CN100493626C - Cell culturing rack material and its preparation - Google Patents
Cell culturing rack material and its preparation Download PDFInfo
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- CN100493626C CN100493626C CNB2006100410177A CN200610041017A CN100493626C CN 100493626 C CN100493626 C CN 100493626C CN B2006100410177 A CNB2006100410177 A CN B2006100410177A CN 200610041017 A CN200610041017 A CN 200610041017A CN 100493626 C CN100493626 C CN 100493626C
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Abstract
The present invention discloses one kind of cell culturing rack material and its preparation process. The cell culturing rack material is prepared with silk as main material, and through degumming, dissolving, purifying and drying to form fimbrin; dissolving together with collagen or gelatin in the same kind of solvent; high voltage electrostatic spinning to obtain 3D netted nanometer fiber non-woven felt of fimbrin base material, collagen, gelatin, etc. and final organic alcohol treatment. The cell culturing rack material has average fiber diameter smaller than 100 nm, average pore size of 1.0-5.0 microns and porosity of 70-90%. Test shows that the nanometer fiber material has no toxicity, no irritation, no sensibilization, excellent tissue compatibility and perforated pore structure, and may be used as cell culturing rack material for repairing human body tissue.
Description
Technical field
The present invention relates to a kind of biomaterial for medical purpose and preparation method thereof, being particularly related to a kind of is base material with the fibroin albumen layer, cell culturing rack material of the nanofiber non-woven mat structure that is composited with the mixture of collagen protein, gelatin or collagen protein, gelatin and fibroin albumen and preparation method thereof belongs to technical field of polymer materials.
Background technology
Before the present invention makes, once adopted natural or synthetic high polymer is made nanofiber nonwoven film as cell culture vector through electrostatic spinning, for example: PCL, PLA, PLGA, collagen protein etc., former three has hydrophobicity, is unfavorable for the adhesion of cell in hydrophilic biotic environment; The latter is the hydrophilic natural high polymer, helps the adhesion growth of cell, but because spinnability and intensity are relatively poor, and cost an arm and a leg, be difficult to really come into operation.
The native protein that fibroin albumen is made up of 18 seed amino acids has bigger similarity with the collagen protein of human body, has the favorable tissue compatibility, and it is a kind of hydrophilic high polymer, helps the adhesion growth of cell in hydrophilic biotic environment; Fibroin albumen derives from silk fiber, and the source is abundant, cheap, easy to prepare.Therefore fibroin albumen is as the ideal basic material of tissue engineering material.
Publication number is that the Chinese invention patent " a kind of porous fibroin membrane and preparation method thereof " of CN1260363A discloses a kind of perforated membrane, it is main film forming raw material with silkworm silk, adopt vacuum freeze-drying method to make porous fibroin membrane, thickness, the porosity that this method makes is lower, the hole up/down perforation is relatively poor, is unfavorable for the growth and the cultivation of cell.
Summary of the invention
Purpose of the present invention overcomes the deficiency that prior art exists, and provides a kind of both good with the human body compatibility, has excellent cell growing environment again, and cell culturing rack material easy to prepare and preparation method thereof.
The technical solution used in the present invention is: a kind of cell culturing rack material, and it is made up of the material of two-layer three-dimensional netted nanofiber non-woven mat structure, and base material is a raw material with the fibroin albumen layer; Compound another layer raw material is selected from the mixture of collagen protein, gelatin, collagen protein and fibroin albumen or the mixture of gelatin and fibroin albumen on it; The average fibre diameter of described nanofiber non-woven mat is 40～100nm, and average pore size is 1.0～5.0 μ m, and porosity is 70～90%; In the weight of entire bracket material, contain 1～5% additive; Described additive is one or more in sodium borate, calcium chloride, the cerous acetate.
Optimized technical scheme is, in the weight of entire bracket material, the content of fibroin albumen is 50～79%, and the content of collagen protein or gelatin is 20～45%.
The preparation method of a kind of cell culturing rack material of the present invention, earlier with silkworm silk through come unstuck, after dissolving, dialysis, purification make the pure silk cellulose solution, drying makes pure fibroin albumen, carries out the processing of following steps again:
A, the mixture of above-mentioned pure fibroin albumen, collagen protein, gelatin or collagen protein, gelatin and fibroin albumen is dissolved in respectively in the polar solvent of the same race makes solution, described polar solvent is a kind of in hexafluoroisopropanol, trifluoroethanol, anhydrous formic acid and 88% formic acid;
B, in the above-mentioned solution for preparing, add 1～5% additive respectively, described additive is one or more in sodium borate, calcium chloride, the cerous acetate, makes mass fraction and be the spinning solution of the mixture of the mixture of 10～20% fibroin albumen, collagen protein, gelatin, collagen protein and fibroin albumen or gelatin and fibroin albumen;
C, elder generation adopt electrostatic spinning process with the fibroin albumen spinning solution, obtain three-dimensional netted nanofiber fibroin albumen non-woven mat on dash receiver;
D, the spinning solution with the mixture of the mixture of collagen protein, gelatin, collagen protein and fibroin albumen or gelatin and fibroin albumen adopts electrostatic spinning process again, obtaining with the fibroin albumen non-woven mat on dash receiver is base material, the nanofiber non-woven mat of the mixture of the three-dimensional netted collagen protein of compound another layer, gelatin, collagen protein and fibroin albumen or the mixture of gelatin and fibroin albumen;
E, with 50% methanol, absolute methanol, dehydrated alcohol or methanol: the ethanol ratio is a kind of in 1: 1 the mixed alcohol, and the nanofiber non-woven mat impregnation process 0.5～1h to above-mentioned gained obtains cell culturing rack material behind the vacuum drying.
In the technique scheme, described electrostatic spinning process condition is: electrostatic pressure 15～34Kv, spinning distance are that 8～20cm, spinning liquid feed quantity are 0.1～5.0ml/h.And can carry out the gamma-ray irradiation sterilization to the cell culturing rack material that makes.
In technical scheme of the present invention, select a kind of solvent in hexafluoroisopropanol, trifluoroethanol, anhydrous formic acid and 88% formic acid for use as the preparation spinning solution, be because they are not only a kind of easy evaporable polar solvent, simultaneously, it also is the cosolvent of fibroin albumen, collagen protein or gelatin, help improving spinning properties and uniformity, two layer components of formed composite nano fiber in conjunction with firmly, do not come off, do not separate.
Additive sodium borate in the spinning solution, calcium chloride, cerous acetate are electrolyte, and it can increase the charge density on the spinning solution surface of injection during electrostatic spinning, thereby increase the drafting force of jetting fluid under electric field, help forming even successive nanofiber.
Compared with prior art, the invention has the advantages that:
1, a kind of cell culturing rack material provided by the present invention is with fibroin albumen and collagen or gelatin-compounded forming, used material is the native protein for being made up of 18 seed amino acids all, with human collagen albumen bigger similarity is arranged, therefore, with human body excellent tissue compatibility, nontoxic, non-stimulated and sensitization are arranged; Especially collagen protein and gelatin, collagen protein is by the synthetic a kind of biopolymer of zooblast, is the Main Ingredients and Appearance of concluding tissue; Gelatin is to form by collagen protein such as the intravital bone of animal, muscle, tendon are refining, has better tissue compatibility with human body.
2, employed native protein is the hydrophilic high polymer, and the hydrophilic biotic environment of cell growth requirement is provided, and helps adhesion, the propagation of cell; Especially collagen and gelatin, the structure and the biological property of their structure and extracellular matrix protein are closely similar, and more can promote cell adhesion, propagation and growth than fibroin, therefore, with the silk fibroin nano-fiber felt is base material, the mixture nanofiber mats of the mixture of compound one deck collagen protein, gelatin, collagen protein and fibroin albumen or gelatin and fibroin albumen on it, not only provide better growing environment to cell, and, greatly reduce the cost of cell culturing rack material, and overcome the defective of collagen or gelatin fiber felt intensity difference.
3, the nanofiber non-woven mat that adopts electrostatic spinning technique to obtain is tridimensional network, have fiber finer, characteristics that specific surface area is big, form the hole of a large amount of up/down perforations between fiber, it is consistent with the structure of extracellular matrix, this structural environment not only helps the growth of cell, and can make cell easily move to internal stent, fibroblast, smooth muscle cell or people's umbilical blood vessels endotheliocyte etc. all there are good adhesion, proliferation function, can be used for the reparation of tissues such as human body skin wound surface or blood vessel.
4, in technical scheme of the present invention, resulting nanofiber mats is carried out organic alcohol handle, on the one hand, can make proteic random-coil conformation partly be converted into the βZhe Die structure, the nanofiber film strength is improved, and dissolubility reduces, and more helps the support as cell culture; On the other hand, handle, can remove solvent residual in the nanofiber mats, remove organic alcohol during vacuum drying again, guaranteed that finished-product material has better cell culture environment through organic alcohol.
Description of drawings
Fig. 1 is scanning electron microscope (SEM) figure of the cell culturing rack material that makes by the technical scheme that the embodiment of the invention 1 is provided;
Fig. 2 is the SEM figure of the cell culturing rack material for preparing by the technical scheme that the embodiment of the invention 2 is provided.
The specific embodiment
The invention will be further elaborated below in conjunction with embodiment.
Embodiment one:
1. the useless mulberry silk of 0.2kg is put into 6L concentration and be 0.05% aqueous sodium carbonate, boil 0.5h, the sericin of most silkworm silk periphery is taken off in reprocessing three times, obtains fibroin, dries under the room temperature;
2. be 1: 8: 2 calcium chloride, water, alcoholic solution with dry fibroin with the 1.2L mol ratio, become silk fibroin protein solution 78 ± 2 ℃ of following heating for dissolving;
3. the silk fibroin protein solution that step 2 is made is poured in the cellulose bag filter, earlier with the tap water dialysis, and the dialysis of reuse deionized water, to remove ethanol and the calcium chloride micromolecule in the solution, reuse multilamellar absorbent carbasus filters, and makes pure silk fibroin solution;
4. getting 100ml pure silk fibroin solution respectively, to pour area into be 20 * 20cm
2The polystyrene plastics dish in, constant temperature and humidity (25 ℃, RH65%) following drying and forming-film, it is stand-by to take off the pack of regenerated silk film;
5. take by weighing 11.7g regenerated silk film, add polar solvent anhydrous formic acid 68g, at room temperature stir 30min, add additive sodium borate 0.2g again, stir into uniform regenerated silk spinning liquid.Spinning liquid mass fraction 14.8%.
6. take by weighing the 3.0g medical gelatin, add polar solvent anhydrous formic acid 18g, at room temperature stir 30min, add additive sodium borate 0.1g again, stir into uniform gelatin spinning liquid.Spinning liquid mass fraction 14.7%.
7. the regenerated silk spinning liquid that step 5 is made is poured micro-injection pump into.By pump spinning liquid is injected in the spin duct.The anode of electrostatic generator is inserted in the spin duct, negative electrode is connected on the metal dash receiver, the electrostatic pressure of regulating electrostatic generator is 30kV, the spinning distance is 15cm, open electrostatic generator and micro-injection pump, it is 1.0ml/h that the adjusting micro-injection pump makes the feed quantity of spinning liquid, and spinning liquid sprays continuously, the polar solvent anhydrous formic acid volatilizees in air, can collect the fibroin nanofiber mats on receiver sheet.
8. pour the gelatin spinning liquid of step 6 gained into micro-injection pump.By pump spinning liquid is injected in the spin duct, carry out electrostatic spinning, the electrostatic pressure 26kV of electrostatic spinning, spinning is apart from 13cm, spinning liquid feed quantity 1.0ml/h.Dash receiver is the established fibroin nanofiber mats of step 7, and promptly composite number layer gelatine nano fiber felt on the fibroin nanofiber mats forms gelatin/fibroin composite nanometer fiber felt 15g.Referring to accompanying drawing 1, by scanning electron microscope (SEM) figure as can be seen, the average diameter of fiber is about that the hole average pore size is about 1.2um between 95nm, fiber, porosity is about 78%.Can make area is 15 * 15cm
2, thickness is about 30 of the non-woven mat of 0.015mm.
9 areas with step 8 gained are 15 * 15cm
2The fiber non-woven felt under room temperature, put into the 30ml absolute methanol and flood 0.5h, fiber felt is taken out put into vacuum drying oven evacuation 24h subsequently, pack and on the high frequency sealing machine, seal with the double-layer plastic bag, sterilize standby with gamma-ray irradiation then.
Embodiment two.
1. it is 0.05% aqueous sodium carbonate that the silkworm silk of 0.1kg being got a foothold is put into 3L concentration, boils 0.5h, and the sericin of most silkworm silk periphery is taken off in reprocessing three times, obtains fibroin;
2. be 1: 8: 2 calcium chloride, water, alcoholic solution with the dry fibroin of step 1 gained with the 0.6L mol ratio, become silk fibroin protein solution 78 ± 2 ℃ of following heating for dissolving;
3. above-mentioned silk fibroin protein solution is poured in the cellulose bag filter, earlier with the tap water dialysis, the dialysis of reuse deionized water, to remove ethanol and the calcium chloride micromolecule in the solution, reuse multilamellar absorbent carbasus filters, and makes pure silk fibroin solution;
4. getting 100ml pure silk fibroin solution respectively, to pour area into be 20 * 20cm
2Stainless steel disc in, at-20 ℃ of following freezing 8h, place vacuum drying 20h in the freezer dryer again, make spongy regenerated silk film.
5. take by weighing 4.68g regenerated silk film, add in the 42.66g trifluoroethanol, at room temperature stirred 30 minutes, add additive cerous acetate 0.06g again, be stirred to CL, the spinning liquid mass fraction is 10%.
6. take by weighing 1.2g type i collagen albumen, add in the 11.34g trifluoroethanol, at room temperature stirred 30 minutes, add additive cerous acetate 0.06g again, be stirred to CL, the spinning liquid mass fraction is 10%.
7. the regenerated silk spinning liquid that step 5 is made is poured micro-injection pump into.By pump spinning liquid is injected in the spin duct.The anode of electrostatic generator is fixed on the metal needle before the spin duct, negative electrode is connected on the metal dash receiver, the electrostatic pressure of regulating electrostatic generator is 30kV, the spinning distance is 15cm, open electrostatic generator and micro-injection pump, regulating micro-injection pump, to make the feed quantity of spinning liquid be 1.5ml/h, and the metal needle of spinning liquid before by spin duct sprays continuously, the polar solvent trifluoroethanol volatilizees in air, can collect the fibroin nanofiber mats on receiver sheet.
8. pour the collagen spinning liquid of step 6 gained into micro-injection pump.By pump spinning liquid is injected in the spin duct, carry out electrostatic spinning, the electrostatic pressure 26kV of electrostatic spinning, spinning is apart from 13cm, spinning liquid feed quantity 1.0ml/h.Dash receiver is the established fibroin nanofiber mats of step 7, i.e. composite number layer collagen nanofiber felt on the fibroin nanofiber mats, form collagen/fibroin composite nanometer fiber felt 6g, referring to accompanying drawing 2, by scanning electron microscope (SEM) figure as can be seen, the average diameter of its fiber is about that the hole average pore size is about 1.0um between 80nm, fiber, porosity is about 70%.Can make area is 15 * 15cm
2, thickness is about 12 of the non-woven mat of 0.020mm.
9 areas with step 8 gained are 15 * 15cm
2The fiber non-woven felt to put into 30ml absolute methanol and dehydrated alcohol under room temperature be that 1: 1 mixed alcohol floods 1h, subsequently fiber felt is taken out and put into vacuum drying oven evacuation 24h, pack and on the high frequency sealing machine, seal with the double-layer plastic bag, standby with the gamma-ray irradiation sterilization then.
Embodiment three.
1. the useless mulberry silk of 0.2kg is put 6L concentration and be 0.05% aqueous sodium carbonate, boil 0.5h, the sericin of most silkworm silk periphery is taken off in reprocessing three times, obtains fibroin, dries under the room temperature;
2. be 1: 8: 2 calcium chloride, water, alcoholic solution with dry fibroin with the 1.2L mol ratio, become silk fibroin protein solution 78 ± 2 ℃ of following heating for dissolving;
3. the silk fibroin protein solution that step 2 is made is poured in the cellulose bag filter, earlier with the tap water dialysis, and the dialysis of reuse deionized water, to remove ethanol and the calcium chloride micromolecule in the solution, reuse multilamellar absorbent carbasus filters, and makes pure silk fibroin solution;
4. getting 100ml pure silk fibroin solution respectively, to pour area into be 10 * 20cm
2The polystyrene plastics dish in, constant temperature and humidity (25 ℃, RH65%) following drying and forming-film, it is stand-by to take off the pack of regenerated silk film;
5. take by weighing 8g regenerated silk film, add polar solvent anhydrous formic acid 59.8g, at room temperature stir 30min, add additive sodium borate 0.15g again, stir into uniform regenerated silk spinning liquid; Spinning liquid mass fraction 12%.
6. take by weighing 4.0g medical gelatin and regenerated silk film 1.2g, add polar solvent anhydrous formic acid 39.2g, at room temperature stir 30min, add additive sodium borate 0.15g again, stir into uniform gelatin-fibroin spinning liquid; Spinning liquid mass fraction 12%.
7. the regenerated silk spinning liquid that step 5 is made is poured micro-injection pump into.By pump spinning liquid is injected in the spin duct.The anode of electrostatic generator is inserted in the spin duct, negative electrode is connected on the metal dash receiver, the electrostatic pressure of regulating electrostatic generator is 30kV, the spinning distance is 15cm, open electrostatic generator and micro-injection pump, it is 1.0ml/h that the adjusting micro-injection pump makes the feed quantity of spinning liquid, and spinning liquid sprays continuously, the polar solvent anhydrous formic acid volatilizees in air, can collect the fibroin nanofiber mats on receiver sheet.
8. pour the gelatin spinning liquid of step 6 gained into micro-injection pump.By pump spinning liquid is injected in the spin duct, 7 carry out electrostatic spinning set by step, dash receiver is the established fibroin nanofiber mats of step 7, i.e. composite number layer gelatin-fibroin nanofiber mats on the fibroin nanofiber mats, form gelatin-fibroin/fibroin composite nanometer fiber felt 13.5g, the average diameter of fiber is about that the about 1.0um of hole average pore size, porosity are about 75% between 89nm, fiber.Can make area is 15 * 15cm
2, thickness is about 22 of the non-woven mat of 0.020mm.
9 areas with step 8 gained are 15 * 15cm
2The fiber non-woven felt under room temperature, put into the 30ml absolute methanol and flood 0.5h, fiber felt is taken out put into vacuum drying oven evacuation 24h subsequently, pack and on the high frequency sealing machine, seal with the double-layer plastic bag, sterilize standby with gamma-ray irradiation then.
Claims (4)
1, a kind of cell culturing rack material is characterized in that: it is made up of the material of two-layer three-dimensional netted nanofiber non-woven mat structure, and base material is a raw material with the fibroin albumen layer; Compound another layer raw material is selected from the mixture of collagen protein, gelatin, collagen protein and fibroin albumen or the mixture of gelatin and fibroin albumen on it; The average fibre diameter of described nanofiber non-woven mat is 40～100nm, and average pore size is 1.0～5.0 μ m, and porosity is 70～90%; In the weight of entire bracket material, contain 1～5% additive; Described additive is one or more in sodium borate, calcium chloride, the cerous acetate.
2, cell culturing rack material according to claim 1 is characterized in that: in the weight of entire bracket material, the content of fibroin albumen is 50～79%, and the content of collagen protein or gelatin is 20～45%.
3, a kind of preparation method of cell culturing rack material as claimed in claim 1, earlier with silkworm silk through come unstuck, after dissolving, dialysis, purification make the pure silk cellulose solution, drying makes pure fibroin albumen, it is characterized in that carrying out the processing of following steps again:
A, the mixture of the mixture of above-mentioned pure fibroin albumen, collagen protein, gelatin, collagen protein and fibroin albumen or gelatin and fibroin albumen is dissolved in respectively in the polar solvent of the same race makes solution, described polar solvent is a kind of in hexafluoroisopropanol, trifluoroethanol, anhydrous formic acid and 88% formic acid;
B, in the above-mentioned solution for preparing, add the additive of 1～5% weight respectively, described additive is one or more in sodium borate, calcium chloride, the cerous acetate, makes mass fraction and be the spinning solution of the mixture of the mixture of 10～20% fibroin albumen, collagen protein, gelatin, collagen protein and fibroin albumen or gelatin and fibroin albumen;
C, elder generation adopt electrostatic spinning process with the fibroin albumen spinning solution, obtain three-dimensional netted nanofiber fibroin albumen non-woven mat on dash receiver;
D, the spinning solution with the mixture of the mixture of collagen protein, gelatin, collagen protein and fibroin albumen or gelatin and fibroin albumen adopts electrostatic spinning process again, obtaining with the fibroin albumen non-woven mat on dash receiver is base material, the nanofiber non-woven mat of the mixture of the three-dimensional netted collagen protein of compound another layer, gelatin, collagen protein and fibroin albumen or the mixture of gelatin and fibroin albumen;
E, with 50% methanol, absolute methanol, dehydrated alcohol or methanol: a kind of than in the mixed alcohol of 1:1 of ethanol to the nanofiber non-woven mat impregnation process 0.5～1h of above-mentioned gained, obtains cell culturing rack material behind the vacuum drying;
The electrostatic spinning process condition is described in above-mentioned steps c and the steps d: electrostatic pressure 15～34Kv, spinning distance are that 8～20cm, spinning liquid feed quantity are 0.1～5.0ml/h.
4, the preparation method of a kind of cell culturing rack material according to claim 3 is characterized in that: with the cell culturing rack material that makes again through gamma-ray irradiation.
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| CN102813965B (en) * | 2012-08-09 | 2015-04-01 | 东华大学 | Multilayer well-organized silk fibroin tissue engineering scaffold and preparation method thereof |
| CN104288835B (en) * | 2013-10-12 | 2016-08-03 | 上海市第六人民医院 | A kind of albumen compound rest that may be used for urethra reconstruction and preparation method thereof |
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| CN104307038B (en) * | 2013-10-12 | 2017-02-01 | 上海市第六人民医院 | Gelatin-containing protein composite scaffold and preparation method thereof |
| CN103877622B (en) * | 2014-03-26 | 2016-04-20 | 中山大学 | A kind of Electrospun nano-fibers-ECM coupled biomaterial and its preparation method and application |
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| CN113308937A (en) * | 2021-05-28 | 2021-08-27 | 陕西科技大学 | Cell culture paper and preparation method and application thereof |
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