryoplasm is more even, and nuclear membrane is clear.The kytoplasm Mitochondria mostly is oval, and what have is stock shape, and ridge is abundanter.Visible endoplasmic reticulum is distributed in kytoplasm more equably.
In Fig. 9 and Figure 10, optical microphotograph Microscopic observation: HE dyeing is lower shows that loaded Chitosan/Gelatin Microspheres surface and inside are mesh-like structure, short texture, and fibre bundle is tiny, arranges more regularly, and interior cavity is connected as seen.The prompting of HE coloration result: cell can be grown at microsphere surface, and can enter growth inside.Again verified that we are connected in constructed three-dimensional porous loaded Chitosan/Gelatin Microspheres surface and inside.
In Figure 11, control group is plane cultivation group k, and not containing three-dimensional porous loaded Chitosan/Gelatin Microspheres, simple HepG2 cell lines is in culture dish bottom adherent growth; Experimental group is Mixed culture group g, contains three-dimensional porous loaded Chitosan/Gelatin Microspheres, in the culture dish through anti-adherent processing, with the HepG2 cell lines mix suspending, cultivates.
Carrying out respectively three-dimensional porous loaded Chitosan/Gelatin Microspheres and HepG2 cell lines mix suspending cultivates and the plane cultivation, at different time sections difference collecting cell, extract cell RNA, real-time fluorescence quantitative PCR is analyzed albumin (ALB) gene expression dose, find to cultivate after 24 hours, experimental group (g-alb) ALB expression is 3.36 times of control group (k-alb); After Mixed culture 72 hours, experimental group ALB expression is 1260.69 times of control group; Descend gradually afterwards, but still maintain higher level, in the time of the 6th day, to express be still 36 times of control group to experimental group ALB, and difference all has statistical significance.It the results are shown in Figure shown in 9, illustrates that three-dimensional porous loaded Chitosan/Gelatin Microspheres and HepG2 cell lines mix suspending are cultivated can rebuild hepatocellular polarity, maintains liver cell three-dimensional environment in vivo, brings into play hepatocyte function.
Respectively by ALT in gained cultured cell supernatant, LDH, urea level send automatic clinical chemistry analyzer to detect, result shows that control group and experimental group do not have significant difference, supernatant albumin (alb) ELISA result does not show both yet, and there were significant differences, trace it to its cause, because three-dimensional porous loaded Chitosan/Gelatin Microspheres water content after nutrient solution soaks own is just abundanter, so be difficult to accurately control the cumulative volume of nutrient solution in follow-up process, thereby cause and detect the difference that ALT, LDH, urea, alb can not accurately reflect experimental group and control group from supernatant; The advantage that simultaneously mix suspending is cultivated shows later stage 7-14 talent, and under the condition of culture of plane because the restriction of culture space can only be carried out 7 days with interior observation.
Above said content is the basic explanation under conceiving for the present invention only, and, according to any equivalent transformation that technical scheme of the present invention is done, all should belong to protection scope of the present invention.
Claims (8)
1. the preparation method of a three-dimensional porous loaded Chitosan/Gelatin Microspheres is characterized in that comprising the following steps:
(1), the preparation of a freeze-drying microballoon
By 1.5% shitosan acetic acid solution and the 2% gelatin B aqueous solution by volume, i.e. 1.5% shitosan acetic acid solution: the 2% gelatin B aqueous solution is that 5:1 mixes mutually, in the syringe that the suction aperture size is 0.7mm, utilize the high-pressure electrostatic principle to splash into snap frozen balling-up in liquid nitrogen through high-voltage pulse microcapsules forming instrument; Control needle point and metal guide wire loop in freezing balling-up process apart from 0mm-5mm, metal guide wire loop and liquid nitrogen container mouth, apart from 5mm-10mm, arrange high-voltage pulse microcapsules forming instrument voltage 60, frequency 90, and pulsewidth 6, Portable-type micro-injection pump speed is 90mm/h; Carry out freeze-drying after balling-up, controlling freeze temperature is-60 ℃ to-80 ℃, obtains the loaded Chitosan/Gelatin Microspheres 1 of a freeze-drying after freeze-drying 24-48h again; Described gelatin B takes from the ox-hide skin, by being that alkaline process obtains, and has more carboxyl on described gelatin B strand, and its isoelectric point is in pH=4.7～5;
(2), the preparation of secondary freeze-drying microballoon
The ethanolic solution that microballoon 1 use that step (1) is made contains saturated tripolyphosphate (TPP) 85%, hierarchy of control pH is 5-6, again after crosslinking curing 24-72h, stop cross-linking reaction, enter-20 ℃ of refrigerator overnight, freeze dryer on next day, controlling freeze temperature is-60 ℃ to-80 ℃, obtains the microballoon 2 of secondary freeze-drying after freeze-drying 24～48h;
(3), the preparation of three three-dimensional porous loaded Chitosan/Gelatin Microspheres of freeze-drying
The abundant aquation of microballoon 2 that step (2) is made, add 2% water-soluble cross-linker carbodiimide/N-hydroxyl succinyl (EDC/NHS) aqueous solution and the 2% gelatin A aqueous solution, after 38 ℃ of water-baths, lucifuge reaction 24-48h, wash away unreacted EDC/NHS and gelatin A, enter-20 ℃ of refrigerator overnight, carry out freeze-drying for the third time next day, controlling freeze temperature is to obtain three-dimensional porous loaded Chitosan/Gelatin Microspheres 3 after-60 ℃ to-80 ℃ freeze-drying 24-48h; Described gelatin A takes from porcine skin, by acid system, obtains, and described gelatin A strand contains more amino, and isoelectric point is in pH=7～9.
2. the preparation method of a kind of porous chitosan/gelatine microsphere as claimed in claim 1, it is characterized in that the shitosan of a freeze-drying in step (the 2)/amount of gelatin B microballoon 1 and the proportioning of saturated tripolyphosphate (TPP) the 85% ethanolic solution amount by microballoon 1: saturated tripolyphosphate (TPP) 85% ethanolic solution is 0.01g:50mL.
3. the preparation method of a kind of three-dimensional porous loaded Chitosan/Gelatin Microspheres as claimed in claim 1, the proportioning that it is characterized in that microballoon 2, carbodiimide/N-hydroxyl succinyl and gelatin in step (3), by the amount of microballoon 2: 2% carbodiimide/N-hydroxyl succinyl aqueous solution volume: 2% gelatin A aqueous solution volume is 0.01g:40mL:10mL; Wherein, in carbodiimide/N-hydroxyl succinyl aqueous solution, carbodiimide: N-hydroxyl succinyl mass ratio is 4:1.
4. as the three-dimensional porous loaded Chitosan/Gelatin Microspheres of claim 1, the described preparation method's gained of 2 or 3 arbitrary claim, it is characterized in that microsphere diameter is at 300-800um, surface apertures 50-200um, and described microsphere surface is connected with inner aperture, the closer to microballoon inside, aperture is larger.
5. three-dimensional porous loaded Chitosan/Gelatin Microspheres as claimed in claim 4 is as the application of hepatocyte cultures.
6. the application that three-dimensional porous loaded Chitosan/Gelatin Microspheres as claimed in claim 4 is cultivated as HepG2 cell lines.
7. carry out the method for hepatocyte cultures with three-dimensional porous loaded Chitosan/Gelatin Microspheres as claimed in claim 4, it is characterized in that:
When carrying out hepatocyte cultures, three-dimensional porous loaded Chitosan/Gelatin Microspheres and liver cell combined inoculation to be cultivated, cultivation temperature is 37～38 ℃, 95%O
2, 5%CO
2controlling rotating speed on shaking table is 30-40rpm/min, suspends and cultivates 1 hour, and static cultivation 1 hour, suspend and cultivate 1 hour again on shaking table, after repeating 3 times, continuously suspends and cultivates with 30-40rpm/min low speed after adding the proper amount of fresh nutrient solution; Every cultivation was changed nutrient solution after 24 hours, and continued to maintain the suspension of 30-40rpm/min low speed and cultivate, collecting cell after a week; Described liver cell and porous chitosan/gelatine microsphere with magnitude relation, be cell concentration 1 * 10
5individual/mL nutrient solution, porous chitosan/gelatine microsphere is paved with 12 orifice plate bottom 50% areas.
8. three-dimensional porous loaded Chitosan/Gelatin Microspheres as claimed in claim 7 carries out the method for hepatocyte cultures, it is characterized in that cultivation temperature is 37 ℃.
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| CN112250892A (en) * | 2020-10-22 | 2021-01-22 | 苏州新丝原生物科技有限公司 | Gelatin microsphere and preparation method and application thereof |
| CN114921343B (en) * | 2022-06-28 | 2023-09-05 | 中国科学院苏州生物医学工程技术研究所 | Cell gel microsphere generating device based on high-voltage pulse electric field |
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