CN104001219A - Method for sintering co-loading cell porous microsphere scaffold through subcritical carbon dioxide - Google Patents
Method for sintering co-loading cell porous microsphere scaffold through subcritical carbon dioxide Download PDFInfo
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Abstract
The invention discloses a method for sintering a co-loading cell porous microsphere scaffold through subcritical carbon dioxide. An ammonium bicarbonate aqueous solution serves as an aqueous phase, an organic solution of PLGA serves as an oil phase, the aqueous phase is dispersed in the oil phase, and water-in-oil single emulsion is formed through homogenization; the single emulsion is dispersed in a PVA aqueous solution to obtain water-in-oil-in-water multiple emulsion; stir is continued to make dichloromethane volatilize so as to obtain cured porous microspheres; the cured porous microspheres are collected, washed and freeze-dried; a mold is filled with the free-dried porous microspheres and a certain amount of cells together, and under the condition of subcritical carbon dioxide, the porous microsphere scaffold is obtained through sintering. According to the method for sintering the co-loading cell porous microsphere scaffold through subcritical carbon dioxide, under the mild conditions, the co-loading cell porous microsphere scaffold is obtained through a one-step method, and activity of the cells is ensured while the porous form of the surface of the microspheres is ensured.
Description
Technical field
The present invention relates to the preparation method of three-dimensional tissue's engineering rack, specifically relate to a kind of method that subcritical carbon dioxide sintering carries cell porous microsphere support altogether.
Background technology
The more ripe tissue construction thinking of the outer research of Present Domestic is the mode of (Top-down) from top to bottom, first prepare biomimetic scaffolds, again seed cell is inoculated on timbering material, carries out tissue construction by means such as perfusion technique, interpolation somatomedin, mechanical stimuluss.Main preparation methods comprises: solvent casting method, be separated/freeze-drying, and 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, the easy inactivation of bioactie agent, and cell compatibility is poor.The more important thing is the growth rhythm that cannot accurately control cell, be difficult to form the tissue of complex micro structure feature.And the structure thinking of modular organization engineering is the mode of (Bottom-up) from bottom to top, simulate the body of the people in natural situation, many tissues are exactly to be formed by little piece of tissue, as: fascicula forms muscle, lobules of liver forms liver etc., by the design forming of microstructure features being intended to ecological microstructure unit to build modular construction, then in order to build bulk tissue.
Sintering microsphere support technology is utilized modularization idea just, carries out tissue construction.First prepare the single microsphere of different component feature, recycle different sintering processings material is plastified, make to be bonded together each other between microsphere, form support, common sintering processing has thermal sintering, solvent sintered etc.Ultimate principle, for microsphere surface is melted by high temperature or solvent, is wound around adjacent microsphere strand, and when after temperature reduction or organic solvent volatilization, polymer molecular chain solidifies again, forms permanent cohering.(the Aronin CEP such as Aronin, Sadik KW, Lay AL, et al.Comparative effects of scaffold pore size, pore volume, and total void volume on cranial bone healing patterns using microsphere-based scaffolds.Journal of Biomedical Materials Research Part A, 2009, 89 (3): 632-641) with Poly(D,L-lactide-co-glycolide (poly (lactic-co-glycolic acid), PLGA) be material, 80 DEG C of sintering 3h in copper mould, prepare solid microsphere sintering support, (the Shi X such as Shi, Wang Y, Varshney RR, et al.Microsphere-based drug releasing scaffolds for inducing osteogenesis of human mesenchymal stem cells in vitro.European Journal of Pharmaceutical Sciences, 2010,39 (1-3): 59-67) seal dexamethasone with PLGA, beta-glycerophosphate and ascorbic acid, taking acetone/ethanol mixed solvent as agglutinant, prepare the solid microsphere component support that carries altogether three kinds of medicines.But high temperature and organic solvent be not only bad for the activity of protein medicaments, and residual organic solvent is difficult to remove, and cell compatibility is poor.Although attempting additive method, people prepare PLGA microsphere support, as (CN103212116A) such as Wang Yingjun discloses a kind of method that room temperature low pressure seasoning is prepared PLGA porous microsphere support; D Michael etc. (US8669107B2) disclose a kind of method that subcritical sintering carries cell PLGA microsphere support altogether, but all cannot realize under the prerequisite that ensures microsphere surface pattern and can carry altogether cell, or in the situation that carrying cell altogether, for cell provides the sites of sticking more, make cell to single microsphere unit growth inside, avoid because mass transfer is obstructed, cause vascularization regeneration to be obstructed and apoptotic problem.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, a kind of method that provides subcritical carbon dioxide sintering to carry altogether cell porous microsphere support, realize a step structure that carries altogether cell porous microsphere support, having reduced conventional stent needs the step of secondary inoculation cell, avoid the impact of high temperature and organic solvent, in retaining microsphere surface pattern, can also ensure that cell is to microsphere 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 is added in organic solvent dichloromethane, obtain uniform oil phase by stirring;
B) ammonium bicarbonate is added in distilled water, obtain uniform water by stirring, wherein ammonium bicarbonate concentration is 5%～20% (w/v);
C) water step b being obtained is distributed in the oil phase that step a obtains, and by homogenizing homogenization, forms water in oil single emulsion, and wherein the volume ratio of water and oil phase is 1/1～1/20;
D), under 100～1000rpm stir speed (S.S.), single emulsion dispersion that step c is obtained is to the double emulsion that obtains W/O/W in PVA aqueous solution;
E) continue to stir emulsion 2～12h with stir speed (S.S.) in steps d, make the dichloromethane volatilization in oil phase, obtain curing porous microsphere;
F) collect curing porous microsphere, with deionized water wash, subsequently lyophilizing;
G) by the porous microsphere of lyophilizing 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 to process 10～1800s under the subcritical carbon dioxide conditions of 20～40 DEG C, sinters into and carries altogether cell porous microsphere support.
In one embodiment: in step a, in described oil phase, the concentration of PLGA is 1%～15% (w/v), and PLGA molecular weight is 20～100kDa, and in PLGA, the mol ratio of LA and GA is 25/75～75/25.
In one embodiment: in step c, the condition of described homogenizing homogenize is: stir speed (S.S.) 3000～15000rpm, mixing time 2～10min.
In one embodiment: in steps d, in described PVA aqueous solution, PVA concentration is 0.1%～2% (w/v).
In one embodiment: described PLGA replaces by the one in PCL or PLA-PET.
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 is added in organic solvent dichloromethane, obtain uniform oil phase by stirring;
B) ammonium bicarbonate is added in distilled water, obtain uniform water by stirring, wherein ammonium bicarbonate concentration is 5%～20% (w/v);
C) water step b being obtained is distributed in the oil phase that step a obtains, and by homogenizing homogenization, forms water in oil single emulsion, and wherein the volume ratio of water and oil phase is 1/1～1/20;
D), under 100～1000rpm stir speed (S.S.), single emulsion dispersion that step c is obtained is to the double emulsion that obtains W/O/W in PVA aqueous solution;
E) continue to stir emulsion 2～12h with stir speed (S.S.) in steps d, make the dichloromethane volatilization in oil phase, obtain curing porous microsphere;
F) collect curing porous microsphere, with deionized water wash, subsequently lyophilizing;
G) by the porous microsphere of lyophilizing 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 to process 10～1800s under the subcritical carbon dioxide conditions of 20～40 DEG C, sinters into and carries altogether microorganism porous microsphere support.
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. the method that a kind of subcritical carbon dioxide sintering provided by the present invention carries cell porous microsphere support altogether, realize one-step method preparation and carried altogether cell porous microsphere support, bring following technique effect: a) with first sintering 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 be grown, and easily produces mass transfer and is obstructed, and causes vascularization regeneration to be obstructed and apoptosis, thereby causes tissue necrosis; And in the present invention, carry altogether cell and sintering support settles at one go, cell enters internal stent in the time of sintering support, can be on the surface of support and inside survive simultaneously and grow, the problem of having avoided mass transfer to be obstructed; C) compare traditional solid microsphere sintering support, porous microsphere sintering support has higher porosity, is beneficial to mass transfer and cell proliferation, and the higher specific surface area of porous microsphere is for cell attachment provides more site, is beneficial to efficient structure tissue.
2. the method that a kind of subcritical carbon dioxide sintering provided by the present invention carries cell porous microsphere support altogether, does not adopt high temperature and organic solvent, avoided the destruction to microsphere surface pattern, and organic solvent-free is residual, does not affect cells growth activity; Owing to not there is not the use of pyroprocess and organic solvent, the present invention also can be applied to field of medicaments, can not exert an influence to pharmaceutically active and safety.
3. the method that a kind of subcritical carbon dioxide sintering provided by the present invention carries cell porous microsphere support altogether, using subcritical carbon dioxide as agglutinant, volatile, noresidue, pollution-free, clean environment firendly more compared to existing technology.
Brief description of the drawings
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 altogether cell porous microsphere surface topography map.
Fig. 4 be embodiment 2 prepare carry altogether cell porous microsphere support shape appearance figure.
Fig. 5 be embodiment 2 prepare carry altogether cell porous microsphere support laser confocal microscope photo, in figure, white arrow is denoted as after subcritical carbon dioxide sintering, active good cell.
Fig. 6 be embodiment 3 prepare carry altogether cell porous microsphere support laser confocal microscope photo, in figure, white arrow is denoted as after subcritical carbon dioxide sintering, active good cell.
Detailed description of the invention
Illustrate content of the present invention below by embodiment:
Embodiment 1
Concrete implementation step is as follows:
A) under room temperature, PLGA is added in organic solvent dichloromethane, obtain uniform oil phase by stirring, wherein the concentration of PLGA is 6.25% (w/v), PLGA molecular weight is 40kDa, and LA in PLGA (lactic acid) is 50/50 with the mol ratio of GA (hydroxyacetic acid);
B) ammonium bicarbonate is added in distilled water, obtain uniform water by stirring, wherein ammonium bicarbonate concentration is 10% (w/v);
C) water 2.5ml step b being obtained is distributed in the oil phase 15ml that step a obtains, and by homogenizing homogenization, forms water in oil single emulsion, and wherein the volume ratio of water and oil phase is 1/6; The condition of described homogenizing homogenize is: stir speed (S.S.) 8000rpm, mixing time 4min;
D), under 200rpm stir speed (S.S.), in PVA (polyvinyl alcohol) aqueous solution of 0.5% (w/v) that single emulsion dispersion that step c is obtained is 400ml to volume, obtain the double emulsion of W/O/W;
E) continue to stir emulsion 4h with 200rpm stir speed (S.S.), make the dichloromethane volatilization in oil phase, obtain curing porous microsphere;
F) collect curing porous microsphere, use deionized water wash 3 times, lyophilizing 48h subsequently, obtains the well dried porous PLGA microsphere of surface topography;
G) porous microsphere of lyophilizing being inserted in politef mould, is 2MPa at pressure, and temperature is to process 10min under the subcritical carbon dioxide conditions of 25 DEG C, sinter porous microsphere support into, please refer to Fig. 1-2, the porous microsphere rack surface pattern obtaining is good, and average pore size is greater than 20 μ m.
Embodiment 2
Concrete implementation step is as follows:
A) under room temperature, PLGA is added 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 40kDa, and in PLGA, the mol ratio of LA and GA is 50/50;
B) ammonium bicarbonate is added in distilled water, obtain uniform water by stirring, wherein ammonium bicarbonate concentration is 10% (w/v);
C) water 2.5ml step b being obtained is distributed in the oil phase 12.5ml that step a obtains, and by homogenizing homogenization, forms water in oil single emulsion, and wherein the volume ratio of water and oil phase is 1/5; The condition of described homogenizing homogenize is: stir speed (S.S.) 8000rpm, mixing time 4min;
D), under 200rpm stir speed (S.S.), in the PVA aqueous solution of 0.5% (w/v) that single emulsion dispersion that step c is obtained is 400ml to volume, obtain the double emulsion of W/O/W;
E) continue to stir emulsion 4h with 200rpm stir speed (S.S.), make the dichloromethane volatilization in oil phase, obtain curing porous microsphere;
F) collect curing porous microsphere, use deionized water wash 3 times, lyophilizing 48h subsequently, obtains the well dried porous PLGA microsphere of surface topography;
G) by the porous microsphere of lyophilizing and 1 × 10
6individual mouse fibroblast cell mix homogeneously, inserts in politef mould jointly, is 3MPa at pressure, temperature is to process 4min under the subcritical carbon dioxide conditions of 25 DEG C, sinter into and carry altogether cell porous microsphere support, please refer to Fig. 3-4, the porous microsphere rack surface pattern obtaining is good; Subsequently by support by AO/EB (acridine orange/ethidium bromide) dyeing, and detect cytoactive with laser confocal microscope, as shown in Figure 5, in figure, white arrow is denoted as active good cell to testing result.As can be seen here, the present invention is obtained and is carried altogether cell porous microsphere sintering support by one-step method, in guaranteeing microsphere surface porous pattern, can also ensure cytoactive.
Embodiment 3
Concrete implementation step is as follows:
A) under room temperature, PLGA is added 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 40kDa, and in PLGA, the mol ratio of LA and GA is 50/50;
B) ammonium bicarbonate is added in distilled water, obtain uniform water by stirring, wherein ammonium bicarbonate concentration is 10% (w/v);
C) water 2.5ml step b being obtained is distributed in the oil phase 8ml that step a obtains, and by homogenizing homogenization, forms water in oil single emulsion, and wherein the volume ratio of water and oil phase is 1/3.2; The condition of described homogenizing homogenize is: stir speed (S.S.) 5000rpm, mixing time 3min;
D), under 200rpm stir speed (S.S.), in the PVA aqueous solution of 0.1% (w/v) that single emulsion dispersion that step c is obtained is 400ml to volume, obtain the double emulsion of W/O/W;
E) continue to stir emulsion 4h with 200rpm stir speed (S.S.), make the dichloromethane volatilization in oil phase, obtain curing porous microsphere;
F) collect curing porous microsphere, use deionized water wash 3 times, lyophilizing 48h subsequently, obtains the well dried porous PLGA microsphere of surface topography;
G) by the porous microsphere of lyophilizing with contain 2 × 10
6500 μ l cell suspension mix homogeneously of individual mouse fibroblast cell, insert in politef mould jointly, are 3MPa at pressure, and temperature is to process 2min under the subcritical carbon dioxide conditions of 25 DEG C, sinters into and carries altogether cell porous microsphere support; Proceeded to subsequently in the culture medium of 1ml, dye 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 testing result.As can be seen here, the present invention is obtained and is carried altogether cell porous microsphere sintering support by one-step method, and cytoactive is good.
Embodiment 4
Concrete implementation step is as follows:
A) under room temperature, PLGA is added in organic solvent dichloromethane, obtain uniform oil phase by stirring, wherein the concentration of PLGA is 1% (w/v), and PLGA molecular weight is 100kDa, and in PLGA, the mol ratio of LA and GA is 75/25;
B) ammonium bicarbonate is added in distilled water, obtain uniform water by stirring, wherein ammonium bicarbonate concentration is 20% (w/v);
C) water 2.5ml step b being obtained is distributed in the oil phase 50ml that step a obtains, and by homogenizing homogenization, forms water in oil single emulsion, and wherein the volume ratio of water and oil phase is 1/20; The condition of described homogenizing homogenize is: stir speed (S.S.) 15000rpm, mixing time 2min;
D), under 1000rpm stir speed (S.S.), in the PVA aqueous solution of 2% (w/v) that single emulsion dispersion that step c is obtained is 400ml to volume, obtain the double emulsion of W/O/W;
E) continue to stir emulsion 2h with 1000rpm stir speed (S.S.), make the dichloromethane volatilization in oil phase, obtain curing porous microsphere;
F) collect curing porous microsphere, use deionized water wash 3 times, lyophilizing 48h subsequently, obtains the well dried porous PLGA microsphere of surface topography;
G) by the porous microsphere of lyophilizing with contain 2 × 10
450 μ l cell suspension mix homogeneously of individual mouse fibroblast cell, insert in politef mould jointly, are 0.5MPa at pressure, and temperature is to process 30min under the subcritical carbon dioxide conditions of 20 DEG C, sinters into and carries altogether cell porous microsphere support.
Embodiment 5
Concrete implementation step is as follows:
A) under room temperature, PLGA is added in organic solvent dichloromethane, obtain uniform oil phase by stirring, wherein the concentration of PLGA is 15% (w/v), and PLGA molecular weight is 20kDa, and in PLGA, the mol ratio of LA and GA is 25/75;
B) ammonium bicarbonate is added in distilled water, obtain uniform water by stirring, wherein ammonium bicarbonate concentration is 5% (w/v);
C) water 2.5ml step b being obtained is distributed in the oil phase 2.5ml that step a obtains, and by homogenizing homogenization, forms water in oil single emulsion, and wherein the volume ratio of water and oil phase is 1/1; The condition of described homogenizing homogenize is: stir speed (S.S.) 3000rpm, mixing time 10min;
D), under 100rpm stir speed (S.S.), in the PVA aqueous solution of 1% (w/v) that single emulsion dispersion that step c is obtained is 400ml to volume, obtain the double emulsion of W/O/W;
E) continue to stir emulsion 12h with 100rpm stir speed (S.S.), make the dichloromethane volatilization in oil phase, obtain curing porous microsphere;
F) collect curing porous microsphere, use deionized water wash 3 times, lyophilizing 48h subsequently, obtains the well dried porous PLGA microsphere of surface topography;
G) by the porous microsphere of lyophilizing with contain 2 × 10
71000 μ l cell suspension mix homogeneously of individual mouse fibroblast cell, insert in politef mould jointly, are 7.38MPa at pressure, and temperature is to process 10s under the subcritical carbon dioxide conditions of 40 DEG C, sinters into and carries altogether cell porous microsphere support.
In addition, those skilled in the art can learn, the present invention has utilized the PLGA can be swelling by subcritical carbon dioxide, cause the characteristic that material surface plastifies and is bonded together, therefore, other can, by the swelling material that causes that surface plastifies and coheres 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 sintering of the present invention carries cell porous microsphere altogether.
Those skilled in the art can learn, of the present invention with the once sintered cell that is shaped to porous microsphere support of material, and its requirement is can keep under undercritical conditions active; Therefore, other can keep active organism under undercritical conditions, the microorganisms such as such as Nitrobacter, Rhodopseudomonas, Penicillium, yeast cells, magnetotactic bacteria, are all suitable for the method that subcritical carbon dioxide sintering of the present invention carries cell porous microsphere altogether.
The above, only for preferred embodiment of the present invention, therefore can not limit according to this scope of the invention process, the equivalence of doing according to the scope of the claims of the present invention and description changes and modifies, and all should still belong in the scope that the present invention contains.
Claims (6)
1. the method that subcritical carbon dioxide sintering carries cell porous microsphere support altogether, is characterized in that: comprising:
A) PLGA is added in organic solvent dichloromethane, obtain uniform oil phase by stirring;
B) ammonium bicarbonate is added in distilled water, obtain uniform water by stirring, wherein ammonium bicarbonate concentration is 5%～20% (w/v);
C) water step b being obtained is distributed in the oil phase that step a obtains, and by homogenizing homogenization, forms water in oil single emulsion, and wherein the volume ratio of water and oil phase is 1/1～1/20;
D), under 100～1000rpm stir speed (S.S.), single emulsion dispersion that step c is obtained is to the double emulsion that obtains W/O/W in PVA aqueous solution;
E) continue to stir emulsion 2～12h with stir speed (S.S.) in steps d, make the dichloromethane volatilization in oil phase, obtain curing porous microsphere;
F) collect curing porous microsphere, with deionized water wash, subsequently lyophilizing;
G) by the porous microsphere of lyophilizing 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 to process 10～1800s under the subcritical carbon dioxide conditions of 20～40 DEG C, sinters into and carries altogether cell porous microsphere support.
2. the method that a kind of subcritical carbon dioxide sintering according to claim 1 carries cell porous microsphere support altogether, it is characterized in that: in step a, in described oil phase, the concentration of PLGA is 1%～15% (w/v), PLGA molecular weight is 20～100kDa, and in PLGA, the mol ratio of LA and GA is 25/75～75/25.
3. the method that a kind of subcritical carbon dioxide sintering according to claim 1 carries cell porous microsphere support altogether, is characterized in that: in step c, the condition of described homogenizing homogenize is: stir speed (S.S.) 3000～15000rpm, mixing time 2～10min.
4. the method that a kind of subcritical carbon dioxide sintering according to claim 1 carries cell porous microsphere support altogether, is characterized in that: in steps d, in described PVA aqueous solution, PVA concentration is 0.1%～2% (w/v).
5. the method that a kind of subcritical carbon dioxide sintering according to claim 1 carries cell porous microsphere support altogether, is characterized in that: the one in described PLGA PCL or PLA-PET replaces.
6. the method that subcritical carbon dioxide sintering carries microorganism porous microsphere support altogether, is characterized in that: comprising:
A) PLGA is added in organic solvent dichloromethane, obtain uniform oil phase by stirring;
B) ammonium bicarbonate is added in distilled water, obtain uniform water by stirring, wherein ammonium bicarbonate concentration is 5%～20% (w/v);
C) water step b being obtained is distributed in the oil phase that step a obtains, and by homogenizing homogenization, forms water in oil single emulsion, and wherein the volume ratio of water and oil phase is 1/1～1/20;
D), under 100～1000rpm stir speed (S.S.), single emulsion dispersion that step c is obtained is to the double emulsion that obtains W/O/W in PVA aqueous solution;
E) continue to stir emulsion 2～12h with stir speed (S.S.) in steps d, make the dichloromethane volatilization in oil phase, obtain curing porous microsphere;
F) collect curing porous microsphere, with deionized water wash, subsequently lyophilizing;
G) by the porous microsphere of lyophilizing 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 to process 10～1800s under the subcritical carbon dioxide conditions of 20～40 DEG C, sinters into and carries altogether microorganism porous microsphere support.
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