CN104001219B - Subcritical carbon dioxide sintering carries the method for cell porous microsphere support altogether - Google Patents
Subcritical carbon dioxide sintering carries the method for cell porous microsphere support altogether Download PDFInfo
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- CN104001219B CN104001219B CN201410264713.9A CN201410264713A CN104001219B CN 104001219 B CN104001219 B CN 104001219B CN 201410264713 A CN201410264713 A CN 201410264713A CN 104001219 B CN104001219 B CN 104001219B
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Abstract
The invention discloses the method that a kind of subcritical carbon dioxide sintering carries cell porous microsphere support altogether, using ammonium bicarbonate aqueous solution as aqueous phase, the organic solution of PLGA, as oil phase, by above-mentioned Aqueous dispersions in oil phase, forms water in oil single emulsion by homogeneous homogenize; The emulsion of water-in-oil-in-water will be obtained in above-mentioned single emulsion dispersion to the PVA aqueous solution; Continuing stirring makes methylene dichloride volatilize, and obtains the porous microsphere solidified; Collect the porous microsphere of solidification, washing, freeze-drying; The cell of the porous microsphere of freeze-drying and some amount is inserted in mould jointly, under the condition of subcritical carbon dioxide, sinters porous microsphere support into.The present invention under mild conditions, is obtained by single stage method and carries cell porous microsphere sintering support altogether, while guaranteeing microsphere surface porous pattern, ensure cytoactive.
Description
Technical field
The present invention relates to the preparation method of three-dimensional tissue's engineering rack, specifically relate to the method that a kind of subcritical carbon dioxide sintering carries cell porous microsphere support altogether.
Background technology
Present Domestic studies the mode that more ripe tissue construction thinking is (Top-down) from top to bottom outward, namely first biomimetic scaffolds is prepared, again seed cell is inoculated on timbering material, carries out tissue construction by perfusion technique, means such as interpolation somatomedin, mechanical stimulus etc.Main preparation methods comprises: solvent casting method, and be separated/freeze-drying, fiber bonding method, 3 D-printing, melt molding method etc.The subject matter that this building mode exists is: Determination of Residual Organic Solvents is high, and biologically active factors easy in inactivation, cell compatibility is poor.The more important thing is the growth rhythm that accurately cannot control cell, be difficult to the tissue forming complex micro structure feature.And the Constructed wetlands of modular organization engineering is the mode of (Bottom-up) from bottom to top, the body of the people under simulation its natural environment, many tissues are exactly formed by little tissue block, as: fascicula forms muscle, liver lobule forms liver etc., by intending ecological microstructure unit to the design forming of microstructure features to build modular construction, then in order to build bulk tissue.
Sintering microsphere support technology utilizes modularization idea just, carries out tissue construction.First prepare the single microsphere of different components feature, recycle different sintering processing to plasticize, make to be bonded together each other between microballoon, form support, common sintering processing has thermal sintering, solvent sintered etc.Ultimate principle, for be melted by microsphere surface by high temperature or solvent, makes adjacent microballoon molecular chain be wound around, and after temperature reduces or organic solvent volatilizees, polymer molecular chain solidifies again, forms permanent cohering.(the AroninCEP such as Aronin, SadikKW, LayAL, etal.Comparativeeffectsofscaffoldporesize, porevolume, andtotalvoidvolumeoncranialbonehealingpatternsusingmicro sphere-basedscaffolds.JournalofBiomedicalMaterialsResear chPartA, 2009, 89 (3): 632-641) with Poly(D,L-lactide-co-glycolide (poly (lactic-co-glycolicacid), PLGA) be material, 80 DEG C of sintering 3h in copper mould, prepare solid microsphere sintering support, (the ShiX such as Shi, WangY, VarshneyRR, etal.Microsphere-baseddrugreleasingscaffoldsforinducingo steogenesisofhumanmesenchymalstemcellsinvitro.EuropeanJo urnalofPharmaceuticalSciences, 2010,39 (1-3): 59-67) encapsulate dexamethasone with PLGA, beta-glycerophosphate and xitix, with acetone/ethanol mixed solvent for agglutinant, prepare the solid microsphere component support carrying three kinds of medicines altogether.But high temperature and organic solvent are not only bad for the activity of protein medicaments, and residual organic solvent is difficult to remove, and cell compatibility is poor.Although people attempt additive method prepare PLGA microsphere support, as (CN103212116A) such as Wang Yingjun discloses a kind of method that Room-temperature low-pressure desiccating method prepares PLGA porous microsphere support; DMichael etc. (US8669107B2) disclose a kind of method that subcritical sintering carries cell PLGA microsphere support altogether, but all cannot realize can carrying cell altogether under the prerequisite ensureing microsphere surface morphology, or when carrying cell altogether, site is sticked for cell provides more, make cell to single microballoon unit growth inside, avoid because mass transfer is obstructed, cause vascularization to regenerate and be obstructed and apoptotic problem.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, provide the method that a kind of subcritical carbon dioxide sintering carries cell porous microsphere support altogether, achieve a step of carrying cell porous microsphere support altogether to build, decrease the step that conventional stent needs secondary inoculation cell, avoid the impact of high temperature and organic solvent, while reservation microsphere surface morphology, can also ensure that cell is to microballoon growth inside, and keep active.
One of the technical solution adopted for the present invention to solve the technical problems is:
Subcritical carbon dioxide sintering carries a method for cell porous microsphere support altogether, comprising:
A) PLGA being added in organic solvent dichloromethane, obtaining uniform oil phase by stirring;
B) add in distilled water by bicarbonate of ammonia, obtain uniform aqueous phase by stirring, wherein ammonium bicarbonate concentration is 5% ~ 20% (w/v);
C), in the oil phase that the Aqueous dispersions obtained by step b obtains to step a, by homogeneous homogenization, form water in oil single emulsion, wherein the volume ratio of aqueous phase and oil phase is 1/1 ~ 1/20;
D) under 100 ~ 1000rpm stir speed (S.S.), single emulsion dispersion that step c obtains is obtained in the PVA aqueous solution double emulsion of water-in-oil-in-water;
E) continue to stir emulsion 2 ~ 12h with stir speed (S.S.) in steps d, the methylene dichloride in oil phase is volatilized, obtain the porous microsphere solidified;
F) porous microsphere of solidification is collected, with deionized water wash, freeze-drying subsequently;
G) by the porous microsphere of freeze-drying and 2 × 10
4~ 2 × 10
7individual cell or 50 ~ 1000 μ L contain 2 × 10
4~ 2 × 10
7the cell suspension of individual cell is inserted in mould jointly, is 0.5 ~ 7.38MPa at pressure, and temperature is process 10 ~ 1800s under the subcritical carbon dioxide condition of 20 ~ 40 DEG C, sinters into and carries cell porous microsphere support altogether.
In one embodiment: in step a, in described oil phase, the concentration of PLGA is 1% ~ 15% (w/v), and PLGA molecular weight is the mol ratio of LA and GA in 20 ~ 100kDa, PLGA is 25/75 ~ 75/25.
In one embodiment: in step c, the condition of described homogeneous homogenize is: stir speed (S.S.) 3000 ~ 15000rpm, churning time 2 ~ 10min.
In one embodiment: in steps d, in the described PVA aqueous solution, PVA concentration is 0.1% ~ 2% (w/v).
In one embodiment: the one in described PLGA PCL or PLA-PET replaces.
Two of the technical solution adopted for the present invention to solve the technical problems is:
Subcritical carbon dioxide sintering carries a method for microorganism porous microsphere support altogether, comprising:
A) PLGA being added in organic solvent dichloromethane, obtaining uniform oil phase by stirring;
B) add in distilled water by bicarbonate of ammonia, obtain uniform aqueous phase by stirring, wherein ammonium bicarbonate concentration is 5% ~ 20% (w/v);
C), in the oil phase that the Aqueous dispersions obtained by step b obtains to step a, by homogeneous homogenization, form water in oil single emulsion, wherein the volume ratio of aqueous phase and oil phase is 1/1 ~ 1/20;
D) under 100 ~ 1000rpm stir speed (S.S.), single emulsion dispersion that step c obtains is obtained in the PVA aqueous solution double emulsion of water-in-oil-in-water;
E) continue to stir emulsion 2 ~ 12h with stir speed (S.S.) in steps d, the methylene dichloride in oil phase is volatilized, obtain the porous microsphere solidified;
F) porous microsphere of solidification is collected, with deionized water wash, freeze-drying subsequently;
G) by the porous microsphere of freeze-drying and 2 × 10
4~ 2 × 10
7individual microorganism or 50 ~ 1000 μ L contain 2 × 10
4~ 2 × 10
7the microorganism suspension of individual microorganism is inserted in mould jointly, is 0.5 ~ 7.38MPa at pressure, and temperature is process 10 ~ 1800s under the subcritical carbon dioxide condition of 20 ~ 40 DEG C, sinters into and carries microorganism porous microsphere support altogether.
Mould described in the present invention is on existing market and can buys or customized product.
The technical program is compared with background technology, and its tool has the following advantages:
1. a kind of subcritical carbon dioxide sintering provided by the present invention carries the method for cell porous microsphere support altogether, achieve single stage method preparation and carry cell porous microsphere support altogether, bring following technique effect: a) with first sinter support in conventional art again compared with secondary inoculation cell, step of the present invention is more succinct, operates easier; B) in conventional art after secondary inoculation cell, cell cannot inwardly grow, and easily produces mass transfer and is obstructed, and causes vascularization to regenerate and is obstructed and apoptosis, thus cause tissue necrosis; And carry altogether in the present invention cell with sintering support settle at one go, cell sinter support time enter internal stent, can survive simultaneously on the surface of support and inside and grow, avoiding the problem that mass transfer is obstructed; C) compare traditional solid microballoon sintering support, porous microsphere sintering support has higher porosity, be beneficial to mass transfer and cell proliferation, and the higher specific surface area of porous microsphere provides more site for cell attachment, is beneficial to and efficiently builds tissue.
2. a kind of subcritical carbon dioxide sintering provided by the present invention carries the method for cell porous microsphere support altogether, do not adopt high temperature and organic solvent, avoid the destruction to microsphere surface morphology, and organic solvent-free remains, and does not affect cells growth activity; Owing to there is not the use of pyroprocess and organic solvent, the present invention also can be applied to field of medicaments, can not have an impact to pharmaceutical activity and security.
3. a kind of subcritical carbon dioxide sintering provided by the present invention carries the method for cell porous microsphere support altogether, using subcritical carbon dioxide as agglutinant, volatile, and noresidue is pollution-free, compared to existing technology more clean environment firendly.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described.
Fig. 1 is porous microsphere surface topography map prepared by embodiment 1.
Fig. 2 is porous microsphere support shape appearance figure prepared by embodiment 1.
Fig. 3 be embodiment 2 prepare carry cell porous microsphere surface topography map altogether.
Fig. 4 be embodiment 2 prepare carry cell porous microsphere support shape appearance figure altogether.
Fig. 5 be embodiment 2 prepare carry cell porous microsphere support laser confocal microscope photo altogether, in figure, white arrow is denoted as after subcritical carbon dioxide sintering, active good cell.
Fig. 6 be embodiment 3 prepare carry cell porous microsphere support laser confocal microscope photo altogether, in figure, white arrow is denoted as after subcritical carbon dioxide sintering, active good cell.
Embodiment
Content of the present invention is illustrated below by embodiment:
Embodiment 1
Concrete implementation step is as follows:
A) under room temperature, PLGA is added in organic solvent dichloromethane, uniform oil phase is obtained by stirring, wherein the concentration of PLGA is 6.25% (w/v), PLGA molecular weight is that in 40kDa, PLGA, LA (lactic acid) is 50/50 with the mol ratio of GA (oxyacetic acid);
B) add in distilled water by bicarbonate of ammonia, obtain uniform aqueous phase by stirring, wherein ammonium bicarbonate concentration is 10% (w/v);
C) the aqueous phase 2.5ml obtained by step b is distributed in the oil phase 15ml that step a obtains, and by homogeneous homogenization, form water in oil single emulsion, wherein the volume ratio of aqueous phase and oil phase is 1/6; The condition of described homogeneous homogenize is: stir speed (S.S.) 8000rpm, churning time 4min;
D) under 200rpm stir speed (S.S.), the single emulsion dispersion obtained by step c is the double emulsion obtaining water-in-oil-in-water in PVA (polyvinyl alcohol) aqueous solution of 0.5% (w/v) of 400ml to volume;
E) continue to stir emulsion 4h with 200rpm stir speed (S.S.), the methylene dichloride in oil phase is volatilized, obtain the porous microsphere solidified;
F) collect the porous microsphere of solidification, with deionized water wash 3 times, freeze-drying 48h subsequently, obtains the porous PLGA microballoon that surface topography is well dried;
G) inserting in Teflon mould by the porous microsphere of freeze-drying, is 2MPa at pressure, and temperature is process 10min under the subcritical carbon dioxide condition of 25 DEG C, sinter porous microsphere support into, please refer to Fig. 1-2, the porous microsphere rack surface pattern obtained is good, and mean pore size is greater than 20 μm.
Embodiment 2
Concrete implementation step is as follows:
A) under room temperature, added to by PLGA in organic solvent dichloromethane, obtain uniform oil phase by stirring, wherein the concentration of PLGA is 6.25% (w/v), and PLGA molecular weight is the mol ratio of LA and GA in 40kDa, PLGA is 50/50;
B) add in distilled water by bicarbonate of ammonia, obtain uniform aqueous phase by stirring, wherein ammonium bicarbonate concentration is 10% (w/v);
C) the aqueous phase 2.5ml obtained by step b is distributed in the oil phase 12.5ml that step a obtains, and by homogeneous homogenization, form water in oil single emulsion, wherein the volume ratio of aqueous phase and oil phase is 1/5; The condition of described homogeneous homogenize is: stir speed (S.S.) 8000rpm, churning time 4min;
D) under 200rpm stir speed (S.S.), the single emulsion dispersion obtained by step c is the double emulsion obtaining water-in-oil-in-water in the PVA aqueous solution of 0.5% (w/v) of 400ml to volume;
E) continue to stir emulsion 4h with 200rpm stir speed (S.S.), the methylene dichloride in oil phase is volatilized, obtain the porous microsphere solidified;
F) collect the porous microsphere of solidification, with deionized water wash 3 times, freeze-drying 48h subsequently, obtains the porous PLGA microballoon that surface topography is well dried;
G) by the porous microsphere of freeze-drying and 1 × 10
6individual mouse fibroblast cell mixes, and jointly inserts in Teflon mould, is 3MPa at pressure, temperature is process 4min under the subcritical carbon dioxide condition of 25 DEG C, sinter into and carry cell porous microsphere support altogether, please refer to Fig. 3-4, the porous microsphere rack surface pattern obtained is good; Dyeed by AO/EB (acridine orange/ethidium bromide) by support subsequently, and detect cytoactive with laser confocal microscope, as shown in Figure 5, in figure, white arrow is denoted as active good cell to detected result.As can be seen here, the present invention is obtained by single stage method and carries cell porous microsphere sintering support altogether, while guaranteeing microsphere surface porous pattern, can also ensure cytoactive.
Embodiment 3
Concrete implementation step is as follows:
A) under room temperature, added to by PLGA in organic solvent dichloromethane, obtain uniform oil phase by stirring, wherein the concentration of PLGA is 6.25% (w/v), and PLGA molecular weight is the mol ratio of LA and GA in 40kDa, PLGA is 50/50;
B) add in distilled water by bicarbonate of ammonia, obtain uniform aqueous phase by stirring, wherein ammonium bicarbonate concentration is 10% (w/v);
C) the aqueous phase 2.5ml obtained by step b is distributed in the oil phase 8ml that step a obtains, and by homogeneous homogenization, form water in oil single emulsion, wherein the volume ratio of aqueous phase and oil phase is 1/3.2; The condition of described homogeneous homogenize is: stir speed (S.S.) 5000rpm, churning time 3min;
D) under 200rpm stir speed (S.S.), the single emulsion dispersion obtained by step c is the double emulsion obtaining water-in-oil-in-water in the PVA aqueous solution of 0.1% (w/v) of 400ml to volume;
E) continue to stir emulsion 4h with 200rpm stir speed (S.S.), the methylene dichloride in oil phase is volatilized, obtain the porous microsphere solidified;
F) collect the porous microsphere of solidification, with deionized water wash 3 times, freeze-drying 48h subsequently, obtains the porous PLGA microballoon that surface topography is well dried;
G) by the porous microsphere of freeze-drying and containing 2 × 10
6500 μ l cell suspensions of individual mouse fibroblast cell mix, and jointly inserting in Teflon mould, is 3MPa at pressure, and temperature is process 2min under the subcritical carbon dioxide condition of 25 DEG C, sinters into and carries cell porous microsphere support altogether; Proceeded to subsequently in the substratum of 1ml, dyeed by AO/EB, and detect cytoactive with laser confocal microscope, as shown in Figure 6, in figure, white arrow is denoted as active good cell to detected result.As can be seen here, the present invention is obtained by single stage method and carries cell porous microsphere sintering support altogether, and cytoactive is good.
Embodiment 4
Concrete implementation step is as follows:
A) under room temperature, added to by PLGA in organic solvent dichloromethane, obtain uniform oil phase by stirring, wherein the concentration of PLGA is 1% (w/v), and PLGA molecular weight is the mol ratio of LA and GA in 100kDa, PLGA is 75/25;
B) add in distilled water by bicarbonate of ammonia, obtain uniform aqueous phase by stirring, wherein ammonium bicarbonate concentration is 20% (w/v);
C) the aqueous phase 2.5ml obtained by step b is distributed in the oil phase 50ml that step a obtains, and by homogeneous homogenization, form water in oil single emulsion, wherein the volume ratio of aqueous phase and oil phase is 1/20; The condition of described homogeneous homogenize is: stir speed (S.S.) 15000rpm, churning time 2min;
D) under 1000rpm stir speed (S.S.), the single emulsion dispersion obtained by step c is the double emulsion obtaining water-in-oil-in-water in the PVA aqueous solution of 2% (w/v) of 400ml to volume;
E) continue to stir emulsion 2h with 1000rpm stir speed (S.S.), the methylene dichloride in oil phase is volatilized, obtain the porous microsphere solidified;
F) collect the porous microsphere of solidification, with deionized water wash 3 times, freeze-drying 48h subsequently, obtains the porous PLGA microballoon that surface topography is well dried;
G) by the porous microsphere of freeze-drying and containing 2 × 10
450 μ l cell suspensions of individual mouse fibroblast cell mix, and jointly inserting in Teflon mould, is 0.5MPa at pressure, and temperature is process 30min under the subcritical carbon dioxide condition of 20 DEG C, sinters into and carries cell porous microsphere support altogether.
Embodiment 5
Concrete implementation step is as follows:
A) under room temperature, added to by PLGA in organic solvent dichloromethane, obtain uniform oil phase by stirring, wherein the concentration of PLGA is 15% (w/v), and PLGA molecular weight is the mol ratio of LA and GA in 20kDa, PLGA is 25/75;
B) add in distilled water by bicarbonate of ammonia, obtain uniform aqueous phase by stirring, wherein ammonium bicarbonate concentration is 5% (w/v);
C) the aqueous phase 2.5ml obtained by step b is distributed in the oil phase 2.5ml that step a obtains, and by homogeneous homogenization, form water in oil single emulsion, wherein the volume ratio of aqueous phase and oil phase is 1/1; The condition of described homogeneous homogenize is: stir speed (S.S.) 3000rpm, churning time 10min;
D) under 100rpm stir speed (S.S.), the single emulsion dispersion obtained by step c is the double emulsion obtaining water-in-oil-in-water in the PVA aqueous solution of 1% (w/v) of 400ml to volume;
E) continue to stir emulsion 12h with 100rpm stir speed (S.S.), the methylene dichloride in oil phase is volatilized, obtain the porous microsphere solidified;
F) collect the porous microsphere of solidification, with deionized water wash 3 times, freeze-drying 48h subsequently, obtains the porous PLGA microballoon that surface topography is well dried;
G) by the porous microsphere of freeze-drying and containing 2 × 10
71000 μ l cell suspensions of individual mouse fibroblast cell mix, and jointly inserting in Teflon mould, is 7.38MPa at pressure, and temperature is process 10s under the subcritical carbon dioxide condition of 40 DEG C, sinters into and carries cell porous microsphere support altogether.
In addition, those skilled in the art can learn, present invention utilizes PLGA can be swelling by subcritical carbon dioxide, material surface is caused to plastify and the characteristic be bonded together, therefore, other can be plastified and the material cohered by the swelling surface that causes of subcritical carbon dioxide, include but not limited to PCL (polycaprolactone), PLA-PET (PLA-PEG copolymer) etc., are all suitable for the method that subcritical carbon dioxide of the present invention sintering carries cell porous microsphere altogether.
Those skilled in the art can learn, of the present invention with the once sintered cell being shaped to porous microsphere support of material, are to keep active under undercritical conditions to its requirement; Therefore, other can keep active organism under undercritical conditions, the microorganisms such as such as Nitrobacter, Rhodopseudomonas, Penicillium, yeast cell, magnetotactic bacteria, are all suitable for the method that subcritical carbon dioxide of the present invention sintering carries cell porous microsphere altogether.
The above, be only present pre-ferred embodiments, therefore can not limit scope of the invention process according to this, the equivalence change namely done according to the scope of the claims of the present invention and description with modify, all should still belong in scope that the present invention contains.
Claims (4)
1. subcritical carbon dioxide sintering carries a method for cell porous microsphere support altogether, it is characterized in that: comprising:
A) PLGA being added in organic solvent dichloromethane, obtaining uniform oil phase by stirring; In described oil phase, the concentration of PLGA is 1% ~ 15% (w/v), and PLGA molecular weight is the mol ratio of LA and GA in 20 ~ 100kDa, PLGA is 25/75 ~ 75/25;
B) add in distilled water by bicarbonate of ammonia, obtain uniform aqueous phase by stirring, wherein ammonium bicarbonate concentration is 5% ~ 20% (w/v);
C), in the oil phase that the Aqueous dispersions obtained by step b obtains to step a, by homogeneous homogenization, form water in oil single emulsion, wherein the volume ratio of aqueous phase and oil phase is 1/1 ~ 1/20; The condition of described homogeneous homogenize is: stir speed (S.S.) 3000 ~ 15000rpm, churning time 2 ~ 10min;
D) under 100 ~ 1000rpm stir speed (S.S.), single emulsion dispersion that step c obtains is obtained in the PVA aqueous solution double emulsion of water-in-oil-in-water;
E) continue to stir emulsion 2 ~ 12h with stir speed (S.S.) in steps d, the methylene dichloride in oil phase is volatilized, obtain the porous microsphere solidified;
F) porous microsphere of solidification is collected, with deionized water wash, freeze-drying subsequently;
G) by the porous microsphere of freeze-drying and 2 × 10
4~ 2 × 10
7individual cell or 50 ~ 1000 μ L contain 2 × 10
4~ 2 × 10
7the cell suspension of individual cell is inserted in mould jointly, is 0.5 ~ 7.38MPa at pressure, and temperature is process 10 ~ 1800s under the subcritical carbon dioxide condition of 20 ~ 40 DEG C, sinters into and carries cell porous microsphere support altogether.
2. a kind of subcritical carbon dioxide sintering according to claim 1 carries the method for cell porous microsphere support altogether, and it is characterized in that: in steps d, in the described PVA aqueous solution, PVA concentration is 0.1% ~ 2% (w/v).
3. a kind of subcritical carbon dioxide sintering according to claim 1 carries the method for cell porous microsphere support altogether, it is characterized in that: the one in described PLGA PCL or PLA-PEG replaces.
4. subcritical carbon dioxide sintering carries a method for microorganism porous microsphere support altogether, it is characterized in that: comprising:
A) PLGA being added in organic solvent dichloromethane, obtaining uniform oil phase by stirring;
B) add in distilled water by bicarbonate of ammonia, obtain uniform aqueous phase by stirring, wherein ammonium bicarbonate concentration is 5% ~ 20% (w/v);
C), in the oil phase that the Aqueous dispersions obtained by step b obtains to step a, by homogeneous homogenization, form water in oil single emulsion, wherein the volume ratio of aqueous phase and oil phase is 1/1 ~ 1/20;
D) under 100 ~ 1000rpm stir speed (S.S.), single emulsion dispersion that step c obtains is obtained in the PVA aqueous solution double emulsion of water-in-oil-in-water;
E) continue to stir emulsion 2 ~ 12h with stir speed (S.S.) in steps d, the methylene dichloride in oil phase is volatilized, obtain the porous microsphere solidified;
F) porous microsphere of solidification is collected, with deionized water wash, freeze-drying subsequently;
G) by the porous microsphere of freeze-drying and 2 × 10
4~ 2 × 10
7individual microorganism or 50 ~ 1000 μ L contain 2 × 10
4~ 2 × 10
7the microorganism suspension of individual microorganism is inserted in mould jointly, is 0.5 ~ 7.38MPa at pressure, and temperature is process 10 ~ 1800s under the subcritical carbon dioxide condition of 20 ~ 40 DEG C, sinters into and carries microorganism porous microsphere support altogether.
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