CN102172498A - 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, reacting in a dark place, 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 800 mu m and the surface aperture of 50-200 mu m, wherein the surface of each microsphere is communicated with the internal aperture. 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 shitosan/gelatine microsphere and preparation method thereof and the application in hepatocyte cultures.Relate to the big pore property that utilizes gelatin and gelatin characteristic more specifically, prepare a kind of three-dimensional porous shitosan/gelatine microsphere and the application in hepatocyte cultures in conjunction with generator for microcapsules-electrostatic method, ion condensation method and three desivacs as the good cell epimatrix.
Background technology
Shitosan is a kind of boiomacromolecule that is obtained through deacetylated reaction by chitin; it is the alkaline polysaccharide of the unique positively charged of nature found up to now; nontoxic; have excellent biological compatibility 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 mainly is made up of the amino acid sequence glycine-proline-carboxyl proline of uniqueness as the hydrolysate of collagen, 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 characteristic of the big pore property and the good extracellular matrix of gelatin conduct of gelatin, generator for microcapsules-electrostatic method, ion condensation method and desivac combine and can make satisfactory three-dimensional porous shitosan/gelatine microsphere, be used 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 a kind of three-dimensional porous shitosan/gelatine microsphere according to the method described above with shitosan, gelatin, and used it for hepatocyte cultures.Although the existing both at home and abroad article of preparing microballoon is delivered, used material, prepare the method for microballoon and the microballoon that makes and the present invention and all be very different.At first be on material is selected, selected shitosan and gelatin main material as the preparation microballoon, although also useful shitosan or (with) the gelatin article of preparing microballoon delivers, but the method that adopts mainly is 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 control liquid drop forms oil-in-water or Water-In-Oil suspension, adding glutaraldehyde is main crosslinking agent reaction balling-up, washing, obtain microballoon after the drying, the microballoon that obtains mostly is solid microsphere, add the loose structure that the microballoon that obtains behind the pore-foaming agent neither be real, but inside hole independently, do not communicate with the external world, and the easy adhesion of the microballoon that emulsion process obtains, and crosslinking agent glutaraldehyde itself is poisonous, also increased certain risk factor, at present the microballoon prepared of emulsion process 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 the alkaline solution, as NaOH, NaOH-methyl alcohol etc., by centrifugal, filtration is carried out separation and purification to microballoon, obtain microballoon after the washing, but the microsphere surface that this method obtains is shrinkage forms groove, the surface does not communicate with inside, i.e. blind hole).Although the bibliographical information of desivac is also arranged, mostly be directly and pour in the mould the membranaceous or cylindric support of preparation after the direct freezing freeze-drying after solution mixed into; Or it is the support of freeze-drying is crosslinked with NaOH, glutaraldehyde etc., but because the shitosan after the freeze-drying or (with) the gelatin materials space structure changes, very easily water-soluble, can not well keep original shape after crosslinked, membranaceous support is used for skin tissue engineering or the preparation mould rack is used for bone tissue engineer so generally be used to prepare, do not see sphere is arranged, diameter communicates with inner aperture on 50-200um, surface at 300-800um, surface apertures and the closer to the report of the big more three-dimensional porous microballoon in aperture, microballoon inside.
The present invention directly splashes into snap frozen balling-up in the liquid nitrogen with 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 plain conductor ring distance, plain conductor ring and liquid nitrogen container mouth distance, high-voltage pulse microcapsules forming instrument voltage, frequency, pulsewidth, the size of control shitosan/gelatin B solution droplets such as portable micro syringe pump speed is controlled the initial size of microballoon, and in subsequent step by adjusting the crosslinking agent tripolyphosphate, the concentration and the pH value of carbodiimide/N-hydroxyl succinyl, crosslinking time, freezing temperature, three freeze-drying modes obtain keeping spherical-like morphology, enlarged the aperture, surperficial and inner aperture communicates, transparent, be beneficial to the microballoon of cell adhesion.Therefore, in selection, concentration of material and the proportioning of material, the application facet for preparing the concentration, crosslinking time of use, the crosslinking agent of two kinds of crosslinking agents in method (promptly controlling the factor of microballoon size) that microballoon uses, the preparation process, freezing temperature, freeze-drying mode, three-dimensional porous microballoon or the like all should be the claimed content of the present invention.
Summary of the invention
One of purpose of the present invention provides a kind of three-dimensional porous shitosan/gelatine microsphere.
Two of purpose of the present invention provides the preparation method of above-mentioned a kind of three-dimensional porous shitosan/gelatine microsphere.
Three of purpose of the present invention provides the above-mentioned application process of a kind of three-dimensional porous shitosan/gelatine microsphere in hepatocyte cultures.
Be the composition of the present invention according to the 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 microsphere surface and internal void equably, three-dimensional environment in the 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 is to cell high density in bioartificial liver (BAL) and the hepatocyte transplantation, high activity, large-scale culture provides certain theoretical foundation and experimental basis, promotes its clinical practice and development at BAL.
Technical scheme of the present invention
Make three-dimensional porous shitosan/gelatine microsphere by generator for microcapsules-electrostatic method, ion condensation method and three desivacs.
The preparation method of above-mentioned three-dimensional porous shitosan/gelatine microsphere, the process flow diagram of its preparation process as shown in Figure 2, it specifically may further comprise the steps:
(1), the preparation of a freeze-drying microballoon
1.5% shitosan acetic acid solution and 2% gelatin B (are taken from 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, sucking aperture size is in the syringe of 0.7mm, syringe is installed on the portable micro syringe pump, the terminal positive pole that connects high-voltage pulse microcapsules forming instrument of syringe needle, the plain conductor ring of syringe needle below connects the negative pole of high-voltage pulse microcapsules forming instrument, the conductor loop below is a liquid nitrogen container, open high-voltage pulse microcapsules forming instrument successively, portable micro syringe pump switch, make that under the high-pressure electrostatic effect drop that forms splashes into snap frozen balling-up in the liquid nitrogen, as Figure 1-1;
Control syringe needle point and plain conductor ring are apart from 0mm-5mm in the freezing balling-up process, and plain conductor ring and liquid nitrogen container mouth are provided with high-voltage pulse microcapsules forming instrument voltage 60 apart from 5mm-10mm, frequency 90, and pulsewidth 6, portable micro syringe pump speed is 90mm/h;
Carry out freeze-drying after the balling-up again, the control freeze temperature is-60 ℃ to-80 ℃, obtains the shitosan/gelatine microsphere 1 of a freeze-drying behind the freeze-drying 24-48h;
Described gelatin B takes from the ox-hide skin, by being that alkaline process obtains, and has more carboxyl on the described gelatin B strand, and its isoelectric point is in pH=4.7～5;
(2), the preparation of secondary freeze-drying microballoon
The proportioning of the amount of the shitosan/gelatine microsphere 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 shitosan/gelatine microspheres of freeze-drying
The microballoon 2 abundant aquations that step (2) is made, add 2% water-soluble cross-linker carbodiimide/N-hydroxyl succinyl (EDC/NHS) and 2% gelatin A and (take from the pigskin skin, sigma company), behind 38 ℃ of water-baths, lucifuge reaction 24～48h, unreacted EDC/NHS of flush away and gelatin A, go into-20 ℃ of refrigerator overnight, carry out freeze-drying for the third time next day, the control freeze temperature is promptly to obtain three-dimensional porous shitosan/gelatine microsphere 3 of the present invention behind-60 ℃ to-80 ℃ freeze-drying 24-48h;
Described gelatin A takes from the pigskin skin, obtains by acid system, and described gelatin A strand contains more amino, and isoelectric point is in pH=7～9.
Beneficial effect of the present invention
Three-dimensional porous shitosan/gelatine microsphere 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 big pore property of gelatin under the prerequisite that does not change microsphere diameter, to prepare macroporous microsphere, provide binding site efficiently with the microballoon after this good cell epimatrix modification of gelatin 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 plain conductor ring distance, plain conductor ring and liquid nitrogen container mouth are apart from the concentration of speed, crosslinking agent tripolyphosphate and the carbodiimide/N-hydroxyl succinyl of the parameter (as voltage, frequency, pulsewidth) of, high-voltage pulse microcapsules forming instrument, portable micro syringe pump, pH value, crosslinked time, freezing temperature, the mode of freeze-drying etc. can be controlled the size and the aperture of microballoon.
In addition, the three-dimensional porous shitosan/gelatine microsphere of gained of the present invention owing to increased specific area, has improved the density that cell is cultivated, thereby has improved the oxygen supply of cell and the exchange of metabolite; Cell is grown in the microsphere surface hole of loose structure, has reduced the damage of shearing force pair cell when suspending cultivation; Three-dimensional porous characteristic makes liver cell pass through the 3 D stereo growing environment of lobuli hepatis in the span analogue body, and reconstituted cell polarity is kept form and the specific function of liver cell in vivo the time, reaches in vitro culture to go out high density, highly active liver cell.Can be used for pair cell quantity and quality has the bioartificial liver of requirement for height, hepatocyte transplantation.
Description of drawings
Fig. 1 is the present invention's structural representation of preparing microballoon under the high-pressure electrostatic effect (among the figure: 1 for syringe, 3 for shitosan/gelatin mixed liquor, 4 for syringe needle, 5 for plain conductor ring, 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 for portable micro syringe pump, 2);
Fig. 2 is the process flow diagram of the preparation process of three-dimensional porous shitosan/gelatine microsphere;
Fig. 3 is the sem photograph of a shitosan/gelatine microsphere after the freeze-drying;
Fig. 4-1 is the sem photograph of three three-dimensional porous shitosan/gelatine microspheres after the freeze-drying;
Fig. 4-2 is sem photographs of three three-dimensional porous shitosan/gelatine microspheres after the freeze-drying;
Fig. 5 is the growth conditions figure of observer's hepatoma cell strain HepG2 on three-dimensional porous shitosan/gelatine microsphere under the light microscope;
Fig. 6 is the growth conditions figure of observer's hepatoma cell strain HepG2 on three-dimensional porous shitosan/gelatine microsphere under the fluorescence inverted microscope;
Fig. 7 is the sem photograph after human hepatoma cell strain HepG2 and three-dimensional porous shitosan/gelatine microsphere mix suspending are cultivated;
Fig. 8 is the transmission electron microscope picture after human hepatoma cell strain HepG2 and three-dimensional porous shitosan/gelatine microsphere mix suspending are cultivated;
Fig. 9, Figure 10 are respectively human hepatoma cell strain HepG2 and three-dimensional porous shitosan/gelatine microsphere HE dyeing back 40 times and 100 times of enlarged drawings under light microscope;
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
Also in conjunction with the accompanying drawings the present invention is further described below by embodiment, but does not limit the present invention.
The used human hepatoma cell strain HepG2 of the present invention takes 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 is taken from the pigskin skin, and is Powdered, cell cultivate level), gelatin B(B type gelatin deacetylation:, take from the ox-hide skin), carbodiimide/N-hydroxyl succinyl (EDC/NHS) is purchased in sigma;
Tripolyphosphate (Alfa Aesar);
The complete medium (RP-1640) that contains 10% calf serum (Gibco);
Poly 2-hydroxyethyl meth acrylate (poly (2-hydroxyethyl Methacrylate), poly-HEMA) (sigma);
Acridine orange (sigma);
Acetate (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 micro syringe pump (Angel 5805 types, the clean electronics in last Hai'an);
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 shitosan/gelatine microsphere
(1), the preparation of a freeze-drying microballoon
Prepare 1.5% shitosan (with the preparation of 1% acetate) and 2% gelatin B (preparing) respectively with distilled water, behind the mixing, be 1.5% shitosan acetic acid solution by volume: the 2% gelatin B aqueous solution is that 5:1 mixes mutually, sucking the syringe needle internal diameter behind the removal bubble is in the 5ml syringe of 0.7mm, connect generator for microcapsules-electrostatic equipment (showing) as Fig. 1, (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) splashes in the liquid nitrogen, after treating that microballoon is sink at the bottom of the liquid nitrogen container, microballoon shifted out put on the iron screen cloth that immersing in liquid nitrogen is crossed, last freeze dryer, the control freeze temperature is-60 ℃ to-80 ℃, close freeze dryer after 24-48 hour and take out, obtain the shitosan/gelatine microsphere 1 after the freeze-drying one time;
(2), the preparation of secondary freeze-drying microballoon
The microballoon usefulness that step (1) is made contained 85% ethanolic solution crosslinking curing of tripolyphosphate (TPP) after 24-72 hour, control pH value is 5-6, remove the crosslinking agent on upper strata, add water and fully wash, microballoon is moved in the culture dish, siphon away excessive moisture as much as possible, go into-20 ℃ of refrigerator overnight, freeze dryer on next day, the control 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, reaction is after 24-48 hour in 38 ℃ of water-baths, add washing and remove unreacted EDC/NHS and gelatin A, move in the culture dish, siphon away excessive moisture as far as possible, go into-20 ℃ of refrigerator overnight, freeze dryer on next day, the control freeze temperature is-60 ℃ to-80 ℃, obtains three-dimensional porous shitosan/gelatine microsphere of the present invention after 24-48 hour.
Fig. 1 is the structural representation for preparing microballoon in the step (1) under the high-pressure electrostatic effect.
Fig. 3 is the sem photograph of the shitosan/gelatine microsphere 1 after the freeze-drying of step (1) gained,
Microsphere diameter is at 300-500um, and its aperture is 10um-45um.
Fig. 4-the 1st, the scanning of the three-dimensional porous shitosan/gelatine microsphere after three freeze-drying of step (3) gained
Electronic Speculum figure, microsphere diameter are at 300-800um, and its aperture is 50um-200um.
Fig. 4-2 is sem photographs (Fig. 4-1 enlarged drawing) of three three-dimensional porous shitosan/gelatine microspheres after the freeze-drying, and the aperture is 151um.
From Fig. 3, Fig. 4-1, Fig. 4-2 as can be seen after three freeze-drying the aperture of microballoon have after than a freeze-drying
Significantly enlarge, promptly its aperture is after the freeze-drying 4.4 times～5 times.
Application Example 1
A kind of three-dimensional porous shitosan/gelatine microsphere and human hepatoma cell strain HepG2 Mixed culture
(1), the recovery of human hepatoma cell strain HepG2
After from liquid nitrogen container, taking out frozen pipe, put into the vibration of 38 ℃ of water baths, thawed in 1 minute, add (GIBCO) mixing of complete medium (RP-1640) that 1ml contains 10% calf serum immediately, slowly add the 9ml nutrient solution again, after mixing piping and druming, centrifugal (1000rpm/min, 5min), after the supernatant discarded, add an amount of nutrient solution among the pellet, behind the mixing, be seeded in the culture dish.Change liquid every other day and remove not adherent cell and metabolite, reach 2-3 and be used for subsequent experimental after generation;
(2), the preparation of three-dimensional porous shitosan/gelatine microsphere
After the above-mentioned abundant aquation of three-dimensional porous shitosan/gelatine microsphere (being that the prompting aquation was finished at the bottom of microballoon was sink to test tube) through gained after three freeze-drying, suction is anhydrated, add 75% ethanol disinfection 30min,, add nutrient solution RP-1640 in the aseptic microballoon and soak standby with aseptic double-distilled water flush away ethanol;
The consumption of nutrient solution RP-1640 promptly places the shared volume of test tube by three-dimensional porous shitosan/gelatine microsphere: RP-1640 nutrient solution volume is that 1:5 calculates;
(3), three-dimensional porous shitosan/gelatine microsphere and human hepatoma cell strain HepG2 Mixed culture
Trypsinization human hepatoma cell strain HepG2, collecting cell, it is centrifugal that (1000rpm/min 5min), abandons supernatant, adds the 5mlRP-1640 nutrient solution among the pellet, and counting is adjusted cell concentration 1 * 10
5Individual/ml; In 12 orifice plates (lining poly-HEMA) of anti-adherent processing, add the three-dimensional porous shitosan/gelatine microsphere after nutrient solution soaks, confluent cultures ware bottom 50%, the nutrient solution that as far as possible exhausts adds cell suspension 1ml (cell concentration 1 * 10 in every hole
5Individual/ml, promptly TCS is 1 * 10
5Individual), on shaking table, suspend and cultivated 1 hour, static cultivation 1 hour, suspend on the shaking table and cultivate 1 hour (so repeating 3 times), low speed (30-40rpm/min) suspension was cultivated after every Kong Xu added the 1ml fresh medium, cultivated and changed nutrient solution after 24 hours, after this keeping low speed (30-40rpm/min) suspension cultivates, changed liquid in per 24 hours, low speed (30-40rpm/min) suspends and cultivates, and cell density is 4.2 * 10 in the time of 7 days
5Individual, cell density is 2.8 * 10 in the time of 13 days
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 cell is because of the growing space deficiency in the time of the 5th day, it is floating that cell begins to pile up, then cell death).
Fig. 5 is the growth conditions figure of observer's hepatoma cell strain HepG2 on three-dimensional porous shitosan/gelatine microsphere under the light microscope, as can be seen from the figure three-dimensional porous shitosan/gelatine microsphere is transparent spherical, visible cell is grown on three-dimensional porous shitosan/gelatine microsphere, cell is bright to be spherical, and after birth is complete.
Fig. 6 is the growth conditions figure of observer's hepatoma cell strain HepG2 on three-dimensional porous shitosan/gelatine microsphere under the fluorescence inverted microscope, as can be seen from the figure cell is spherical after acridine orange dyeing, can demonstrate the profile of three-dimensional porous shitosan/gelatine microsphere simultaneously behind the cell dyeing indirectly, it is spherical that microballoon is, and local cells forms sphere aggregates.
Fig. 7 (Fig. 7-1,7-2) is the sem photograph after human hepatoma cell strain HepG2 and three-dimensional porous shitosan/gelatine microsphere mix suspending are cultivated, the as can be seen from the figure spheric profile of three-dimensional porous shitosan/gelatine microsphere, 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 shows) between cell and the cell.
Fig. 8 is the transmission electron microscope picture after human hepatoma cell strain HepG2 and three-dimensional porous shitosan/gelatine microsphere mix suspending are cultivated, and many irregular microvillus appear in the surface that as seen contacts with nutrient solution among the figure, and quantity is abundant, the sinus hepaticus face in the 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.Mitochondria mostly is oval in the kytoplasm, and what have is stock shape, and ridge is abundanter.As seen endoplasmic reticulum is distributed in the kytoplasm more equably.
Light microscope is observed down among Fig. 9 and Figure 10: HE dyeing shows that down shitosan/gelatine microsphere surface and inside are mesh-like structure, short texture, and fibre bundle is tiny, arranges rule, and as seen interior cavity is connected.The prompting of HE coloration result: cell can be grown at microsphere surface, and can enter growth inside.Verified that once more we are connected in constructed three-dimensional porous shitosan/gelatine microsphere surface and inside.
Control group is plane cultivation group k among Figure 11, does not promptly contain three-dimensional porous shitosan/gelatine microsphere, and simple human hepatoma cell strain HepG2 is in culture dish bottom adherent growth; Experimental group is Mixed culture group g, promptly contains three-dimensional porous shitosan/gelatine microsphere, cultivates with human hepatoma cell strain HepG2 mix suspending in the culture dish through anti-adherent processing.
Carrying out three-dimensional porous shitosan/gelatine microsphere and human hepatoma cell strain HepG2 mix suspending respectively 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 the Mixed culture 72 hours, experimental group ALB expression is 1260.69 times of control group; Descend gradually afterwards, but still maintain higher level, to express still be 36 times of control group to experimental group ALB in the time of the 6th day, and difference all has statistical significance.It the results are shown in shown in Figure 9, illustrates that three-dimensional porous shitosan/gelatine microsphere and human hepatoma cell strain HepG2 mix suspending are cultivated to rebuild hepatocellular polarity, keeps liver cell three-dimensional environment in vivo, brings into play hepatocyte function.
Respectively with ALT in the gained cultured cell supernatant, LDH, urea level send automatic clinical chemistry analyzer to detect, the 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, be because three-dimensional porous shitosan/gelatine microsphere is just abundanter through the water content in nutrient solution immersion back own, so in follow-up process, be difficult to accurately control the cumulative volume of nutrient solution, thereby cause and from supernatant, detect the difference that ALT, LDH, urea, alb can not accurately reflect experimental group and control group; The advantage of mix suspending cultivation simultaneously shows later stage 7-14 talent, and the plane condition of culture is down because the restriction of culture space can only be carried out 7 days with interior observation.
Above said content only is the basic explanation of the present invention under conceiving, 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 shitosan/gelatine microsphere is characterized in that may further comprise the steps:
(1), the preparation of a freeze-drying microballoon
With 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, sucking aperture size is in the syringe of 0.7mm, utilizes the high-pressure electrostatic principle to splash into snap frozen balling-up in the liquid nitrogen through high-voltage pulse microcapsules forming instrument;
Control needle point and plain conductor ring are apart from 0mm-5mm in the freezing balling-up process, and plain conductor ring and liquid nitrogen container mouth are provided with high-voltage pulse microcapsules forming instrument voltage 60 apart from 5mm-10mm, frequency 90, and pulsewidth 6, portable micro syringe pump speed is 90mm/h;
Carry out freeze-drying after the balling-up again, the control freeze temperature is-60 ℃ to-80 ℃, obtains the shitosan/gelatine microsphere 1 of a freeze-drying behind the freeze-drying 24-48h;
Described gelatin B takes from the ox-hide skin, by being that alkaline process obtains, and has more carboxyl on the described gelatin B strand, and its isoelectric point is in pH=4.7～5;
(2), the preparation of secondary freeze-drying microballoon
Microballoon 1 usefulness that step (1) is made contains the ethanolic solution of saturated tripolyphosphate (TPP) 85%, hierarchy of control pH is 5-6, again behind the crosslinking curing 24-72h, stop cross-linking reaction, go into-20 ℃ of refrigerator overnight, freeze dryer on next day, the control freeze temperature is-60 ℃ to-80 ℃, obtains the microballoon 2 of secondary freeze-drying behind freeze-drying 24～48h;
(3), the preparation of three three-dimensional porous shitosan/gelatine microspheres of freeze-drying
The microballoon 2 abundant aquations 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, behind 38 ℃ of water-baths, lucifuge reaction 24-48h, unreacted EDC/NHS of flush away and gelatin A, go into-20 ℃ of refrigerator overnight, carry out freeze-drying for the third time next day, the control freeze temperature is promptly to obtain three-dimensional porous shitosan/gelatine microsphere 3 of the present invention behind-60 ℃ to-80 ℃ freeze-drying 24-48h;
Described gelatin A takes from the pigskin skin, obtains by acid system, 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 amount of the proportioning of the amount of shitosan/gelatin B microballoon 1 of a freeze-drying in the step (2) and saturated tripolyphosphate (TPP) 85% ethanolic solution by microballoon 1: saturated tripolyphosphate (TPP) 85% ethanolic solution is 0.01g:50ml.
3. the preparation method of a kind of three-dimensional porous shitosan/gelatine microsphere as claimed in claim 1, the proportioning that it is characterized in that microballoon 2, carbodiimide/N-hydroxyl succinyl and gelatin in the step (3), promptly 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 shitosan/gelatine microsphere of claim 1, the described preparation method's gained of 2 or 3 arbitrary claims, it is characterized in that microsphere diameter at 300-800um, surface apertures 50-200um, and described microsphere surface is connected with inner aperture, the closer to microballoon inside, the aperture is big more.
5. three-dimensional porous shitosan/gelatine microsphere as claimed in claim 4 is as the application of hepatocyte cultures.
6. the application that three-dimensional porous shitosan/gelatine microsphere as claimed in claim 4 is cultivated as human hepatoma cell strain HepG2.
7. carry out the method for hepatocyte cultures with three-dimensional porous shitosan/gelatine microsphere as claimed in claim 4, it is characterized in that:
When carrying out hepatocyte cultures, three-dimensional porous shitosan/gelatine microsphere and liver cell combined inoculation to be cultivated, cultivation temperature is 37～38 ℃, 95%O
2, 5%CO
2The control rotating speed is 30-40rpm/min on shaking table, suspend to cultivate 1 hour, and static cultivation 1 hour suspends on the shaking table again and cultivated 1 hour, repeats 3 times after, continuous adding behind the proper amount of fresh nutrient solution with the cultivation that suspends of 30-40rpm/min low speed; Every cultivation was changed nutrient solution after 24 hours, and continued to keep the suspension of 30-40rpm/min low speed and cultivate, collecting cell after the week;
Described liver cell and porous chitosan/gelatine microsphere be cell concentration 1 * 10 with magnitude relation
5Individual
/ ml nutrient solution, porous chitosan/gelatine microsphere are paved with 12 orifice plates bottom, 50% area.
8. three-dimensional porous shitosan/gelatine microsphere 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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